WO2020243768A1 - Material for a bioresorbable implant, bioresorbable implant and method of making a bioresorbable implant - Google Patents

Abstract

A material for a bioresorbable implant and/or a bioresorbable implant is described. The material and implant comprise a collagen solution or a collagen and one or more magnesium wire to provide a collagen-based composite membrane; wherein the one or more magnesium wire is coated and/or surface modified with tannic acid. A method of making the bioresorbable implant and optionally a method of surgery comprising inserting the bioresorbable implant are also described. The one or more wires may be further coated and/or surface modified with Magnesium, optionally in the form of Mg-phenolic networks. The coating may advantageously result in pH neutralization and/or passivation of the one or more magnesium wires. The coating may be with a Mg-polyphenolic film and may form a porous oxide layer. The one or more wire may comprise a one layer; two layer; three layer or higher ordered layered pattern.

Description

MATERIAL FOR A BIORESORBABLE IMPLANT.

BIORESORBABLE IMPLANT AND METHOD OF MAKING A BIORESORBABLE IMPLANT

FIELD OF THE INVENTION

[0001] The present invention relates to a material for a bioresorbable implant, a bioresorbable material and a method of making a bioresorbable implant. More particularly, this invention relates to a material for a bioresorbable implant, a bioresorbable implant and a method of making a bioresorbable implant comprising an improved surface.

BACKGROUND TO THE INVENTION

[0002] Tooth loss is a common dental problem with most of the people during their life. Periodontal disease, poor oral hygiene, dental cavities, and accident are the main reasons for tooth loss. Statistically, about 69% of adults who are 35 to 44 years of age have lost at least one permanent tooth in their life. Additional ly, more than one quarter of adults have lost all of their permanent teeth by the age of 74 (1,2).

[0003] Placement of dental implants is conventionally known as a cost effective method for supporting the jawbone when a single or some of a natural tooth is missing (2). Conventional dental implants are metal frames that are placed into the jawbone to provide a suitable support for mounting the artificial tooth. Approximately 100,000 to 300,000 dental implants are placed each year, which is about equal to the numbers of placement of hip and knee joints implants (3). Commercial ly, the global dental implants market is expected to be $6.81 billion by 2024.

[0004] The density of jawbone in the tooth loss area is a critical ly important parameter that should be considered by the surgeon before any cl inical operations. Dental implants require dense bone to support their structure. In some cases when a tooth is extracted from the jaw, the bone surrounding the socket area is unable to fil l and consequently the height and width of the alveolar ridge bone will continue to decrease, especially for people who suffer from osteoporosis. Hence, in such cases reconstruction of the height and width of the alveolar ridge is vitally necessary before the placement of dental implant.

[0005] Ridge augmentation (RA) is conventionally acknowledged as the gold standard dental operation performed before the placement of dental implants in order to regenerate the natural contour of the jawbone to accommodate an implant properly (4). Guided bone regeneration (GBR) is known as one of the popular treatment modalities for alveolar ridge reconstruction (5,6). In this method, graft materials are utilized for the fill ing and reshaping of the alveolar ridge, which is wrapped and protected by the barrier membrane. The role of the barrier membrane is critical ly important and affects the success of ridge augmentation directly.

[0006] There are some difficulties with the application of any type of barrier membrane which influences their performance. Because they have some valuable properties, like proper bioactivity and angiogenesis capacity, biodegradable membranes may be preferable compared to non-biodegradable membranes. However, there are stil l some weaknesses in terms of mechanical properties such as, poor bending stiffness, weak tearing resistance and poor anti-collapsing behaviour. The provision of stronger biodegradable membranes is desirable.

[0007] A significant challenge to the uti l ization of barrier membranes as a cover for the graft is that it often tends to collapse in the grafted space, which can cause damage and consequently decrease the volume of the graft in the ridge defect area and prevent proper formation of new bone which is required for a desirable contour of the gums and jaw. There is no need for concern of the membrane collapsing for osseous defects which have proper walls to maintain tenting of the barrier membrane. This kind of defect which is referred to as a ‘favourable osseous defect’ can prepare adequate space for new bone regeneration Whereas, in an‘unfavourable alveolar ridge defect’ the barrier membrane will definitely collapse in the graft volume (7).

[0008] Conventionally, stabil ization screws are used to prevent collapsing of a barrier membrane in cl inical appl ications. However, this method is not successful enough to provide a desirable ridge contour for implantation. As a complementary approach and especially in lateral RA, tent screws may be used for a better contour formation. This kind of screw is placed on the host bone to maintain an adequate height and/or width for the graft volume. The main problems with the util ization of tent screws are potential difficulty in proper positioning as well as the necessity of second surgery to remove them (7,8).

[0009] Similarly, titanium (Ti) mesh is often appl ied in RA of vertical and compound defects (Class II and Class I I I). This technique was first used in 1985 by Dr Boyne for augmentation of the atrophic alveolus to deduct graft resorption without using a barrier membrane (9,10). This approach has been widely applied since then. After the new bone reconstruction process, the titanium mesh needs to be removed which sometimes causes difficulties because of the integration of titanium with newly formed bone (11).

[0010] Titanium-reinforced membranes are a newer design to strengthen the barrier membrane against collapse in larger alveolar ridge defects. They are often used in vertical RA, especially when the soft tissue closure is not achievable. The membrane is made of non-resorbable material such as, expanded polytetrafluoroethylene (e-PTFE) and dense polytetrafluoroethylene (d-PTFE) (12,13). This means a secondary surgery should be done to extract the membrane. Regardless of the cost, which is more than resorbable membranes, membrane exposure is a significant clinical problem, which has been reported for Ti-reinforced membranes (13).

[0011] There appears to be a need for one or more alternative designs for bioresorbable barrier membranes, which may be use in RA (especial ly Class I) with one or more of an anti-collapsing structure; and the abi l ity to maintain graft volume safely. It would be advantageous if these alternative one or more designs maintained the advantages of Ti mesh and/or Titanium-reinforced membranes and/or mitigate the above-mentioned disadvantages,

[0012] Based on biodegradability, different types of barrier membranes are classified into two main categories including: resorbable and non-resorbable membranes. Both have their advantages as well as more suitable applications. The size and class of an alveolar ridge defect may guide selection of different types of membrane in terms of biodegradabil ity (13).

[0013] In one preferable situation, a resorbable barrier membrane should serve as a barrier during the ridge augmentation process and, after completion of bone regeneration integrate with the surrounding soft tissue (14). Resorbable membranes are more advantageous compared with non-resorbable membranes in terms of the el imination of secondary surgery to membrane removal after heal ing which results in less morbidity, lower rate of compl ications, and clinical costs reduction (15).

[0014] However, compared to non-resorbable membranes, resorbable membranes are not very successful to serve proper space maintenance and some additional devices, such as tenting screws may need to be uti lized to maintain sufficient space and prevent collapse of these membranes into the defect zone within the reconstruction process (13, 16). In addition, a resorbable membrane should be capable of utilisation in cases that could be covered by a soft tissue flap, which includes mostly lateral ridge defects (Class I).

[0015] Different materials have been used to make resorbable membranes. However, due to possessing advantageous characteristics when compared to other materials such as, polylactic acid/polyglycolic acid copolymer (PGLA), collagen based resorbable membranes have been uti lized more widely. Col lagen, especially collagen type I, is the most avai lable protein in the human body - constituting about one third of the whole-body weight. Collagen is a highly conserved molecule and is a major component of human connective tissue (17). This molecule is conducive to cell adhesion, moti lity, proliferation, and biodegradation (18). These properties allow collagen to function properly in vivo through its resultant bio- and cyto-compatibi l ity which thereby facilitates tissue integration (19). Moreover, collagen type I has considerable angiogenic capacity, which has a prominent role in successful tissue regeneration (20).

[0016] Although natural collagen membrane provides advantages in terms of good cel l response, rapid vascularization, and drug del ivery, it is weak in terms of stiffness and its application in, at least, dentistry, is mostly l imited to non-load bearing sites. Furthermore, its fast in vivo degradation time fai ls to provide the structural integrity necessary for bone regeneration (14,21).

[0017] One conventional cross-l inking approach to improve the mechanical properties of collagen membrane is to compact col lagen solutions and enhance the collagen fiber packing density which could increase the mechanical properties of the membrane (22). The electrochemical compaction is known as an effective method to fabricate dense collagen fibers. This method which is based on the principles of isoelectric focusing by using pH gradients induced by electrolysis of water molecules has been widely uti l ized to synthesize aligned col lagen fibers for tendon, cartilage, nerve, and vascular tissue engineering applications (22).

[0018] In 2015, Anowarul Islam et al (19) proposed a computer aided electrochemical compaction (ELCOM) approach for bio-fabrication of pure collagen meshes with controlled macro-porosity. This method was flexible enough to produce multi layered collagen sheet with controlled pore size, shape and desired pattern which could affect both mechanical properties and bioactivity of final product.

[0019] Furthermore, in some cases it is necessary to shape the membrane into a special geometry to position it properly in the defect site. The existing resorbable collagen membranes are not capable of being deformed permanently. Whereas, Ti-reinforced non- resorbable membranes can be deformed permanently, which is beneficial to provide better space maintenance to match the outer surface of the membrane with a natural bone geometry in the alveolar ridge.

[0020] Additionally, tearing is one the most common reasons for the failure of the collagen membranes during the placement or the regeneration period of the damaged tissue. Tearing may occur due to excessive load appl ied by the surgeon or incorrect fixation method in the loading areas (14). Such kind of failure wi l l definitely lead to unsuitabil ity for, or at least significantly reduced performance in, RA surgery. Hence, fabrication of a new generation of col lagen membranes which are tolerant and resistant enough to tearing may be desirable,

[0021] In general, tearing resistance, bending stiffness and shapeabil ity are three main specifications that are desired to be improved in crossl inked resorbable collagen membranes in order to mitigate their disadvantages and increase their application in a variety of ridge defects.

[0022] Metal lic wires have been widely used in a wide range of industries in different applications. Wire drawing is a conventional metal forming process which has been applied to fabricate metall ic wire. Briefly, this process continuously decreases the cross- sectional area of a specimen by pull ing it through a single (in single pass drawing) or series (in the multi-pass drawing) of conical die(s) (23).

[0023] The application of this wire drawing process in the production of biocompatible wire with different materials has been extensively investigated. Drawn wire from titanium and its alloys, stainless steels, and Nitinol has been used in different medical devices such as, cardiovascular stents (24), coronary guide catheters (25), coil occlusion of the aneurysm (26), Kirschner wires (K-wire) appl ied to orthopaedic fracture surgery (27), orthodontic arch wires (28), ligature wire (29), surgical sutures and staples (30). Based on the application, such devices might be implanted either for short or long ti me in the human body and regardless of the duration time, after finishing their mission, they should be removed from the body by a secondary operation. Because in some cases, if they are kept for more time, they will lead to compl ications such as harsh inflammations (31).

[0024] In order to decrease the cl inical expenses and increase the quality of life of patients by reducing any secondary surgeries for removal of metal implants as well as minimising any negative consequences, a new generation of metal lic biomaterials has been introduced as a temporary support. These kinds of materials called biodegradable metals (BMs), are required to be strong enough during the target tissue heal ing process and gradually resorb in the human body (32, 33), During the degradation process, some corrosion products wil l be released as ions in body fluid. The presence and the release of these ions means they are required to be nontoxic to the human body, especially to the surrounding host tissues (34). To that aim, different biodegradable alloys have been utilised. However, magnesium (Mg), iron (Fe), zinc (Zn), and their al loys as biodegradable implant metals are three main types that have found application as compared to other elements such as calcium (Ca) and strontium (Sr) (33). In addition to the structural role of Mg, Fe, Zn, and their alloys as an implant device, they play important roles in many metabolic reactions and biological mechanisms of the body and consequently reduce the healing duration (35). This is the reason these materials are named nutrient metals (NMs) (34, 36). Studies have been conducted to increase the performance of these nutrient alloys by enhancing their initial mechanical properties and mitigating their potential drawbacks such as, weak corrosion resistance to convert them from mechanical replacement devices to nutrient implants.

[0025] As an implantation device, N Ms in the shape of a wire were firstly applied in 1878 by Edward C. Huse. He used pure Mg wire as a ligature to stop bleeding vessels. Although Huse reported the first clinical application of Mg wire, the Austrian surgeon Prof. Dr. Payr is known the most pioneering person in the field of biodegradable Mg implants. H is valuable results and consequently his inspiring articles publ ished from 1892 to 1905 led to the uti lization of nutrient wires in both human and animal models by other researchers (37). Andrews in 1917 (38), and Seelig in 1924 (39) were early fol lowers in the investigation of in vivo experiments in animals of pure Mg wire. From that time, a considerable number of feasibi lity studies have been done to propose Mg, Fe and more recently Zn and their alloys as an ideal candidates in special clinical devices such as, cardiovascular stents (40), and orthopaedic implants (23,41). However, there are still some challenges including improvement of one or more of the surface and bulk properties such as one or more of: corrosion resistance; bioactivity; strength; ductility; and toxicity,

[0026] Magnesium as a mineral is the fourth most abundant cation element in the body (42). It has a role in many metabolic processes including enzymatic reaction, formation of apatite, and bone cells adsorption (43). About half of the total amount of magnesium in our body is stored in bone tissue (44). Wu et al. reported that Magnesium ions (Mg²⁺) govern a stimulating effect on new bone formation by increasing the prol iferation and differentiation of osteogenic (stem) cells via osteogenesis signall ing pathways in vitro (45). The release of Mg²⁺ is related to the chemical reaction of magnesium in the human body fluids such as body plasma as an aqueous solution which produces magnesium hydroxide and hydrogen gas evolution (46).

[0027] In terms of structural aspects, Mg and its biocompatible al loys may be more advantageous in bone applications in comparison with other metallic materials, ceramics and biodegradable polymers (BPs). Because the mechanical properties of Mg are more similar to those of natural cortical bone compared to other materials resulting in a decrease in the stress shielding effect and prevention of consequent bone resorption in the implanted sites (47, 48). That is the reason magnesium is the most attractive NM in orthopaedic devices and implants as a suitable substitution for non-bioresorbable materials.

[0028] Numerous previous studies have shown that both the functional behaviour and mechanical, chemical, as well as, physical properties of biomaterials can be affected by different surface modification techniques like coating deposition (49). Although plenty of coating methods like the electrodeposition (50), sol-gel preparation and dip coating (51), chemical conversion (52), anodizing combined with biomimetic coating (53), vapour deposition techniques (54), spin coating (55), and alkali treatment (56) have been proposed to control the rapid corrosion rate of Mg and its alloys, few of them are effective and flexible enough for the formation of a uniform coating with desired thickness on the nonflat surfaces such a wires. Also, the provision of a proper adhesion between substrate and coating layer is another point that should be considered, Final ly, prevention of deterioration, as much as possible, of the mechanical properties of the substrate is the third influencing parameter in the selection of an adequate coating method. To summarise, conventional opinion is that only sol-gel preparation combined with dip coating and alkal i treatment can provide all of the three requisite parameters, which makes them suitable and cost-effective methods for the coating of Mg-based wires (49, 57).

[0029] The composition of the covering films is another critically important parameter in the coating of Mg-based nutrient wires. An ideal coating material for the bone applications should be able to balance an enhanced biocompatibility and osteointegration properties of Mg-based substrate simultaneous with an increase in the corrosion resistance.

[0030] Bone grafting is an operation aimed at replacing diseased or injured bones with grafting materials. The bone graft could be taken from the patient (autograft) or donors (al lograft) or synthetic materials may be uti lized (58). Even though both of autograft and allograft have some significant advantages, cl inical problems with autografts including: additional surgery on the donor site causing pain and morbidity, as wel l as size and geometry limitations for the source of bone, loss of biological factors, weakening during the preparation process, inconsistency with host tissue and disease transmission in the application of allograft, there is a growing demand for synthetic materials as bone substitutes (59).

[0031] Among the wide range of synthetic materials, calcium phosphate cement (CPC) is known as the most similar synthetic material to natural bone which is clinically suitable for the repair of bone defects in a variety of orthopaedic and dental applications (60, 61). Although CPCs are biodegradable, biocompatible, osteoconductive and have proper compression strength, because of their low bending and tensile strength and low fracture toughness their applications are l imited to non-load bearing areas such as small cranial and maxi llo-facial surgeries (62, 63).

[0032] Poly-lactic acid (PLA) is known as one of the most attractive biodegradable polymers with valuable biological properties and is widely used is as an implant material in different bone appl ications. However, there are two main problems with PLA including relatively reduced mechanical properties and causing a reduction in the pH in biological environment which prevent from its further development.

[0033] US Patent Publication No. 20180071427, to Botiss Biomaterials, GmbH, names Oliver Bielenstein and Drazen Tadic as inventors, describes a bioresorbable magnesium or magnesium mesh membrane covered with a collagen-containing preparation disposed on a support, used as an implant and in particular as a wound dressing.

[0034] PCT/US2015/020338 to the University of Pittsburgh of the Commonwealth

System of Higher Education is publ ished as WO 2015142631 and names Charles Sfeir and Andrew Brown as inventors. This document discloses a magnesium-polymer composite useful as medical implants and wound healing compositions. Collagen is listed as a suitable polymer.

[0035] European Patent No. EP2229189, to the University of Hong Kong, and its fami ly member US Patent Application No. 13/413,215 published as US Patent Publication No. 2012/0209402, list the inventors as Chi Keung Yuen and Wing Yuk Ip. This document is directed to resorbable medical implants comprising magnesium which may be surface modified by immersion into a solution such as, a collagen solution. The implants may be used in the generation and/or regeneration of soft tissues, membranous tissues or organs such as, ligaments and tendons.

[0036] EP1951270 is the European Application that matured from

PCT/US2006/060055, publ ished as W02007/048099, in the name of Organogenesis, Inc and naming Ginger Abraham and Andrew N ixon as inventors. This document describes collagen constructs with antimicrobial properties comprising a sheet-like layer of purified collagenous tissue matrix derived from a tissue source, such as the tunica submucosa of smal l intestine or a processed intestinal collagen layer derived from the tunica submucosa of small intestine, treated with an antimicrobial agent.

[0037] US7495076 lists the inventors as Jennifer L. Gu and Edward Lee. This document is directed to pharmaceutical formulations of collagen peptide chelated mineral products such as, a calcium/magnesium col lagen chelate, for supporting mineral homeostasis.

[0038] US20170143872 to Tepha, Inc, l ists the inventors as Skander Limen, Bhavin

Shah and Said Rizk. This document describes perforated collagen coated polymeric meshes useful for soft tissue repair, regeneration and remodel ling including for hernia repair, mastopexy, treatment of urinary incontinence, pelvic floor reconstruction and ligament and tendon repair. Although cross-linking is described, the use of tannic acid does not appear to be disclosed.

[0039] WO2008134541 to the Musculoskeletal Transplant Foundation lists Arthur A.

Gertzman and M ichael Schuler as the inventors. This document discloses a composite material comprising a biological material, derived from a living or a once living source, and a reinforcement material that may be collagen.

[0040] PCT/EP2011/059954, published as WO2011/157758, to Innotere GmbH, lists the inventors as Berthold Nies and Stefan Glorius. This document is directed to a bone implant comprising a magnesium-containing metallic material and inorganic bone cement. The metallic material may be present in the form of wires or membranes.

[0041] PCT/I B2008/001922, published as W02009/047598, which lists the Appl icant and inventor as Fernando Briceno Rincon, is directed to col lagen membranes originating from the intestinal submucosa reinforced with a titanium mesh.

[0042] PCT/KR2018/008866 published as WO/2019/031776 to Osstemimplant Co.,

Ltd., lists the inventors as Da Young Noh and J in Rae Kim. This document discloses a dental composite in which a plurality of collagen layers have different biodegradation rates.

[0043] The publication entitled“Preparation of Hydroxyapatite/Tannic Acid Coating to Enhance the Corrosion Resistance and Cytocompatibility of AZ31 Magnesium Alloys” indicates that the hydroxyapatite/tannic acid coating effectively protects the alloy against corrosion in simulated body fluid and that cell proliferation and cell morphology observations results shows the coating was not toxic (Coatings 2017, 7, 105).

[0044] The publication entitled“Preparation and properties of tannic acid cross-l inked collagen scaffold and its application in wound healing” describes a bio-durable porous scaffold of collagen with good biocompatibility and enhanced wound healing potential that is prepared through a casting technique using tannic acid (TA) as crosslinker ( J Biomed Mater Res B Appl. Biomater. 2013; 101(4):560-7). [0045] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge.

