CN110624103B

CN110624103B - Biomaterial device and topical composition for the treatment of skin abnormalities

Info

Publication number: CN110624103B
Application number: CN201910828939.XA
Authority: CN
Inventors: 邱丹尼; 塞尔吉奥·马德里加尔·卡巴洛
Original assignee: Haizhicui Biotechnology Co ltd
Current assignee: Haizhicui Biotechnology Co ltd
Priority date: 2018-09-06
Filing date: 2019-09-03
Publication date: 2023-12-15
Anticipated expiration: 2039-09-03
Also published as: KR20200029363A; JP2020058785A; KR102232371B1; CN110624103A; JP6933393B2

Abstract

A device for Guiding Tissue Regeneration (GTR) includes a matrix of chitosan and variable collagen tissue (MCT), wherein the chitosan is electrostatically bound to the MCT to form an MCT-chitosan composite. MCT can be isolated from invertebrate marine organisms such as sponges, jellyfish, molluscs and echinoderms. The MCT-chitosan composite may be formulated as a biofilm, 3D-sponge, hydrogel or electrospun nanofiber, or the MCT-chitosan composite may be coated on the surface of a biomaterial. The device may include a wound dressing and a tissue sponge (including 3D sponges). Applications include tissue engineering and wound healing, burns and other related guided tissue regeneration applications. MCT and MCT-chitosan composites contained in a pharmaceutically acceptable topical carrier also have cosmeceutical applications for treating scars as well as skin discoloration and various pigmentation problems (including liver spots/chloasma).

Description

Biomaterial device and topical composition for the treatment of skin abnormalities

Cross Reference to Related Applications

The present application claims priority from U.S. non-provisional patent application serial No. 16/123,986 filed on month 9 of 2018, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a biomaterial device for Guiding Tissue Regeneration (GTR). More particularly, the present invention relates to tissue sponges, wound dressings, cosmeceutical compositions and other devices and topical compositions comprising variable collagenous tissues (MCTs) for achieving GTR. More particularly, the present invention relates to such devices comprising a complex of MCT and chitosan biopolymer (CHT) for achieving GTR. The invention also relates to the treatment of burns, wounds, ulcers and other lesions and related skin diseases by applying MCTs alone or as a complex in a biomaterial device to promote GTR. The invention also relates to the treatment of skin abnormalities such as scars and skin discoloration (including chloasma discoloration) by the use of MCT and/or MCT-chitosan complexes in cosmeceutical formulations.

Background

Natural polymers have been used in a number of pharmaceutical applications and medical device technologies. A natural polymer chitosan (sometimes referred to herein as CHT) has been used to prepare nanoparticles, microspheres, hydrogels, films, fibers and tablets. Chitosan has been used to prepare potential drug delivery systems such as oral, nasal, parenteral, transdermal and ophthalmic formulations. Chitosan has also been used to prepare wound dressings and tissue sponges (Kumar et al, chem. Comment 2004, 104, 6017-6084). However, chitosan formulations and materials suffer from a number of drawbacks, including limited stability, biodegradability and tensile strength. Materials such as modified chitosan and synthetic composites have been tested for many of the same uses to evaluate chitosan, but many of these materials have similar drawbacks, including insufficient biocompatibility.

Thus, there is a need for new materials that are biocompatible and biodegradable, and have suitable stability and mechanical properties as well as performance for human and other mammalian treatments and therapies. These new materials and compositions preferably have advantages over chitosan alone, such as additional and/or improved biocompatibility, high stability, and improved physical and biological properties. The ability to use these materials as tissue sponges, wound dressings, cosmeceuticals, and/or systems for delivering therapeutic agents would further aid researchers in the fields of biomedical engineering, biomaterials, and tissue engineering.

Disclosure of Invention

Embodiments of the present invention provide biodegradable and biocompatible variable collagen tissue (MCT) and MCT-chitosan composites. These composites can be formed into a variety of materials such as hydrogels, biofilms, three-dimensional sponges, and nanofibers. MCT-chitosan composites are stronger and have better mechanical properties than known chitosan materials. The MCT component of the complex increases biocompatibility, cell attachment, physical and chemical stability, and improves the mechanical, antibacterial, and hemostatic properties of the chitosan component, thereby significantly increasing the effectiveness of the complex in therapeutic applications.

Accordingly, in one aspect, the present invention provides a composition comprising MCT or a matrix of MCT and Chitosan (CHT), wherein the MCT is isolated from a marine invertebrate. In the matrix, CHT can be linked to MCT by electrostatic interactions (such as hydrogen bonding and dipole-dipole interactions) to form MCT-CHT composites. In one aspect, the MCT-chitosan complex comprises a polyelectrolyte cross-linked structure between GAGs and collagen in MCT and its interactions with N-glucosamine units on chitosan. The variable collagen tissue may comprise collagen and glycosaminoglycans (GAGs).

In one aspect, MCT may comprise collagen and chondroitin sulfate. The composition or MCT used to form the composition may consist essentially of type I collagen. The chitosan may have a degree of deacetylation of about 60% to about 99%. The average molecular weight of chitosan may be about 20kDa to about 400kDa. In some embodiments, the mass ratio of MCT in the composite may be 100:0 to 10:90 of the mass ratio of chitosan in the composite. In some embodiments, the mass ratio of MCT is about 100:0 to 50:50 of the mass ratio of chitosan.

By selecting the appropriate marine invertebrate sources (e.g., sponges, jellyfish, molluscs, and echinoderms) and isolation procedures, the amount and ratio of MCT obtained with higher yields of collagen and glycosaminoglycans can be controlled. For example, MCT with a higher content of type I collagen, fibrillar collagen, which is a key structural composition of several connective tissues, can be isolated and used in the compositions described herein. In addition, the isolated MCT can be biased or controlled according to the nature of the types of fibrillar collagen (I, II, III, V, XI) and glycosaminoglycans (chondroitin sulfate, hyaluronic acid) and their structural heterogeneity.

In one embodiment, MCTs may be isolated from marine invertebrates (such as sponges, jellyfish, molluscs, and echinoderms). In more specific embodiments, the MCT will be isolated from marine invertebrate echinoderms, such as sea urchins and sea cucumbers. In a more specific embodiment, the MCT will be isolated from sea cucumber.

In another embodiment, the MCT may be composed of collagen. In more specific embodiments, the collagen may be type I, type II, type III, type V or type XI fibrillar collagen. In a more specific embodiment, the fibrillar collagen is type I. Type I fibrillar collagen can be selectively isolated by the isolation methods described herein.

In another embodiment, MCT may comprise collagen and glycosaminoglycans. In some embodiments, the glycosaminoglycan may include chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate, and/or dermatan sulfate, or a mixture of both components. In another embodiment, the glycosaminoglycans in the MCT will comprise chondroitin sulfate and/or hyaluronic acid or a mixture of both components.

MCT and MCT-chitosan compositions may be hydrogels, biofilms, 3D-sponges, or nanofibers. The nanoparticles may be formulated into various therapeutic agent (such as oral solution, IV solution, or aerosol) delivery systems. The biofilm may be formed in wound healing, surface coatings or packaging materials. The 3D-sponge may be used as a sponge, such as for tissue engineering and wound dressing templates. The nanofibers may be formulated as a wound dressing template or as a surface coating agent.

In some embodiments, the MCT and MCT-CHT composites may be crosslinked by physical and/or chemical means. Physical crosslinking may be accomplished by radiation treatment (UV, gamma) and/or heat treatment. Chemical crosslinking may be achieved by adding a crosslinking agent to the MCT or MCT-chitosan complex, and the amount of crosslinking agent used in crosslinking the biomaterial may be about 0.1 to about 1.0% relative to the content of MCT. Crosslinking agents that may be used include glutaraldehyde, ethyl-dimethyl-carbodiimide (EDC) -N-hydroxysuccinimide (NHS), riboflavin, genipin, and the like.

MCT and MCT-chitosan composites can also be formulated into a biofilm of 3D-sponge with improved water absorption, thermal stability, vapor permeability, and cell adhesion. In such embodiments, the biofilm and/or 3D-sponge would be suitable as a sponge for tissue engineering and as a wound dressing template for surgical and medical applications.

Aspects of the invention also provide methods of delivering a bioactive agent to a mammal, the methods comprising administering to the mammal an MCT or an MCT-chitosan composite as described herein. MCT and MCT-chitosan composites may form nanoparticles, nanofibers, hydrogels, biofilms, or 3D-sponges, which encapsulate bioactive agents (e.g., drugs or nutrients). Examples of drugs, vitamins and nutrients that may be incorporated into the formulation include lipids such as fatty acids (including omega-3 and omega-6 fatty acids), fat-soluble vitamins (e.g., vitamins A, D, E and/or K), antibiotics (e.g., amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin, metronidazole, penicillin, tetracycline, vancomycin, etc.), probiotics (e.g., lactic acid bacteria, bifidobacteria, etc.), active skin compounds (e.g., retinoic acid, tranexamic acid, hydrogen peroxide, hydroquinone, cysteamine, azelaic acid, tyrosinase inhibitors, etc.), micronutrients (e.g., beta-carotene and/or ascorbic acid, proteins and polypeptides). In some embodiments, MCTs and other nutritional supplements may also be included in the MCT-chitosan composite matrix or in a composition that includes the MCT-chitosan composite matrix.

By varying the amounts of the components and the method of making the MCT and the composite, the MCT and MCT-chitosan composites can be tailored to degrade over a range of rates under a variety of conditions. Accordingly, aspects of the present invention also provide methods of making MCT and MCT-chitosan composites. Aspects of the invention further provide for the use of the compositions described herein for the manufacture of biomedical devices and medicaments useful for treating bacterial and/or fungal infections, burns, diabetic foot, and inflammatory disorders in mammals, such as humans.

In a further aspect, MCT and MCT-chitosan composite biomaterials may be provided as therapeutic cosmetics or cosmeceuticals and by combining MCT or MCT-chitosan complexes with a pharmaceutically acceptable topical carrier, including but not limited to solutions, suspensions, liquids, gels, ointments, lotions or creams, to promote collagen formation, scar healing, wound healing, reduction of liver spots and chloasma, and other skin related benefits.

Drawings

The following drawings form a part of the specification and are included to further demonstrate certain embodiments or aspects of the present invention. Embodiments of the invention may be best understood in some cases by reference to the detailed description set forth herein in connection with the accompanying drawings. The description and drawings may highlight a particular example or aspect of the invention. However, one of ordinary skill will understand that portions of the examples or aspects may be used in combination with other examples or aspects in accordance with embodiments of the invention.

In the drawings:

figures 1A and 1B depict a comparative structure between a general collagen structure and a general MCT structure.

Figure 2 depicts the overall structure of glycosaminoglycans.

Figures 3A and 3B depict a comparative morphology of collagen fibril structure between bovine collagen and MCT.

Fig. 4 depicts the general structure of chitosan.

Figures 5A and 5B are schematic diagrams of MCT-chitosan composites prepared according to embodiments.

Figure 6 is a schematic representation of the preparation of MCT-chitosan complex biofilm according to an example.

Figure 7 is a schematic representation of the preparation of MCT-chitosan complex 3D-sponges according to the examples.

Figure 8 is a schematic representation of the preparation of MCT-chitosan composite hydrogels according to examples.

Fig. 9A is a schematic diagram of the preparation of MCT-chitosan electrospun nanofibers and fig. 9B is a photograph of the resulting nanofibers according to the examples.

FIG. 10A depicts the effect of a cross-linking agent (glutaraldehyde 0.1% v/v) on the mechanical properties of an MCT-CHT composite biofilm, and FIG. 10B depicts the swelling behavior of an MCT-CHT composite biofilm according to an embodiment.

Fig. 11A and 11B depict an illustrative representation of an MCT-chitosan biofilm and its potential application as a wound dressing template, and fig. 11C and 11D depict morphological characterization of MCT-chitosan composite electrospun nanofibers by Scanning Electron Microscopy (SEM).

