WO2020120805A2 - Échafaudages hétérogènes et procédés de fonctionnalisation de surfaces - Google Patents

Échafaudages hétérogènes et procédés de fonctionnalisation de surfaces Download PDF

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WO2020120805A2
WO2020120805A2 PCT/EP2019/085447 EP2019085447W WO2020120805A2 WO 2020120805 A2 WO2020120805 A2 WO 2020120805A2 EP 2019085447 W EP2019085447 W EP 2019085447W WO 2020120805 A2 WO2020120805 A2 WO 2020120805A2
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Prior art keywords
layer
bioceramic
scaffold
weight
tropocollagen
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PCT/EP2019/085447
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English (en)
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WO2020120805A3 (fr
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Anna Mas VINYALSLAND
Salvador Borrós GÓMEZ
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Institut Químic De Sarrià Cets Fundació Privada
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Publication of WO2020120805A2 publication Critical patent/WO2020120805A2/fr
Publication of WO2020120805A3 publication Critical patent/WO2020120805A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/24—Materials or treatment for tissue regeneration for joint reconstruction

Definitions

  • the present invention relates to scaffolds comprising bioceramics and hydrogels, used in the treatment of osteoarthritis.
  • the present invention also relates to methods for immobilising hydrogels onto substrates.
  • Osteoarthritis is the most common form of arthritis and it is considered one of the most disabling diseases in developed countries. It affects 240 million people globally and its prevalence is increasing. The increase of its risk factors suggest that the increasing prevalence will continue, together with consequential personal and societal economic costs due to healthcare use. Its symptoms cause a significant impact in day- to-day function, loss of independence and reduced quality of life. There are no drugs approved which prevent, treat, or restrain OA. The available treatments focus on reducing symptoms; including pain, without providing a cure for OA. Thus, there is a need for treatments that target the illness itself, providing a means for tissue repair that obviates the need for invasive surgical procedures
  • Present methodologies seek to reproduce the bioceramic gradient that is present in the cartilage layer by preparing multilayer structures wherein each layer has a different amount of a bioceramic material, such that a bioceramic gradient exists ab initio.
  • Such scaffolds comprising hydroxyapatite bioceramic layers are disclosed in e.g. WO2010/084481 and W02006/092718.
  • the methodology for making such scaffolds uses iterative freezing techniques (e.g. lyophilization), comprising sequentially freezing suspensions comprising different ratios of hydroxyapatite and collagen to form a multilayer structure with a step gradient of stiffness.
  • the scaffolds of the art suffer from poor mechanical integrity, due to an absence of strong intermolecular interactions between the respective layers. Instead, the individual layers of the scaffolds of the art are only weakly adhered to each other during the freezing step.
  • the present invention improves on the scaffolds of the art by providing a scaffold that spontaneously forms a continuous bioceramic gradient when placed in
  • a continuous bioceramic gradient forms across the scaffold of the invention when it is placed in vivo, such that it effectively mimics the entire articular junction.
  • the scaffolds of the art also direct differentiation of mesenchymal stem cells into osteoblasts and chondrocytes, according to the stiffness gradient that results from the continuous bioceramic gradient across the scaffold.
  • the scaffold of the invention comprises a bioceramic layer and a collagen hydrogel layer.
  • the inventors have found that scaffolds incorporating a bioceramic with a mean crystalline domain size of 1 to 275 nm will spontaneously form a continuous bioceramic gradient when placed in physiological fluids.
  • the mean crystalline domain size of the bioceramic layer is a critical parameter that dictates the level to which the calcium phosphate from the bioceramic layer undergoes dissolution, migration from the bioceramic layer, and recrystallization in the collagen hydrogel layer when placed in physiological fluids.
  • the present invention provides a scaffold comprising a first layer and a second layer adjacent to the first layer, wherein the first layer comprises a bioceramic and the second layer comprises collagen, wherein the bioceramic has a mean crystalline domain size of 1 to 275 nm.