SUMMARY OF THE INVENTION

[0046] Generally, embodiments of the present invention relate to a material for a bioresorbable implant, a bioresorbable implant and a method of making a bioresorbable implant.

[0047] In a broad form, the invention relates to a material for a bioresorbable implant, a bioresorbable implant and a method of making a bioresorbable implant comprising an improved surface. The improved surface may comprise an exposed surface.

[0048] In one aspect, although it need not be the only or indeed the broadest form, the invention provides a material for a bioresorbable implant and/or a bioresorbable implant comprising:

a col lagen solution or a col lagen and one or more magnesium wire to provide a collagen-based composite membrane;

the one or more magnesium wire is coated and/or surface modified with tannic acid.

[0049] In a second aspect, the invention provides a method of making a bioresorbable implant, the method comprising:

combining a collagen solution or a col lagen and one or more magnesium wire to provide a collagen-based composite membrane; and

coating and/or surface modifying the one or more magnesium wire with tannic acid.

[0050] In a third aspect, the invention provides a method of surgery comprising inserting a bioresorbable implant of the first aspect or a bioresorbable implant made according to the method of the second aspect. The method of surgery may comprise a guided tissue regeneration. The method of surgery may comprise ridge augmentation.

[0051] According to any of the above aspects, the one or more wires may further be coated and/or surface modified with Magnesium. The coating may be with a magnesium- phenolic network. The coating and/or surface modification with Magnesium may result in one or more of: pH neutral ization and passivation of the one or more magnesium wire. The coating may be with a Mg-polyphenolic fi lm with passivating and pH neutralizing effects. The film may comprise a thin fi lm. [0052] According to any one of the above aspects, the coating and/or surface modification may form a porous oxide layer. The coating may comprise a thickness of 1 to 20 pm; 2.5 to 15 pm; or 5 to 10 pm. The thickness of the coating may be 1.0; 1.5; 2,0; 2,5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10.0; 10.5; 11.0; 11.5; 12.0;

12.5; 13.0; 13.5; 14.0; 14.5; 15.0; 15.5; 16.0; 16.5; 17.0; 17.5; 18.0; 18.5; 19.0; 19.5 or 20 pm.

[0053] According to any one of the above aspects, the one or more wire may comprise a one layer; two layers; three layers or higher ordered layered pattern. Each wire may comprise one or more lines joined by a linker. The one or more lines may comprise neighbouring lines in a opposite directions. The one or more lines may be boustophedonic. The linker between each line may be disposed at a right angle, an acute angle, an obtuse angle, an oblique angle or may comprise an arc between the two neighbouring lines. The linker may be selected to form a desired shape.

[0054] According to any one of the above aspects, the one or more wire may comprise a wavy pattern such as, a two-layered wavy pattern. The two-layered pattern may comprise one or more wire in one layer perpendicular to one or more wire in the other layer. The wavy pattern may comprise a sinusoidal wave. Parallel wires may be equidistant throughout the periodicity of the wave or may be symmetrical throughout the periodicity of the wave. The symmetry may comprise a distance between two neighbouring wires increasing to a maxima and then decreasing to a minima in each period of the wave,

[0055] According to any one of the above aspects, the one or more wire may comprise an auxetic pattern.

[0056] According to any one of the above aspects, the one or more wire may comprise a circular cross-section.

[0057] According to any one of the above aspects, the one or more wire may comprise a diameter of 140 to 500 pm; 150 to 500 pm; 200 to 600 pm or 100 to 700 pm. The one or more wire may comprise a diameter of 100; 110; 120; 130; 135; 140; 142; 144; 146; 148;

150; 152; 154; 156; 158; 160; 170; 180; 190; 200; 210; 220 230; 240; 250; 260; 270; 280;

290; 300; 310; 320; 330; 340; 350; 360; 370; 380; 390; 400; 410; 420; 430; 440; 450; 460;

470; 480; 490; 500; 510; 520; 530; 540; 550; 560; 570; 580; 590; 600; 610; 620; 630; 640;

650; 660; 670; 680; 690; 700 pm.

[0058] According to any one of the above aspects, the thickness of the material or implant may be 0.5 to 2.0 mm; 0.4 to 2.5 mm; 0.2 to 3.0 mm. The thickness may comprise 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; or 3.0 mm.

[0059] According to any one of the above aspects, the one or more wires may be comprised in a high dose; medium dose; or low dose with a value of 0.5; 1; and 2 mm for

“S” respectively.“S” is the distance between two neighbouring lines of a wire in one layer. The one or more boustophedonic wire or layer may comprise a series of lines which may be parallel and/or equidistant.

[0060] According to any one of the above aspects, the mesh design may be based on a computational simulation conducted to optimise the mechanical properties.

[0061] According to any one of the above aspects, the magnesium wire may comprise a two dimensional and or three dimensional arrangement such as, a lattice or mesh, The mesh may comprise a weave. The weave may comprise one or more of a plain, twil led, five-heddle, and plain dutch weave.

[0062] According to any one of the above aspects, the bioresorbable implant may comprise a barrier membrane. The bioresorbable implant may comprise ridge augmentation.

[0063] According to any one of the above aspects, the bioresorbable implant may be biodegradable.

[0064] According to any one of the above aspects, the resorption and/or biodegradation of the implant may provide a nutrient, The nutrient provided may comprise magnesium.

[0065] According to any one of the above aspects, the malleability and/or shapeabil ity may be suitable for and/or improved compared to other materials and implants.

[0066] According to any one of the above aspects, the material and implant may comprise a sufficient mechanical stability resistance to col lapse and provide an anti-tearing property.

[0067] According to any one of the above aspects, the coating and/or surface modification may comprise a coating and/or surface modification solution comprising Tannic acid and/or Magnesium. The concentration of the Magnesium in the coating and/or surface modification solution may comprise 0.6 to 3.6; 0.5 to 4.0; 0.3 to 5.0 mg/ml. The concentration may comprise 0.6; 0.7; 0.8; 0.9; 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3.0; 3.1; 3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9; 4.0; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; or 5.0 mg/ml. [0068] According to any one of the above aspects, the thickness of the coated and/or surface modified one or more wire may comprise 1.5 to 3.0; 1,2 to 3.5 or 1.0 to 4.0 the thickness of the uncoated and/or unmodified one or more wire, The thickness ratio of the coated and/or surface modified one or more wire : uncoated and/or unmodified one or more wire may comprise 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1,6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3.0; 3.1; 3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9 or 4.0:1.

[0069] According to any one of the above aspects, the dosage of the implant may comprise low; medium and high dosages.

[0070] According to any one of the above aspects, the tannic acid modified and/or coated magnesium may comprise one or more of better absorption; increased adhesion of collagen; enhanced corrosion resistance and enhanced biocompatibility.

[0071] According to any one of the above aspects, the one or more wire may comprise a maximum thickness of 600miti.

[0072] According to any one of the above aspects, the one or more wire may comprise a reduced amount of wire.

[0073] According to any one of the above aspects, the one or more wire may comprise a wavy pattern with d_v=2.5 mm.

[0074] According to any one of the above aspects, the collagen may comprise type-1 collagen. The type-1 collagen may comprise bovine, porcine or human collagen. The human collagen may comprise an allograft material. The collagen may be crosslinked by one or more of: glutaraldehyde vapour crossl inking; dehydrothermal crosslinking; genipin crossl inking and electrochemical compaction. The collagen may be a recombinant collagen such as, a recombinant human col lagen.

[0075] According to any one of the above aspects, the material or implant may comprise ease of cutting. The cut may be by dentist; dental hygienist or dental assistant or other health professional or clinician such as, cutting and sizing with scissors and bending with dental tweezers, are also design criteria for the membrane.

[0076] Of significant advantage is the testing to date which has shown the mechanical properties of the designed patterns are superior to the commercial ly used titanium mesh.

[0077] According to any one of the above aspects, one or more of the fol lowing may be comprised: an a) addition of a high concentration of collagen and freeze drying; b) soaking in phosphate buffered saline (PBS); c) mechanical compression; 4) freeze drying; 5) crossl inking; and 6) steril ization with gamma or ultraviolet (UV) radiation or 70% ethanol. [0078] According to any one of the above aspects, the coating and/or surface modification may comprise a dip coating method. The dip coating method may comprise a 1 mg/ml solution of tannic acid combined with differential concentrations of magnesium chloride in a sodium hydroxide solution and washing with sodium hydroxide.

[0079] According to any one of the above aspects, the one or more wire may comprise a shape obtained through a model ling procedure. The modelling procedure may comprise Finite Element Method (FEM).

[0080] According to any one of the above aspects, the bioresorbable implant according to any above aspect may comprise a dental; orthopaedic; and cosmetic implant. The dental implant may comprise a ridge augmentation implant. The orthopaedic implant may comprise an implant for one or more of fracture; general bone repair; bone reconstruction or bone augmentation. The cosmetic implant may comprise of facial augmentation including, nose, cheek and forehead.

[0081] According to any one of the above aspects, the implant may harness the regenerative power of periosteum.

[0082] According to any one of the above aspects, the coated implant may comprise a controlled degradation. The controlled degradation may comprise a low rate of degradation. The low rate may allow blood flow to clear any hydrogen gas associated with degradation.

[0083] According to any one of the above aspects, the coated implant may prevent, substantially prevent, mitigate or substantially mitigate formation of hydrogen gas associated with degradation. The coated implant may comprise an improved or substantially improved biocompatibi lity.

[0084] Further aspects and/or features of the present invention wil l become apparent from the fol lowing detailed description.

BRI EF DESCRIPTION OF THE DRAWINGS

[0085] In order that the invention may be readily understood and put into practical effect, reference will now be made to embodiments of the present invention with reference to the accompanying drawings, wherein like reference numbers refer to identical elements, The drawings are provided by way of example only, wherein:

[0086] Figure 1(a): Schematic of a deformed membrane derived from the histological images of the posterior ridge augmentation in a canine mandible (8). [0087] Figure 1(b): deformed geometry of the collagen membrane assumed for FEM simulations.

[0088] Figure 2: Load and boundary conditions considered for Ti mesh.

[0089] Figure 3: Load and boundary conditions considered for Ti mesh.

[0090] Figure 4: The initial geometry, load, and boundary conditions.

[0091] Figures 5(a) and 5(b): A schematic of one example of a magnesium wire scaffold that was used in biocompatibi l ity testing according to one embodiment of the invention, showing a top view (5(a)) and a perspective view (5(b)).

[0092] Figures 6(a), 6(b), 6(c), 6(d): Example patterns for weaving wire mesh.

[0093] Figures 7(a) and 7(b): One-layered patterns: (a) straight (b) wavy.

[0094] Figures 8(ai), (aii), (b), (ci) and (cii): Two-layered patterns: (a) straight, i) cross-section view, i i) perspective view; (b) wavy, perspective view with inset showing a magnification of the encircled region; (c) auxetic, i) perspective view, ii) magnification.

[0095] Figure 9: The deflection contour of: (a) pure Ti, (b) pure collagen membrane,

(c) wire-reinforced collagen membrane with one layered wavy pattern when d =0.625mm,

(d) wire-reinforced collagen membrane with two-layered auxetic pattern when d_a=2.5mm

(e) wire-reinforced collagen membrane with two-layered wavy pattern when d =0.625mm,

(f) wire-reinforced collagen membrane with one- layered straight pattern when d_s=0.625mm.

[0096] Figure 10: The first three buckl ing mode shapes of Ti mesh; (a) first mode, (lΐ) (top); (b) second mode, (12) (middle); (c) third mode, (l3) (bottom).

[0097] Figures ll(ai), (ai i), (bi), (bi i), (ci) and (cii): The comparison between the first three mode shapes of a wire mesh with one-layered straight pattern when d_s=lmm and two-layered wavy pattern when d =2.5mm: (a) the first mode shape, (b) the second mode shape, (c) the third mode shape.

[0098] Figures 12(a), (b) and (c): Comparison of the distribution of the maximum tensi le stress of a col lagen membrane reinforced with: (a) two-layered auxetic pattern when da=2.5mm, (b) two-layered wavy pattern when d =2.5mm, (c) one-layered straight pattern when ds=lmm.

[0099] Figures 13(a), (b) and (c): The first three buckling mode shapes of bended Ti mesh; (a) first mode, (lΐ); (b) second mode, (l2); (c) third mode, (l3).

[0100] Figure 14: The fabricated setup for the tensi le testing of wires. [0101] Figure 15: an infographic showing a protocol for analysing the degradation behaviour of wires immersed in blood plasma according to one embodiment of the invention.

[0102] Figure 16: bar graphs showing the UTS (a) and elongation at break (b) results wherein the bars in each group are from left to right, as shown in the key at the top, WO; Wl-C; Wl-R; W2-C; W2-R; W3-C; W3-R; W4-C; W4-R.

[0103] Figure 17: Schematic of the 2D spiral scaffold and straight samples prepared for the in vitro biological tests.

[0104] Figure 18: In vitro investigation on the OIM property of M MW using extracted RAW cells culture medium after 24 h activation by LPS and I FN-g (qPCR: quantitative polymerase chain reaction, ELISA: enzyme-linked immunosorbent assay, SEM: scanning electron Microscopy, A&R: alizarin red staining, CM: extracted condition medium, OM : osteogenic medium).

[0105] Figure 19: Cel l viability assessment in the presence of MMWs at 1, 3 and 5 days of culture, detected by MTT. a) RAW cells, b) BMSCs, *** Indicates statistically significant differences (p < 0.05) compared to the control group at each time-point, respectively; morphology of the cel ls cultured observed by SEM in the presence of M MWs at 1, 3 and 5 days: c) RAW cel ls, d) BMSCs.

[0106] Figure 20: Comparison between M MW3 and M MW4 samples. Variation of the two parameters in the cultured medium at the same time-points to MTT tests: a) concentration of Mg, b) the pH value. Comparison between SEM micrographs of: c) the cluster structure formed on Mg wire substrate in MMW4 immersed in the cultured medium d) the normal oxide layer of Mg wire formed on the surface of MMW3; EDX spectrum and weigh% elemental quantitative data representative of: e) the cluster structure, f) the normal oxide layer.

[0107] Figure 21: Comparison between different groups of mineralization level and concentration of different elements in the OM . Al izarin red staining of ECM of BMSCs after 15 days of culture in the OM: a) 0.675X images, b) 10X images; c) The quantification of Alizarin red in each group. *** Indicates significant differences (p < 0.05) compared to the control group; d) Changes in the concentrations of Mg, Ca, K, and Na elements in the osteogenic medium during 15 days mineralization.

[0108] Figure 22: Dose effects of M MW on inflammatory activity of RAW cells under inflammatory environment using iFNy & LPS. a) relative expressions of inflammatory- related genes, b) release of inflammatory cytokines by RAW cells. *** Indicates statistically significant differences (p < 0.05) compared to the control (+) group; c) SEM macrographs of the morphology of RAW cells observed by SEM on CDHA at 12 h of post-activation: first panel: general view, second and third panels: an example of an expanded and elongated RAW cel l in each group. Control (+) and Control (-) represents iFNy & LPS activated and non-activated RAW cel ls in the absence of M MW, respectively.

[0109] Figure 23: Influence of OI M property of the MMWs on osteogenic activity of BMSCs. Alizarin red staining of ECM of BMSCs after 15 days of culturing in the prepared conditioned medium including RAW cel ls conditioned medium and the OM with 1:1 ratio: a) 0.675X images, b) 10X images; c) the corresponding amount of quantified Alizarin red; d) relative expressions of osteogenesis-related genes. *** Indicates statistically significant differences (p < 0.05) compared to the control group.

[0110] Figure 24: Mechanical testing of different sizes of M MW. The prepared samples were immersed in blood plasma and mounted on the designed sample holder.

[0111] Figure 25: Schematic of the assembly of MPNs on Mg-based alloys.

[0112] Figure 26: Changes in the mechanical properties and surface morphology of different sizes of M MWs during 4 weeks immersion in blood plasma a) UTS, b) elongation at break, c) the cross section view of after the failure; d) SEM images from surface morphological changes of the M MWs during the degradation in the blood plasma; e) the detai led SEM image showing the different portions of the degraded M MW, f) the corresponding EDX spectrum and weigh% elemental quantitative data of the CL and IOL layers.

[0113] Figure 27: Comparison between the surface morphology and thickness of the NaOH and different MPN-coated samples. SEM macrographs of: a) the surface morphology of the coated samples in different process parameters using secondary electron detector (SED); b) the cross-section of the coated samples using backscattered electron scanning.

[0114] Figure 28: Surface characterization of the Raw, NaOH, and different MPN- coated samples. XPS results a) the wide scan spectrum, b) the corresponding Mg 2p high resolution spectrum; c) the average surface roughness (Sa); d) the water contact angle, significance is shown as *** and +++ p <0.05 compared Raw and NaOH samples respectively.

[0115] Figure 29: Comparison between electrochemical behaviour of different samples, a) Nyquist EIS spectra between, b) PDP curves; c) equivalent circuit model used for interpretation of the corrosion behaviour of the coated samples. [0116] Figure 30: Table showing corresponding parameters determined from curve fitting of the EIS and PDP results.

[0117] Figure 31. SEM images of the morphology of MC3TC-E1 cells cultured on different samples at Day 1 and Day 3 of incubation.

[0118] Figure 32: Average absorbance of MC3T3-E1 cells after 1, 3 days incubation with different culture medium. ***, ···, ■■■, +++, and ### Indicates statistically significant differences (p <0.05), differences from the control group, 3.6%Mg@TA, 2.4%Mg@TA, 1.2%Mg@TA, and NaOH at each time-point respectively.

[0119] Figure 33: Confocal images of the cytoskeletal arrangement of MC3TC-E1 cells incubated with different culture medium at Day 1 and 3.

[0120] Figure 34: Comparison between the changes in the UTS of the Raw and MPN- coated M MWs with ϋ=300miti immersed in blood plasma for 4 weeks. *** Indicates statistically significant differences (p < 0.05) compared to the raw samples at each time- point.

[0121] Figure 35: (a) Schematic of the conducted rat cranial bone defects surgery. Two 5 mm diameter critical sized defects were made on the skull; (b) comparison between uncoated and coated Mg implants in terms of the formation of subcutaneous hydrogen gas cavity in the implantation area; c) Front and back views of the reconstructed micro-CT images obtained at 4 weeks after implantation surgery. The blue circle indicates the initial size of the defects,

[0122] Figure 36: Evolution of Fh gas during immersion of Mg samples: a) comparison between the uncoated and coated Mg-disks with MPNs coating method; b) comparison between the three different doses of pure Mg wires applied in this technology.

[0123] Figure 37: An examples of fabrication steps for making Mg-wire reinforced collagen composite membrane.

[0124] Figure 38: General image from the fabricated Mg-wire reinforced collagen composite membrane using wavy pattern.

[0125] Skilled addressees wi ll appreciate that elements in the drawings are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the relative dimensions of some elements in the drawings may be distorted to help improve understanding of embodiments of the present invention. DETAI LED DESCRIPTION OF THE I NVENTION

[0126] Embodiments of the present invention relate to a material for a bioresorbable implant, a bioresorbable implant and a method of making a bioresorbable implant. More particularly, this invention relates to a material for a bioresorbable implant, a bioresorbable implant and a method of making a bioresorbable implant comprising an improved surface.

[0127] Although in one embodiment, the present invention will be described with reference to a membrane barrier, the present invention is not so limited.

[0128] A lack of sufficient bone density, as well as a proper volume and contour in the alveolar ridge, is one of the most prominent risk factors endangering the success of the mounting operation of dental implants. In some cases, the socket area is not able to fil l after tooth extraction surgery. Furthermore, because of bone resorption especially for elderly patients, the height and width of the alveolar ridge bone may not be sufficient to support a dental implant. In both types of cases,“ridge augmentation” is conventional ly acknowledged as the most effective cl inical approach to regenerate the contour and the density of the crest of the alveolar ridge for the post placement of dental implants.