Figure 12 is a schematic of a different MCT-chitosan composite material for GTR applications.

Figures 13A-13C depict the physical appearance of MCT-chitosan (MCT/CHT) composite 3D-sponges prepared at different MCT/CHT mass ratios according to the examples.

Figure 14 depicts TGA thermograms of MCT-chitosan complex 3D-sponges formulated at different MCT/CHT mass ratios.

Figure 15 depicts the water absorption capacity of different MCT-chitosan composite 3D-sponges formulated at different MCT/CHT mass ratios.

Fig. 16A (no cells added) and fig. 16B (cells added) are SEM micrographs showing adsorption of ADSC cells on MCT-CHT 3D-sponge.

Figure 17 depicts proliferation curves of ADSC cells cultured on MCT-chitosan complex 3D-sponge during 15 days incubation.

Figure 18 depicts ATR-FTIR spectra of composite electrospun nanofibers (ESNF) prepared with MCT-chitosan complexes of different mass ratios.

Figure 19 depicts thermal analysis of chitosan, MCT, and MCT-chitosan complexes by TGA.

Fig. 20A to 20C are SEM micrographs of chitosan, fig. 20D to 20F are SEM micrographs of MCT-chitosan ESNF, with a scale of 10 μm (fig. 20A and 20D), a scale of 2 μm (fig. 20B and 20E) and a scale of 200nm (fig. 20C and 20F), wherein circles indicate the presence of droplets associated with poor electrospinning processes in chitosan ESNF, showing the improvement of MCT-chitosan composites for obtaining electrospun nanofibers.

Figure 21 depicts proliferation of L929 fibroblasts co-cultured with chitosan, MCT, and MCT-chitosan complex ESNF.

Figures 22A-22C are SEM micrographs showing cell adhesion to chitosan (figure 22A), MCT-chitosan complex (figure 22B), and MCT (figure 22C) ESNF after 7 days of incubation.

Figure 23 depicts gel electrophoresis analysis (SDS-PAGE) showing the major protein bands of MCT compared to collagen samples extracted from calves and chickens.

Figure 24 depicts gel electrophoresis analysis (SDS-PAGE) showing the efficacy of MCT separation process from sea cucumber as indicated by the consistency of the protein bands of MCT between batches.

Fig. 25A depicts FTIR-infrared spectra of MCT collagen compared to calf collagen, showing the chemical structural differences between the two samples, and fig. 25B depicts a comparative FTIR spectrum showing the efficacy of the MCT separation process from sea cucumber, as shown by the consistency of the FTIR chemical curves of MCT between batches.

Figure 26 depicts thermogravimetric analysis (TGA) of collagen samples showing the difference in thermal behavior of MCT and calf collagen samples.

FIG. 27 shows the distribution of the structural amino acid composition of collagen present in variable collagen tissue extracted from sea cucumber and compared with bovine collagen isolated from calf skin.

Figures 28A and 28B are Scanning Electron Microscope (SEM) pictures showing morphology and porosity of MCT-chitosan dressing templates (3D-sponges) fabricated by solvent casting techniques.

Fig. 29A and 29B are Scanning Electron Microscope (SEM) pictures showing the surface morphology and structure of MCT-chitosan nanofiber dressing templates fabricated by electrospinning techniques.

Fig. 30A and 30B are photographs before and after administration showing the effect of scar cream administration according to an embodiment.

Fig. 31A and 31B are additional photographs before and after administration showing the effect of scar cream administration according to an embodiment.

Fig. 32A to 32C are further photographs before and after administration showing the scar cream administration effect according to the embodiment.

Fig. 33A and 33B are further photographs before and after administration showing the effect of scar cream administration according to an embodiment.

Fig. 34A to 34D are further photographs before and after administration showing the scar cream administration effect according to the embodiment.

Figures 35A-35C depict MCT-CHT matrix structures.

Fig. 36 depicts a wound sponge and its characteristics and features according to an embodiment.

Fig. 37 depicts the functionality of a 3D sponge according to an embodiment.

Fig. 38 depicts the functionality of an apparatus according to an embodiment.

Detailed Description

In accordance with aspects of the present application, novel biodegradable, biocompatible composites are described that comprise a combination of variable collagen organization (MCT) and chitosan. MCT and MCT-chitosan complexes are extremely versatile and can be formulated into a variety of biomaterials such as skin patches, three-dimensional sponges, biodegradable sutures, and sponges for cell proliferation in tissue engineering, as well as hydrogels and biofilms for tissue regeneration.

In addition, MCT and MCT-chitosan composites can also be formulated into biofilms of 3D-sponges with improved water absorption, thermal stability, vapor permeability and cell adhesion. In such embodiments, the biofilm and/or 3D-sponge would be suitable as a sponge for guiding tissue regeneration in tissue engineering and as a wound dressing template for surgical and medical applications.

Definition: as used herein, certain terms have the following meanings. As will be understood by those of ordinary skill in the art, all other terms and phrases used in this specification have their ordinary and customary meaning. Such usual and ordinary meaning may be obtained by reference to a technical dictionary such as r.j. Lewis, hawley's Condensed Chemical Dictionary of John Wiley & Sons (holy concise chemical dictionary) 14 th edition, new york, n.y.,2001.

References in the specification to "one embodiment," "an example embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, portion, or characteristic, but every embodiment may not necessarily include the aspect, feature, structure, portion, or characteristic. Furthermore, such phrases may, but do not necessarily, refer to the same embodiment as mentioned in other parts of the specification. Furthermore, when a particular aspect, feature, structure, portion, or characteristic is described in connection with an embodiment, it is submitted that it is within the knowledge of one skilled in the art to effect such aspect, feature, structure, portion, or characteristic in connection with other embodiments whether or not explicitly described.

The term "and/or" means any one of the items, any combination of the items, or all items associated with the term.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds, and thus compound X includes a plurality of compounds X.

The term "about" may refer to a change in a specified value of + -5%, + -10%, + -20%, or + -25%. For example, in some embodiments, "about 50%" may represent a 45% to 55% change. For a range of integers, the term "about" can include one or two integers greater and/or less than the stated integer. Unless otherwise indicated herein, the term "about" is intended to include values near the stated range, such as equivalent weight percentages in terms of the function of the individual ingredients, compositions, or embodiments. In addition, the recited ranges (e.g., weight percent, carbon family, etc.) include each specific value, integer, fraction, or identity within the range. Specific values of ranges and the like listed herein are for illustration only; they do not exclude other defined values or other values within a defined range.

The phrase "one or more" will be readily understood by those of ordinary skill in the art, particularly when read in the context of its use. For example, if, for example, the phenyl ring is di-substituted, one or more substituents on the phenyl ring refer to one to five, or one to up to four.

The term "contacting" refers to touching, making contact, or otherwise causing a close or close-proximity action (including at the molecular level), such as causing a chemical reaction or physical change in a solution or other reaction mixture.

An "effective amount" generally refers to an amount that provides the desired effect. Thus, an effective amount refers to a dose sufficient to enhance the therapeutic effect on the disease state or condition being treated. Thus, the effective amount may vary depending on the patient, the disease, and the affected treatment.

The term "patient" or "subject" refers to any animal, such as mammals, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans.

With respect to MCT, the phrase "substantially comprises collagen" means that MCT comprises at least fibrillar collagen. For example, MCT fibrillar collagen may comprise type I, type II, type III, type V, and/or type XI collagen. In one aspect, MCT fibril collagen will be characterized as type I. Other compounds that may be explicitly included or excluded from the complexes described herein include type II, type III, type V and/or type XI fibrillar collagen or combinations thereof. The term "glycosaminoglycan" refers to molecules comprising long unbranched polysaccharides containing repeating disaccharide units, including, for example, chondroitin sulfate, hyaluronic acid, heparin, keratan sulfate, heparan sulfate, and/or dermatan sulfate.

Variable collagen organization (MCT): echinoderms are marine invertebrates that are widely distributed throughout the ocean and have been used as a food source (e.g., sea cucumbers and sea urchins) for decades. They are also known for their characteristic connective tissue, known as variable collagen tissue (MCT), which is capable of rapidly changing its passive mechanical properties (stiffness and viscosity) under control of the nervous system. MCT is a unique feature of echinoderms and has been described in all five existing categories (IC Wilkie, "Mutable collagenous tissue: overview and perspectives (variable collagen organization: overview and concept)", v.matranga (Ed.), echinodermta. Progress in Molecular and Subcellular Biology (molecular and subcellular biological advances). Marine Molecular Biotechnology (marine molecular biotechnology), volume 5, springer, berlin (2005), pages 221-250. Variable collagen structures are composed of discontinuous collagen fibrils organized into bundles (fibers) by an elastic network of fibrillin microfibrils and interconnected by a stress transfer matrix composed of glycosaminoglycans that bind to fibrils and aggregate fibrils this type of organization has recently been proposed as a source of inspiration for "smart dynamic biomaterials" for tissue engineering and regenerative medicine applications (A.Barbaglio, S.Tricarico, C.Di Benedetto, D.Fassini, A.P.Lima, A.R.Ribeiro, C.C.Ribeiro, M.Sugni, F.Bonasoro, I.C.Wilkie, M.Barbosa, M.D.Candia nevali, "The smart connective tissue of echinoderms: a materializing promise for biotech applications (smart connective tissue for echinoderm: biotechnology applications)," cah.20154-720 (713); di Benedetto, A.Barbaglio, T.Martinello, V.Alongi, D.Fassini, E.Cullor a, M.Patruno, F.Bonasoro, M.A.Barbosa, M.D.Candia canevali, m.sugni, "Production, characterization and biocompatibility of marine collagen matrices from an alternative and sustainable source: the sea urchin Paracentrotus lividus" ("Production, characterization and biocompatibility of marine collagen matrix from alternative sustainable sources (sea urchin (Paracentrotus lividus)), mar.drugs,12 (2014), pages 4912-4933). In particular, sea cucumber membranes (known as MCTs) can provide a sustainable and biocompatible source of natural fibrillar collagen to produce films for regenerative medicine applications. Figure 1 shows the general collagen structure on the left (bovine collagen in this case) and MCT on the right. In the case of MCT, the right hand side of figure 1 shows an exploded view of a portion of the structure with proteoglycan-CAG crosslinks between the interfiber matrices and between the collagen fibrils.

In "blue biomaterial", marine invertebrate collagen itself is used as an effective substitute for the most commonly used mammalian-derived collagen (e.g., bovine collagen shown on the left side of fig. 1). Mammalian-derived collagen is commonly used in a wide range of human applications, from large-scale uses such as the food, pharmaceutical/nutraceutical industries and cosmetics, to more targeted fields such as cell culture and biomedical/clinical applications. However, due to allergic problems, religious and social/lifestyle restrictions, disease transmission related causes (e.g. bovine spongiform encephalopathy) and the high cost of recombinant technology, alternatives to mammalian collagen sources are continually being investigated (T.H.Silva, J.Moreira-Silva, A.L.P.Marques, A.Domingues, Y.Bayon, R.L.Reis, "Marine origin collagens and its potential applications (collagen of marine origin and its potential applications)", mar. Drugs,12 (2014), pages 5881-5901).

Glycosaminoglycans (the general structure of which is shown in FIG. 2 below) are long unbranched polysaccharides consisting of repeating disaccharide units. The repeating unit is composed of an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), an uronic acid (glucuronic acid or iduronic acid) or galactose. Glycosaminoglycans have a high polarity and attract moisture. Thus, they are useful to the body as lubricants or as shock absorbers, mainly on the cell surface or in the extracellular matrix (ECM).