  • the present invention also provides a method of functionalizing a substrate with a collagen hydrogel.
  • the method comprises functionalizing the surface of a substrate with a functionalisation layer, attaching a monolayer of tropocollagen to the functionalisation layer and forming a collagen hydrogel from the tropocollagen monolayer.
  • the method of the invention can be used to immobilise collagen hydrogels onto a variety of surfaces and has application in the field of medical implants, for example odontology, where collagen hydrogels can be immobilised onto dental implants before implantation into a patient.
  • the method of functionalizing a substrate with a collagen hydrogel of the second aspect of the present invention comprises the steps of:
  • a Functionalising a surface of the substrate with a functionalisation layer
  • b Attaching a monolayer of tropocollagen to the functionalisation layer by contacting the functionalisation layer with a first solution comprising tropocollagen;
  • the tropocollagen monolayer that is attached to the surface of the substrate in the method of the second aspect of the invention is formed from tropocollagen and acts as an anchoring point for the formation of the collagen hydrogel layer, facilitating the self-assembly of tropocollagen into higher-order fibrillar structures. Accordingly, the inventors have found that the method of the second aspect of the invention can be used to developed scaffolds with mechanical integrity, due to the presence of strong covalent linkages between the functionalisation layer and the tropocollagen monolayer and robust non-covalent interactions, which result from the self-assembly of the collagen monomers, between the tropocollagen monolayer and the collagen hydrogel. Furthermore, the functionalization layer is covalently attached to the surface of the substrate. In addition, the tropocollagen monolayer facilitates diffusion of the ions of the bioceramic into the collagen layer, for example to form a
  • the present invention provides a scaffold comprising a first layer, a second layer, and a functionalisation layer between the first layer and the second layer, wherein the first layer comprises a bioceramic and the second layer comprises a collagen hydrogel, wherein the functionalisation layer is attached to a surface of the first layer, and wherein the second layer is covalently attached to the functionalisation layer.
  • the present invention also provides a scaffold described herein for use in treating osteoarthritis in a mammal.
  • the first and third aspects of the present invention provide a novel scaffold
  • the scaffold of the invention may further comprise a functionalisation layer between the first and second layer, which is attached to the surface of the bioceramic and is covalently attached to the collagen hydrogel of the second layer.
  • the collagen hydrogel of the second layer may be in the form of higher order structures such as fibrils.
  • the continuous bioceramic gradient does not need to be present in the scaffold of the invention ab initio (e.g. through a method of manufacture, such as preparing a multilayer structure with different amounts of the bioceramic in each layer).
  • the bioceramic in the first layer of the scaffold of the invention undergoes spontaneous diffusion of calcium phosphate when placed in physiological fluids, forming a continuous bioceramic gradient as a result of having a mean crystallite domain size of between 1 to 275 nm, such as 2 to 250 nm, 5 to 240 nm, 10 to 230 nm, 50 to 220 nm, 60 to 200 nm, 80 to 210 nm, 100 to 200 nm, or 120 to 180 nm.
  • calcium and phosphate ions from the bioceramic layer spontaneously diffuse into the collagen hydrogel.
  • the calcium and phosphate ions migrate into the collagen hydrogel layer, where the collagen fibrils act as nucleation points for crystallisation of the calcium and phosphate ions into a bioceramic. Diffusion of the calcium and phosphate ions is greatest to areas of the scaffold nearest the bioceramic layer and least to the areas of the scaffold furthest from the bioceramic layer. Consequently, a continuous bioceramic gradient will form, from an area comprising a high weight percent of bioceramic (i.e.
  • the bioceramic that forms in the collagen hydrogel of the second layer of the scaffold of the invention is hydroxyapatite. In some embodiments of the first aspect of the invention, the bioceramic that forms in the collagen hydrogel of the second layer of the scaffold of the invention is carbonated hydroxyapatite.