[0129] “Guided bone regeneration (GBR)” is known as one of the most effective treatments among different ridge augmentation modalities. In this method, a combination of graft materials and a barrier membrane is applied to prepare a suitable volume and density of bone in the implantation site. Regardless of having proper compatibi lity with surrounding soft tissue, these materials have acceptable integrative properties and are convenient for clinical use. The provision of a physical barrier for good space maintenance is the main role of barrier membranes. It should be sustainable enough to prevent any collapsing during the reconstruction of the alveolar ridge. Collagen-based membranes are more advantageous in terms of bioactivity properties l ike angiogenesis capacity, good cel l response, and drug delivery compared to different types of resorbable and non-resorbable membranes. However, the lack of sufficient bending stiffness resistance and permanent shapeabi lity makes this application challenging in the ridge augmentation surgeries, especially in unfavourable alveolar ridge defects. One or more additional device, such as tenting screws, may have to be util ized to maintain enough space and prevent col lapsing of these membranes into the defect zone within the reconstruction process. If these additional devices are used, a second surgery is then required to remove them. Additional ly, the poor tearing resistance is another difficulty regarding collagen membranes causing their fai lure during placement or because of sl ightly excessive loading conditions. [0130] The inventors’ application of nutrient biodegradable metal l ic wires provides reinforcement of biomaterials which otherwise have poor or moderate mechanical properties, Furthermore, the corrosion products may be beneficial to the bone regeneration process. Another advantage is that the provision of the one or more wire enhances one or more of the shapeabil ity and/or malleabi l ity; the bending stiffness; the anti-collapsing; and the tearing resistance of a collagen membrane by introducing a novel wire reinforced collagen-based composite membranes.

[0131] In one aspect the invention provides a material for a bioresorbable implant and/or a bioresorbable implant comprising a collagen solution or a col lagen and one or more magnesium wire to provide a collagen-based composite membrane. Advantageously, the one or more magnesium wire may be coated and/or surface modified,

[0132] The invention provides a method of making a bioresorbable implant, the method comprising combining a col lagen solution or a col lagen and one or more magnesium wire to provide a collagen-based composite membrane; and coating and/or surface modifying the one or more magnesium wire with tannic acid/Mg.

[0133] Also provided is a method of surgery comprising inserting the bioresorbable implant or the bioresorbable implant made according to the method described herein. The method of surgery may comprise a guided tissue regeneration such as, ridge augmentation.

[0134] The one or more wire may further be coated and/or surface modified with Magnesium. The coating may be with a magnesium-phenolic network. The coating and/or surface modification with Magnesium may result in one or more of: pH neutralization and passivation of the one or more magnesium wire. The coating may be with a Mg- polyphenolic thin fi lm with passivating and pH neutralizing effects.

[0135] Advantageously, as elucidated below, the coating and/or surface modification may form a porous oxide layer.

[0136] The one or more wire may comprise a one layer; two layer; three layer or higher ordered layered pattern. Each wire may comprise one or more l ines joined by a linker. The one or more lines may comprise neighbouring l ines in a opposite directions, The one or more l ines may be boustophedonic. The linker between each l ine may be disposed at a right angle, an acute angle, an obtuse angle, an obl ique angle or may comprise an arc between the two neighbouring l ines. The linker may be selected to form a desired shape. From the teaching herein a skil led person is readily able to select a suitable structure and/or pattern. [0137] The one or more wire may comprise a wavy pattern such as, a two-layered wavy pattern. The two-layered pattern may comprise one or more wire in one layer perpendicular to one or more wire in the other layer. The wavy pattern may comprise a sinusoidal wave. Parallel wires may be equidistant throughout the periodicity of the wave or may be symmetrical throughout the periodicity of the wave. The symmetry may comprise a distance between two neighbouring wires increasing to a maxima and then decreasing to a minima in each period of the wave.

[0138] From the teaching herein, a skil led person is readily able to select a suitable pattern such as, an auxetic pattern.

[0139] The one or more wire may comprise a circular cross-section. From the teaching herein a skilled person is readily able to select a suitable cross-section shape.

[0140] The one or more wire may Comprise a diameter of 140 to 500 pm; 150 to 500 pm; 200 to 600 pm or 100 to 700 pm. The one or more wire may comprise a diameter of 100; 110; 120; 130; 135; 140; 142; 144; 146; 148; 150; 152; 154; 156; 158; 160; 170; 180;

190; 200; 210; 220 230; 240; 250; 260; 270; 280; 290; 300; 310; 320; 330; 340; 350; 360;

370; 380; 390; 400; 410; 420; 430; 440; 450; 460; 470; 480; 490; 500; 510; 520; 530; 540;

550; 560; 570; 580; 590; 600; 610; 620; 630; 640; 650; 660; 670; 680; 690; 700 pm.

[0141] The one thickness of the material or implant may be 0.5 to 2.0mm; 0.4 to 2.5 mm; 0.2 to 3.0 mm. The thickness may comprise 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0;

1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; or 3.0

[0142] The one or more wires may be comprised in a high dose; medium dose; or low dose with a value of 0.5; 1 ; and 2 mm for“S” respectively.“S” is the distance between two neighbouring l ines of a wire in one layer. The one or more boustophedonic wire or layer may comprise a series of lines which may be parallel and/or equidistant.

[0143] The mesh design may be based on a computational simulation conducted to optimise the mechanical properties.

[0144] The magnesium wire may comprise a two dimensional and or three- dimensional arrangement such as, a lattice or mesh. The mesh may comprise a weave. The weave may comprise one or more of a plain, twil led, five-heddle, and plain dutch weave.

[0145] The bioresorbable implant may comprise a barrier membrane. The bioresorbable implant may comprise ridge augmentation.

[0146] The bioresorbable implant may be biodegradable. [0147] The resorption and/or biodegradation of the implant may provide a nutrient. The nutrient provided may comprise magnesium.

[0148] The mal leabi lity and/or shapeabi lity may be suitable for and/or improved compared to other materials and implants.

[0149] The material and implant may comprise a sufficient resistance to col lapse and anti-tearing properties.

[0150] The coating and/or surface modification may comprise a coating and/or surface modification solution comprising Tannic acid and/or Magnesium. The concentration of the Magnesium in the coating and/or surface modification solution may comprise 0.6 to 3.6; 0.5 to 4.0; 0.3 to 5.0 mg/ml. The concentration may comprise 0.6; 0.7; 0.8; 0.9; 1.0; 1.1;

1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3.0; 3.1 3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9; 4.0; 4.1; 4.2; 4.3; 4.4; 4.5; 4.6; 4.7; 4.8; 4.9; or 5.0 mg/ml.

[0151] The thickness of the coated and/or surface modified one or more wire may comprise 1.5 to 3.0; 1.2 to 3.5 or 1.0 to 4.0 the thickness of the uncoated and/or unmodified one or more wire. The thickness ratio of the coated and/or surface modified one or more wire : uncoated and/or unmodified one or more wire may comprise 1.0; 1.1;

1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3.0; 3.1; 3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9 or 4.0:1.

[0152] The dosage of the implant may comprise low; medium and high dosages.

[0153] The tannic acid modified and/or coated magnesium may comprise one or more of better absorption; increased adhesion of collagen; enhanced corrosion resistance and enhanced biocompatibil ity.

[0154] The one or more wire may comprise a maximum thickness of 600mhi.

[0155] The one or more wire may comprise a reduced amount of wire compared to conventional implants.

[0156] The one or more wire may comprise a wavy pattern with dv=2.5 mm.

[0157] The collagen may comprise type-1 collagen. The type-1 collagen may comprise bovine, porcine or human collagen. The human collagen may comprise an al lograft material. The collagen may be crosslinked by one or more of: glutaraldehyde vapour crossl inking; dehydrothermal crosslinking; genipin crosslinking and electrochemical compaction. The collagen may comprise a recombinant collagen such as, a recombinant human collagen. [0158] The material or implant may comprise ease of cutting. The cut may be by dentist or other clinician such as, cutting and sizing with scissors and bending with dental tweezers, are also design criteria for the membrane,

[0159] Of significant advantage is the testing to date which has shown the mechanical properties of the designed patterns are superior to the commercial ly used titanium mesh.

[0160] The invention may further comprise one or more of: an a) addition of a high concentration of collagen and freeze drying; b) soaking in phosphate buffered saline (PBS); c) mechanical compression; 4) freeze drying; 5) crosslinking; and 6) steril ization with gamma or ultraviolet (UV) radiation or 70% ethanol.

[0161] The coating and/or surface modification may comprise a dip coating method. The dip coating method may comprise a 1 mg/ml solution of tannic acid combined with differential concentrations of magnesium chloride in a sodium hydroxide solution and washing with sodium hydroxide.

[0162] The one or more wire may comprise a shape obtained through a model ling procedure. The model l ing procedure may comprise Finite Element Method (FEM).

[0163] The bioresorbable implant according to any above aspect may comprise a dental; orthopaedic; and cosmetic implant. The dental implant may comprise a ridge augmentation implant. The orthopaedic implant may comprise an implant for one or more of fracture; general bone repair; bone reconstruction or bone augmentation. The cosmetic implant may comprise of facial augmentation including, nose, cheek and forehead.

[0164] Advantageously, the implant may harness the regenerative power of periosteum.

[0165] The following non-limiting examples illustrate the invention. These examples should not be construed as l imiting: the examples are included for the purposes of illustration only. The Examples will be understood to represent an exemplification of the invention.

Examples

[0166] In one embodiment, the inventors have provided a design for a reinforced collagen guide membrane using a finite element method (FEM) simulation, Additional ly, the degradation behaviour of the one or more nutrient wires as well as crossl inked collagen membrane in terms of the mechanical properties, corrosion products, and biological feedback has been investigated in vitro. Further, the one or more wire reinforced collagen guide membrane has been fabricated and tested in vitro. The structural design of one or more wire reinforced collagen guide membrane using finite elements method (FEM)

[0167] Overview: There are 3 studies in this section investigating the effects of different parameters including: membrane thickness, membrane material model, and pattern (array) of reinforcement element on the mechanical behaviour (i.e. deflection, stress, and collapsing modes) of the reinforced collagen membrane using Abaqus finite elements software package.

[0168] Typically, defect size in posterior teeth (the width and the height) is bigger than that those of the anterior teeth and consequently more graft materials and larger barrier membrane should be uti lized for the regeneration of the crest of the alveolar ridge in the posterior site. Therefore, as a critical situation the dimensions of the barrier membrane is considered for the augmentation of a posterior tooth. More recently, measurements have shown the average width of the posterior mandibular ridge to be about 10mm and it has been asserted that at the implantation site, the adequate mandibular alveolar ridge width for the primary insertion of dental implants should be more than 6 mm. In this research the width of a lateral defect was assigned 10mm. Furthermore, the height was considered 15 mm. So, the total length of membrane was assumed to be 35mm, including both of loading and fixed areas. Finally, the width of the membrane was selected to be 10 mm which is bigger than the 9 mm total space between adjacent teeth for single tooth implants proposed by others.

[0169] Figure 1(a) depicts the schematic of a mounted membrane in the lateral augmentation procedure in the posterior site of a canine mandible. It is clear that the part of membrane which covers the filler materials is considered as the loading area. In other words, this area should be sustainable enough against collapse during the augmentation process. The remaining part of the deformed member is the non-loading area where there is not any direct loading. These two ends of the membrane could be fixed completely by fixing screws or left free with just a contact with surrounding bone and gum tissues. The same concept is used in FEM simulations (Figure 1(b)).

[0170] FEM simulations were conducted in two different categories in terms of the deformation mode of the membrane: a) before the permanent deformation of composite membrane; and b) after bending (permanent deformation). Depending on which parameter(s) or results were needed, one of the mentioned categories was selected. Furthermore, a wire frame model with the beam element type was considered for the reinforcement pattern which was embedded in a membrane model with a thickness of 0.5mm. It should be pointed that the cross-section of the wire elements was circular with a diameter of 150pm. Final ly, for all the simulations a proper mesh size was selected by performing the mesh independency study and checking the convergence of results to gain the most accurate possible result and least possible computational time and cost.

Study 1: Investigation on the bending stiffness

[0171] Weak bending stiffness is one of the main drawbacks of collagen membranes, In this study the effect of different structure and material designs were compared in terms of their bending stiffness. Only those designs having higher or at least similar bending stiffness to that of titanium thin sheet, which is cl inically used in ridge augmentation, were selected for further investigations.

[0172] Generally, the bending stiffness (K_b) is the resistance of a part against bending moment. Regardless of the length of the member (L) and the boundary condition in terms of the fixation situation, the modulus of elasticity (E) as a mechanical property characteristic and the area moment of inertia (I) as a geometrical parameter are two main factors influencing Kb. In other works, K is considered as a mechanical property which can be derived from the slope of the load-displacement curve of a member which is under bending loading. So:

Kb = F/d wherein“F” and“d” are force and deflection respectively. Hence, one of the effective approaches to compare the bending stiffness of different designs with different structures is to apply a distinct force and then extract the deflection. The less defection, the higher bending stiffness. So, in this study the bending stiffness of the reinforced collagen membrane was investigated by selecting different arrays of wires using deflection contour as a marker. The ideal design for wire reinforced col lagen membrane would be the one that has less consumption of wire and a higher bending stiffness - less deflection under a fixed loading condition.

[0173] Furthermore, K_b could be important for the wire reinforced collagen membrane. This is because during the preparation of the size and shape of the Ti mesh based on the defect size and geometry, dentists bend and deform the Ti mesh with ordinary tweezers. So, the stiffness of the wire reinforced collagen membrane should be near to that of thin Ti mesh to make it convenient for a dentist to bend and shape it in a desired form. So, the collapsing resistance and ease of bend are two important parameters which also seem to contradict each other. Hence, there should be an optimum value for Kb to achieve these properties.

[0174] Table 1 gives information about the dimensions and mechanical properties of the materials used. All of the simulations were performed in the elastic zone of the selected materials (below the yield point). IN vertical force was appl ied to the free edge of the member which is fully fixed from the 10mm of its length. Figure 2 illustrates the loading and boundary conditions were considered for the simulation. Simi lar parameters were applied for the simulations of the wire reinforced collagen membrane with different reinforcement patterns. The results were compared in terms of the max deflection in the vertical direction.

Investigation on the linear bucking behaviour

[0175] Based on the theory of “Mechanics of Materials”, buckling is known as a structural instability that leads to the failure of a structure while subjected to compressive loading. This may happen even when the stresses level has not exceeded the strength of the materials that the structure is made from. General ly, a critical value is defined for the resistance of a structure against buckling. This critical force (F_cr) is generally dependent on the length of a member (L) and the boundary condition, the modulus of elasticity (E) and the area moment of inertia (I). The ratio between applied force (F) and F_cr is defined as the load factor. If the value of load factor is more than one it shows that the structure is stable enough against buckling. If not, the structure becomes unstable and buckling occurs, Hence, having information about the value of F_cr is essential in the design of a structure.

[0176] Linear or elastic buckling analysis is a method used to determine Fcr at which a structure becomes unstable. It is also possible to predict different buckled mode shapes and to extract different critical load factors. To that purpose, FEM assumes buckl ing as an eigenvalue problem which can be solved using a matrix method to extract different eigenvalues (lί=1, 2, 3,...) which correspond to different buckl ing load factors. The lowest value for different eigenvalues is related to the first one (lΐ).

[0177] In this study, linear buckling analysis was conducted to determine the eigenvalues to investigate the effect of the pattern of wire on the stability of col lagen-based membrane. Furthermore, simi lar analysis was done for Ti mesh as reference value. The eigenvalues of wire reinforced col lagen-based composites are desired to be higher or at least equal to those of Ti mesh. Also, the effect of wire pattern on the deformation behaviour of each mode shape was considered. The results of this study would be a proper guidance in the selection of wire patterns which are more stable than others for further investigation.

[0178] In order to perform FEM linear buckling simulations, a unit compressive load (IN) was applied on one edge of the FEM model. The material properties as well as dimensions are the same as those mentioned in Table 1. Figure 3 depicts the loading and boundary conditions assigned for the linear buckling analysis of Ti mesh. Similar loading and clamping conditions were applied for al l of the linear buckling analysis of different wire patterns of col lagen-based membrane model. Finally, the first three eigenvalues (lΐ, l2, l3) with their mode shapes were extracted and compared with the eigenvalues Ti mesh. Investigation on the bending and buckling behaviour of the wire- reinforced collagen membrane

[0179] Two series of FEM analysis was done. At first the bending of membranes was conducted in order to bend them into a shape that dentists mostly use before placement of Ti mesh. In this series of simulations, the distribution of the tensile stress on the upper surface of the membrane was considered as a critical parameter in design of the collagen- based membranes. Then the bent FEM models underwent buckling analysis.

[0180] The results above were the main guidance for this analysis. Only designs which had good results in terms of bending stiffness and a higher load factor of linear buckling were selected for bending and consequent buckling analyses. Figure 4 depicts the initial geometry, load, and boundary conditions considered. A bent geometry was assigned for the initial situation in order to mimic a clinical procedure such as, ridge augmentation, wherein a flat shape would be unlikely. Both smaller edges were clamped in order to represent the clamping mode which occurs during ridge augmentation. Furthermore, a pressing load which resembles the load transferred from surrounding soft tissues because of the suturing force during surgery and its consequent loading throughout the healing period was appl ied. Finally, load factors and mode shapes for different geometries were extracted and compared.

Degradation behaviour of Mg-based wires in terms of the mechanical properties, corrosion products, and biological feedback using in vitro studies

[0181] Overview: 3 studies mainly focused on the degradation behaviour of Mg wires in terms of the change in tensile strength, release of the corrosion products, and biological feedback. The results wi ll be considered as decisive evidence which influence both the design and fabrication of the nutrient wire- re info reed collagen-based membrane.

Study 1: Bioactivity tests [0182] The bioactivity of the pure magnesium wire may be examined by immersion in simulated body fluid (SBF) solution or blood plasma or Dulbecco's Modified Eagle Medium (DM EM) with initial pH of 7,4 for 4 weeks with 1 week intervals whi le temperature will be kept at 37±0.5 °C. The suppl ied pure magnesium wire (uncoated and coated with Tannic Acid/Mg coating) with different diameters in range of 148-500pm may be cut in 7cm in length and ultrasonically cleaned in 100% acetone for 5 min, then in 100% ethanol for 5 min, in order to remove any surface residue. Then, rinsed in deionized (Dl) water. 12 samples may be used for each interval. Therefore, 36 samples may be used for the whole of this study. In each test, the 5BF or blood plasma may be completely removed every day and replaced with a freshly prepared fluid to mimic the natural process in the body and avoid contamination. Each sample may be removed from the SBF or blood plasma, washed twice with disti lled water and dried in room temperature for a short time and then transferred to a freezer kept at -18°C and then transferred into the freeze dryer at - 51°C and near vacuum situation to absorb any remaining moisture. Then weighed by balance and kept in a vacuum desiccator.

[0183] To investigate the effects of the pure magnesium wire on the SBF or blood plasma or DM EM solution, the concentrations of Mg, Ca , K and Na ions in SBF or blood plasma or DMEM may be determined using ICP-OES or ICP-MS machines. Also, any change to the pH of SBF or blood plasma or DMEM solution may be measured by pH meter, Furthermore, to determine if SBF or blood plasma or DMEM solution forms apatite on the surface of the samples, SEM imaging may be conducted. X-ray Photoelectron Spectroscopy (XPS) may be also used to determine the oxidation state and surface chemical composition. Finally, any change in the tensile strength of the wire at each interval may be assessed using a tensile testing method. The results of this study may provide experimental knowledge regarding the bioactivity of pure magnesium wire in vitro.

Cell Proliferation with MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

[0184] Cell prol iferation with MTT assay as a colorimetric assay is a process used for measuring cell metabol ic activity. This experiment may be done to check the effect of the pure magnesium wire scaffolds with a diameter in range of 148-500miti on the balance between cell divisions and cell loss through cell death or differentiation. The control group may be a blank 96 cell culture well seeded by the hbmsc cells. [0185] The pure magnesium scaffolds may consist of two layers. Based on the size of “S”, which is depicted in Figure 5(a), which shows one embodiment of a scaffold used in biocompatibility testing, the scaffold samples may be classified into 3 categories including: high dose, medium dose and low dose scaffolds with a value of 0.5; 1; and 2 mm for“S” respectively.“S” is the distance between two neighbouring lines of a wire in one layer. The boustophedonic wire or layer comprises a series of lines which may be parallel and/or equidistant. As shown in Figures 5(a) and 5(b), in neighbouring layers, the lines may be perpendicular to one another. The linker between each line may disposed at a right angle, an acute angle, an obtuse angle, an oblique angle or may comprise an arc between the two neighbouring lines. The linker may be selected to form a desired shape.