Another advantage of echinoderm MCT is that it is relatively easy to obtain a large number of natural collagen fibrils that retain their original structure (Di Benedetto et al, 2014, cited above). Indeed, most mammalian collagens are commonly used in their hydrolyzed (acid-soluble) form, a property that greatly reduces the mechanical properties of the produced membrane/sponge, and is limited in biomedical applications (e.g., tendon/ligament regeneration or dermis reconstruction) where highly resistant materials with fibrillar three-dimensional tissue are required. Echinoderm MCTs can be used to easily and rapidly produce fibrillar collagen films that have a high similarity in ultrastructural and mechanical properties to the physiological condition of connective tissue. Fig. 3A and 3B show a comparative morphology of collagen fibril structure of bovine collagen (fig. 3A) and MCT (fig. 3B). MCT fibrils cross-link internally through GAGs (glycosaminoglycans) providing greater stability to the macromolecular structure of collagen and reducing its biodegradability, an important aspect of wound healing and an attribute of bovine collagen deficiency. Fig. 35A-35C depict crosslinking of fibrils using GAGs. Fig. 35A shows a macroscopic arrangement of collagen fibrils and illustrates that the aligned fibrous structures are held together by an intra-fibrous cross-linked network driven by interactions between glycosaminoglycans (GAGs) and collagen core proteins. Fig. 35B shows the intra-fiber crosslinked network in more detail. Fig. 35C depicts the multidirectional stability of strong tissue networks, such that in vivo mechanical properties and biodegradation characteristics are improved.

A particular regenerative medicine area in which MCT fibril collagen is suitable for commercial use is Guided Tissue Regeneration (GTR). One of the goals of GTR is to reduce post-operative tissue adhesions, a common and only partially resolved complication that prevents normal tissue regeneration. Adhesions are abnormal attachments or cell mixtures that form between tissues or organs after surgery or due to local inflammation. Until recently, researchers have attempted to provide effective and satisfactory tools to overcome them. In fact, barrier membranes comprising several different biological materials (e.g. chitosan and hyaluronic acid) have been tested for GTR, but none of them show all necessary functional properties, the most important of which is to avoid cell penetration into the underlying anatomical compartment (S.Tang, W.Yang, X.Mao, "Agarose/collagen composite scaffold as an anti-adhesive sheet (Agarose/collagen composite scaffold as anti-adhesive sheet)", biomed. The echinoderm MCT-based films have porosity and three-dimensional structure modified as desired.

Chitosan: chitin is a biopolymer composed of poly-N-acetylglucosamine. Chitin is the second most abundant biopolymer worldwide next to cellulose. It is typically found in the exoskeleton or cuticle (such as the hull of a marine arthropod) of many invertebrates, the cell wall of most fungi and some algae. Chitin is generally insoluble in water, but can be purified by caustic alkali Such as sodium hydroxide, to form soluble cationic polysaccharides, chitosan. The chemical name of chitosan is poly (beta- (1- > 4) -2-amino-2-deoxy-D-glucopyranose). Fig. 4 shows the general chemical structure of chitosan.

Chitosan-based bandages and surgical dressings produced by HemCon Medical Technologies have recently been approved by the U.S. FDA as hemostatic bandages with demonstrated antibacterial properties against a variety of pests, including MRSA and acinetobacter baumannii (acinetobacter baumannii). Bandages and dressings are useful for rapid hemostasis (including extensive arterial bleeding). Both the blood clotting and antimicrobial properties of the material can be attributed to chitosan (see U.S. patent No. 7,482,503 (gregori et al), which is incorporated herein by reference). MCT-chitosan composites can be used to replace chitosan in the compositions described herein while still maintaining useful properties of chitosan (such as mucoadhesion, biocompatibility, and biodegradability).

Chitosan is commercially available from a number of chemical suppliers such as Sigma Aldrich co (Sigma Aldrich corporation of st. Chitosan has various grades, average molecular weights and degrees of deacetylation.

In some embodiments, the chitosan may be "high molecular weight" chitosan. High molecular weight chitosan is chitosan having a number average molecular weight of at least about 100kDa, typically about 170kDa to about 400 kDa. In some embodiments, the high molecular weight chitosan may have a molecular weight of at least about 100kDa, at least about 110kDa, at least about 150kDa, or at least about 200 kDa. In other embodiments, the high molecular weight chitosan may have a molecular weight of about 100kDa to about 400kDa, about 120kDa to about 400kDa, about 150kDa to about 400kDa, about 170kDa to about 400kDa, 100kDa to about 300kDa, about 120kDa to about 300kDa, about 150kDa to about 300kDa, about 170kDa to about 300 kDa. The value of "DA" in FIG. 4 may be any number or range that yields a value that approximates the N-acetyl-D-glucosamine content of chitosan described herein. As one of ordinary skill in the art will readily recognize, chitosan as shown in fig. 4 may also be partially acetylated.

Other embodiments may include low molecular weight chitosan. Low molecular weight chitosan refers to a chitosan molecule having less than 100 monomer units (less than about 18kDa or less than about 20 kDa). The molecular weight of chitosan can be determined by, for example, gel permeation chromatography and capillary viscosity.

Chitosan may have a degree of deacetylation that is typically at least about 60%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. Alternatively, chitosan may be fully deacetylated.

MCT-chitosan composite material: due to the high molecular weight and charge density of MCT and chitosan, chitosan combines with structural components in MCT through electrostatic interactions driven by its positively charged amino groups to allow formation of strong hydrogen bonds and dipole-dipole interactions. These interactions enable the development of stable biomaterials such as hydrogels, biofilms, 3D-sponges and nanofibers. MCT-chitosan composites can be used in GTR to prepare skin patches, cosmeceuticals and dressings for wound healing applications. MCT-chitosan composites can also be used as hydrogels or spongy materials or scaffolds for skin and cartilage tissue culture, as matrices for tissue engineering, and as biocompatible coatings for biomedical devices.

MCT and MCT-chitosan composites provide higher biocompatibility, improved mechanical properties, and excellent biodegradability relative to known synthetic and animal collagen-like devices. In addition to direct biological effects (antibacterial, antifungal and wound healing properties) from MCT-chitosan complexes, biomaterials can also be used in targeted or controlled release systems to encapsulate therapeutic agents for oral, dermal or respiratory delivery.

Nanoparticles and biofilms can be prepared from MCT and chitosan, as described herein. MCTs comprising collagen are particularly suitable for use in the preparation of nanoparticles. Since MCT mainly comprises fibrillar collagen, MCT is very suitable for preparing MCT-chitosan composite hydrogels, biofilms, 3D-sponges and nanofibers, providing composite biomaterials with superior mechanical properties compared to known animal collagen-based biomaterials.

MCT and MCT-chitosan composite biomaterials can be crosslinked during preparation by chemical treatment with biocompatible crosslinkers (glutaraldehyde, ECC/NHS) or by heat treatment under vacuum pressure. Swelling of the composite polymer matrix may result in faster degradation and reduced mechanical properties. Crosslinking reduces or prevents spontaneous expansion of the composite biomaterial to increase mechanical properties and handling.

In some embodiments, MCT and MCT-chitosan composites may include or exclude polymers other than chitosan or MCT. For example, some embodiments include dextran, alginate, and/or cellulose-derived materials (such as hydroxyethyl cellulose); other embodiments exclude some or all of these. Some embodiments include synthetic polymers such as polyvinyl alcohol, polycaprolactone, or polyethylene oxide, while other embodiments do not include some or all of them.

Analysis of MCT, chitosan and combination products: MCT, chitosan and its composite products can be analyzed and evaluated using a variety of methods. These techniques include mass spectrometry, mechanical properties (tensile strength) and swelling properties, scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) to characterize surface morphology, differential Scanning Calorimetry (DSC) for thermal characterization of composite biomaterials. Tensile strength and swelling properties of the composite biomaterial are characterized in ASTM measurements.

Analysis of MCT and MCT-chitosan composites described herein showed that the composites had improved stability, higher drug loading capacity, improved drug release properties, improved cellular uptake, greater porosity, improved tensile strength and thermal stability compared to compositions comprising chitosan alone. The material is also non-cytotoxic in vitro.

Wound healing: MCT and MCT-chitosan composite biomaterials also have valuable properties for wound healing applications because they exhibit enhanced bacteriostatic activity, improved biocompatibility and enhanced mechanical properties relative to pure chitosan. MCT-chitosan complexes showed an increase in chitosan antibacterial activity. Binding of the complex to negatively charged bacterial surfaces Disturbing the cell membrane. These properties can be applied to GTR applications by formulating injectable hydrogels, skin patches, and wound dressing templates, for example, to promote ulcer and burn healing. MCT-chitosan complexes may also be used as hemostatic agents in wound and surgical dressings.

MCT and MCT-chitosan composite biomaterials may be used in a variety of other biomedical applications. MCT-chitosan complexes are useful for surgical applications and regenerative medicine due to their biocompatibility, such as good blood compatibility, enhanced mechanical properties, and cell growth efficiency. The permeability of the MCT-chitosan composite membrane can be controlled by plasma treatment. Thus, such composite membranes can be used for dialysis.

The above-mentioned U.S. Pat. No. 7,482,503 (Gregory et al) describes a method of preparing a wound dressing. The MCT and MCT-chitosan composites described herein can be used in place of the chitosan biomaterials described by gregories et al to prepare wound dressings according to these methods. In addition, MCT-chitosan complexes can be used as coatings for medical devices, such as stents, catheters, and prostheses, to prevent the formation of deleterious biofilms or bacteremia in patients, and also to promote bio-mimicking (bio-localization) and osseointegration. As shown in SEM micrographs, complexation of MCT with chitosan promotes surface modification of collagen fibrils, which increases the porosity of its 3D-sponge scaffold. MCT was also found to improve physical properties of chitosan scaffolds such as tensile strength, swelling and thermal stability, as shown by mechanical analysis and DSC calorimetry.

Tissue engineering: tissue Engineering (TE) studies are based on seeding cells onto porous biodegradable polymer matrices. The main factor in successful seeding is the availability of good biological material, which acts as a temporary matrix or scaffold for cell proliferation and differentiation. Recently, chitosan and its derivatives have been reported as attractive candidates for spongy materials because they degrade with the formation of new tissues, eventually without inflammatory reactions or toxic degradation. In TE applications, the cationic nature of chitosan is primarily responsible for electrostatic interactions with anionic glycosaminoglycans, proteoglycans, and other negatively charged molecules.

As shown by SEM micrographs, complexation of MCT with chitosan promotes surface modification of chitosan films, which increases their 3D-sponge porosity. MCT has also been found to improve physical properties of chitosan biofilms, such as tensile strength, swelling and thermal stability, as shown by mechanical analysis and DSC calorimetry. MCT and MCT-chitosan composite biomaterials can be used to control cell morphology and function and thus can be used as tissue engineering scaffolds or matrices in GTR wound healing applications. MCT and MCT-chitosan composite biomaterials may also be chemically modified for TE applications. For example, the complex may be modified by grafting specific sugars to the MCT backbone. Certain cells can differentially recognize specific sugars, thereby providing specific recognition for antigen presenting cells (such as B cells, dendritic cells, and macrophages).

Cosmeceutical formulation: the invention also provides formulations comprising MCT and MCT-chitosan complexes described herein for use as therapeutic cosmetics (cosmeceuticals). MCT and MCT-chitosan complexes in powder or solution form can be added to a base cosmetic formulation to form pharmaceutical and/or functional cosmetic products. These cosmeceutical compositions may be formulated with a dermatologically and/or pharmaceutically acceptable topical carrier (including but not limited to solutions, suspensions, liquids, gels, ointments, lotions or creams). The composition provides for prolonged release of MCT and CHT to tissues, promotes collagen formation, scar healing, wound healing, reduction of liver spots/chloasma or other skin discoloration, and other benefits to the skin.

Cosmetic compositions may be formulated by standard techniques known to those of ordinary skill, such as those described in U.S. patent No. 9,980,894 (hermmann et al) and U.S. patent No. 9,962,464 (hermmann et al), both of which are incorporated herein by reference.