  • the continuous bioceramic gradient provides the scaffolds of the first and third aspects of the invention improves the mechanical integrity of the bioceramic/collagen hydrogel interface as the scaffolds have a continual stiffness gradient, wherein the scaffold is most stiff in the parts of the scaffold with the highest concentration of bioceramic and least stiff in the parts of the scaffold with the lowest concentration of bioceramic.
  • any external mechanical forces that act upon the scaffold of the invention such as those experienced in the joint of a mammal, are more evenly distributed across the scaffold of the invention.
  • the scaffolds of the art comprise step bioceramic gradients and the individual layers are only weakly adhered to each other during the iterative freezing steps of their manufacture.
  • the scaffolds of the art may therefore undergo delamination, wherein the layers of the scaffold detach from each other, in response to an external mechanical force. Furthermore, the scaffolds of the first and third aspects of the invention effectively mimic the bone phase of the articular junction. Upon formation of the continuous bioceramic gradient, the initial two layer structure of the scaffold of the first and third aspects of the invention may no longer be identifiable. However, the covalent linkages between the bioceramic layer and the functionalisation layer, when present, and between the functionalisation layer and the collagen hydrogel of the second layer are still present after the formation of the continuous bioceramic gradient.
  • the stiffness gradient that forms in the scaffold of the art will facilitate the differentiation of mesenchymal stems cells into either osteoblasts or chondrocytes according to the stiffness of the scaffold.
  • the scaffold of the first and third aspects of the invention can be used as an implant for effective treatment of osteoarthritis as the spontaneous continuous bioceramic gradient that forms in the scaffold of the first and third aspects of the invention mimics the osteochondral interface, and provides a continuous transition zone from the subchondral bone to the hyaline cartilage.
  • the scaffold of the first and third aspects of the invention can therefore be used in an improved treatment of osteoarthritis where it can replace a portion of the articular junction.
  • the scaffold of the third aspect of the invention has mechanical integrity due to the nature of the bonding that exists between the respective layers and therefore have a reduced risk of delamination of the layers when placed in vivo.
  • the functionalisation layer is covalently attached to the collagen hydrogel layer in the scaffold of the third aspect of the invention. Furthermore, the collagen that is covalently attached to the functionalisation layer is attached to the rest of the collagen hydrogel in the second layer through robust non-covalent interactions, which result from the self-assembly of the collagen into higher-order structures, such as fibrils. Finally, the functionalisation layer may be covalently attached to the surface of the substrate.
  • scaffolds of the invention comprise robust
  • Example 5 of the present invention demonstrates how, in the absence of covalent linkages between the bioceramic layer and the collagen hydrogel layer (e.g. the scaffolds of the art), changes in the physiological medium in which the scaffold is placed disrupt the interface between the bioceramic and collagen layers and result in the loss of collagen from the bioceramic layer.
  • the first layer comprises at least 50% by weight of a bioceramic, such as at least 55% by weight, at least 60% by weight, at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 92% by weight, at least 94% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, or at least 99% by weight of a bioceramic.
  • the first layer consists essentially of a bioceramic.
  • the first layer is a bioceramic.
  • the scaffold of the first and third aspects of the invention comprises at least 84% by weight of a bioceramic.
  • the first layer of the scaffold of the first and third aspects of the invention can comprise additional components, including collagen. Accordingly, in some embodiments of the first and third aspects of the invention the first layer comprises up to 16 weight % collagen, such as up to 14 weight % collagen, up to 12 %, up to 10 weight %, up to 8 weight %, up to 6 weight %, or up to 3 weight % collagen. In some embodiments of the first and third aspects of the invention, the first layer comprises bioceramic and collagen in a weight percent ratio of 100:0, or 95:5, or 90:10, or 85:15.
  • the first layer comprises at least 84% of a bioceramic and up to 16% collagen.
  • the bioceramic of the first layer is bone.
  • the bioceramic of the first layer is a bone-like ceramic, or a processed tissue (such as demineralized bone).