[0186] hBMSC (4,000 cells in 100 mI_ of culture media) may be cultured onto the wire scaffolds. Before any experiments, al l scaffolds may be ultrasonical ly cleaned in 100% acetone for 10 min, then in 100% ethanol for 10 min, in order to remove any surface residue. All may receive a final rinse in deionized (Dl) water. Clean samples may be placed under UV radiation for 1 hr for sterilization. After incubation of hBMSC cells for 1, 3 and 5 days on the magnesium wire samples in 96-well plate the medium may be replaced with IOOmI fresh DMEM and mixed with 11 mΐ of MTT stock solution to each well and will be incubated at 37 °C for 4 hr to form formazan, After incubation, the medium may be removed from each well . Then, 0.1 ml of DMSO may be added to dissolve the formazan crystals. The optical density (OD) of the resulting solution may be measured at 490 nm on a microplate reader. Three scaffolds may be used for each time interval. Therefore, 27 samples may be used for this study including 3 dosing categories. Based on the results obtained from this study, three cases are expected to happen. In the first case, there will be no difference between different doses of Mg wires. Therefore, in this case all of the sizes considered of the selected patterns for Mg wire mesh will be acceptable. It means that there will be no concerns regarding the dosing of Mg wires in terms of any negative side effects to the bone regeneration. In the second case, just one (low dose) or two doses (medium and low doses) have acceptable results in terms of cell prol iferation, Consequently, few patterns with acceptable dosing can be applied in the fabrication of wire reinforced collagen membrane. Finally, the last possible case will be in a situation that none of three mentioned doses (high, medium and low) are suitable for hBMSC cell proliferation. In this critical case, further study will be conducted in order to check the cytotoxicity to the hBMSC. All in all, performing this study will provide illustrative evidence regarding how magnesium wire mesh and the amount thereof affects the proliferation of the human hBMSC cells. This information may be important in the proposing a suitable length of wire for the final wire mesh pattern which can even improve cell proliferation.

Cytotoxicity tests

[0187] If the result of the cell proliferation tests is unclear, one or more cytotoxicity and/or viabil ity assay may be conducted as a complementary cel l culture study in order to measure how much the thin magnesium wire may be cytotoxic to the hBMSCs. The size, geometry and number of samples used in this study wi l l be simi lar to those considered above. To conduct this study a LIVE/DEADs cytotoxicity kit will be utilized. The hBMSCs may be seeded and incubated at 37C for 1, 3, and 5 days. Then, the samples may be washed gently with PBS and then 100 mI of working solution may be dropped on each sample. After incubation at 37C for 5 min, all samples may be washed again with PBS and observed using a l ive confocal laser scanning microscope, By aid of this method the live cells and dead ones will be recognised.“How much wire per square centimetre (cm²) is acceptable to prevent its cytotoxicity to the bone-like cells?” is a critically important question that may be answered by doing this study. The response to this question may influence the design of the wire mesh. In other words, the dosing of the wire pattern wil l be final ised based on the l imitations proposed by the results of this study. It is worth mentioning that the control group will be blank 96 cel l culture well seeded by the hBMSC cells. If the results of this study show that there wil l be some concerns regarding the cytotoxicity of any dose of Mg wires to bone-like cells, subsequent action will be undertaken. There will be two reasons regarding the potential cytotoxicity of Mg wires: increase in pH (more than 8) and/or excessive evolution of H2 gas.

[0188] Because of the release of Mg ions in the first implantation of Mg wires to the bone-like cells, the pH of the solution will go up and make the DM EM solution alkaline. Therefore, the consequent action wil l be pH neutralization of the medium in the first implantation of Mg wires to the bone-like cells. As mentioned before, Mg(OH as one of corrosion products of Mg-based alloys has a pH of around 7 and is known as an effective coating material for pH neutralization. Temporary coating of Mg wires with Mg(OHh may be a suitable approach to prevent the increase in pH by control l ing the amount of released Mg²⁺ ions. Additionally, excessive evolution of H2 gas is related to fast degradation rate of Mg wires in the medium solution and may lead to production of insoluble gas bubbles which may be quite harmful for cells to survive and prol iferate. So, temporary passivation of the Mg wire surface wi l l be necessary in this case. Mg(OH can act as passivating coating for Mg-based alloys to control their degradation rate. To summarise, both pH neutral ization and passivation of Mg wires may be achievable by applying Mg(OH)2 on the surface of such kind of wires. To that aim, coating with Mg-polyphenol ic thin film with passivating and pH neutralizing effects is a suitable choice to create a thin temporary film of Mg(OH)2. Consequently, the cytotoxicity test may be repeated for coated samples and compared with the results of uncoated ones. Furthermore, cel l proliferation with MTT wil l be conducted for coated samples as well.

[0189] It should be mentioned that, the effect of Mg wires on the inflammatory reaction of immune cells may be investigated in vitro. Furthermore, as a complementarity study bone-l ike cell differentiation under the degradation media may be investigated as well. The result of this study may provide a better understanding of how much the corrosion products of Mg wire with special dosing in a collagen membrane can potential ly improve osteogenesis.

Fabrication of the wire-reinforced collagen-based membrane

[0190] Overview: There wi ll be 3 studies concentrating on the fabrication and characterization of the wire-reinforced collagen membrane. In other words, these studies are directing to making the fabrication of this type of collagen membrane feasible using practical methods.

Study 1: Fabrication of pure crosslinked collagen membrane

[0191] This study is considered as a primary step in the fabrication of the final prototype of the wire-reinforced col lagen membrane, The bovine collagen type-1 wi l l be used as a base material. Generally, glutaraldehyde vapour crosslinking, dehydrothermal crossl inking, and electrochemical compaction are the most common approaches for the stabi lisation and crossl inking of col lagen type-1. However, an ideal method for this project should be flexible enough for the fabrication of the wire-reinforced collagen membrane. Furthermore, the availabi l ity of faci l ities and equipment is another influencing factor on the selection of the method for the fabrication of the crossl inked collagen membrane.

[0192] The method util ised was selected because of its flexibi lity and simpl icity of the electrochemical compaction. The fabrication method is similar to that mentioned in Refs, (18) and (22). Briefly, purified bovine acid soluble col lagen type-1 in powder form may be dissolved in 0.1 N acetic acid solution at a concentration of 3 rngml^-1. Then, molecular collagen solutions may be dialyzed against ultrapure water and injected between two flat graphite electrodes in circular rubber washers with inner diameter of 14 mm and thickness of 1 mm. After that, an electrical field (3 volts, 45 min) may be applied. Since both cathode and anode electrodes have a simi lar charge, the collagen molecules wi l l be repelled away from the electrodes and will be compacted along the isoelectric point, where the net charge is zero. The electrochemically compacted collagen (ECC) may be removed from the cathode at the end of the electrochemical process, Final ly, the ECC matrices may be incubated in PBS at 37 °C for 4 h to promote fibril formation and improve the structural and mechanical integrity of the matrices (22). Another parallel method for the fabrication of collagen membrane is purring (freezing and then drying with a freeze-drying process) the collagen solution with appropriate volume in glass container. If there are unwanted bubbles, these can be removed by putting the sample briefly under vacuum. After this, it may be rapidly frozen and consequently freeze dried. This wi l l give a fairly open, sponge like coating. It may then be stabilised.

[0193] The crossl inking of the fabricated membranes may be performed using genipin based on the method mentioned in Ref. 22. Crosslinking with l-ethyl-3 (3dimethylaminopropyl) carbodiimide-N-hydroxysuccinimide (EDC-N HS) is another approach that may be performed and its results in terms of mechanical properties, degradation rate, water content, cell response will be compared genipin crossl inking.

[0194] After fabrication of the pure col lagen membranes, degradation behaviour wil l be assessed using biodegradation tests. In brief, the prepared samples wi ll be immersed in PBS solution at 37+0.5 °C and pH 7.4 for up to 3 weeks with 1 week intervals. Each sample will be taken out, washed with disti lled water and half of them wi l l be dried at room temperature, Finally, the tensile mechanical test may be performed to investigate the change in strength and elongation of wet and dried samples after the degradation test. Furthermore, the structural morphology will be done using SEM imaging. To that aim, samples will be fixed and consequent dehydration based on the process mentioned in Ref. (22). Finally, different cell studies proposed herein may also be conducted in order to check the bioactivity and cel l response to the fabricated membranes. The thickness and the porosity of collagen membranes are two main variables which may be considered during all of the mentioned experiments. Collectively, the results of this study as a control group may provide clear guidance for performing consequent experiments including those outlined herein. Additionally, all of the results and any kind of change or improvement in different aspects may be compared with this control group.

Study 2: Fabrication of simple wire reinforced crosslinked collagen membrane

[0195] The main target of this study is the fabrication of the simple wire-reinforced composite membrane in order to prove the main concept of this project. This study may focus on how to produce a composite membrane which provides the desired characteristics as follows:

[0196] Prevent any severe corrosion of Mg wires during fabrication of composite membrane general ly, collagen type I solution is a water-based solution which may be corrosive to Mg wires and which definitely affects mechanical properties even before implantation to the patient’s defect area. Therefore, one of the main targets of this study may be the assessment of how much collagen solution and consequently fabrication method can be corrosive to the Mg wire. If it is severe, then temporary passivation of Mg wires surface with Mg (OHh may be utilised. Mg(OH)2 is known to be an excel lent biomaterial and insoluble in water that can be very simply coated on Mg wires using Mg- polyphenolic thin networks with passivating and bioactive characteristics.

[0197] Attachment of the Mg wire with collagen membrane: The proper attachment between collagen membrane and wire is critically important. The wire should be embedded properly with the col lagen membrane in order to protect it from fast degradation.

[0198] Furthermore, the attachment should be strong enough to resist and/or withstand against peeling of collagen from the wire during the placement by a dentist and throughout the heal ing process.

[0199] The total thickness of membrane: the fabrication method should be accurate enough to produce a membrane with construable thickness. The thick membrane and very thin one may not be suitable for clinical applications. Therefore, there should be a fine cover of collagen on the wire mesh. Moreover, in one embodiment the distribution of the collagen cover should be homogenous.

[0200] The fabrication process of the composite membrane is similar to that procedure described above. The only difference is that the wire may be placed in the collagen solution during the membrane fabrication process. After the fabrication of the composite membrane biodegradation, morphology assessment, and cell culturing may be performed based on the procedure mentioned above. All in all, the result of this study may be illustrative for the fabrication of the first prototype of the nutrient wire-reinforced collagen membrane.

Fabrication of the first prototype of the nutrient wire-reinforced collagen membrane

[0201] All of the results obtained from previous studies may be summarised to design a fabrication method which may serve al l of the specifications outlined. Briefly, the results of the first part of structural design may clarify which kind of pattern(s) for the wire mesh should be assigned as the reinforcement part of the composite collagen-based membrane. The degradation studies may define any pre-protection of wires before placement in the collagen solution to assure a suitable situation in terms of the corrosion rate. Additionally, fabrication studies may prepare enough information regarding the methodology for the fabrication of composite membrane.

[0202] The fabrication process for this study may comprise three steps. At first, the wire mesh with a specific pattern may be prepared based on the procedure mentioned in Ref. 41. In summary, a flat polymeric die will be fabricated using 3D printing. The geometry of the die will be based on the pattern of wire that is going to be prepared. Then, the wire may be passed through the die to create the desired pattern. After that, the prepared wire mesh may be removed from the polymeric die. In the second step the wire mesh may be immersed in the collagen solution. Finally, the collagen-based composite membrane may be fabricated based on the finalized method described above regarding the fabrication of simple wire reinforced crosslinked col lagen membrane. The size of the samples may be similar to those assigned in the above studies.

[0203] After the fabrication process of each sample, the biodegradation and mechanical tests, morphology assessment, and cell studies may be performed based on the procedure mentioned above. Moreover, all of the results may be compared with those obtained in the fabrication study as a control group to investigate the influence of Mg-wire mesh on both biological and structural (mechanical) properties of collagen-based membranes.

[0204] Based on the procedure explained for tensi le testing below, some general static simulations were performed in order to compare Kb using the resulting deflection. It should be mentioned that all of the possible patterns for the design of wire mesh were comprehensively studied. However, just a few of them were selected as suitable candidates for further studies. Having a proper combination of the equivalent area moment of inertia (IEQV), less consumption of wire, and ease of fabrication are the main parameters considered given more weight in the selection of the proper design for the pattern of the wire mesh.

[0205] Conventionally, ordinary patterns including plain, twi l led, five-heddle, and plain dutch weaves are i llustrated in Figures 6(a), 6(b), 6(c) and 6(d), respectively, are the most common for weaving wire mesh. There are two main concerns regarding their application in the context of this project. The first one is related to their mechanical behaviour in response to bending forces. Generally, the degree between warp and weft or shul wires is 9(T. This mitigates the role of weft or shul wire in the strengthening of the wire mesh against bending load. The second concern is the total thickness of these patterns. If the dimeter of the wire is about 150miti, the total thickness of wire mesh for the plain, twilled, five-heddle weaves will be 300pm and 450pm for the dutch weave. Suppose that the fabrication of a composite membrane with a thickness of 500pm is the aim. Then the coating layer above the dutch weave wil l be negligible and hard to fabricate and control. Furthermore, if a pattern with at least two layers of wires is targeted it will be possible to design a pattern as thick as that of the plain, twi l led, five-heddle weaves with more effective and sustainable structure against bending, Al l in all, some patterns with one or two flat layers were assigned for the FEM simulations of this study.

One-layered wire mesh patterns

[0206] The straight and wavy were two main patterns selected as the most appealing potential candidates for fabrication of wire-reinforced collagen composite membranes, Figures 7(a) and 7(b) depict the geometry of these two patterns. It is clear that the distance between the straight lines (ds) which represents the numbers of lines (n) is the main variable for the straight pattern. Similarly, the distance between A and B points (dw) is the main variable for the wavy pattern. Col lectedly, a wide range of member from 0 to 19 was assigned for“n” parameter. Also, 2.5, 1.25, and 0.625mm are three amounts which were considered for“dw” in the wavy pattern.

Two-layered wire mesh patterns

[0207] The two parallel straight layers, two wavy layers and auxetic structure were three main categories for two-layered patterns which were selected for the fabrication of the wire-reinforced collagen composite membranes (see Figure 8(ai) and 8(aii); 8(b); and 8(d) and 8(cii)). The numbers of lines (n) for each layer is the main variable for the straight pattern. The dw and da are the main variables for the wavy and auxetic patterns respectively. Similar to one-layered patterns, n_max=19 and dw & d_a = 0.625, 1.25, 2.5 were assigned to the introduced patterns.

Bending stiffness assessment

[0208] The maximum deflection (5 max) and the total consumed wire (LT) were two main parameters assessed in al l of simulations. An ideal pattern should have less amount for both of 6^max and compared to other patterns. Figures 9(a), 9(b), 9(c), 9(d), 9(e) and 9(f) show the deflection contour of some patterns which compares with the pure collagen membrane and the Ti mesh as well.

[0209] Table 2 summarises the information about d max and the total length of wires consumed (LT) in different patterns. It is clear that in all of the cases d max for wire- reinforced col lagen membrane is near or even less than that of Ti mesh. So, in terms of the bending stiffness, most of the patterns are good enough for intending application.

[0210] Additionally, investigations were made regarding a good balance between the bending stiffness and the amount of wire consumed for the selected patterns. Based on this table, it can be seen that if the one-layered pattern is uti l ised, then the straight pattern wil l provide the best bending stiffness simultaneous with the minimum util ization of wire (6max=0.683mm, LT=484mm). But, if minimizing the wire consumption in a pattern is more important than having the best bending stiffness, two-layered patterns may be a better choice. Furthermore, because of the existing connection points (nodes) between the first and second layers, the pattern will be a more interconnected structure which can be more sustainable against any unpredictable loading condition compare to one-layered patterns, To summarize, a two-layered structure with wavy and/or auxetic patterns may provide both acceptable bending stiffness and logical amount of wire.

[0211] It may be concluded that if the consumption of the wire is the main critical parameter in the selection of the proper pattern, auxetic structure with a higher value for d_a may be a suitable choice. However, finalizing the best pattern in terms of having acceptable bending stiffness may depend on the results of the different studies herein which will propose the critical amount of wire that can be applied as the nutrient reinforcement layer for collagen membrane. Furthermore, the results from the other studies herein may affect the ideal pattern for the reinforcement layer which may be suitable for al l of the necessities considered herein.

Wire Pattern Analysis

[0212] Regarding the methodology expressed herein, some general l inear buckling analysis using FEM was performed in order to assess the first three eigenvalues (lΐ , l2, l3) with their mode shapes and compare with the eigenvalues for Ti mesh. It is worth mentioning that the same patterns’ categories considered above were assigned for this study as well. Figures 10(a), (b) and (c) i l lustrate the first three buckling mode shapes of Ti mesh under compression load. It is clear that lΐ, l2, l3 are 132.41, 271.2, 530.8 respectively. These amounts were considered as the reference values to compare with the different patterns for the nutrient wire-reinforced layer(s).

[0213] Table 3 gives information about the first three eigenvalues as well as the wire consumption of the wire for each pattern. As shown in this table, all of the patterns were more stable than the pure Ti mesh against buckling. However, there are some differences between different patterns that need to be discussed. Firstly, the second and third mode shapes of straight pattern were completely different with those of the wavy or auxetic patterns.

[0214] Figures ll(ai), (ai i), (bi), (bii), (ci) and (cii) compare the first three mode shapes of a one-layered straight pattern with a two-layered wavy pattern. As shown in this figure, the behaviour of both patterns was similar in the first mode shape. Whi le, the second and the third mode shapes were completely different. Secondly, the difference between the amounts of l2 and l3 was another comparable point between straight and wavy patterns. For all straight patterns the amounts for l2 and l3 were close to each other. Whereas, this difference for wavy and auxetic patterns was significantly different and almost doubled from l2 to l3 (see Table 3). Similar behaviour was seen for the pure Ti mesh where l2 and l3 were 271.2 and 530.8.

[0215] All in all, it can be claimed that in a constant length of nutrient wire, wavy and auxetic patterns had the better performance against buckl ing. So, two-layered wire mesh, with those patterns, were more preferable compared with one-layered straight patterns. Moreover, similarities between the behaviour of the collagen reinforced with either wavy or auxetic patterns with the pure Ti mesh was another advantageous parameter in comparison with the straight pattern.

[0216] Regarding the approach which is presented herein the designed patterns were bent to simulate the procedure which is normally performed by dentists before the placement of Ti mesh in the defect area in ridge augmentation surgeries.

[0217] Based on the mechanics of materials, during bending of a member, one side of its horizontal surface is under tensile loading and the other side under compression which leads to tensile and compressive stress, respectively. For a collagen membrane, the tensi le stress is much more critical than the compressive stress. Therefore, if the applied tensi le stress is more than the ultimate tensile strength of the collagen membrane, delamination and consequently tearing wil l happen which results in the failure of the col lagen membrane as a Suitable barrier for guided bone regeneration.“Collprotect” and“Jason” are two most famous brands fabricating collagen membranes for guided bone regeneration, Using the experiments conducted by Ortolani et al. (14) for the collagen membranes of these two brands with average thickness of 0,2-0.3mm, the maximum tensile stress was reported approximately 13MPa. In order to compare the effect of the wire mesh pattern on the distribution of the tensile stress on the outer surface of the collagen composite membrane, the maximum tensile strength was assumed 13MPa. Figures 12(a), (b) and (c) compare the distribution of the stress on the upper surface of a bended reinforced membrane for three different patterns with quite similar amount for Lr. Because of the simpl icity of the straight pattern, the distribution of the stress in this case was the most uniform one. Furthermore, almost all of the points had lower tensi le stress of 13M Pa. Although the auxetic pattern had less uniform stress distribution compared to the straight pattern, the maximum tensile stress was not greater than 13 MPa. The most critical case was related to the two-layered wavy pattern. As shown in Figure 12(b), in three zones the exposed stress to the upper surface of the member exceeded more than 13 MPa. Therefore, in design of wavy patterns with two layers the consideration of the tensile stress on the upper surface of the membrane is quite important. It should be pointed out that in al l of the patterns the ampl itude of the compression stress was higher than the tensile stress.