Examples of drugs, vitamins and nutrients that may be incorporated into the formulation include lipids such as fatty acids (including omega-3 and omega-6 fatty acids), fat-soluble vitamins (e.g., vitamins A, D, E and/or K), water-soluble vitamins (e.g., vitamin C, thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, folic acid, vitamin B12), antibiotics (e.g., amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin, metronidazole, penicillin, tetracyclines, vancomycin, etc.), probiotics (e.g., lactic acid bacteria, bifidobacteria, etc.), active skin compounds (e.g., retinoic acid, tranexamic acid, hydrogen peroxide, hydroquinone, cysteamine, azelaic acid, tyrosinase inhibitors, etc.), micronutrients (such as β -carotene and/or ascorbic acid, proteins and polypeptides).

These compositions and formulations typically contain at least 0.1% MCT or MCT-chitosan composite. Of course, the percentage of the compositions and formulations may vary, and may conveniently be from about 2% to about 60% by weight of a given unit dosage form. The amount of MCT and MCT-chitosan composite in such therapeutically useful compositions is such that an effective dosage level can be obtained.

The cosmeceutical composition and the like may also contain the following substances: binding agents such as xanthan gum, acacia, corn starch or gelatin; excipients such as calcium diphosphate; disintegrants such as corn starch, potato starch, alginic acid and the like; and/or lubricants, such as magnesium stearate. In addition to the above-mentioned types of materials, some specific cosmeceutical compositions may contain a liquid carrier, such as a vegetable oil or polyethylene glycol. The liquid carrier or vehicle may be a solvent or liquid dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof. For example, by maintaining the desired particle size in the case of dispersions or by using surfactants, proper fluidity can be maintained. The action of certain microorganisms may be prevented by various additional antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like). In many cases, it is preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents which delay absorption (for example, aluminum monostearate and gelatin). Of course, any material used to prepare any unit dosage form should be pharmaceutically acceptable and use substantially non-toxic in amounts.

For topical administration, MCT and MCT-chitosan composites may be administered in pure form. However, it is often desirable to apply them to the skin as a composition or formulation, for example, in combination with a dermatologically and/or pharmaceutically acceptable topical carrier (which may be solid or liquid).

Useful solid supports include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), ethanol, or ethylene glycol, or water-ethanol/ethylene glycol mixtures, wherein the MCT or MCT-chitosan composite can be dissolved or dispersed at an effective level, optionally with the aid of a non-toxic surfactant. Adjuvants (such as fragrances and additional antibacterial agents) may be added to optimize the characteristics of a given use. The resulting liquid composition may be applied from absorbent pads for impregnating bandages and other dressings, or sprayed onto the affected area using a pump or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials may also be used with the liquid carrier to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the skin of a user.

Examples of useful dermatological compositions that can be used to deliver MCT or MCT-chitosan composites to skin are known in the art; see, for example, jacquet et al (U.S. patent No. 4,608,392), geria (U.S. patent No. 4,992,478), smith et al (U.S. patent No. 4,559,157), and worth (U.S. patent No. 4,820,508), all of which are incorporated herein by reference.

Aspects of the invention provide methods of treating various conditions associated with GTR in a mammal comprising administering to a mammal having such a condition an effective amount of an MCT or MCT-chitosan complex of one or more embodiments of the invention. Mammals include primates, humans, rodents, dogs, cats, cattle, sheep, horses, pigs, goats, and the like.

The following examples are intended to illustrate aspects of the invention and should not be construed as narrowing the scope of the invention. Those of ordinary skill in the art will readily recognize that these examples suggest other ways in which the invention may be practiced. It should be understood that many variations and modifications may be made while remaining within the scope of the invention.

Examples

Example 1 mct-chitosan complex: preparation, data and applications

Pharmaceutical grade chitosan (by ¹ The degree of deacetylation calculated by H NMR was 92%; the average molecular weight calculated by the specific viscometry was 185 kDa) purchased from Sigma Aldrich (St.Louis, mitsui, U.S.A.). The degree of deacetylation and the average molecular weight distribution can be controlled in the production of MCT-chitosan complexes to provide chitosan with higher or lower degrees of deacetylation and/or higher or lower average molecular weights.

Variable collagen tissue (MCT) was isolated from marine invertebrate echinoderms. Adult specimens of sea urchins, starfish and sea cucumbers were collected and immediately dissected by divers in china, tathik island and japan, etc. Specimens of sea urchin peristomal membranes, starfish pair arm walls and sea cucumber whole body walls were collected and stored at-20 ℃ for the collagen extraction protocols described subsequently by Ferrario c, leggio l, leone r, di Benedetto c, guiletti l, cocc v, ascagni M, bonasoro f, la pora CAM, candia Carnevali MD, "Marine-derived collagen biomaterials from echinoderm connective tissues (Marine-derived collagen biomaterials from echinoderm connective tissue)", mar Environ res (Mar environmental institute), volume 128, pages 46-57. Animal harvesting and experimental procedures were performed according to the laws and regulations of each country. Sea urchins (peristomatal membrane) and starfish (juxta-arm wall) were dissected into small pieces, rinsed in artificial seawater, and placed in hypotonic buffer (10mM Tris,0.1%EDTA) for 12 hours at Room Temperature (RT), and then kept in decellularized solution (10 mm tris,0.1% sodium dodecyl sulfate) for 12 hours at room temperature. After washing several times in Phosphate Buffered Saline (PBS), the samples were placed in a deagglomeration solution (0.5M NaCl,0.1M Tris-HCl pH8.0,0.1M beta-mercaptoethanol, 0.05M EDTA-Na). The MCT suspension obtained was filtered and dialyzed against 0.5M EDTA-Na solution (pH 8.0) at room temperature for 3 hours, and For dH at room temperature ₂ O was dialyzed overnight. The starfish samples were subjected to additional steps in 1mM citric acid (pH 3-4) between the decellularization solution and the deagglomeration solution to remove as much calcium carbonate ossicles as possible present in fresh tissue. All steps were performed under stirring. Sea cucumber MCTs are extracted from the whole body wall according to different protocols. Briefly, the starting tissue was cut into small pieces, placed in PBS and gentamicin (40. Mu.g/mL), and left under stirring at room temperature for at least 5 days to obtain an MCT suspension, and then filtered. The suspensions obtained from the three experimental models were then stored at-80 ℃ until use.

Preparation of MCT-chitosan complex: variable collagen tissue (MCT) was dissolved in 0.5% v/v acetic acid overnight at room temperature and degassed before preparing the biomaterial and the complex with chitosan. The chitosan solution was prepared by dissolving chitosan powder in aqueous acetic acid (0.5% v/v) at Room Temperature (RT). After complete dissolution of the chitosan powder, the solution was filtered and degassed by vacuum filtration. Fig. 5A schematically illustrates dissolution and degassing of a chitosan solution according to an embodiment. The chitosan solution (0.1-0.5% w/v) was then mixed with MCT isolated from echinoderm (2.0-10.0% v/v) at different MCT-CHT molar ratios (100:0, 80:20, 60:40, 50:50, 40:60, 20:80, and 10:90). The solution was stirred at room temperature for 1 hour. Figure 5B further illustrates the preparation of MCT-chitosan complexes according to an example. The concentrations of chitosan and MCT were controlled during the formation of the composite by adding different proportions of the components to provide the desired composition.

Preparation of MCT-chitosan composite biological film: the MCT-chitosan complex solution was cast onto a glass or silicon mold and slowly spread to form a uniform liquid film. The liquid film was then evaporated at 80 ℃ for 24 hours or at 40 ℃ overnight to provide a 2D cast composite biofilm. Figure 6 shows casting of MCT-chitosan biofilm according to an embodiment.

Preparation of MCT-chitosan complex 3D-sponge: MCT-chitosan 3D-sponge was prepared by a crosslinking method driven by heat treatment under vacuum pressure. By casting/freezingA composite sponge (diameter=12 mm, thickness=6 mm) was prepared by the drying technique (step 3 c). 1 gram of 2% w/w chitosan aqueous solution or 0.5% v/v acetic acid solution was mixed with MCT aqueous solution (0.5-2.5% v/v) as shown in FIG. 7. The resulting mixture was poured into a sufficiently sized glass or silicon mold, frozen at-20 ℃ and freeze-dried to remove the solvent, yielding an MCT-chitosan porous 3D-sponge. MCT-chitosan complex 3D-sponges are physically similar to known chitosan sponges. However, the composite 3D sponge has significant additional properties such as higher water retention (swelling), improved mechanical properties, and excellent biocompatibility. For example, MCT-chitosan complex 3D-sponges can be used to provide improved wound and hemostatic (clotting) dressings, because the hemostatic effect of chitosan is increased by the immunostatic nature of the MCT component. The addition of MCT also improved its mechanical properties, cell attachment and growth.

Preparation of MCT-chitosan composite hydrogel: MCT (2% v/v), chitosan (1% w/v) complex solutions were frozen at-20 ℃ and freeze-dried to remove the solvent, leaving behind a powder material. 2 g of the freeze-dried MCT-chitosan complex was dissolved in 100 ml of deionized water and vigorously stirred while gradually increasing the pH by a concentrated NaOH 6N solution, as shown in FIG. 8. Once the solution reaches a sufficient pH (-7.2), a composite hydrogel spontaneously forms and the viscosity of the dispersion increases significantly. In another embodiment, the MCT-chitosan hydrogel may also be manufactured by adjusting the final concentration of High Molecular Weight (HMW) chitosan between 2 and 10% w/v or by mixing the final MCT-chitosan complex with a viscosity enhancing additive such as hydroxyethylcellulose, glycerol or polyethylene glycol, etc.

Preparation of MCT-chitosan composite electrospun nanofiber: MCT-chitosan nonwoven nanofiber mats were prepared by electrospinning techniques, as shown in fig. 9A and 9B. The MCT-chitosan complex was dissolved in deionized water at a chitosan concentration ranging from 0.5 to 2.0 (% w/v). The concentration of the MCT-chitosan complex was adjusted so that its viscosity and conductivity were suitable for electrospinning. Prior to the electrospinning experiments, the dispersion was placed in a refrigerator (4 ℃) overnight to be fully hydrated. Maximum shear rate taking Depending on the tubular geometry of the power law material, the volume flow (Q) is 2.78X10 ^-10 m ³ S, the tube inner radius is 1mm (r=0.5x10 ^- ³ m). To improve the MCT-chitosan complex properties for electrospinning, samples were mixed with polyvinyl alcohol (PVA) as an electrospinning aid. PVA (10% w/v) was dissolved in water at 80℃under vigorous stirring for 4 hours. A dispersion of MCT-chitosan complex and PVA mixed at 100: 00. 60: 40. 50: 50. 40:60 and 0:100 mass ratio. MCT-chitosan/PVA blend samples (5 mL) were electrospun using an electrospinning apparatus and a 30kV power supply (Gamma High Voltage Research, aomond beach, florida, usa). The distance between the tip and the collector was set at 20cm, the voltage 20kV, and the solution was pumped at 1 mL/h. The nanofibers were collected on aluminum foil and stored in a desiccator for further characterization as shown in fig. 9A.

Characterization of MCT-Chitosan biofilm: the mechanical properties of MCT-chitosan composite biofilms were evaluated by comparing tensile strength and swelling behaviour. Swelling is the first step in the physical degradation of the biofilm. Rapid swelling facilitates rapid and uncontrolled release of active compounds (e.g., drugs and/or pesticides) from a biofilm matrix. Glutaraldehyde is generally added as a cross-linking agent in the production of chitosan biofilms to slow down the swelling rate. The disadvantage of using glutaraldehyde in the hydrogel formulation is the reduced tensile strength of the biofilm. MCT-chitosan composite biofilms were cast according to the methods described previously. Furthermore, crosslinked biofilms were formulated by first immersing pre-cast chitosan or MCT-chitosan complex biofilms in glutaraldehyde solution (0.10% v/v) for 30 minutes, then thoroughly washing with deionized water, then drying at 80 ℃ for 2 hours.