  • the bioceramic of the first layer is b- trichlorophosphate (b-TCP), a-trichlorophosphate (a-TCP), hydroxyapatite, dicalcium phosphate (DCP), dicalcium phosphate dehydrate (DCPD), tetracalcium phosphate (TTCP), octacalcium phosphate (OCP), or mixtures thereof.
  • the bioceramic of the first layer is b-TCP, for example Cerasorb® available from Curasan AG (Germany).
  • the bioceramic of the first layer is hydroxyapatite, for example bovine hydroxyapatite such as Bio-Oss® available from Garlich (USA), or synthetic hydroxyapatite available from Plasma Biotal Ltd. (U.K.).
  • the bioceramic of the first layer is biphasic calcium phosphate (BCP), comprising a mixture of b-TCP and hydroxyapatite, such BCPs including BONIT matrix sterile® available from Dyna Dental, Netherlands, BONIT matrix unsterile® available from Dyna Dental, Netherlands, or Straunmann® available from Straumann®,
  • BCP biphasic calcium phosphate
  • the bioceramic of the first layer is biphasic calcium phosphate (BCP) comprising a-TCP and hydroxyapatite.
  • BCP biphasic calcium phosphate
  • the first layer is a bioceramic formed by compressing a commercially available bioceramic, such as those listed herein, into a disk, using a hydraulic press (e.g. 10 tonne).
  • a hydraulic press e.g. 10 tonne
  • mesenchymal stem cells are present in the bioceramic.
  • physiological fluids e.g. in vivo
  • mesenchymal stems cells migrate from the first layer into the collagen hydrogel layer. Once migrated, the mesenchymal stems cells colonise the scaffold and undergo differentiation depending on the mechanical properties (i.e. stiffness) of the area of the scaffold to which the cells have migrated.
  • the scaffold can be seeded with mesenchymal stem cells prior to implantation into a patient. Accordingly, in some embodiments of the scaffold of the invention, the scaffold further comprises mesenchymal stem cells. Crystallinity of the first layer
  • the crystalline domain size of the bioceramic is a critical parameter for ensuring that the scaffold has bioactivity (e.g. undergoes dissolution of the bioceramic phase and recrystallization of the bioceramic phase in the collagen hydrogel layer) once placed in physiological fluids.
  • bioceramic materials having a mean crystalline domain size of 1 to 275 nm result in optimum dissolution of the bioceramic layer, with the concomitant migration of the calcium and phosphate ions and formation of the continuous bioceramic gradient in the scaffold.
  • the bioceramic of the first layer of the scaffold has a mean crystalline domain size of 2 to 250 nm, such as 5 to 240 nm, 10 to 230 nm, 50 to 220 nm, 80 to 210 nm, 100 to 200 nm, or 120 to 180 nm.
  • Example 4 demonstrates bioceramics having a mean crystalline domain size of 1 to 275 nm, and particularly 60 to 200 nm, such as 120 to 180 nm, undergo dissolution when immersed in physiological fluids, as evidenced by the increase in both the pH and the concentration of calcium ions of the simulated body fluid (SBF) in which disks of each bioceramic material are immersed.
  • the mean crystalline domain size dictates the dissolution rate of the bioceramics and is optimum within the recited ranges. For all bioceramics, the majority of the observed pH increase occurs within the first 24 hours of the disk being immersed in physiological fluid.
  • the scaffolds of the first and third aspects of the invention will undergo dissolution of the bioceramic layer, followed by subsequent recrystallization of the bioceramic in the fibrils of the collagen hydrogel, which act as nucleation points for the recrystallization process.
  • bioceramics for use in the invention were tested and were found to retain mechanical integrity at the end of the experiment. Whilst it is necessary that the bioceramic material in the scaffold undergoes dissolution so that the continuous bioceramic gradient may form, the scaffolds must also be suitably stable under physiological conditions such that the bioceramic layer maintains structural integrity post-implantation. Accordingly, the bioceramics for use with the first and third aspects of the invention are stable under physiological conditions and therefore can be used in heterogeneous scaffolds.