[0218] Another important property of auxetic structures is synclasticity, which leads to different behaviour compared to conventional patterns. When a conventional pattern with non-auxetic structure is subjected to a bending load, it wil l be shaped to a “saddle” geometry which is not good in terms of space making ability of a membrane. This is because some areas of the membrane wi ll collapse to the defect areas and miss the ideal contour of the jaw-bone in the defect area. Whereas, auxetic patterns adapt with a“domeshaped” geometry which prevents the collapsing of the membrane due to the bending. Furthermore, the energy absorption of auxetic patterns are much higher than conventional patterns. Col lectively, auxetic patterns are promising enough for space making purposes of the membranes.

[0219] After the bending simulations, buckl ing analysis were performed. All of simulations were done under the same boundary conditions as previously described. A pressure load was applied on all the outer surfaces of bent membrane. Figures 13(a), (b) and (c) depict the first three modes of shapes of bent Ti mesh. It is clear that in the first mode shape, collapsing mostly occurred at the vertical part of the mesh. Whereas in the second and third mode shapes, expansion and wrinkling occurred in both vertical and horizontal parts of Ti mesh.

[0220] It should be pointed out that, in this part of the study, just the three patterns that had the lowest consumption of wires were compared. If the anti-collapsing behaviour of these patterns are sustainable enough other similar patterns with higher LT would be expected to be safer against buckling and consequent buckling. Table 4 summarises the data regarding the first three eigenvalues of three different patterns with similar LT. It is clear that the first eigenvalue (lΐ ) of all of these three patterns was higher than that of Ti mesh which was 16.32. Furthermore, the two-layered wavy and auxetic patterns had the highest and lowest eigenvalues respectively. It is also shown that the difference between lΐ for the two-layered wavy pattern with one-layered straight pattern was negligible. Whereas, this difference became larger for l2 and l3 and reached to 3% and 27% correspondingly. Additionally, the amount for l3 of one-layered straight pattern was the lowest one among all of patterns which is mostly related to the weakness of this pattern against complicated mode shapes. Another important point worth mentioning is the higher risk of one-layered patterns compared to two-layered ones. Suppose that one or two straight lines are damaged because of any reasons such as inhomogeneous corrosion. In this case resistance of membrane against collapsing will be mitigated more in comparison to two layered patterns.

Conclusion

[0221] Based on the results obtained from the studies herein, it may be concluded that in a constant amount for LT, two-layered wavy patterns are more advantageous to others in terms of bending stiffness, col lapsing behaviour. Furthermore, if the less wire is targeted, wavy pattern with d =2.5mm should be suitable. It is worth mentioning that the suitable size (not pattern) wil l be finalised based on the results of the degradation behaviour of Mg wires in biological situations.

Dosing:

[0222] In order to conduct experiments related to dosing, most of the work regarding the materials and experimental setup have already prepared and fabricated as follows:

[0223] Nutrient wire: Pure magnesium wire with diameter of 150pm has been supplied. Also, some disks with diameter of 6mm and 1mm thickness were also fabricated for some primary studies and concept approval test and decrease the waste of nutrient wires.

[0224] Experimental setup for tensile testing of wires: Because of the diameter of the wire, which is in sub-mi llimetre scale, ordinary experimental setup for tensile testing of such wires is not suitable. Because of the stress concentration on the gripping areas, the fracture of the wire will occur at an incorrect point, Therefore, there should be a special clamping method to prevent any stress concentration of the thin wire during the tensi le testing. Furthermore, the clamping force should be high enough to prevent sliding of wire during testing. To that that aim, and based on the logics of wire testing, an experimental setup 100 was designed and fabricated. As shown in Figure 14, the wire samples wi ll be placed on two clamping pads 114. In the clamping area, rubber pads were used in order to create a sufficient friction force preventing wire sliding. Then the clamped wire will be passed through round disks to el iminate the stress concentration. Also shown is a load cel l connection 112; a wire sample 200; and a basement connection 118.

[0225] Simulated body fluid (SBF) or blood plasma or DMEM : the SBF or blood plasma or DM EM solution was prepared for bioactivity tests. The preparation process of SBF solution was based on the method mentioned in Ref. (64).

[0226] hBMSC cell extraction: the hBMSC cel ls were obtained from humans undergoing knee replacement surgery. The ethics approval for the use of human bone samples for this experiment was granted by the QUT approval number of 1400001024 (St Vincent’s Health & Aged Care (SVHAC) Human Research Ethics Committee (HREC) with approval number 15/04). The bone samples were col lected under sterile condition, which were minced and washed with PBS solution, The samples were later transferred to basal culture media (DM EM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. hBMSC cel l growth was observed after 7-14 days. Then the cel ls were expanded for further use. Finally, they were frozen at -8(T C and then kept in nitrogen gas for the future usage.

Fabrication:

[0227] The porcine col lagen type-1, in the shape of sponges, were supplied and kept at 4° C. Furthermore, the primary studies have been started regarding the preparation of the collagen solutions and fabrication of the collagen membranes.

Fabrication of wire reinforced collagen membrane

[0228] As extensively mentioned in the methodology sections, temporary coating of Mg wires with Mg oxides is an effective method for passivation and pH neutralization of the surface. Because of the importance of this coating to the results, some primary experiments have been done to establish a suitable coating protocol.

[0229] Recently, naturally occurring polyphenol groups with tannic acid (TA), and metal ions such as Fe³⁺ have been introduced as a simple and cost-effective method for deposition of metal-phenolic networks on different substrates like bulk materials, nanomaterials, and bio-interfaces (see 65, 66). Fi l m deposition occurs due to the adsorption of the polyphenols and simultaneous cross-linking of TA by metal ions.

[0230] And finally, nice three dimensionally stabilized metal-phenol ic networks (MPNs) may be created on the surface of substrate. Depending on the desired thickness for metal-phenolic networks this process can takes from a few minutes to few hours. Additionally, the coating cycle may be repeated a plural ity of times. [0231] In most of the research work done until now, there was not any chemical reaction between the substrate and the tannic acid solution. Whereas, Mg-based alloys are active materials than definitely participate in watery solutions. Therefore, the detailed coating method including concertation and content of solution as well as coating time should be specified for Mg-based substrate to prevent any excessive corrosion and consequent degradation. The target polyphenolic fi lm should include 3D networks of Mg oxides to serve passivating and pH neutralizing effects.

[0232] To that aim, four groups of samples made from Mg in shape of disks with 6mm in diameter and the thickness of 1mm were prepared. Table 5 provides information about the specification of the coating solution as well as coating time and time intervals which were considered for each group. Mg disk without coating was considered as the control group. In the second group, only sodium hydroxide (NaOH) was added to ultra-purified water to prepare an alkal ine solution with pH of 10. Because of the alkaline content of this solution it was predicted that some oxide particles will be appear on the surface of samples. In the third group of samples lmg/ml TA was added to the prepared NaOH solution and reduced the pH to 8. Because of the active nature of Mg, it was expected that there would be a reaction between phenol groups of TA with Mg²⁺ ions released from the Mg disk because of the corrosion and formed Mg polyphenol networks with the Mg oxides composition. In the last group 0,5ml/mg Magnesium chloride hexahydrate (MgCl2.(6H20)) was added. Basically, in all of the previous work the chloride solution of the target metal was used to create the metal l ic polyphenol ic networks. For instance, to create Fe(OH)3, FeCh should be added to TA. That is the reason for, in the last group of samples, addition of MgCl₂.(6H₂0) to TA. Furthermore, it was expected that by adding MgCl₂.(6H₂0), less reaction would occur between TA and Mg disks and consequently less corrosion would result. All of the prepared samples were immersed in the prepared solutions for 4 hours. After 2 hours the solution was replaced with the fresh solution, to increase the efficiency of the coating process. Final ly, all samples were removed and washed gently with NaOH solution (pH=10) and dried gently and immediately transferred to a vacuum desiccator to prevent any undesired corrosion of prepared samples. Then, they were removed and coated with gold coating and final ly primary surface morphology was conducted using SEM.

[0233] In future studies, all of the concluded points will be investigated to increase the efficiency of Mg-polyphenolic networks deposition by increasing the film density on the surface and preventing excessive cracking of the substrate. Furthermore, other semi qualitative and quantitative methods such as EDX and XPS wil l be conducted to quantify the coated samples.

[0234] Degradation Behaviour: All of the studies mentioned in the Dosing studies will be conducted step by step in order to have a comprehensive knowledge about the behaviour of magnesium wire during the degradation using in vitro studies. Then, bioactivity of the Mg wires wil l be investigated using SBF or blood plasma or DM EM solution. Ion release, changing in tensile strength and surface morphology are the main parameters that may be investigated during the degradation of Mg wires in SBF solution or blood plasma or DMEM. Afterwards, MTT assay wi l l be performed to check the proliferation of hBMSC cells. In this study, the high, medium and low doses may be investigated in order to check the sensitivity of the pattern design in terms of the consumption of wire. If the results for all of the three dosing categories are critical, a cytotoxicity assay will be done in order to find the critical value for dosing of Mg wire per cm². The output of this study is il lustrative data for the final isation of the pattern design of the reinforcement layer.

[0235] Fabrication: Based on the approaches presented in the Fabrication section, the fabrication process of the wire-reinforced collagen-based membranes may be establ ished. Briefly, the collagen solution protocol may be establ ished by some trials. Then, the setup for electrochemical compaction may be prepared in the near future. Afterwards, the collagen membrane without Mg reinforcement layer will be fabricated. In the next step, the wire-reinforced collagen membrane, without concern about the wire mesh pattern, may be prepared. Final ly, after addressing all of the problems with the previous steps, the first prototype for the wire-reinforced collagen-based composite membrane may be fabricated. It is worth mentioning that for each step, there may be a different assessment and experiment, in order to monitor the progress of this aim. Furthermore, if any surface modifications {i.e. pH neutral ization and passivation using thin film of MgO or Mg(OH)2)for Mg wires is conducted, the same approach will be conducted for the Fabrication studies”.

[0236] Structural Design: If necessary, the amount considered for d_s, d_w, and d_a for the patterns of the reinforcement layer may be modified based on the results obtained from the other studies. Because the limitations for the acceptable dosing of Mg wires in terms of cytotoxicity or inflammatory reaction may be clarified in these studies. Furthermore, imported data for performing FEM simulations may be revised based the experimental results obtained from the other studies. New Design of Collagen-based Barrier Membrane for Ridge Augmentation

[0237] Investigations have been conducted on: “mechanical stability”; “osteo- compatibility” and“osteo-immune system response” properties of different doses of micro Mg wires.

[0238] Figure 15 is an infographic explaining the protocol for study of the degradation behaviour of wires in blood plasma. The time points included week 0; week 1; week 2; week 3 and week 4. Figures 16(a) and (b) shows graphs i l lustrating the results of this degradation testing. Figure 16(a) shows tensile testing to obtain load-displacement curves so that UTS may be extracted.

Investigate dose effects on the biocompatibility and osteo-immunomodulatory of Mg wires as the nutrient reinforcement component in the Nutrient biodegradable barrier membrane

[0239] OVERVIEW: The results from FEA computer simulation showed that the initial mechanical properties of all the considered patterns for the Mg wire-based reinforcement compartment were higher than those of Ti mesh. Flowever, since the Mg wire-based reinforcement compartment is a biodegradable material; the acceptable dose range for the amount of Mg wires needs to be finalized by investigating the influence of the“dose” of Mg wires on the biocompatibility of such fi lament material. Furthermore, without wanting to be bound by any one theory, based on the hypothesis, Mg wires not only can play a reinforcement role in the composite collagen membrane but also can serve a nutrient effect stimulating the bone formation process by releasing Mg²⁺ ions during their degradation. However, the excessive release of Mg²⁺ ions could result in the formation of excessive H2 gas bubbles and consequently could lead to oxygen deficiency and pH increase around the implantation area, which eventually causes undesired complications. The amount and rate of delivered Mg²⁺ ions are crucial factors in the application of Mg- based wires in bone regeneration and have not been investigated in detai l. The effects of the dose of Mg micro wires (M MWs) on biocompatibi l ity and osteo-immunomodulation were thoroughly investigated in vitro by considering a direct dynamic interaction between such wires and cells, The release of Mg²⁺ ions from MMWs were investigated for the dose- dependency of viability and polarization of both bone marrow stromal cells (BMSCs) and macrophages using MTT assay simultaneously by tracking the changes in the Mg concentration and the pH value of the culture medium as well as morphological changes of these cells in the presence of different doses of MMWs. Additional ly, the osteo- immunoregulatory (OI M ) role of M MWs was further investigated using qPCR, ELISA kit, and SEM imaging. Moreover, both direct and indirect (extracted medium) impact on osteogenesis of Mg²⁺ ions released from M MWs were evaluated using Alizarin red staining, qPCR together with the continuous measuring of the concentration of Mg, Ca, Na, and K in the osteogenic medium (OM). The results of this section suggested that applying the proper dose of Mg wires can significantly decrease pro-inflammatory-related cytokines and stimulate the osteogenesis process. It is worth mentioning that the proposed dose range in this section covered the amount of Mg wires required in the designed patterns per square centimetre achieved from computer simulation.

[0240] I NTRODUCTION : It is proven that the release of Mg ions (Mg²⁺) and Mg oxides wil l induce new bone formation. Additionally, the anti-inflammatory property of Mg-based al loys was reported, which showed creation of a microenvironment suitable for bone regeneration, However, the main chal lenge with the cl inical application of Mg-based implants is their fast degradation rate, which generates large amounts of hydrogen gas (H2) bubbles. In less vascularized tissues like bone, the flow rate is not sufficient to clear these H2 bubbles, resulting in the formation of H2 cavities and consequently causing oxygen deficiency and preventing feeding nutrients in the local microenvironment. Furthermore, Mg-implant degradation would excessively increase the local pH level, which can deteriorate the condition and endanger the cells required in bone regeneration.

[0241] These mentioned obstacles, however, can be tackled by manipulating the size/dose effect of the amount of the filament appl ied in bone implants such as defect space-making mesh. A bone scaffold or a space-making mesh made from thick wires with a diameter in mill imetre-scale has more surface area (more Mg per single surface area) and definitely more formation of H₂ bubbles. Whereas, micro-scale Mg filament with much lighter mass and negligible surface area (as compared to bigger wire) can, therefore, decrease the excessive H₂ gas formation. Until now, only a few studies have investigated the effect Of MMWs on bone regeneration, but didn’t consider the dose effects of MMWs. The rest of the research has been focused on the fabrication, characterization, and degradation behaviour of such wires or using MMWs as a reinforcement structural element for other biomaterials; however, the dose-effect of Mg on bone heal ing is largely unknown.

[0242] In the osteo-immunology research field, it is proven that there is a close relationship between the condition of the immune system and the initiation of the bone healing process in the defect area. Accordingly, Osteo-immunomodulation (OI M) is known as a vital property for bone biomaterials, which describes how bone implants interact with the immune system to create a condition suitable for bone regeneration. It is proven that to initiate the osteogenesis, bone biomaterials should effectively regulate the immune cells by decreasing the pro-inflammatory response to improve bone tissue regeneration. Due to playing multiple roles in the bone heal ing metabolism, macrophages are the most attractive immune cell lines used in evaluation of the OI M property of bone-related biomaterials, Apart from a limited number of research works, such as on OIM property Mg-based implants, the dose dependency of OI M property of such implant materials like MMWs has not been ful ly understood yet.

[0243] In this section, the dose effects of Mg wires on biocompatibility and OIM were thoroughly investigated in vitro. The calculated value for LT in was used to consider an initial guess for the dose range of Mg wires for conducting the biological studies. However, the considered dose range in this section was wider than that of the dose achieved from the results in the simulation part. The dose dependency of viability and morphological changes of both bone marrow stromal cells (BMSCs) and macrophages (RAW cells) on the amount of Mg wire was applied per cm². After narrowing the dose range to the one with no significant cytotoxicity, the immunomodulatory effect of the total applied Mg wires on RAW cel ls under the inflammatory condition was dynamical ly evaluated, as well as the effect of this immunomodulation on osteogenic differentiation of BMSCs. Moreover, both direct and indirect (extracted medium) osteogenesis were conducted to evaluate the difference in the nutrient effect of the dose of the applied Mg wires in bone mineralization.

[0244] MATERIALS AND METHODS: Dose rage of MMWs - Apart from the geometry of the deigned patterns, the amount of wire per 100mm² (1cm²) was used as an initial guess for choosing a proper dose range for biological studies planned in this section. Based on the achieved results from simulation part, the dose of Mg wire for all the considered patterns was in the range of 3.5-llmg/cm² (mg/lOOmm²). As there was no similar research in the literature that investigated the dose-dependency of the biocompatibility and OIM, in order to have a compressive study regarding the dose effects of MMWs on the bone biological feedbacks, a wider dose range of 2.5-30mg/cm² was considered in the experiments of this section,

[0245] Preparation of MMWs: Drawn pure magnesium wires (purity: Mg > 99.98%) with initial diameter of 4.0 mm were continuously processed by cold drawing passes in order to fabricate pure magnesium micro wires with D=300pm, and then kept in a vacuum desiccator to prevent oxidation. Before using the fabricated micro wires for any experiments, they were ultrasonical ly cleaned in 100% acetone (10 min), in 100% ethanol (10 min) and finally rinsed in deionized (Dl) water in order to remove any surface contaminations.

[0246] In vitro biological tests : In all in vitro experiments in which the interaction between Mg wires and cells was planned, MMWs were cut into specific lengths and formed in a shape of a 2D spiral scaffold (Figure 17), and placed in a tilt position to serve a dynamic interaction between the material and the cultured cel ls. Generally, for al l experiments, at least three samples were used per group per time-point unless otherwise stated (n=3). Cells were treated with 2D spiral M MW samples with dose of 2.5, 5, 15, and 30 mg/lOOmm² (MMW1, MMW2, M MW3, and MMW4, respectively).

[0247] Cell viability assessment. To assess the effects of the dose of released Mg²⁺ and hh on both BMSCs and Raw cells, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT, Sigma Aldrich, Australia) assay was performed. BMSCs were col lected from the harvested human bone marrow of the patients who had undergone knee replacement surgeries, with ethics numberl400001024 (St Vincent’s Health & Aged Care (SVHAC) Human Research Ethics Committee (HREQ).

[0248] Cells were suspended (with densities as follows: 1*10⁴ cells/ml for BMSCs and 8*10³ cells/ml for RAW cel ls, respectively) in culture medium (Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Austral ia) supplemented with 10% fetal bovine serum (FBS) as well as 1% penicillin-streptomycin (PS) on coverslips and cultured under 5% C0₂ at 37°C for 6h. MMW1, M MW2, M MW3, MMW4 samples were then placed in the cell culture wells. At the end each time-point (i.e., day 1, 3, and 5), M MW samples were removed. Cel ls were washed by PBS, then 10% v/v MTT solution (5 mg/mL, suspended in culture medium) was added to each well and incubated at 37°C and 5% CO2 for another 4h. Afterward, the culture medium was replaced completely with dimethyl sulfoxide (DMSO, Fisher Scientific, U K) and incubated at room temperature for 10 min (protected from light). A microplate reader (Benchmark Plus, Bio-Rad, USA) was used to measure the absorbance. Furthermore, paral lel samples with MTT assay were prepared to measure the Mg concentration and the pH value of the culture medium inductively coupled plasma optical emission spectroscopy (ICP-OES 700, Agilent, USA) and a pH meter (Thermo Fisher Scientific, USA), respectively, to study the variation of pH value during the time of culture. All procedures were repeated three times.

[0249] Cell morphological analysis: To study the effects of M MW1, MMW2, MMW3, M MW4 samples on cel l morphological changes, BMSCs and RAW cells were used to monitor cell morphology, adhesion, and proliferation in the presence of MMWs. The same time-points and cell seeding densities considered for the Cel l viabil ity assessment test were used. Cells were seeded on coverslips and kept under 5% CO2 at 37°C. After six hours (h), the M MW samples were placed in the cell culture wells, MMW1, M MW2, MMW3, M MW4 samples were compared against the control group in three different time-points (day 1, 3 and 5). The culture medium solution was replaced every two days from the initial placement of M MWs. At the end of each time-point, the culture medium and the samples were removed, and the coverslips were washed with PBS and fixed with 3% glutaraldehyde for 30 min, then washed again with PBS three times. Afterward, the samples were dehydrated according to previous protocols. Subsequently, the samples were coated with a thin layer of gold using a sputter (EM SC005 Gold Coater, Leica). Finally, the cel l morphology was observed using scanning electron microscopy (SEM, J EOL 7001F, Japan). Al l procedures were repeated three times.