Evaluation of mechanical properties: tensile strength measurements were made on a universal mechanical tester (model TEST 108 from the french GT TEST, equipped with TEST Winner 920 software) with a 10 mm/min crosshead speed and a 2kN static load cell. The biofilm was cut into standard tensile specimens from a dumbbell-shaped knife (H3 type) of dimensions 17mm x 4mm x 0.08mm (length x width x thickness). At least five samples of each type of biofilm were tested after an appropriate shelf life (3 weeks and 20 weeks) in a humidity chamber (CIAT, france) at 50±3% rh and 23±2 ℃. The maximum Tensile Stress (TS) was calculated by dividing the maximum load of the broken film by the cross-sectional area. MCT-chitosan composite biofilms exhibit higher tensile strength than chitosan biofilms alone. Figures 10A and 10B show the mechanical behavior of different MCT-chitosan composite biofilms (mass ratios of 50:50 and 100:0, respectively) compared to chitosan alone (0:100). Data are shown as [ mean ± SD; n=5 ]. The addition of a cross-linking agent (glutaraldehyde) reduces the tensile strength of all biofilms relative to uncrosslinked biofilms.

Evaluation of swelling behavior: the swelling degree of the chitosan and MCT-chitosan composite biofilm was evaluated by gravimetric methods. First, the sample is weighed on an analytical balance (W _d ) Each dried biofilm. After weighing, the biofilm was immersed in distilled water at room temperature for 60 minutes. The biofilm was then removed from the water and weighed at 5, 10, 20, 30, 40, 50 and 60 minutes (W _s ). Each biofilm sample was quickly removed from the water bath and blotted with tissue to remove excess water before weighing on a high precision balance. After weighing, the biofilm was returned to water. The swelling (%) of each biofilm sample was then calculated according to the following equation:

swelling degree (%) = [ (Ws-Wd)/Ws ] ×100

The results indicate that MCT-chitosan composite biofilms exhibit lower swelling rates and lower overall swelling compared to chitosan biofilms alone. Figure 10B shows the swelling degree of MCT-chitosan hydrogel as a function of time.

These results indicate that the MCT-chitosan composite acts as a strong cross-linker or equivalent thereof, reducing the swelling of the composite while increasing the tensile strength of the biofilm. MCT provides properties superior to those of glutaraldehyde crosslinkers in chitosan biofilms. MCT is thus a suitable, reliable and biocompatible "green substitute" for glutaraldehyde for use in formulating biofilms for packaging, repair and surgical biomaterials.

Characterization of MCT-chitosan composite wound dressing templates (biofilm, 3D-sponge and electrospun nanofibers): MCT-chitosan composite wound dressing templates (biofilm, 3D-sponge and electrospun nanofibers) were characterized according to their chemical characteristics using attenuated total reflectance fourier transform infrared spectroscopy (Nicolet 4700 ATR FT-IR, thermo Scientific, gland island, new york, usa) and thermal characterization by thermogravimetric analysis (TGA, Q100, TA Instruments, utah, usa). Thermal analysis (DSc and TGA) was performed in a nitrogen atmosphere (20 mL/min) at 5 ℃/min over a temperature sweep range of 20-400 ℃. Figures 11A and 11B illustrate MCT-chitosan biofilms and their potential application as templates for wound dressing. The morphology of the nanofibers was examined using SEM (Leo 1530-FE, zeiss, cambridge, UK). The average fiber diameter was determined by analyzing at least 20 fibers in the SEM image using ImageJ software. Fig. 11C and 11D show surface morphology.

Advantages of MCT-chitosan composite biological material compared with chitosan biological material: MCT-chitosan composite biomaterials (figure 12) provided significantly improved properties for various applications compared to chitosan biomaterials. The MCT-chitosan composite biomaterial can be prepared into nano particles, hydrogel, a biological film, 3D-sponge or electrospun nano fibers. Each of these forms of biomaterials can be used for a variety of target applications, and each composite biomaterial has significant advantages over chitosan biomaterials, as summarized in table 1 below.

Table 1. Improvement of MCT-chitosan complexes compared to chitosan and collagen biomaterials for GTR applications.

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Example 2 MCT-Chitosan Complex 3D-sponge for GTR, wound healing and tissue engineering

Chitosan (CHT) is reported to be biocompatible and bioabsorbable. In particular, CHT is considered a good wound healing accelerator. On the other hand, collagen (MCT) is one of the most widely used matrix biomaterials in tissue engineering. Highly porous MCT single 3D sponges have been used to support the in vitro growth of many types of tissues. The mixed 3D-sponge biomaterials were prepared by mixing CHT and MCT (isolated from sea cucumber samples) at different mass ratios using previously developed methods involving solvent casting and freeze-drying. MCT/CHT hybrid 3D-sponges were characterized in terms of their water absorption capacity, mechanical properties, thermal behavior (TGA) and morphology (SEM). The hybrid 3D sponge exhibits improved stability, greater porosity, increased thermal stability and mechanical properties, and higher biodegradability compared to a single 3D sponge. Cell culture incubations with adipose tissue-derived stem cells (ADSCs) and SEM imaging showed that MCT/CHT hybrid 3D-sponges allowed ADSCs to adhere, diffuse and grow in vitro.

Preparation of MCT-chitosan hybrid 3D-sponge: chitosan (CHT, 2.0% w/v) was dissolved in acetic acid (0.1% v/v) and slowly mixed with aqueous collagen solution (MCT, 5% w/v) at MCT-CHT 100: 0. 80: 20. 60: the molar ratio of 40 and 50:50 produced a hybrid solution. MCT-chitosan hybrid 3D-sponges were made by pouring each solution into a glass mold, solvent casting and freeze drying for 48 hours. The sponges were cut into small pieces (12 mm diameter and 3mm thickness) for further characterization and cell proliferation studies and stored in a desiccator under controlled relative humidity.

Physical and chemical characterization of MCT-chitosan hybrid 3D-sponges: optical microscopy imaging of the fabricated hybrid sponges was collected on an inverted microscope (LIB-305, USA) at 4 Xmagnification. The morphology of MCT-chitosan hybrid 3D-sponges was examined on a scanning electron microscope (SEM, JSM-5200, jeol, usa) at a magnification of 20 kx. The tilt angle of each sample was 30 degrees. Thermogravimetric analysis (TGA) was performed on a TGA-7 instrument (Perkin Elmer, USA). A sponge sample (5-10 mg) was poured into an aluminum rack and analyzed under a nitrogen atmosphere (10 mL/min) at a heating rate of 10 ℃/min according to a temperature program set between 50 and 600 ℃. By using a universal tensile tester (Tensilon RTG, japan) load cell with a maximum force of 250N under ambient conditions (20 ℃ and 50% relative humidity, RH) Uniaxial mechanical compression test of MCT/CHT hybrid 3D-sponges (n=5 per condition). Compression (mm) and load (N) were collected at a crosshead speed of 5 mm/min. The compressive elastic modulus is calculated as the tangential slope of the stress-strain curve in the initial linear region of the compression curve. Compressive strength was calculated at 15% strain (in the region where the stress-strain curve is linear in all samples). The dried 3D-sponge sample had a cylindrical shape with a diameter of 12mm and a thickness of 3mm, as measured by an electronic micrometer (DMH Series 293, mitotoyo, japan).

Cell attachment and proliferation studies: adipose tissue-derived stem cells (ADSCs) were isolated from live horses. ADSC (-10) ⁵ Cells/cm ² ) Placed on top of each MCT-chitosan hybrid 3D-sponge. Tissue culture plate (polystyrene) wells served as controls. Cultures were placed in an incubator for 1 day and after removal washed with Phosphate Buffered Saline (PBS) and trypsinized. Aliquots of the resulting dissociated cell suspensions were counted on a coulter particle counter (Model 0646,Coulter Electronics (coulter electronics), hai-geria, florida, usa). Only counts between 8 and 32 μm in diameter were used. After 1, 3, 7 and 10 days of culture, cell proliferation was determined by cell counting as described above. In this experiment, six replicate samples were examined. The ligated and/or expanded ADSC cells were fixed with glutaraldehyde (2.5% v/v) in 0.1M PBS (pH 7.4) for 30 min, and then rinsed with 0.1M PBS. The fixed cell samples were freeze-dried and coated with gold sputtering, and morphological analysis was performed by scanning electron microscopy (SEM, hitachi Model S-2460N, manufactured by Tokyo Hitachi, japan).

Data and statistical analysis: all data are reported as mean ± standard deviation of at least three replicates. Statistical analysis was performed using JMP Pro (version 10.0.0; sas institute, karst, north carolina, usa) setting p=0.05. Results were analyzed using a two-way ANOVA model in which the interaction between the independent variables "sample" and "concentration" was used to evaluate significant differences.

Results and discussion

MCT-chitosan hybridized 3D-spongePhysical and chemical characterization: chitosan is physically bound to collagen through hydrogen bonding interactions driven by its available amines and hydroxyl groups. This interaction allows the development of stable Biomaterials such as nanoparticles, biofilms, biofoam and tissue sponges (madrig-carball et al, polymer-liposome nanoparticles obtained by the electrostatic bio-adsorption of natural polymers onto soybean lecithin liposomes (Polymer-liposome nanoparticles obtained by electrostatic bioadsorption of natural polymers to soy lecithin liposomes), intl.J. nanoparticles 5 (3) (2012) 196-209; madrig-carball et al, protein-loaded chitosan nanoparticles modulate uptake and antigen presentation of hen egg-white lysozyme by murine peritoneal macrophages (Protein-loaded chitosan nanoparticles regulate the uptake and antigen presentation of egg white lysozyme by murine peritoneal macrophages), intl.J. nanoparticles 3 (2) (2010) 179-191; ma et al, "A preliminary in vitro study on the fabrication and tissue engineering applications of a novel chitosan bilayer material as a sponge of human neofetal dermal fibroblasts (novel chitosan bilayer material as a preliminary in vitro study of the manufacture and tissue engineering application of sponges for human fetal fibroblasts)", biomaterials,22 (4) (2001), pages 331-336). Figure 13A shows a prepared MCT-chitosan hybrid 3D-sponge. As shown in FIG. 13A, the hybrid 3D sponges shown were manufactured with different MCT/CHT mass ratios (50:50, 60:40, 80:20 and 100:0). Fig. 13B shows an optical microscope image of the sponge, and fig. 13C is an SEM micrograph of each manufactured 3D-sponge. In FIG. 13C, the scale bar is 500. Mu.m.

SEM micrograph in figure 13C shows the change in surface morphology of chitosan monolongs when combined with MCT. This change was demonstrated by the addition of MCT to the MCT/CHT hybrid 3D-sponge matrix with a decrease in apparent pore size. Thus, MCT interactions with chitosan appear to provide greater crosslink density, which may be driven by more potential hydrogen bonding interactions available between the two macromolecules, thus increasing molecular alignment and compactness.

FIG. 14 is a graph of thermal analysis of MCT-chitosan hybrid 3D sponge by thermogravimetric analysis (TG) showing thermal behavior of MCT/CHT (50:50) hybrid 3D-sponge between MCT/CHT (100:0) and MCT/CHT (0:100) composite sponge. The hybrid 3D sponge containing MCT shows better thermal stability than chitosan single sponge, the average decomposition temperature is 300 ℃, and the thermal stability is improved by 15 times compared with that of CHT single sponge.

Biological 3D sponges require sufficient mechanical properties to maintain their integrity after implantation. Thus, compression tests were performed on MCT-chitosan hybrid 3D-sponges to obtain stress-strain mechanical curves and calculate the elastic modulus and compressive stress (at 15% strain), respectively, as shown in table 2. The results show that mechanical properties are positively affected by the addition of MCT to the chitosan 3D-sponge matrix. The hybrid MCT/CHT (50:50) 3D-sponge showed an approximately 85-fold increase in Young's modulus under compression compared to the MCT/CHT (100:0) sponge. In addition, the compressive strength (15% strain) of the MCT/CHT (60:40) hybrid 3-D sponge system was found to increase by about 78-fold. The observed increase in mechanical strength may be associated with the formation of an internal hydrogen-bond driven polymer network between collagen and chitosan that promotes mechanical stabilization of the matrix and thus promotes a suitable 3D-sponge for potential implantation purposes.