  • physiological conditions it is meant any conditions that occur where collagen is found in nature. Such conditions include a temperature of from 20 to 40 °C, such as 22 to 40 °C, 25 to 40 °C, 30 to 40 °C, 32 to 38 °C, or 35 to 37 °C, and at a pH of from 6-8.
  • the attachment of the tropocollagen monolayer can be performed using the method of the first and third aspects of the invention at pH 7 and 37 °C.
  • the mean size of the crystalline domains of the bioceramics for use in the scaffolds of the first and third aspects of the invention is measured by powder X-ray diffraction (PXRD).
  • the samples are first ground into powder. PXRD analysis of the
  • bioceramic powders was carried out with an X-Ray diffractometer (Bruker D-5005) using a Cu-Ka radiation generated at 40kV and 100 mA.
  • Figures 8 to 10 are PXRD spectra of the bioceramics for use in the scaffolds of the present invention.
  • the Scherrer equation (I) is employed to calculate the mean size of the crystalline domains from the PXRD spectra obtained.
  • t is the mean size of the crystalline domains.
  • K is the dimensionless shape factor.
  • K 0.89.
  • l is the X-ray wavelength used in the analysis.
  • Q is the Bragg angle.
  • At least 50% of the bioceramic present in the first layer of the scaffold is crystalline. In some embodiments of the first and third aspects of the invention, at least 60%, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the bioceramic present in the first layer of the scaffold is crystalline.
  • the second layer of the scaffold of the first and third aspects of the invention comprises collagen.
  • the second layer of the scaffold of the first and third aspects of the invention can be a collagen hydrogel.
  • the second layer comprises at least 50% by weight of collagen, such as at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, or at least 95% by weight of collagen.
  • the collagen for use in the second layer of the scaffold of the first and third aspects of the invention can be Type I collagen, Type II collagen, Type III collagen, or combinations thereof.
  • the collagen for use in the second layer of the scaffold can be Fibricol®.
  • the collagen hydrogel of the second layer comprises Type I collagen.
  • the collagen of the hydrogel of the second layer of the scaffold of the invention can be in the form of higher-order structures, such as fibrils.
  • a continuous bioceramic gradient spontaneously forms in the scaffold of the first and third aspects of the invention when it is placed in
  • the second layer of the scaffold of the first and third aspects of the invention does not comprise a bioceramic. Accordingly, in some embodiments of the first and third aspects of the invention, the second layer does not comprise a bioceramic. If desired, the second layer can comprise a bioceramic. The presence of a bioceramic in the second layer will not prevent the formation of a continuous bioceramic gradient throughout the scaffold of the invention.
  • the second layer of the scaffold comprises at least 1 % by weight of a bioceramic, such as at least 2% by weight, at least 3% by weight, at least 5% by weight, or at least 10% by weight. In some embodiments of the first and third aspects of the invention, the second layer comprises less than 30% by weight of a bioceramic, such as less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 3% by weight, or less than 2% by weight of a bioceramic.
  • the second layer of the scaffold comprises from 1 % to 16 % by weight of a bioceramic, such as from 2% to 14 %, from 3% to 12 %, from 4% to 10 %, or from 5 % to 8 % by weight of a bioceramic.
  • the bioceramic of the first layer is b-trichlorophosphate (b-TCP), a-trichlorophosphate (a-TCP), hydroxyapatite, dicalcium phosphate (DCP), dicalcium phosphate dehydrate
  • DCPD tetracalcium phosphate
  • TTCP tetracalcium phosphate
  • OCP octacalcium phosphate
  • the bioceramic in the second layer is bone. In some embodiments of the first and third aspects of the invention, the bioceramic of the first layer is a bone-like ceramic.