[0250] Osteogenic differentiation of BMSCs: Based the results of cell viability tests and morphological change assessment, three doses with no significant cytotoxicity (i.e., MMW1, M MW2, and M MW3) were used to investigate the stimulating effect of M MWs on osteogenesis. BMSCs were seeded at the density of 6xl0⁴ cells/cm². In 24 h-post seeding, cells were treated with osteogenic medium (OM, culture medium with 50 pg/mL ascorbic acid, lOmM b-glycerophosphate and 100 nM dexamethasone). The OM was refreshed every three days. After 15 days, the M MW samples were removed. Subsequently, the cells were washed twice with PBS and fixed with 4% of PFA for 15 min at RT. Then, the cells were stained using 40 mM alizarin red solution (ARS, Sigma- Aldrich, USA) with an adjusted pH range of 4.1-4.2 for 20 min with very gentle shaking. After aspiration of the unincorporated dye, the wells were washed four times with dFhO. The images were taken by a l ight microscope (Nikon ECLIPSE TS100). Final ly, the amount of bounded Alizarin red was quantified by dissolving the stained extracel lular matrix (ECM) of BMSCs into 10% CPC solution, which was consequently measured using a plate reader (Benchmark Plus, Bio-Rad, USA).

[0251] Furthermore, in order to track the amounts of the released Mg²⁺ ions from MMWs during the osteogenic process, the extracted medium, at every third day, was analysed by inductively coupled plasma optical emission spectroscopy ( ICP-OES 700, Agilent, USA). In addition to Mg, the amounts of calcium (Ca), potassium (K), and sodium (Na) in the extracted medium were also measured, to track any possible changes in these elements during the osteogenic process (in comparison with their initial concentration in the OM before cell culture). All procedures above were repeated three times. [0252] Investigate the OIM effect of different doses ofMMW: Response of RAW cells to graded doses of M MWs under the inflammatory condition: Graded doses of M MWs (were appl ied to investigate their effects on the response of RAW cells under inflammatory stimulation. RAW cells (3*10⁵ cells/ml) were stimulated with 100 ng/mL of lipopolysaccharide (LPS, R&D Systems, USA) and 100 ng/mL interferon gamma (I FN-g, R&D Systems, USA). The MMW-samples were placed in the cell culture wel ls. After stimulation for 24h, the MMWs were removed, and the cells were gently washed with PBS three times, and then cultured 12h in serum free medium for collection of conditioned medium (CM). Inflammation-stimulated cells without M MWs (treated with LPS and I FN-g only, Group Control (+)), and cells cultured in normal culture medium (control (-)) were prepared simultaneously for comparison. A schematic view of all the experiments conducted in this section is shown in Figure 18.

[0253] To investigate the effects of MMWs on inflammatory response of RAW cells, quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) were performed , to detect the mRNA levels of pro-inflammatory genes, the levels of secreted inflammatory cytokines, and the morphological changes, respectively. For qPCR analysis, samples were harvested by TRIzol reagent (Ambion™, Life Technologies Pty Ltd., Australia), and RNA extraction was performed following the manufacturer’s protocol. The amount of 1000 ng of total RNA was used for cDNA synthesis using DyNAmo™ cDNA Synthesis Kit (Genesearch, QLD, Australia). The mRNA levels of pro-inflammatory-related genes ( I L-6, iNOS, CD86) were detected using a QuantstudioTM Real-Time PCR machine (Appl ied Biosystems, Foster City, California, USA) with SYBR Green qPCR Master Mix (Life Technologies). The housekeeping reference gene GAPDH was used for normalizing qPCR data. Relative gene expression was normal ized against GAPDH and calculated as previously described. Al l experiments were following the M IQE guidel ines and replicated three times.

[0254] Additionally, the concentrations of I L-6 and I L-Ib in the harvested CM were quantified using Uncoated ELISA kits (R&D Systems, China) following the manufacturer’s protocol (replicated three times).

[0255] To observe the morphological changes, RAW cells (seeded on coverslips) were fixed, dehydrated, and gold coated, and SEM imaged.

[0256] Investigate the effect of RAW cell-derived CM on osteogenic differentiation of BMSCs: The harvested RAW cell-CM was centrifuged (5000 rpm, 10 min) and fi ltered with a 0.2-pm fi lter (Millipore Corporation, Billerica, MA) to remove cell debris, and stored at -80°C for future analysis. To study the effect of the harvested CM in osteogenesis, BMSCs were treated with the CM combined with fresh OM at ratio of 1:1 for 3/15 days, respectively (Figure 18). In 3-day-osteogenesis, BMSCs were harvested by TRIZOL for RNA extraction. An amount of 1000 ng RNA was used for cDNA synthesis, and qPCR was performed to detect the rriRNA levels of osteogenic markers (COL-I, RUNX2, and OPN) following the same procedures described before. GAPDH housekeeping reference gene was used, and relative gene expression was normalized against GAPDH and calculated as described before. In 15 day-osteogenic differentiation, cells were fixed and processed with 40 mM Al izarin red staining solution.

[0257] Statistical analysis : Statistical analyses were performed via one-way analysis of variance (ANOVA) using SigmaPlot verl3.0 (Systat Software, Inc, USA) software package. The reported data in graphs were presented as mean ± standard deviation (SD) (n=3 unless otherwise stated). The level of significance for all tests was set at p < 0.05.

[0258] RESULTS AND DISCUSSION: Viabi l ity and morphological changes of RAW cells and BMSCs treated with M MWs: The viabil ity of RAW and BMSC cells with different doses of MMWs were compared with that of the control groups. As shown in Figures 19(a) and (b) for both RAW cells and BMSCs, M MW4 was the only dose resulting in a significantly lower absorbance value, as compared to the control groups (p <0.05) at all the time-points. Whereas, the cells treated with MMW3 showed no significant difference in comparison with the controls, Additionally, MMW1/2 significantly enhanced the metabolic activity in the consequent time-points compared to the controls (i.e., p <0.05 for RAW cells: M MW1/2 at day 5 and p <0.05 for RAW cells: M MW2 at day 3 and 5). These differences between different M MW groups can be due to differences in the concentration of the Mg (Figure 20(a)) resulted from the immersion of different doses of MMWs in the medium, which led to increase of the pH value compared to the control group (Figure 20(b)). It is found that culture medium with the pH value in the range of 8.4- 8.5 (the purple horizontal dashed li nes shown in Figure 20(b)) can effectively enhance the viability of BMSCs in vitro. Meanwhi le, pH value over that range decreases cell viabi lity, In this study, the concentration of Mg as wel l as pH values at different time-points of culture medium with different MMWs were tracked. It is obvious that the trend of change in the concentration of Mg (Figure 20(a)) is consistent with that of the pH values (Figure 20(b)) in all the considered time-points. All the groups represented a simi lar behaviour from day 1 to day 5, in that the pH values increased from day 1 to day 3 and then decreased at day 5 (day 5-pH was a bit higher than that of day 1). The degradation of Mg would result in the formation of a more temporary oxide passivating layer, which can be the reason for decreasing pH values after day 3. The pH value of Group M MW4 was much higher than 8.5, which should be the reason for the observed reduction in the metabolism of cells. Medium with MMW1/2/3 showed a moderate increase of pH value within the proposed optimum range.

[0259] The morphology of RAW cells and BMSCs cultured with M MWs were then observed by SEM. As shown in Figure 19(c), on day 1, RAW cells in all the groups showed suitable attachment and spreading (Figure 19(d)); however, the M MW4- microenvironment was inappropriate for the attachment of BMSCs (Figure 19(d)). On day 3, extra increase of the pH value in the MMW4 group (Figure 20(b)) indicated that this dose of MMW would not be biocompatible for both RAW and BMSC cells (Figure 19(c), (d)), Accordingly, the morphology of the cells in MMW4 showed they were unhealthy and unable to spread and proliferate, which explained the not considerable changes in the metabolism of MMW4 samples from day 3 to day 5 Figure 19(a), (b)). By contrast, MMW1-3 delivered a proper environment for the cel ls, owing to the suitable pH value range. On day 5, the cel ls cultured with MMW1-3 expanded actively by creating more fi lopodia to initiate the proliferation (Figure 19(c), (d)), which is in agreement with the increase of the metabolism from day 3 to day 5 (shown in Figure 19(a), (b)).

[0260] To further investigate the reason for dose-dependent biocompatibi lity of MMWs, the surface morphology and surface chemical analysis of the M MW3 and M MW4 groups were conducted after for 5 days immersion in the culture medium. Formation of flower-like clusters with needle spikes on the surface of the immersed wires in cultured medium, can be further evidence for the difference in the biological environment created by the MMW4 group (Figure 20(c)). As shown in Figure 20(d), even in M MW3 there was no sign for the deposition of such a big cluster as formed on the M MW4. It seems that the excessive increase of pH value in M MW prepares an oversaturated condition for the included ions in the cultured medium and leads to formation of insoluble salts with magnesium ions. As shown in the EDX profiles (Figure 20(e), (f)), the cluster structure, which entirely covered the Mg substrate in M MW4, mainly included Mg and 0, which formed such a needle structure made from Mg oxide. Meanwhile, the normal oxide layer contained 0, Mg, Ca, P and Cl elements formed in the surface of M MW3.

[0261] Overal l, M MW4 could not provide a suitable level of biocompatibility, which significantly reduced the viability of both RAW cells and BMSCs. Therefore, this group of samples were not considered for the following experiments in this study. [0262] The effect of MMWs at graded doses on the mineralization of BMSCs: The dose effects of Mg-based implants on the mineralization of BMSCs have been investigated in two different methodologies. In the first one, a chemical solution containing different concentrations of Mg was used. For instance, Yoshizawa S., et al. used magnesium sulphate (MgS04) at concentrations of 0.8, 5, 10, and 20 mM were applied in osteogenesis in vitro to study the role of Mg²⁺ ions in mineralization. They reported that mineral ization of ECM was enhanced with MgSC at 5 and 10 mM. In the second methodology, which is more common for designing new Mg-based al loys, to investigate the biocompatibility of the degradation products from the Mg-based alloys, the Mg-based samples were immersed in CM and after a certain time, the medium that contains Mg²⁺ ions was used for culturing bone-like cells to study mineralization. In both the mentioned approaches, the concentration of the Mg in CM during mineralization tests was kept constant, which resembles a static condition. In this research, the direct effects of the three different doses of M MWs (MMW1, 2 and 3) on the mineral ization of BMSCs, was dynamical ly investigated using Alizarin Red staining. It could be predicted that the release of Mg from MMWs may not be constant during the osteogenic process, and the changes in the concentration of Mg element in the OM were monitored. Furthermore, the concentrations of Ca, K, and Na (basic elements) in the OM were tracked. As shown in Figure 21(a), (b); with the increase of the dose of M MW, stronger Al izarin red staining was observed in comparison with the control group (without M MW). Accordingly, the quantitative data (resulted from dissolving the red stained nodules), showed that the mineralization of BMSCs cultured with M MW2 and MMW3 was significantly enhanced compared to the control group (p<0.05). Whereas, there was no significant difference between the M MW1 and the control groups (Figure 21(c)).

[0263] As shown in Figure 21(d), the concentration of the released Mg from M MW- included groups was not constant during osteogenesis. Apart from the appl ied dose of MMW, Mg concentration in the OM was rapidly increased from day 0 to day 3, and then decreased sharply from day 3 to day 6, Afterwards, it was gradual ly reduced from day 6 to day 15. The delivered Mg from immersion of M MW3 in the OM was in a wide range of 137-425 ppm during days 3-15. While, in the same condition, it was in a more limited range of 122-40 and 135-90 ppm for M MW1 and MMW2 groups, respectively. Interestingly, the trend of Ca changes from day 0 to day 3 in al l the MMW groups was completely opposite, with the trend explained for Mg. It decreased sharply from day 0 to day 3. Then, apart from the fluctuations, Ca content was almost levelled up after day 9, while the included Ca in the control group was almost constant simi lar to the trend observed for the concentration of Mg. These obvious differences between the changes of Mg and Ca contents in the OM in M MW2 and MMW3 with the control ones, can be the main reason for delivering significant different levels of mineralization. Because reduction in Ca content in extracted OM from M MW groups, in comparison with the constant level of Ca in the control group in driftnet time-points, can be considered logical evidence for taking more Ca by the cel ls for ECM mineral ization, it is consistent with the proven key role of Mg to Ca absorption in bone-related dietary field. The trends of changes in the contents of K and Na in the considered M MW groups were almost the same, fluctuating approximately in the range of 230-241 ppm for K and 3880-4340 for Na (the blue dashed lines and arrows in Figure 21(d)), respectively. Furthermore, less fluctuation was observed in the changes of both K and Na contents in the control group.

[0264] Taken together, M MWs in a certain dose range (M MW2 and MMW3) could successfully induce BMSC mineralization by delivering a proper concentration of Mg²⁺ ions, which can effectively contribute to the absorption of more Ca by the bone-related cells to stimulate ECM mineralization.

[0265] The dose effects of MMWs on the inflammatory response of RAW cells: Macrophages play an important role in innate immunity. They can polarize to pro- inflammatory or anti-inflammatory phenotypes with the release of inflammatory/antiinflammatory cytokines, In the current study, the responses of macrophages (RAW cells) treated by M MW at different doses under an inflammatory environment were assessed (Figure 22(a), (b)). It can be observed that MMWs significantly downregulated the mRNA levels of pro-inflammatory markers (iNOS, I L-6 and CD-86) in comparison with the control (+) group (p < 0.05). Conversely, the expression of anti-inflammatory marker I L- 10 was significantly upregulated by applying the M MWs. The obvious dose-dependent effects could be found in the decrease/increase of mRNA levels of pro-/anti-inflammatory cytokines, respectively (I L6/I L-10, Figure 22(a)). Figure 22(b) shows the concentrations of released pro-inflammatory cytokines ( IL-Ib and IL-6). Simi lar to the trend observed in Figure 22(a), the concentrations of I L-I b and IL-6 were significantly decreased in comparison with the control (+) group (p < 0.05). Additionally, morphological changes would happen in immune cel ls (such as RAW cells) in the inflammatory environment compared to the non-stimulated cells. In Figure 22(c), the SEM images of non-activated (with IFNy and LPS), as well as activated RAW cells with M MWs are compared. Generally, activated cel ls showed induced expansion and elongation compared to the non- activated ones, which is in accordance with the previous reported results. The cel ls cultured with M MWs were expanded and elongated more, where M MW3 led to the most expanded and elongated RAW cells among al l the considered groups. In addition, the shapes of cel ls in MMW-groups were different from that in the Control (+) group. This result suggests that the relationship between phenotype and morphology of macrophages still needs further investigation. Although the pro-inflammation is considered as a vital reaction initiating the bone healing process, its continuation can cause a negative effect on the heal ing process. It is demonstrated that presence of pro-inflammatory cytokines at high levels prevented osteogenic differentiation. Recently, it has been reported that downregulation of the pro-inflammatory cytokines may stimulate the bone heal ing process. Therefore, in addition to the beneficial effect of the M MWs in mineral ization (Figure 21), MMWs can effectively inhibit the inflammatory response of macrophages, which should improve bone regeneration.

[0266] MMW-directed immunoregulation on RAW cells effectively induced osteogenesis in vitro: As discussed in the previous section, M MWs played an effective role in directing the polarization of the RAW cells towards a less-inflammatory phenotype. To verify if this regulation can be beneficial for the osteogenesis process, the RAW cel ls extracted CM was used during osteogenesis of BMSCs. Figures 36(a) and (b) show that compared with the CM from Control (+)-RAW cells, CM originated from M MW-treated cells, significantly induced mineralization of BMSCs, as shown by the staining i mages and the quantitative results (Figure 23(a), (b) and (c)) (p<0.05). This increase is in accordance with the Mg dose levels in M MWs. Moreover, expression of the osteogenic markers, including collagen type I (COL-I), Runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) was significantly upregulated in BMSCs treated with the M MW-CM, as compared to the control group treated with the Control (+)-CM (Figure 36d) (p<0.05). The results in Figure 23 further verify that M MWs at graded doses, directed a proper immuno-environment, which led to induced expression of osteogenic-related markers, and then eventually induced the mineralization of BMSCs, These results are consistent with the previous studies, which reported the important relationship between inflammatory molecules and osteogenic factors.

[0267] All in all, the above results provide convincing evidence for the suitable OIM property of M MWs, which can potentially modulate the immune system in the local environment and consequently facilitate osteogenesis in bone tissue repair. [0268] CONCLUSION: The obtained results in this section, demonstrated that the total dose of the M MWs played an important role in biocompatibility by altering the viability of both macrophages and BMSCs. Additionally, choosing MMW at a proper dose could induce the mineral ization process by releasing a suitable dose Mg²⁺ ions, which could alter the absorption of more Ca by BMSCs. Moreover, the suitable doses of the MMWs have shown an excellent 01 M property by modulating the immune-environment under inflammatory condition to benefit the osteogenic activities of BMSCs. We observed that Mg wires with dose of 2.5, 5 mg/100mm² (mg/cm²) significantly increased the viability of the cel ls. Interestingly, the trend of Mg and Ca concentration in the extracted OM during osteogenesis tests were almost opposite: the more the released Mg, the less the concentration of Ca, which can be convincing evidence for the important role of the released Mg²⁺ ions from MMWs on the absorption of more Ca by BMSCs, led to significantly enhance the mineralization process with doses of 5, 15 mg/100mm². It was also seen that the dose range of 2.5-15 mg/lOOmm² showed suitable osteo- immunoregulatory property of M MWs and resulted in downregulating pro- inflammatory- related cytokines and upregulating osteogenesis-related genes significantly.

[0269] In summary, our findings in this section may give a better understanding of the nutrient effect of MMWs in bone biomaterials via investigation of the dependency of osteoimmunology and osteogenesis in vitro to the dose of M MWs. The optimized dose range of 2,5-15 mg/100mm² in this section, del ivered proper biocompatibility and osteoimmunology, and covers the dose range of 3.5-11 mg/100mm² resulted from the designing stage of the Mg-reinforcement pattern presented in computer simulation part of this patent. However, as far as MMWs are biodegradable material, their structural properties are changed during their degradation.

Investigate the degradation behaviour of Mg wires and develop a surface modification strategy:

[0270] Overview : It is critically important that the M MWs should be inherently degradable biomaterials, providing proper mechanical stability during the healing process, especially during the first few weeks after the RA surgery. The fracture of even a single wire in the Mg wire-based component can potentially cause the failure of the reinforcement layer(s) and may finally result in the col lapse of the composite membrane into the defect area and missing the desired natural contour of the ridge area, which is the main purpose of an RA surgery. Therefore, this section mainly focused on the degradation behaviour of MMWs over time to assess the mechanical stability of“single” Mg filament. For that purpose, the degradation behaviour {i.e., mechanical stabil ity and morphological changes) of different sizes of Mg wires during four weeks of immersion in the body fluid was studied first. The results achieved from mechanical testing illustrated that the M MWs with the thickest considered diameter in (i.e., D=500pm) were still mechanical ly stable after four weeks. Whereas, the thinner wires were not that stable. Therefore, if the application of thinner MMWs is desirable for making the Mg reinforcement layer, as a pilot study, we tried to propose a new surface modification method using Mg phenolic networks (MPNs) that can deliver more durable mechanical stability for thin M MWs with D<300pm. Additionally, the effects of this surface modification approach on the surface morphology, corrosion resistance, degradation behaviour, and osteo-compatibi l ity (in vitro) of the Mg substrate, were thoroughly investigated so that it can be considered as a platform for any future studies related to the surface modification (passivation) of the Mg- based implant biomaterials. The results showed that the developed surface modification method can be considered as a cost-effective, straightforward, and flexible approach for coating of the tiny objects such as M MWs.