TABLE 2 Young's mechanical modulus results report mean.+ -. SD (n=5) for different MCT-chitosan hybrid 3D-sponges

Swelling properties are important in sponges and promote hydration and cell growth. MCT-chitosan 3D sponges showed intermediate behavior between MCT and two simple sponges of chitosan, with one with the highest proportion of chitosan showing the highest water absorption capacity. These results can be explained by the presence of more available hydrogen bonding sites in chitosan biomolecules than in collagen biomolecules due to the reduced rotation and mobility of the four-level structure driven functional groups of collagen.

Figure 15 shows the water uptake behavior obtained for different MCT-chitosan hybrid 3D-sponges. In figure 15, data are expressed as mean ± SD, n=3 and (×) =p <0.05 compared to MCT/CHT (100:0) single 3D-sponge at the same time point. Figure 15 also includes pictures inserted for illustration purposes to show swelling behavior of MCT/CHT (50:50) hybrid 3D-sponges. The graph of FIG. 15 shows the similarity below 70% relative humidity for all different hybrid 3D-sponges. Meanwhile, as the proportion of MCT in the hybrid 3D-sponge matrix increases after reaching 85% relative humidity, a significant difference was observed between the systems, showing a 250-fold range of water absorption differences, with MCT/CHT (50:50) hybrid 3D sponges showing the highest water absorption capacity (approaching 300%), and MCT/CHT (100:0) single 3D sponges showing the lowest water absorption capacity (values approaching 50%). The MCT/CHT (80:20) and MCT/CHT (60:40) showed water absorption capacity between the MCT/CHT (100:0) and MCT/CHT (50:50).

The ability of the composite sponge to retain water is an important aspect of evaluating its characteristics and suitability for skin tissue engineering. The water binding capacity of MCT-chitosan sponges can be attributed to their hydrophilicity and their maintenance of three-dimensional structure. Chitosan and MCT have abundant hydrophilic groups (such as hydroxyl, amino and carboxyl) that are capable of retaining water in their microstructure. MCT appears to promote increased hydrophilicity at higher relative humidity, resulting in higher water absorption capacity. The water absorption values obtained for MCT-chitosan sponges were consistent with similar experiments previously reported (Ma et al, 'Chitosan porous sponges with improved biostability for skin tissue engineering (chitosan porous sponges with improved biostability for skin tissue engineering)', biomaterials. Elsevier,24 (26) (2003), pages 4833-4841; chhabra et al, "Optimization, characterization, and efficacy evaluation of 2%chitosan sponge for tissue engineering and wound healing (2% chitosan sponges Optimization, characterization and efficacy assessment for tissue engineering and wound healing)," Journal of pharmacy & bioallied sciences, medknow Publications,8 (4) (2016), page 300).

Adipose tissue-derived stem cells (ADSCs) grown on MCT-chitosan hybrid 3D-sponges: to study between ADSC and MCT-chitosan hybrid 3D-spongeA porous structure of about 12 mm in diameter and 3 mm in thickness was used for the interaction. After 72 hours of incubation, ADSCs reached a cell coverage of greater than 90% on the sponge. SEM images of sections of MCT/CHT (100:0) 3D-sponges (fig. 16A and 16B) showed that, 72 hours after cell seeding, ADSCs adhered and spread on the porous MCT-chitosan (100:0) 3D-sponge surface and completely merged with each other, making the intercellular connections invisible (fig. 16B) compared to the sponge system without cells attached (fig. 16A). In fig. 16A and 16B, the scale bar indicates 10 μm. The surface of the porous sponge is filled with cells and membranes which can secrete ECM pellets from the cells (Lin, li and Su, "Three-dimensional chitosan sponges influence the extra cellular matrix expression in Schwann cells (Three-dimensional chitosan sponge affects extracellular matrix expression in schwann cells)", materials Science and Engineering C (materials science and engineering C), 42 (2014), pages 474-478; ji et al, "Biocompatibility study of a silk fibroin-chitosan sponge with adipose tissue-derived stem cells in vitro (study of biocompatibility of a silk fibroin-chitosan sponge with adipose tissue-derived stem cells in vitro)", experimental and Therapeutic Medicine (experimental and therapeutic medicine), 6 (2) (2013), pages 513-518).

Figure 17 shows the proliferation level of MCT-chitosan (50:50) composite 3D-sponge after a 15 day incubation period, where legend (∈s) represents MCT/CHT (100:0) 3D-sponge, legend (o) represents MCT/CHT (0:100) single 3D sponge, legend (Δ) represents MCT/CHT (50:50) composite 3D-sponge, where data is expressed as mean ± SD, n=5, (=p <0.05 compared to MCT/CHT (0:100) single 3D-sponge at the same time point. Three curves depict 3D sponges MCT/CHT (0:100), MCT/CHT (100:0) and MCT/CHT (50:50) composite 3D-sponges. MCT/CHT (50:50) composite 3D-sponges showed a significant increase in cell attachment and proliferation compared to MCSC/CHT (0:100) and MCT/CHT (100:0) 3D sponges, starting with 3 days of ADSC incubation.

The surface of polystyrene dishes is known to have good cell adhesion and to exhibit rapid cell coverage during incubation (Jeong Park et al, "Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration (platelet-derived growth factor releasing chitosan sponge for periodontal bone regeneration)", biomaterials (Biomaterials), 21 (2) (2000), pages 153-159). The extent of cell attachment and proliferation means that MCT-chitosan hybrid 3D-sponges have good cell adaptation. Examination of cell proliferation on the sponge indicated a statistically significant difference between the experimental and control groups after 3 days of ADSC incubation. This may be due to the adaptation process after the ADSC has been added to the sponge. The fact that the difference between samples began to be apparent after 3 days of incubation may be due to the fact that ADSCs demonstrated an initial slow proliferation rate on the sponge, which then returned to some more normal proliferation rate after three days. In addition, MCT/CHT (50:50) composite 3D-sponges showed significantly increased cell attachment and proliferation. For an incubation period of 15 days, a high degree of cell attachment and proliferation of about 50 times was observed from MCT/CHT (50:50) composite 3D sponge compared to MCT/CHT (0:100) and MCT/CHT (100:0) 3D sponge. MCT is known to stimulate proliferation, chemotaxis and collagen synthesis of osteoblast-like cells and ligament fibroblasts (Zhang et al, "Novel chitosan/collagen sponge containing transforming growth factor- β1DNA for periodontal tissue engineering)", biochemical and Biophysical Research Communications (communication of biochemical and biophysical studies), 344 (1) (2006), pages 362-369. In addition, MCT has been reported to enhance progenitor cell proliferation (Costa-Pinto et al, "Adhesion, promotion, and osteogenic differentiation of a mouse mesenchymal stem cell line (BMC 9) seeded on novel melt-based chitosan/polyester 3D pore front-ges (Adhesion, proliferation and osteogenic differentiation of mouse mesenchymal stem cell lines seeded on novel fused chitosan/polyester 3D porous sponges)", tissue engineering. Part A (Tissue engineering A), 14 (6) (2008), pages 1049-1057). The combination of MCT and chitosan may be very beneficial to increase the cell proliferation response.

Conclusion(s): in contrast to the rapid degradation of collagen, chitosan slowly biodegrades in vitro. Modification of collagen 3D-sponge with chitosan leads to machineryImprovement in strength, thermal stability, biocompatibility and biodegradability. MCT-chitosan hybrid 3D-sponges provide multi-dimensional structures for ADSCs both on the surface and inside, have spatial features of cell attachment, migration and proliferation, and promote cell growth. After 72 hours of incubation, ADSCs were found to coalesce and form a complete cell layer on the sponge surface, such that the surface was almost covered and only a few wells were visible, and some cells migrated within the wells. MCT-chitosan hybrid 3D-sponges support ADSC attachment, proliferation and differentiation. SEM images show that the large surface area of the porous sponge allows ADSCs to adhere, diffuse and grow on the sponge. The flattened morphology and excellent diffusion in and around the interconnected porous structure indicate strong adhesion of cells and cell growth. Thus, MCT-chitosan hybrid 3D-sponges exhibit biocompatibility for ADSC attachment and are therefore good candidates for potential application in tissue engineering.

Example 3 electrospun MCT-chitosan composite nanofibers as biocompatible sponges for cell proliferation in wound healing and tissue engineering applications

Electrospun nanofibers (ESNF) were prepared from MCT-chitosan composites. Polyvinyl alcohol (PVA) is used as an auxiliary agent. MCT-chitosan/PVA mixed solutions of different volume ratios (100:0, 80:20, 60:40, 40:60, 20:80, and 0:100) were prepared and adjusted to be similar in terms of viscosity and conductivity suitable for electrospinning. The morphology of ESNF was checked using Scanning Electron Microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and Differential Scanning Calorimeter (DSC). The chemical composition and thermal properties used to characterize Nanofibers (NF) were studied. The ability of NF to support fibroblast proliferation was studied in vitro using an optimized MCT-chitosan/PVA solution. The results show that ESNF based on MCT-chitosan is very suitable for fibroblast growth and is obviously superior to PVA ESNF. The results also show that MCT-chitosan supports cell proliferation better than chitosan alone.

Preparation of MCT-chitosan complexes for electrospinning: MCT-chitosan powder was swollen in deionized water with vigorous stirring until a uniform dispersion (1% v/v) was obtained. The dispersion was placed in a refrigerator (4 ℃) overnight to fully hydrate before the characterization experiment. MCT-chitosan composite componentThe dispersions were characterized by measuring their viscosity by stress sweep test in a rheometer (C-VOR, bohlin Instruments, malva, uk) having cone-plate geometry and conductivity at 25 ℃, with a conductivity meter (Orion Star a215, thermoFisher, voltherm, ma) having an electrode conductivity constant of 0.7265cm ^-1 。

Electrospinning of MCT-chitosan composites: MCT-chitosan complex was mixed with aqueous acetone (30% v/v) with vigorous stirring until a homogeneous dispersion was obtained. The concentration of the composite solutions was adjusted so that their viscosity and conductivity were similar and suitable for electrospinning. Prior to the electrospinning experiments, the dispersion was placed in a refrigerator (4 ℃) overnight to be fully hydrated. From the power law material in the tubular geometry, the maximum shear rate for each MCT-CHT composite sample was calculated, with a volumetric flow rate (Q) of 2.78x10 ^-10 m ³ S, the tube inner radius is 1mm (r=0.5x10 ^-3 Rice).

In order to improve the composite performance of the electrostatic spinning, the sample is mixed with PVA as an electrostatic spinning aid. PVA (10% w/v) was dissolved in 80℃water for 4 hours with vigorous stirring. The MCT-chitosan complex and PVA blend dispersion were mixed at volume ratios of 100:00, 60:40, 50:50, 40:60, and 0:100. MCT-chitosan/PVA blend samples (5 mL) were electrospun using an electrospinning apparatus and a 30kV power supply (Gamma High Voltage Research, aomond beach, florida, usa) (see fig. 9A and 9B). The distance between the tip and the collector was set at 20cm and the solution was pumped at 1 mL/h. Nanofibers were collected on aluminum foil and stored in a desiccator for further characterization.

Characterization of MCT-Chitosan ESNF: ESNF was characterized according to its chemical characteristics using an attenuated total reflection fourier transform infrared spectrometer (Nicolet 4700 ATR FT-IR, thermo Scientific, gland island, new york, usa) and thermal characterization by thermogravimetric analysis (TGA, Q100, TA Instruments, lington, utah, usa). TGA analysis was performed at 5℃per minute in a temperature sweep range of 100-400℃under nitrogen atmosphere (20 mL/min). Examination of sodium using SEM (Leo 1530-FE, zeiss, cambridge England)Morphology of rice fiber. The average fiber diameter was determined by analyzing at least 20 fibers in the SEM image using ImageJ software.

Cell proliferation assay: cell proliferation was determined by MTT cell proliferation assay against viable cell numbers. Briefly, ESNF previously collected under sterile conditions was placed in different wells in a sterile cell culture plate with medium, and 3mL of fibroblast suspension (L929, 1.5×105) was added to each treatment well. The cell culture plates were placed in an incubator at 37℃for 3 days, 7 days and 14 days, respectively. After incubation, the medium was removed and MTT solution was added to each treatment well at 1:10 dilution with fresh medium. Plates were incubated at 37 ℃ for 4 hours and absorbance was measured at 560nm using a microplate reader (SpectraMax Plus, molecular Devices, sanyverer, california). After the cell growth experiment (day 7), ESNF was collected and washed with medium, fixed with glutaraldehyde (2.5% v/v) at 4℃for 2 hours, and coated with gold prior to imaging with SEM.

Statistical analysis: statistical analysis was performed using Assistant VR software (Statistics, allington, tex.). Experimental data are expressed as mean ± SD values. To compare the control and experimental groups, the data were analyzed by a generalized linear model followed by least mean square (SAS; karst, north carolina). At P<At 0.05, the difference was considered statistically significant.

Figure 18 shows ATR-FTIR spectra of ESNF for MCT/CHT (0:100) (indicated by solid lines) and MCT/CHT (100:0) (indicated by dashed lines "- - -") and two different MCT/CHT complexes (60:40, indicated by dotted lines "…", 40:60, indicated by dashed lines "- - - - - - - - - -"). The arrows in the figure represent the changes in the FTIR spectra of nanofibers associated with the addition of MCT to chitosan. In particular, it can be seen that at 1650cm ^-1 And 1000cm ^-1 The absorption tendency at this point increases. These correspond to carbonyl (solid arrow) and carbon-oxygen (dashed arrow) telescoping frequencies, respectively, associated with the polysaccharide properties of chitosan. In general, the differences between CHT and MCT are apparent in the figures. For both complexes, there is dispersion at some locations in the figure, but in many placesAt a large number of overlaps, this is not surprising given the relatively similar proportions of CHT and MCT.

Figure 19 shows TGA thermograms of MCT/CHT complexes (100:0, 60:40, 40:60, and 0:100) showing positive effects on chitosan thermal stability due to MCT addition. MCT-chitosan (60:40) complexes showed improved thermal stability over chitosan alone. The MCT-chitosan (60:40) complex showed an average degradation temperature of about 320℃while chitosan alone showed an average temperature of about 280 ℃

Fig. 20A to 20F show SEM micrographs of chitosan electrospun nanofibers and MCT-chitosan electrospun nanofibers. In fig. 20A and 20D, the scale is 10 μm; in the drawings 20BAnd in FIG. 20E, the scale is 2 μm, and in FIGS. 20C and 20F, the scale is 200nm. The dashed circles indicate the presence of droplets associated with poor electrospinning processes in chitosan ESNF and show improvement in ESNF for MCT-chitosan complexes.

FIG. 21 shows proliferation of L929 fibroblasts co-cultured with chitosan, MCT and MCT-CHT complex (50:50) ESNF. The figure shows data from an MTT cell proliferation assay using PVA ESNF as a control. The mean percentage ± standard deviation of living cells was from three experiments performed at different times. Fig. 22A-22C are SEM micrographs showing cell adhesion to chitosan, MCT-chitosan complex, and MCT, respectively.

Conclusion(s): MCT-chitosan composite nanofibers were successfully produced by electrospinning using PVA (10% w/v) as an adjuvant. And setting electrostatic spinning parameters for manufacturing the MCT-chitosan composite nanofiber, and optimizing the mass ratio of the MCT to the chitosan to 50:50.ATR-FTIR analysis showed the presence of MCT-chitosan components in ESNF. Comparison of the thermal stability of MCT-chitosan complex ESNF with that of chitosan alone shows that adding MCT to chitosan improves the thermal stability of ESNF. The fibroblast proliferation results showed that MCT-chitosan ESNF was suitable for cell growth and significantly better than either chitosan or MCT ESNF alone after 7 days of incubation. SEM imageA large surface area like that of MCT-chitosan ESNF was shown to allow good adhesion, diffusion and growth of L929 fibroblasts. The results indicate that MCT-chitosan ESNF improves the biocompatibility and activity of fibroblast attachment and thus can be used to develop wound dressing templates for tissue engineering, regenerative medicine and wound healing dressings for the treatment of tissue burns.

The following discussion of fig. 36-38 provides an overview of the structure and application of GTR devices and advantages of those devices according to embodiments.

Fig. 36 shows a 3D sponge according to an embodiment. In this figure, the sponge contains MCT consisting of collagen and glycosaminoglycans, which are the main components of the new skin. Neodermis is new tissue formed during wound healing. The collagen-GAG structure of MCT promotes integrin binding during the healing process. As previously mentioned, MCT is derived from marine sources, including marine invertebrates in one aspect, echinoderms in more particular aspects, and sea urchins and/or sea cucumbers in more particular aspects. The ultrastructure of this MCT has some similarities to human connective tissue, pointing to beneficial GTR effects, such as the formation of new skin.

The sponge in fig. 36 also provides moisture management to promote wound closure and healing. In one aspect, the sponge has a gelling effect that promotes patient comfort during healing by a cooling, soothing effect. The cross-linking treatment of MCT made the sponge effective for up to 30 days because the resulting structure exhibited low physical degradation and resistance to proteolytic enzymes. In particular, the resulting structure targets and inactivates excess enzymes, such as Matrix Metalloproteinases (MMPs), which can degrade proteins. This targeting and inactivation promotes and improves wound closure and healing.

Fig. 37 depicts a 3D-sponge according to some embodiments. According to embodiments, the sponge may consist of MCT or of a matrix of MCT-CHT. This material in the sponge supports tissue and vascular ingrowth. Other aspects of the sponge structure include absorbent gel-forming compositions that maintain a moist environment and control exudates, thereby promoting healing and tissue regeneration.

Figure 38 depicts a GTR device, which may be composed of MCT or MCT-CHT matrix, that is sutured into an opening in the skin as part of the treatment. The figure shows the suture around the device, as well as the new skin formed under the device as part of the healing process. Figure 38 is a partial view showing an exploded view of the new skin showing MCT alone or in a complex matrix of MCT and CHT, and showing the integrin binding site.

EXAMPLE 4 cosmeceutical formulation

The following formulations illustrate representative pharmaceutical dosage forms useful for the therapeutic or prophylactic administration of MCT and MCT-chitosan (MCT-CHT) compositions described herein using dermatologically and/or pharmaceutically acceptable topical carriers, including but not limited to solutions, suspensions, liquids, gels, ointments, lotions, or creams. In the following examples, a gel or cream is provided:

The preparation is prepared in the following separate stages: part a of the mixture was prepared by dispersing carbomers in water and then stirring in the other components. All components of part B were mixed together and heated to 70 ℃. Then combining part A and part B, adding triethanolamine and perfume (part C). The obtained cream is stable, smooth, has good moisture retention and good feel to the skin.

Formulations were prepared by mixing all the components together.

The preparation is prepared in the following separate stages: part a of the mixture was prepared by dispersing carbomers in water and then stirring in the other components. All components of part B were mixed together and heated to 70 ℃. Then combining part A and part B, adding triethanolamine and perfume (part C). The obtained cream is rich in excellent moisture retention and does not give greasy feel to the skin.

(iv) Scar cream composition

The preparation procedure is as follows:

1-150 mg vitamin E was weighed and mixed with the paste-based dispersion under gentle stirring.

2-1000 mg of MCT-CHT lyophilized powder (formulated at a mass ratio of MCT: CHT of 100:0 to 70:30) was weighed and dissolved in 5.00mL of acetic acid (0.5M) prepared in distilled water (pH 3.2).

3-200 mg of Moringa oleifera/vitamin C extract was weighed and dissolved in MCT-CHT aqueous solution.

4-50 mg of astaxanthin was weighed and dissolved in an aqueous solution containing MCT-CHT and moringa oleifera/vitamin C extract.

5-weighing 100mg of Lac Regis Apis, and mixing with aqueous solution containing MCT-CHT, moringa oleifera/vitamin C extract and astaxanthin.

6-mixing an aqueous solution containing MCT-CHT, moringa oleifera/vitamin C extract and astaxanthin with a paste base containing vitamin E under continuous gentle stirring until a homogeneous dispersion is achieved.

7-the homogenized scar cream formulation is poured into a suitable glass container and stored at room temperature.

The above formulations may be prepared by conventional procedures well known in the pharmaceutical arts. It will be appreciated that the aforementioned cosmeceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate different amounts and different types of MCT-CHT complexes as active ingredients.

The scar cream composition just described is administered to several patients. Fig. 30A and 30B show the results of administration before and after one patient. Fig. 30A is a photograph of a five year scar from caesarean section prior to administration of the scar cream listed above. Fig. 30B is a photograph of the same scar two weeks after daily administration of the scar cream. As can be seen in fig. 30B, the scar after treatment is shorter and has a different color, closer to the surrounding skin.

Fig. 31A and 31B show the results of administration before and after another patient. Fig. 31A is a photograph of a scar 15 years under the left knee joint prior to application of the scar cream listed above. Fig. 31B is a photograph of the same scar after application of the scar cream. As can be seen in fig. 31B, the scar has a different color, closer to the color of the surrounding skin.

Fig. 32A to 32C show the results before and after administration to yet another patient. Fig. 32A is a photograph of a 25 year appendectomy scar prior to administration of the scar cream listed above. Fig. 32B is a photograph of the same scar after daily administration, and fig. 32C is a photograph of the same scar after 8 days of daily administration of scar cream according to the example. As can be seen from fig. 32B and 32C, the scar after treatment is shorter and has a different color, closer to the surrounding skin.

Fig. 33A and 33B show the results of administration before and after another patient. Fig. 33A is a photograph of a scar from six months of knee surgery, and fig. 33B is a photograph of the same scar seven days after daily application of the scar cream listed above. As can be seen from fig. 33A and 33B, the scar after treatment is less obvious than before and has a different color, a color closer to the surrounding skin. The patient does not have side effects or hyperplasia.

Fig. 34A-34D show the progressive results of treating scars in another patient. Fig. 34A shows a photograph of a burn scar, fig. 34B and 34C show photographs of a scar during daily administration of the scar cream listed above, and fig. 34D shows photographs of a scar seven days after treatment. From these figures it is evident that the treated scar is substantially healed and has a color that is closer to the color of the surrounding skin than the pre-treatment scar appearance.

In addition to the foregoing therapeutic examples, scarring may occur in various areas of the body, including in and around the eyelid, for example, due to injury, surgery, plastic surgery, or other repair and/or healing procedures. The scar cream is also used for the treatment, and good effect is achieved.

As part of the foregoing work and as an example of fabrication, a work was performed to determine and display structural differences between MCTs extracted from sea cucumber and bovine collagen isolated from calf skin tissue. The amino acid composition analysis of the collagen samples was determined in (Cui f, li z, zhang y, dong p, fu X, gao X, "Characterization and Subunit Composition of Collagen from the Body Wall of Sea Cucumber (characterization and subunit composition of collagen of sea cucumber, stichopus japonicus body wall)", food Chem (Food chemistry) 100 (3) (2007):1120-5). Briefly, collagen samples were hydrolyzed with 6M HCl at 110 ℃ for 24 hours, and then the main amino acid composition of the hydrolyzate was analyzed using a seum amino acid analyzer S433D (seum, munich, germany).