[0271] Introduction·. Apart from the nutrient effect of Mg-based bone implant materials such as Mg wires, they reduce clinical expense by mitigating any secondary removal surgeries. Ideally, such degradable implants should be strong enough during the target tissue heal ing process, and be able to be gradually resorbed in the human body. The reduction in the mechanical stabil ity due to the fast degradation rate, is one of the main challenges regarding the application of Mg-based biomaterials. This can endanger the adequate load shielding over the tissue regeneration duration, and especially in the subject study, lack of enough mechanical stability during the augmentation shows to be unfavourable in most, probably causing collapse of the barrier membrane into the defect area and risking its main structural mission, that is, to supply sustainable contour-making property during the healing process. Therefore, it is vitally important to supply a balance between the degradation time of Mg wires and the healing period of damaged tissue. To that aim, the inventors initially focused on the degradation behaviour of three different sizes (150-500pm) of the bare MMWs during immersion tests in the simulated body fluid. Based on the results achieved from the mechanical testing and morphological changes, the biggest considered size (500mhi) served almost proper and durable mechanical tensile strength, even after four weeks of degradation in the considered solution. Whereas, the fi lament with D=150 and 300mhi was no longer strong after 2, and 3 weeks, respectively. Although the main focus of this study was not fabrication of the Mg wire-reinforced collagen composite membranes, we tried to predict any potential issues during the fabrication of such membranes and propose possible solutions that could be applied to address them. One of the main concerns during the fabrication of the Mg wire reinforced collagen composite membranes is damaging of Mg wires in contact with the collagen solution, because collagen solution is inherently an acidic solution that can damage such tiny wires during the process of making a Mg wire-reinforced col lagen membrane. Therefore, the inventors tried to develop a surface passivation method to control the degradation of M MWs by enhancing their corrosion resistance. Additionally, del ivering a porous hydrophi lic surface with nano-sized phenolic networks using this surface modification strategy may mitigate any concerns regarding the separation or sliding of the embedded Mg wires in the collagen, in the future application of the concept of this research project.

[0272] Surface modification has been used as the most effective approach to passivate the surface of Mg-based alloys and enhance their corrosion resistance. Biomimetic degradable coatings such as calcium orthophosphate, collagen, and bio-composite coatings have been shown to be suitable approaches not only to delay the corrosion of Mg, but also to generate a biocompatible interface for bone formation. However, the main drawback of these surface passivation methods is that the positive role of Mg oxides in bone mineral ization becomes negligible. Therefore, deposition of Mg(OH>2 and MgO coatings by various methods such as ion beam deposition, physical vapor deposition, anodization and pulse DC magnetron sputtering has been proposed to protect Mg alloys. But, most of these methods require special and expensive equipment to deposit Mg oxides on the Mg- based substrate. Furthermore, some of them can be applied only to substrates with simple geometries. Therefore, a cost-effective, simple, flexible, and efficient method to coat Mg- based substrates with Mg oxides could offer significant advantages over the methods mentioned above.

[0273] Metal-phenol ic networks (MPNs) were recently introduced as a flexible and cost-effective surface modification method for deposition of multiple types of nanoscale bio-interfaces on various substrates. MPNs are generally formed by dynamic chemical reactions between metal ions and phenolic molecules using tannic acid (TA) or phytic acid, which are the two main natural polyphenol products. Chen et al proposed a Mg-ion- integrated phytic acid coating to form 3D networks of Mg oxides. They found that a bonelike structure formed on the Mg substrate, which can effectively improve the corrosion resistance and osteo-compatibility, simultaneously stimulates the bioactivity of bone-like apatite precipitation and osteoblast cel l adhesion and proliferation in vitro. Although some studies have investigated MPNs as surface coatings on Mg alloys, the effects of experimental parameters such as the type and concentration of the Mg ion source and phenolic molecules, the number of coating cycles, and the characteristics of the intermediate layer on the morphology, density, homogeneity, and stability of the deposited fi lm are not ful ly understood. Furthermore, the effects of those parameters on the degradation behaviour, release of Mg ions, corrosion resistance, cytotoxicity, and cel l differentiation need to be elucidated in order to obtain an osteo-compatible surface. The present section, discussing the results degradation behaviour of the different sizes of bare MMWs, is investigated first. Then, the effects of deposition of MPNs on the Mg filaments on the corrosion resistance, mechanical stabi lity, the surface morphology and characteristics, and biocompatibility of single MMWs were fully investigated.

[0274] Materials and methods: Investigation on changes in mechanical stability and surface morphology of the bare MMWs: Mechanical stability and surface morphology of three different sizes of M MWs (150, 300, and 500 pm) were investigated by immersing the straight M MW-samples (Figure 24) in blood plasma solution for 4 weeks at 37°C. The blood plasma was collected via centrifuging the fresh whole human blood using Allegra X- 15R centrifuge (Beckman Coulter, USA) at 200 g for 20 minutes at room temperature fol lowing the previous protocol. The extracted plasma was then transferred to a cell culture flask containing Mg macro wire samples and inculcated at 37°C and 5% CO², M MW samples were cut in lengths of 50mm and were kept in a straight shape. Blood plasma solution was replaced every two days with fresh solution. The check time points were set as Week 1, Week 2, Week 3 and Week 4. At the end of each interval, M MW samples were extracted, washed with PBS and fixed with 3% glutaraldehyde for 30 min, then washed again with PBS three times. Immediately after the fixating and washing of the sample, tensi le mechanical testing was conducted using a horizontal micro tester (250 MTS Tytron Microforce Tester, USA) to quantify the ultimate tensile strength (UTS) and elongation to fai lure. In order to mitigate the stress concentration in the clamping area, the samples were placed in a sample holder, as illustrated in Figure 24. Before the tensile tests, the two notched side supports in the sample holder were gently cut. Three samples per interval per group of wires with the same diameter were used. After the tensi le tests, the surface morphology of the fracture cross section was observed using SEM (J EOL 7001F, Japan). In paral lel, some MMW samples were prepared and dehydrated to conduct SEM imaging and energy dispersive X-Ray analysis (EDX, SEM, J EOL 7001F, Japan) to assess the morphology and chemical composition changes in the side surface of the samples.

[0275] Mg-phenolic Networks Deposition·. Substrate Preparation: To prevent waste of Mg wires, during the optimization of the coating process, the Mg-based disk shape samples, D= 6 mm, and a thickness of 1 mm were machined from the extruded bil lets. The disks were polished with SiC papers down to 4000 grit. Then, all the samples were ultrasonically cleaned in 100% acetone (10 min), in 100% ethanol (10 min) and final ly rinsed in deionized (Dl) water to remove any surface contamination. It is worth mentioning that after final izing the coating parameters, the final coated samples were prepared with different sizes of M MWs.

[0276] Coating procedure of substrate with Mg-phenolic Networks ( MPNs j: The coating procedure is summarised schematically in Figure 25. The M PNs were coated on disk samples in two steps. In the first step, the prepared Mg-2.8%Zn disks were immersed in NaOH with a pH of 10 for 90 min, A uniform nano-size film of Mg oxides [both Mg(OH and MgO] can form on Mg substrates immersed in NaOH solution. Then the samples were dip-coated in a prepared solution containing 1 mg/mL of TA (analytical grade, purity = 99%, Sigma-Aldrich, USA) combined with MgCh (analytical grade, purity= 99%, Thermo Fisher Scientific, Austral ia) at four concentrations (0.6, 1.2, 2.4, and 3.6 mg/mL) in NaOH solution (pH=10). After 30 min, the samples were removed and washed three times with a NaOH solution to remove any salty liquid from their surfaces. In the second step, this procedure was repeated using fresh solution to perform the second and third cycles and to form a uniform MPN surface. To reduce undesirable oxidation, the coated samples were kept in a vacuum desiccator. The NaOH + 1% TA + 3.6% MgCh, NaOH + 1% TA + 2.4% MgCI², NaOH + 1% TA + 1.2% MgCI₂, NaOH + 1% TA + 0.6% MgCh, and NaOH coating solutions and the samples coated with them are denoted as 3.6%Mg@TA, 2.4%Mg@TA, 1.2%Mg@TA, 0,6%Mg@TA, and NaOH, respectively.

[0277] Surface morphology assessment of Mg-Phenolic Film: To observe the surface morphology of the coated samples after the coating procedure mentioned in the previous section, the samples were coated with a thin layer (~10nm) of gold using a sputter (EM SC005 Gold Coater, Leica). Then, the surface morphology of the different samples was observed using scanning electron microscopy (SEM, J EOL 7001F, Japan). Secondary electron detector (SED) was used to capture detailed shape and surface information. Parallel samples were prepared to observe the cross-section view of the coated samples. To that aim, the samples were mounted inside of the epoxy resin and after the cutting and polishing procedures, the cross-section of the samples was coated with a very thin layer (~4nm) of platinum using a sputter (EM ACE600 Platinum Coater, Leica). Final ly, the cross-section view of the coating layer of the different samples was captured using SEM (SEM, J EOL 7001F, Japan). Back scattered-electron (BSE) imaging was applied to extract chemical contrast information.

[0278] Surface Characterisation of Mg-Phenolic Film: The chemical composition of the sample surfaces was examined by X-ray photoelectron spectroscopy (XPS) using the XPSpeak 4.1 package to analyse the high-resolution spectra and fit the peaks to bonding states. The wettability of the surfaces was assessed by conducting a simple water contact angle test at room temperature and 50% relative humidity using the sessile-drop method on an inclined plate. The roughness of the samples was quantified by atomic force microscopy (AFM) in a scanning range of 20 ^c 20 gm², Each experiment was repeated at least three times for statistical analysis.

[0279] Corrosion Resistance Assessment. The effect of the Mg— phenol ic coating on the corrosion resistance was examined by electrochemical impedance spectroscopy (EIS) with an electrochemical unit (VSP, Bio-Logic Science I nstruments, France) sample as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum foi l as the counter-electrode. Phosphate-buffered saline (PBS) solution was used as the electrolyte. The side and bottom surfaces of the disk samples were covered by si l icon glue, and only the top surface (28,3 mm²) was exposed to the electrolyte. All the tests were performed at 37 ± 0.5°C. The EIS data were recorded from 200 kHz to 100 mHz with a 10 mV sinusoidal perturbing signal. Accordingly, the potentio-dynamic polarization (PDP) tests were conducted at a scanning rate of 1 mV/s. Then, the Tafel method with linear extrapolation of the active polarization zone in the cathodic polarization section of PDP curves was applied to extract the current density ( l_COrr) and corrosion potentials (E_Corr). The EC-Lab software package (VI 1, Bio-Logic Science Instruments, France) was applied to analyse the EIS and PDP results.

[0280] Degradation Behaviour: The degradation behaviour was examined by immersing the samples in PBS solution (pH = 7.4) for three weeks at 37 °C. The surface/volume ratio was 56 mm²/mL. The time checkpoints were set as day 1, day 3, week 1, week 2, and week 3. The PBS solutions were refreshed every two days with 500 pL of a fresh solution. At the end of each interval, the concentration of Mg²⁺ ions released into the extracted PBS solution was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES 700, Agi lent, USA) after measuring the pH value of the extracted solution using a pH meter (Thermo Fisher Scientific, USA) in each time checkpoint. In paral lel, simi lar samples were prepared to measure the evolution of H² gas during the degradation of the samples in PBS with a test area of 0.283 cm² using an eudiometer. For surface morphology assessment, the samples were ultrasonically washed with distilled water for 5 min to remove the salty crystals deposited on the surface from the PBS solution. Scanning electron microscopy (SEM) imaging and energy dispersive X-ray spectroscopy (EDX) were used to assess the surface morphology and chemical composition of the samples, respectively. Three samples were tested for each interval.

[0281] In vitro Biocompatibility Tests : To study the in vitro osteo-compatibi lity of the coated samples, the cell morphology, adhesion, and proliferation of the MC3T3-E1 osteoblast cell line (Sigma-Aldrich, USA) on the coated samples were monitored. A suspension Of 8xl0³ cells in IX Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum as well as 1% pericyte growth supplement was seeded on the surfaces of the samples, which were held under 5% CO2 at 37 °C. After the samples were cultured for three days, the culture medium was extracted, and all the samples were washed with PBS, fixed with 3% glutaraldehyde for 30 min, and then washed again three times with PBS. Next, the samples were dehydrated according to previous protocols. Finally, the morphology of the cells was observed using SEM (J EOL 7001F, Japan).

[0282] Although the main purpose of the proposed passivating coating in this section was to enhance the corrosion resistance of the Mg substrate, that coating should serve a degree of degradability of the substrate to release Mg²⁺ ions and Mg oxides surrounding the implantation area. It is known that release of Mg ions (Mg²⁺) and Mg oxides would induce the viabil ity of the bone-like cells. To investigate that property, an MTT assay was conducted to assess the cel l viability, Each sample was first soaked in 150 mI_ of the prepared culture medium for 24 h at 37 °C and 5% CO2. Then, the extracted medium was diluted with lxDMEM to a 1:1 ratio and used for subsequent experiments, including fixed cell confocal imaging and viability assessment, For confocal imaging, 1.6 x 10⁴ MC3T3- E1 cells were seeded on a round coverslip slide. At the same time, 8 x 10³ MC3T3-E1 cel ls were seeded on a 96-well plate. Three wel ls were used per group for each experiment. Fresh medium was used as the control groups in both experiments. The cells were cultured for one and three days at 37 °C and 5% CO2. At the end of each timeline, the viabil ity of each group of samples was assessed, and the absorbance was measured. [0283] To investigate the cytoskeletal arrangement of the cel ls using multiphoton confocal microscopy, similar samples in different groups were prepared. Briefly, at the end of each time-point, the culture medium was extracted, and the MC3T3-E1 cells were washed with lx PBS and fixed with 4% paraformaldehyde. Next, 0.1% Triton-X was added to permeabilize the cells (1 h). Then, the cells were kept in TRITC-conjugated phalloidin for 1 h. Finally, the cel l nuclei were stained by incubation in 4' 6-diamidino-2-phenylindole for 10 min at room temperature.

[0284] The effect of MPN coating on mechanical stability of MMWs: To investigate the effect of the established coating method in this section using MPNs on the improvement in the mechanical stabi l ity of the MMWs, single Mg wire samples with D=300 miti were prepared and coated with MPNs with 3,6% concentration of Mg in the coating solution. Then, the changes to the mechanical stabi lity of the samples were assessed over four weeks of immersion in blood plasma via mechanical testing in three different time-points (WO, W2, and W4) similar to the procedure conducted for the bare Mg wires shown in Figure 24).

[0285] Statistical analysis: Statistical analyses were performed via one-way analysis of variance (ANOVA) using a SigmaPlot verl3.0 (Systat Software, Inc, USA) software package.

[0286] RESULTS AND DISCUSSIONS: Evaluation of ultimate tensi le strength (UTS) and elongation at break of different sizes of the bare MMW using tensile mechanical testing.

[0287] Figures 26(a) and (b) shows the changes of UTS and elongation at break of the MMW during four weeks of immersion in blood plasma, respectively. It is found that, among the considered sizes only D=50(^m stayed sustainable after four weeks degradation. However, the resulted average UTS was decreased by 66% compared to its strength at week 0. Samples with D=150 and 300pm were just stable for 2 and 3 weeks with 81 and 85% reduction in their UTS, in comparison with week 0, respectively. The elongation to break had almost a similar trend as the ones of UTS. Decreasing of the effective diameters of the wires and surface cracking seemed to be the two most influencing factors for such changes in the mechanical properties of the experiment MMWs. Figure 26(c) shows SEM macrographs of the fracture cross section of the M MWs after tensi le tests. Apart from the formation of brittle oxide and cluster layers on the core of Mg (which would not affect the total strength of the M MW), the effective diameter of the MMW, which was still Mg metal, was decreased. [0288] Morphological assessment and chemical analysis of the surface of the bare MMW: Figure 26(d) represents the morphological changes of the surface of the MMWs. It is clear, that there was no defect such as cracks or voids on the surface of the wires at week 0. After immersion, samples in the blood plasma cracking initiated on the surface of the samples at week 1, due to the reaction between the MMWs and blood plasma. There was no significant change in the morphology of the samples from week 1 to week 2. Unti l week 2, the oxide layer was formed on the Mg core (MC). Meanwhi le, the cluster layer was formed on the top of the previous oxide layer of week 3. As shown in Figure 26(e), the intermediate oxide layer (IOL) and the cluster layer (CL) were marked with purple and yellow colours, respectively It should be noted that the M MWs with O=150miti were completely destroyed after four weeks, making it hard to gently transfer them for SEM imaging, which led to separating wires to the smal ler parts, The detai led view of the changes on the cross-section morphology of the sample from the MC portion to the CL is shown in Figure 26(e). Additionally, the corresponding EDX profiles for the IOL and CL layers is given in Figure 26(f); in that the CL mainly consisted Mg, 0, and P, while the IOL contains Ca, Na, and Cl.

[0289] Characterization of MPNs film: Surface morphology: The surface morphologies of the coated samples after the coating processes are presented in Figure 27(a). The initial step of the coating process using the NaOH solution produced a uniform nano-size porous fi lm with tiny scales on the surface. The effect of Mg concentration on the morphology after three coating cycles is obvious. The tiny scales on the intermediate layer of step 1 were successfully thickened at the lowest Mg concentration (0.6%), The tiny scales formed in step 1 acted as an intermediate passivation layer that reduced Mg substrate corrosion by allowing Mg ions to react with TA to form the 3D phenol ic structure. Additionally, the gaps between the tiny scales were almost filled in 0.6%Mg@TA. When the initial concentration of Mg was increased from 0.6% to 1.2%, the gaps in the intermediate layer were completely fi lled by M PNs. For the 2.4% Mg group, cauliflower-like structure was formed and ultimately became connected to form a uniform secondary layer as the concentration increased to 3.6%. Moreover, the image to 0.6%Mg@TA sample shows the formation of cracks on the surfaces of the coated samples after three cycles of coating at this concentration. The micro- and nano-cracks indicated release of Mg ions from the substrate and subsequent corrosion. This result indicated that Mg ions at the initial concentration of 0.6% did not react with the free oxygen ions of TA. The remaining oxygen ions would react with the substrate, resulting in a lack of Mg ions and fewer M PNs. Figure 27(b) compares the thickness of the coating layer in different samples using backscattered electron imaging. There was no significant difference between the NaOH- and 0.6%Mg@TA-coated samples. Moreover, the thickness of the coating layer was increased from 6 pm to 7.5, 9, and 10 pm by increasing the initial concentration of Mg during step 2 of the coating process from 0.6% to 1.2%, 2.4%, and 3.6%, respectively.

[0290] Chemical Composition Analysis of Mg-phenolic Film: Figure 28(a) shows the chemical composition of the NaOH- and 3.6%Mg@TA-coated samples, as representative of the results of steps 1 and 2 of the coating procedure (Figure 25). C, 0, Mg, and Zn were present in both coatings. Figure 28(b) shows the high-resolution Mg 2p spectra of the coating after steps 1 and 2. The Mg 2p spectra of the NaOH sample showed that step 1 introduced a mixture of MgO and Mg(OH)², By contrast, MgO was the main component of the 3.6%Mg@TA sample owing to the chemical reaction between free O² ions from TA and Mg²⁺ ions, which led to the formation of cauliflower-like MPNs.

[0291] Surface Roughness: Figure 28© summarizes the average roughness (Sa) of various samples observed by AFM in a scanning range of 20 x 20 pm². The average S_a value was approximately 10 nm for the polished raw samples and increased to 23 nm after step 1 of coating. The roughness of the samples completely coated with M PNs increased to 88, 90, 107, and 116 nm for the 3.6%Mg@TA, 2.4%Mg@TA, 1.2%Mg@TA, and 0.6%Mg@TA samples, respectively. The roughness values of the 2.4%Mg@TA and 3.6%Mg@TA samples were quite similar. Moreover, the surface roughness decreased as the initial concentration of Mg increased, owing to smoothing at higher concentrations.

[0292] Wettability: The results of wettability assessment are shown in Figure 28(d), The average water contact angle of the raw samples was -88°. The NaOH coating decreased the water contact angle by 41%, compared to that of the raw samples (p <0.05). It is also clear that regardless of the initial Mg concentration, the addition of MPNs enhanced the hydrophilicity. Hydrophilic surfaces with water contact angles of 20-40° can reportedly provide the highest levels of cell attachment. As shown in Figure 28(d), all the coated samples exhibited moderate water contact angles of 21-27°, which were significantly different from that of the raw sample (p <0.05). Further, the deposited MPNs also significantly increased the hydrophi l icity compared to that of the NaOH coating (p <0.05).

[0293] Corrosion Resistance Analysis: Figures 29(a) and (b) shows the EIS and potentio-dynamic polarization (PDP) curves. The Nyquist impedance spectra clearly shows the kinetics of electrochemical corrosion of the samples. Using the equivalent circuit and obtained E IS curves, curve fitting analysis was performed using the EC-Lab software; the corresponding data for each element of the circuit were extracted and are summarized in Figure 30. R_ct is known to have the largest effect on the impedance of the corrosion reaction. As shown in Figure 30, the Rct value of the 0.6%Mg@TA sample was even lower than that after the first step of coating (i.e., coating with NaOH), possibly owing to cracking during the coating process (Figure 27(a)). The increase in the Mg concentration caused an increase in R_ct. Therefore, it can be concluded that the porous coating of MPNs with the optimal Mg concentration can significantly enhance the corrosion resistance of Mg-based alloys. The obtained curves from PDP tests (Figure 29(b)) and the corresponding values for the E_COrr and Icon· extracted from the polarization curves using Tafel method (Figure 30) had almost the same trend similar to EIS curves (Figure 29(a)) and the Rct value among the considered samples (Figure 30). The 2.4%Mg@TA and 3.6%Mg@TA samples delivered the lowest value for the corrosion current density ( I con) among all the samples (Figure 30). The value of the Ico for these samples is almost five times lower than that of the raw sample. As far as Icon is directly proportional to the corrosion activities, we can claim that the MPNs with proper coating procedure delivers proper electrochemical stability and promotes anti-corrosion performance of the Mg-based substrate kinetically. Moreover, the free corrosion potential ( E_COn) of the coated samples shifted toward the more positive potentials, indicating the thermodynamic reaction activity of the coated samples in PBS was more suppressed compared to the uncoated sample.

[0294] Taken together, all these results can be considered as convincing evidence to prove that performing MPNs strategy with proper initial Mg concentration during the coating procedure can serve a proper corrosion protection for the Mg-based substrate, Furthermore, the Rct and Icon- values of the 2.4%Mg@TA and 3.6%Mg@TA samples were almost the same (Figure 30), indicating that initial Mg concentrations exceeding 2.4% would not further improve corrosion resistance.

[0295] Morphology Assessment of MC3TC-E1 Cells: The morphological changes in the seeded MC3T3-E1 cel ls on the samples were investigated, using SEM imaging to compare the effects of the Mg-phenolic coating on the adhesion and proliferation of the bone-like cells. Figure 31 presents SEM micrographs showing the morphology of MC3T3- E1 cel ls cultured on the samples at the end days 1 and 3. On day 1, the 3.6%Mg@TA, 2.4%Mg@TA, and 1.2%Mg@TA samples showed high osteo-compatibility, with cel l attachment and suitable spreading owing to the stable moderately al kal ine pH and suitable surface wettability of the protective coating. On day 3, the morphology of cells cultured on the samples indicated cell prol iferation. Few cells became attached to the raw and 0.6%Mg@TA samples, indicating that they could not provide a suitable biocompatible physiological environment for cell prol iferation. By contrast, the 3.6%Mg@TA, 2.4%Mg@TA, 1.2%Mg@TA, and NaOH-treated samples exhibited improved biocompatibility, with more cells and acceptable levels of prol iferation. Specifical ly, the 3.6%Mg@TA and 2.4%Mg@TA samples showed the best osteo-compatibil ity among all the groups. The above results were consistent wettability results, and the results of corrosion resistance experiment. That is, the 3.6%Mg@TA and 2.4%Mg@TA samples with the proposed porous osteo-compatible MPNs exhibited acceptable corrosion resistance by mitigating the negative effect of excessive release of H2 bubbles and exhibited suitable wettability for adhesion and prol iferation of bone-like cel ls.

[0296] Viability Assessment of MC3TC-E1 Cells: Surface properties, such as roughness and wettability, the pH of the culture medium, the concentration of Mg ions, and oxygen deficiency due to the release of H2 gas bubbles can be considered to be the four main parameters directly affecting the in vitro viability, in particular the medium-term cytocompatibility of the Mg alloys. Indirect viabi lity assessment is a common method of evaluating the in vitro medium-term osteoblastic cytocompatibi l ity of such al loys and their capacity to mitigate the effects of these surface properties, and increase the pH, H2 gas evolution. That is, indirect viabil ity assessment can provide better information about the medium-term influence of the degradation of Mg-based al loys on the surrounding physiological environment in vitro. Therefore, to focus on the effect of the release of Mg²⁺ ions on the viabil ity of osteoblast-l ike cel ls, indirect viability assessment of extracted medium mixed with lx DMEM at a 1:1 ratio was performed. The average pH values of the medium collected from the 3.6%Mg@TA, 2.4%Mg@TA, 1.2%Mg@TA, 0.6%Mg@TA, NaOH-treated, and raw samples were 8.12, 8.18, 8.25, 8.39, 8.30, and 8.41, respectively. Figure 32 shows the results of MTT assay after 1 and 3 days of cell culture with the prepared culture medium for each group. The absorbance of al l the samples obviously differed significantly from that of the control group (p < 0.05) on day 1. This finding confirms the stimulating effect of Mg corrosion products (especial ly Mg²⁺ ions) on the proliferation of bone-like cells reported by Wu et al. Furthermore, Galow et al have reported that culture medium with alkal ine pH enhances the viability of osteoblast-like cells in vitro. They claimed that higher pH values of up to 8.4 cause the higher viability. [0297] The increase in the absorbance of al l the groups from day 1 to day 3 indicates enhancement of the proliferation mechanisms of the cel ls. The confocal images of the cel ls on day 3 are shown in Figure 33. The cells clearly proliferated well. The differences in cel l proliferation among the groups can be explained in terms of the difference in the medium pH.

[0298] In summary, the indirect viabil ity test showed that the dose of Mg ions from uncoated Mg-based alloys (i.e., the raw sample) resulted in an alkaline pH in vitro, enhancing proliferation. Furthermore, the 3.6%Mg@TA and 2.4%Mg@TA samples have not only an effective coating of M PNs and the most osteo-compatible surface among all the groups, which results in reduced cytotoxicity, but also can still release corrosion products, improving the viabi lity of the biological fluid in the zone around the implantation area.

[0299] Improvement in the Mechanical Stability of the MPN-coated MMWs (Pilot study): Figure 34 compares the changes in the UTS values of the uncoated (Raw) and MPN-coated MMWs. It is clear from the results of WO that no reduction happened in the UTS values during the coating process. Therefore, the M PN was non-destructive to the substrate. Moreover, there was a significant difference between the strength of the uncoated (Raw) and MPN-coated M MWs samples, even after two weeks of immersion in the blood plasma (p<0.05). Furthermore, the raw samples had no mechanical stability at W4, whereas the M PN-coated MMWs were stil l mechanical ly stable by serving an average llOM Pa for the UTS value. Overall, it can be concluded that the MPN coating strategy was effective in terms of improving the mechanical stability of the MMWs.

[0300] Conclusion·. This section mainly focused on the degradation behaviour of MMWs. The mechanical stability tests and morphological changes during one-month immersion in the body fluid showed that the mechanical stability of smaller bare M MWs was not stable till the end of the considered time point (4 weeks). Therefore, in the case of the utilization of thinner M MWs as the fi lament material for making Mg-reinforcement layer(s), we tried to improve the mechanical stabi l ity of such wires using MPNs strategy, which effectively improved the corrosion resistant of Mg-based substrate. The proposed cost-effective, flexible, and simple approach in this section could effectively protect the surface of such tiny wires against undesirable corrosion. It was observed that the concentration of Mg ions in the coating solution was the main factor affecting the performance of the coating. The corrosion resistance increased with increasing concentration of Mg ions in the coating solution. The results from material characterization tests showed that there was significant improvement in the mechanical stabil ity delivered by the MPN-coated samples. Furthermore, SEM images of the morphology of osteoblastlike cells showed that the Mg-based substrate with a suitable coating of MPNs had an osteo-compatible surface that significantly enhanced cell adhesion and prol iferation in the first few days after cell seeding. Additionally, indirect medium-term viability assessment showed that the medium extracted from even the samples with highest Mg concentration in the passivation coating (i.e., 3.6%Mg@TA and 2.4%Mg@TA) enhanced the viability of the bone-like cells. Overall, passivation of M MWs using the M PN method can passivate the surface of such wires with proper corrosion resistance and osteo-compatibility. Additionally, delivering a porous hydrophilic surface with Nano-sized phenolic networks, using this surface modification strategy, can mitigate any future concerns regarding the separation or sliding of the embedded Mg wires in the collagen in the future application of the concept of this research project.

In vivo Investigation of the performance of MPN coating:

[0301] In Vivo Degradation and Bone Healing Situation: After conducting the implantation surgeries and during 4 weeks period unti l final surgery for harvesting skul l bone blocks, all rats were in good health and wound healing without any severe side effects.

[0302] Observation of subcutaneous hydrogen gas cavity: In the case of the severe degradation rate of Mg-based implants, subcutaneous hydrogen gas cavity can be easi ly observed in the surrounds of the implantation area. Regardless of the size of the Mg-based implant, the formation of such cavities can be considered as convincing evidence for a fast degradation rate and improper corrosion resistance which can endanger the biocompatibility of such biomaterials. Based on the literature, taking photographs from the shaved skin covered the defects in the calvarial model has been used for comparing the degradation behaviour of different Mg-based implants. Fig. 35(b) compares the formation of subcutaneous hydrogen gas pockets in uncoated and coated Mg implants after 4 weeks from implantation surgery. An obvious gas cavity formed in the implantation area of uncoated Mg disc. Whereas, no obvious gas pocket formed in the surrounding of coated Mg disc with Mg phenolic networks which indicates the passivating coating layer controlled the degradation of Mg implant at a low rate that can give enough time to the blood flow to clear the created hydrogen gas bubbles in the implantation area and prevent accumulation of those bubbles. Preventing or at least mitigating the formation of Fh gas cavity can increase derivation of sufficient oxygen to the defect area and improve the biocompatibility of Mg-based implants.

[0303] Surgical procedure: Figure 35(a) shows the surgical procedure for in vivo animal studies. The rats were sedated in a chamber with 4% isoflurane in 100% O2 and then anesthetized by intraperitoneal injection of 15 mg/kg Zoletile and 10 mg/kg Rompun. Under local anaesthesia with 2% l idocaine hydrochloride containing 1:100,000 epinephrine, after disinfection with povidone iodine, a middle skin incision was made on a shaved skin area on the skull, and a full -thickness flap was reflected. Under copious saline irrigation, two standardized round defects each 5 mm in diameter were created on the left and right lateral parietal bones of the rat skull using a trephine bur. After the two critical- size calvarial bone defects, a Mg disk with a diameter of 5 mm and thickness of 0.8 mm was implanted in the right defect, and the left defect was used as the sham group. Then, the periosteum and shaved skin were repositioned and sutured properly. After a healing period of 4 weeks, five rats in each group were euthanized in a CO2 chamber. Subsequently, block sections of the rat skull were collected and fixed in a 4% paraformaldehyde solution.

[0304] Micro-CT Findings: Figure 35© compares the focused front and back views of the reconstructed micro-CT images of defect area uncoated, coated Mg implant as wel l as sham control group obtained at 4 weeks after implantation surgery. Regardless of the volume of the bone formed in surrounding of Mg implant, the difference in the degradation of uncoated and coated samples is obvious. In the uncoated Mg implant, some localized corrosion pits and/or holes were formed indicating more severe corrosion rate in comparison with the coated implant with Mg phenolic networks which there was no obvious void or holes. It can be considered as a logical reason for the accumulation of hydrogen gas in the uncoated (Figure 35(b)). The higher the degradation rate, the more probable the s formation of subcutaneous gas pocket.

Investigate the formation of hydrogen gas (H2):

[0305] Two groups of samples were prepared to measure the evolution of H2 gas during the degradation of the samples in PBS using an eudiometer.

[0306] The first group of samples were coated with MPNs and uncoated Mg disk shape samples immersed in PBS solution with test area of 0.283 cm² , and thickness of 1 mm. Figure 36(a) compares the amount of released H2 gas from the surface of the samples in 3 different time-points (Day 1, 3, and 7). It is clear that the coated samples with Mg- phenolic networks (MPNs) could successfully decrease the release of H2 gas compared to the uncoated samples which is convincing evidence for the performance of the developed surface modification method in this project.

[0307] In the second group of samples, the three different doses of pure Mg wires applied in this technology (i.e., High dose (HD), medium dose (MD), and low dose (LD)) were prepared to compare the formation of H2 gas. Figure 36(b) shows the results from these wire samples. It is obvious that there is already l ittle to no gas formation in the optimized dose range for Mg wires in this technology. Therefore, if they combine the wire with the developed MPNs coating method there is expected even less concern with H2 gas formation.

[0308] All in all, the results of this figure show that the optimized dose range between 2.5-15mg/cm² can practically mitigate any concerns regarding evolution of excessive H2 gas affecting the biocompatibi lity of this technology. Furthermore, regardless of the dose and shape of Mg, the developed surface modification methods in this technology can delivery proper performance in terms of decreasing the evolution of H2 gas.

[0309] As schematically illustrated in Figure 37 one the fabrication steps towards making Mg-wire reinforced collagen composite membranes includes the following steps: an a) addition of a high concentration of collagen (3-5mg/ml) and freeze drying; b) soaking in phosphate buffered saline (PBS) as an optional step; c) mechanical compression using mechanical compression machine; 4) freeze drying; 5) crossl inking; and 6) steril ization with gamma or ultraviolet (UV) radiation or 70% ethanol . Figure 38 shows a general overview from the fabricated composite membrane based on the mentioned steps.

[0310] Given the need to improve the process of degradation, as well as the mechanical properties, of resorbable col lagen membranes, the inventors have applied one or more of physical, chemical and enzymatic processes to provide one or more of an increased degradation time and good tensile properties by cross-linking the natural collagen fibres. This is a surprising advantage because, even though it had been claimed that resorbable cross-linked collagen membranes could decrease tissue biocompatibi lity, Garcia et al. (67) has reported that GBR procedures through resorbable collagen membranes achieve volumetric bone gains with no statistical significance between cross- linked and the non-cross-linked membranes.

[0311] Conventional biodegradable materials generally have poor mechanical properties and are poor at maintaining the requisite space. This il lustrates further advantages of the present technology which in some embodiments does not have one or more of these shortcomings. [0312] Additionally, biodegradable graft materials are preferable over non- biodegradable materials which general ly have the disadvantages of: a higher rate of complications and morbidity; membrane exposure; secondary surgery; and higher cost.

[0313] Advantageously, the invention makes use of magnesium. Magnesium ions are known to increase the proliferation and differentiation of osteogenic cells and magnesium hydroxide is known to enhance bone formation and temporarily decrease the bone resorption. Magnesium ions are also known to reduce the inflammation in the defect area.

[0314] Advantageously, the coating provides pH neutralization and surface passivation which increases cell proliferation with reduced cytotoxicity and mitigates corrosion in the collagen solution, while also providing a hydrophil ic surface to increase attachment of the col lagen to the membrane.

[0315] Yet another advantage is that the treatment of bone defects may harness the regenerative power of periosteum, a dense layer of vascular connective tissue enveloping the bones, using autologous periosteum.

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TABLES

Table 1: The dimensions and mechanical properties of the materials used in the study 1 of Aim 1 (17,116).

Table 2 Comparison between the max deflection and wire consumption in different patterns.

Table 3 Comparison between the first three eigenvalues and wire consumption in different patterns.

Table 4 Comparison between the first three eigenvalues in three different patterns with similar LT.

Table 5 General specification of different conditions considered for coating of Mg oxides on Mg disks.

Claims

CLAIMS The Claims defining the invention are as follows:

1. A material for a bioresorbable implant and/or a bioresorbable implant comprising: a collagen solution or a collagen and one or more magnesium wire to provide a collagen-based composite membrane; wherein the one or more magnesium wire is coated and/or surface modified with tannic acid.

2. A method of making a bioresorbable implant, the method comprising: combining a collagen solution or a col lagen and one or more magnesium wire to provide a collagen-based composite membrane; and coating and/or surface modifying the one or more magnesium wire with tannic acid.

3. A method of surgery comprising inserting a bioresorbable implant of Claim 1 or a bioresorbable implant made according to the method of Claim 2.

4. The material, implant or method according to any one of the preceding cla ims wherein the one or more wires are further coated and/or surface modified with Magnesium.

5. The coating and/or surface modification with Magnesium according to Claim 4 results in pH neutralization and/or passivation of the one or more magnesium wire.

6. The coating according to Claim 4 or Claim 5 wherein the coating is with a magnesium-phenolic network.

7. The material, implant or method according to any one of the preceding cla ims wherein the coating and/or surface modification forms a porous oxide layer.

8. The material, implant or method according to any one of the preceding claims wherein the one or more wire comprises a one layer; two layer; three layer or higher ordered layered pattern.

9. The material, implant or method according to any one of the preceding cla ims wherein each wire comprises one or more lines joined by a linker.

10. The material, implant or method according to Claim 9 wherein the one or more lines may comprise neighbouring lines in opposite directions.

11. The material, implant or method according to any one of the preceding claims wherein the linker between each line is disposed at a right angle, an acute angle, an obtuse angle, an obl ique angle or may comprise an arc between the two neighbouring l ines.

12. The material, implant or method according to any one of the preceding claims wherein the one or more wire may comprise a wavy pattern such as a two-layered wavy pattern.

13. The material, implant or method according to any one of the preceding cla ims wherein the one or more wire comprises a two-layered pattern comprising one or more wire in one layer perpendicular to one or more wire in the other layer.

14. The material, implant or method according to any one of the preceding cla ims wherein the one or more wire comprises a wavy pattern comprising a sinusoidal wave.

15. The material, implant or method according to any one of the preceding claims wherein the one or more wire comprises an auxetic pattern.

16. The material, implant or method according to any one of the preceding claims wherein the one or more wire may comprise a diameter of 140 to 500 mhi; 200 to 600 pm or 100 to 700 pm.

17. The material, implant or method according to any one of the preceding cla ims wherein the one or more wires are comprised in a high dose; medium dose; or low dose with a value of 0.5; 1; and 2 mm for“S” respectively.“

18. The material, implant or method according to any one of the preceding cla ims wherein the mesh design is based on a computational simulation conducted to optimise the mechanical properties.

19. The material, implant or method according to any one of the preceding claims wherein the magnesium wire comprises a two dimensional and or three dimensional arrangement such as, a lattice or mesh.

20. The material, implant or method according to any one of the preceding claims wherein the bioresorbable implant comprises a barrier membrane.

21. The material, implant or method according to any one of the preceding cla ims wherein the bioresorbable implant is biodegradable.

22. The material, implant or method according to any one of the preceding claims wherein the resorption and/or biodegradation of the implant provides a nutrient.

23. The material, implant or method according to any one of the preceding cla ims wherein the mal leability and/or shapeabi lity is suitable for and/or improved compared to other materials and implants.

24. The material, implant or method according to any one of the preceding cla ims wherein the coating and/or surface modification comprises a coating and/or surface modification solution comprising Tannic acid and/or Magnesium.

25. The material, implant or method according to Claim 24 wherein the concentration of the Magnesium in the coating and/or surface modification solution comprises 0.6 to 3.6; 0.5 to 4.0; 0.3 to 5.0 mg/ml.

26. The material, implant or method according to any one of the preceding claims wherein the thickness of the coated and/or surface modified one or more wire comprises 1.5 to 3.0; 1.2 to 3.5 or 1.0 to 4.0 the thickness of the uncoated and/or unmodified one or more wire.

27. The material, implant or method according to any one of the preceding claims wherein the dosage of the implant comprises low; medium and high dosages.

28. The material, implant or method according to any one of the preceding cla ims wherein the tannic acid modified and/or coated magnesium comprises one or more of better absorption; increased adhesion of collagen; enhanced corrosion resistance and enhanced biocompatibility.

29. The material, implant or method according to any one of the preceding cla ims wherein the collagen comprises type-1 collagen.

30. The material, implant or method according to any one of the preceding claims wherein the material or implant comprises ease of cutting.

31. The material, implant or method according to any one of the preceding cla ims wherein the one or more wire comprises a shape obtained through a modelling procedure.

32. The material, implant or method according to any one of the preceding claims wherein the bioresorbable implant comprises a dental; orthopaedic; or cosmetic implant.

33. The material, implant or method according to any one of the preceding claims wherein the coated implant comprises a control led degradation such as, low rate of degradation to al low blood flow to clear any hydrogen gas associated with degradation,

34. The material, implant or method according to any one of the preceding claims wherein the coated implant prevents, substantially prevents, mitigates or substantially mitigates formation of hydrogen gas associated with degradation.