Table 3 below shows the amino acid composition of different MCT samples isolated from sea cucumber and bovine (calf skin) collagen isolated from calf skin. Analysis of the calf skin collagen samples 1-4 in the following table was performed in a study as can be found in the following paper: X.Cheng, Z.Shao, C.Li, L.Yu, M.A.Raja, C.Liu, "Isolation, characterization and Evaluation of Collagen from Jellyfish Rhopilema esculentum Kishinouye for Use in Hemostatic Applications (Isolation, characterization and evaluation of collagen from jellyfish for hemostatic applications)", PLOS One,2017,0169731; Y.Han, J-R.Ahn, J-W.Woo, C-K.Jung, S-M.Cho, Y-B.Lee, S-B.Kim, "Processing Optimization and Physicochemical Characteristics of Collagen from Scales of Yellowfin Tuna (Thunnus albacares) (optimization of processing and physicochemical properties of the collagen of tuna (Thunnus albacares) flake)," Fisheries and Aquatic Sciences (fishery and aquatic science) ", volume 13, stage 2, 2010, pages 102-111; H.Li, B.L.Liu, L.Z.Gao, H.L.Chen, "Studies on bullfrog skin collagen (study of bullfrog skin collagen)", food Chemistry (Food Chemistry), volume 84, stage 1, month 1 in 2004, pages 65-69; P.Kittiphattanabawon, S.Nalinanon, S.Benjakul and H.Kishimura, "Characteristics of Pepsin-Solubilised Collagen from the Skin of Splendid Squid (logo for collagen) (pepsin-dissolving collagen properties from the skin of Splendid requires)", "Journal of Chemistry, volume 2015, page 482354,8.

TABLE 3 amino acid composition

Table 3 shows the amino acid composition of variable collagen organization (MCT) and Bovine Collagen (BC). The major amino acids of MCT are glycine (19.0%), glutamic acid (14.0%), proline (12.0%), alanine (9.0%), aspartic acid (9.0%), arginine (8.0%), and hydroxyproline (6.7%), similar to that found in bovine collagen shown in table 4 (see references).

The primary structure of type I collagen is characterized by a domain containing a continuously repeating Gly-X-Y sequence (where X is mainly proline and Y is mainly hydroxyproline) and very short N-and C-terminal regions (known as telopeptides (15 to 26 amino acid residues)). Gly-X-Y repeats in the alpha 1 chain play an important role in the formation of triple helices in the secondary structure. See the reference to the gel K for a reference,aigner T.2003. Collagen Structure, function and biosynthesis Adv Drug Deliver Rev (advanced drug delivery overview) 55 (12): 1531-46; gd mez-Guill enM, gim enezB, lopez-CallFunctional and bioactive properties of ero M, montero m.2011 from alternative sources of collagen and gelatin: overview. Food Hydrocolloid (food colloid) 25 (8): 1813-27. As the lowest molecular weight amino acid, glycine residues arranged in the center of the triple helix can help the helix structure fold tightly [3 ] ]. See Fraser R, macRae T, suzuki e.1979. Chain conformation J Mol Biol 129 (3) in collagen molecules: 463-81. Glycine is therefore the main amino acid in bovine collagen. According to the previous references, glycine content in bovine collagen ranged from 14 to 33%, approximately one quarter of the total amino acids, which is consistent with MCT glycine content (19%).

Fig. 23 and 24 show the results of purity analysis of the collagen matrix of marine collagen compared to calf and chicken collagen and demonstrate that marine collagen is as safe as calf and chicken collagen and therefore sufficiently safe for the application just described. These figures show a triple helix structure with the broad blue band at the bottom being the alpha helix and the top being the beta or gamma helix. MCT1 and MCT2 in these figures represent different MCT batches, showing the reproducibility of the results.

In fig. 25A, the spectrum of bovine collagen is shown by a solid line. Peaks indicated by "+" in the figures show hydrolysis of calf collagen to make it soluble. The comparative curve shows MCT by dashed lines, showing no hydrolysis peaks. Figure 25B shows comparable FTIR spectra, demonstrating reproducibility of the results with respect to the efficacy of the MCT separation process of sea cucumber, with MCT1 indicated by solid lines and MCT2 indicated by dashed lines. Figure 25B shows the high consistency of MCT chemistry across lots.

Figure 26 shows the thermogravimetric analysis (TGA) results of collagen samples and shows the difference in thermal behavior of MCT samples. In fig. 26, the reduced stability is shown as the line is towards the bottom of the curve. The threads of calf collagen show increased instability at 280 ℃, while the threads of MCT1 and MCT2 show stability even at 400 ℃. The curve shows that MCT has better thermal stability than calf collagen, indicating an improved ability to store MCT-based products.

Imino acids (proline and hydroxyproline) are important amino acids that constitute the Gly-X-Y repeat sequence in the alpha chain because they are able to maintain the stability of the collagen triple helix with its pyrrolidine ring. See Wong, dw.1989. Mechanisms and theory of food chemistry, new york: van Nostrand Reinhold. The content of proline and hydroxyproline in MCT was 12.0% and 7.0%, respectively, and the total subunit amino acid content was 20.0%, slightly lower than the bovine collagen value, as shown in figure 27.

The proline and hydroxyproline content was found to be related to ambient temperature. See Zhong M, chen T, hu C, ren c.2015, isolation and characterization of trepang stichopus japonicus body wall collagen, food Chemistry (Food Chemistry) 80 (4): C671-C679. However, the ratio of glycine to imino acid (Hyp/Pro) content was lower when compared to bovine collagen, thus indicating that the imino acid stabilized the glycine-based triple helix more effectively in MCT.

Collagen stability can be affected by amino acid composition, especially imino acids. Proline (PRO) and Hydroxyproline (HYP) can form hydrogen bonds with the pyrrolidine ring to protect the collagen space structure, while the hydroxyl group of hydroxyproline forms hydrogen bonds with adjacent chains to improve the stability of the triple helix. Analysis of the amino acid composition of MCT showed that the high presence of glycine and imino acids, suitable for forming stable triple helices of collagen, showed lower degradation rates than bovine collagen.

Figure 27 is a bar graph of GLY, HYP, PRO and imido (sum of HYP and PRO) of MCT1 and MCT2 samples compared to several calf collagen samples. A very advantageous aspect of MCT is that the ratio of GLY to imido acid is almost 1: 1. the reproducibility is consistent. In contrast, even the most favorable calf collagen samples, in the middle of the figure, do not show a near 1 like the MCT1 and MCT2 samples: 1. High levels of GLY indicate lower stability compared to the imino acids. Although high GLY content is advantageous, GLY with 1 of imino acid: the 1 ratio indicates higher stability.

Figures 28A and 28B and figures 29A and 29B are Scanning Electron Microscope (SEM) photographs of MCTs formed in two different ways. Figures 28A and 28B show the results of forming MCT-chitosan complexes using solvent casting techniques. Figure 28A shows the structural morphology of an MCT-chitosan dressing template (e.g., a 3D sponge). Fig. 28B shows a high degree of porosity of the structure. Fig. 29A and 29B show the results of forming an MCT-chitosan dressing template using an electrospinning technique. As shown in fig. 28A and 28B, respectively, fig. 29A shows the morphology of the structure, and fig. 29B shows the high degree of porosity of the structure. Porosity is an important attribute that directs tissue regeneration to promote cell attachment and growth.

All cited publications, patents, and patent documents are incorporated by reference herein as if individually incorporated by reference. In addition, various aspects of the invention have been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made within the spirit and scope of the invention.

Claims

1. A topical composition for treating skin abnormalities, the composition comprising:

variable collagen organization (MCT) and chitosan, said chitosan being present in a mass percentage greater than 0 and less than or equal to 90, characterized in that said chitosan is electrostatically bound to said MCT to form a matrix, said matrix of chitosan and MCT being in the form of an MCT-chitosan composite; and a pharmaceutically acceptable topical carrier; the MCT comprises collagen and glycosaminoglycans (GAGs).

2. The composition of claim 1 for use in the treatment of scars.

3. The composition of claim 1 for promoting eyelid recovery.

4. The composition of claim 1 for use in treating skin discoloration.

5. The composition of claim 4 for use in the treatment of chloasma.

6. The composition of claim 1, wherein the pharmaceutically acceptable topical carrier is selected from the group consisting of a solution, a suspension, a gel, an ointment, a lotion, or a cream.

7. The composition of claim 6, wherein the pharmaceutically acceptable topical carrier is a gel.

8. The composition of claim 6, wherein the pharmaceutically acceptable topical carrier is a cream.

9. The composition of claim 1 wherein the MCT is isolated from an invertebrate marine organism selected from the group consisting of sponges, jellyfishes, molluscs and echinoderms.

10. The composition of claim 9 wherein the MCT is isolated from an echinoderm selected from the group consisting of sea urchins and sea cucumbers.

11. The composition of claim 1 wherein the MCT consists of fibrillar collagen selected from the group consisting of type I, type II, type III, type V, or type XI or a mixture of two or more selected from the group consisting of type I, type II, type III, type V, and type XI.

12. The composition of claim 1, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate and hyaluronic acid or from a mixture of chondroitin sulfate and hyaluronic acid.

13. The composition of claim 1 wherein the mass ratio of MCT to chitosan is selected from the group consisting of 10:90, 20:80, 40:60, 50:50, 60:40, 80:20, 90:10.

14. The composition of claim 1 wherein the MCT-chitosan complex comprises a polyelectrolyte cross-linked structure between GAGs and collagen in MCT and N-glucosamine units on chitosan.

15. Use of a product for the preparation of a treatment of skin abnormalities, comprising the application to the area to be treated of variable collagen tissue (MCT) and chitosan, via a pharmaceutically acceptable topical carrier, said chitosan being present in a mass percentage greater than 0 and less than or equal to 90, characterized in that said chitosan is electrostatically bound to said MCT to form a matrix, and said matrix of chitosan and MCT is in the form of an MCT-chitosan composite, said MCT comprising collagen and glycosaminoglycans (GAGs).

16. The use according to claim 15 for the treatment of scars.

17. The use according to claim 15 for promoting eyelid recovery.

18. Use according to claim 15 for the treatment of skin discoloration.

19. The use according to claim 15 for the treatment of chloasma.

20. The use according to claim 15, wherein the pharmaceutically acceptable topical carrier is selected from the group consisting of a solution, a suspension, a gel, an ointment, a lotion or a cream.

21. The use according to claim 15, wherein the pharmaceutically acceptable topical carrier is a gel.

22. The use according to claim 15, wherein the pharmaceutically acceptable topical carrier is a cream.

23. The use of claim 15, wherein the administering further comprises administering a matrix of chitosan and MCT, wherein the MCT is isolated from an invertebrate marine organism selected from the group consisting of sponges, jellyfishes, molluscs, and echinoderms.

24. The use of claim 23, wherein the administering further comprises administering a matrix of chitosan and MCT, wherein the MCT is isolated from an echinoderm selected from the group consisting of sea urchins and sea cucumbers.

25. The use according to claim 15, wherein the administering further comprises administering a matrix of chitosan and MCT.

26. The use of claim 15, wherein the administering further comprises administering a matrix of chitosan and MCT, wherein the MCT consists of fibrillar collagen selected from the group consisting of type I, type II, type III, type V, or type XI or a mixture of two or more selected from type I, type II, type III, type V, and type XI.

27. The use according to claim 25, wherein the glycosaminoglycan is selected from the group consisting of chondroitin sulfate and hyaluronic acid or from a mixture of chondroitin sulfate and hyaluronic acid.

28. The use of claim 15, wherein the administering further comprises administering a matrix of chitosan and MCT, wherein the mass ratio of MCT to chitosan is selected from the group consisting of 10:90, 20:80, 40:60, 50:50, 60:40, 80:20, 90:10.

29. The use of claim 25, wherein the administering further comprises administering a matrix of chitosan and MCT, wherein the MCT-chitosan complex comprises a polyelectrolyte cross-linked structure between GAGs and collagen in MCT and N-glucosamine units on chitosan.