  • the collagen in the collagen hydrogel of the second layer of the scaffold has a concentration of about 4 mg mL ⁇ 1 . In some embodiments of the first and third aspects of the invention, the collagen in the collagen hydrogel of the second layer of the scaffold has a concentration of from 0.1 - 20 mg mL ⁇ 1 , such as from 0.5 - 15 mg mL ⁇ 1 , from 1 - 10 mg mL ⁇ 1 , from 2 - 8 mg mL ⁇ 1 , from 3 - 6 mg mL ⁇ 1 , or from 3 - 5 mg mL ⁇ 1 .
  • the second layer of the first and third aspects of the invention may comprise additional components.
  • Said additional components include polysaccharides, such as glycosaminoglycans (GAGs).
  • GAGs are long unbranched polysaccharides consisting of a repeating disaccharide unit, wherein one of the units is an amino sugar.
  • Examples of GAGs include heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and hyaluronic acid, one or more of which may be incorporated into the second layer of the scaffold of the invention.
  • the second layer comprises chondroitin sulfate, hyaluronic acid, or combinations thereof.
  • chondroitin sulfate and/or hyaluronic acid assist in the self-assembly of the collagen hydrogel and promote the differentiation of mesenchymal stems cells into either osteoblasts or chondrocytes.
  • self-assembly it is meant a spontaneous process by which molecular units, e.g. a polymer, form ordered structures through non- covalent interactions.
  • the second layer comprises other polysaccharides, including one or more of dextran, heparan, alginate, agarose, carrageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, chitosan and various sulfated polysaccharides such as heparan sulfate, or dextran sulfate.
  • additional components that may be incorporated into the second layer of the scaffold of the invention include DNA, RNA, low molecular weight nucleic acids, proteins, polypeptides, peptides, hormones, growth factors, cytokines, metabolites and cells.
  • the collagen hydrogel second layer of the scaffold of the first and third aspects of the invention may comprise collagen that are higher-order collagen structures, such as fibrils.
  • the collagen hydrogel of the first and third aspects of the invention therefore has mechanical integrity even in the absence of covalent linkages between the collagen monomers (e.g. crosslinks).
  • the second layer of the scaffold of the invention is uncrosslinked.
  • the collagen hydrogel of the second layer of the invention may be free of crosslinking agents. By uncrosslinked it is understood that the collagen hydrogel does not comprise covalent linkages between collagen monomers.
  • an uncrosslinked collagen hydrogel may still comprise a statistically small amount of covalent linkages between the collagen monomers (i.e. a de minimis amount), but that the majority of the collagen monomers present in the hydrogel will be free of covalent linkages to other collagen monomers.
  • cross-linking of the collagen hydrogel of the second layer is not required to provide structural integrity to the scaffolds of the invention.
  • the collagen hydrogel in the second layer can be crosslinked. Accordingly, in some embodiments of the invention, the collagen hydrogel is at least partially crosslinked.
  • the collagen hydrogel in the second layer of the invention comprises a residue of a crosslinking agent, such as 1 - ethyl-3-[3- dimethylaminopropyljcarbodiimide hydrochloride (EDAC) or glutaraldehyde.
  • a crosslinking agent such as 1 - ethyl-3-[3- dimethylaminopropyljcarbodiimide hydrochloride (EDAC) or glutaraldehyde.
  • the scaffolds of the first and third aspects of the present invention do not require that a bioceramic gradient be introduced ab initio during the preparation of the scaffold. Instead, a continuous bioceramic gradient forms spontaneously when the scaffolds of the invention are placed in physiological fluids. Accordingly, in some embodiments of the first and third aspects of the invention the first layer of the scaffold comprises up to 100% of a bioceramic and the second layer of the scaffold comprises 0% bioceramic.
  • the first layer comprises at least 90 % of a bioceramic and the second layer comprises 0% bioceramic, such as at least 80 % of a bioceramic in the first layer and 0%
  • bioceramic in the second layer at least 70 % of a bioceramic in the first layer and 0% bioceramic in the second layer, at least 60 % of a bioceramic in the first layer and 0% bioceramic in the second layer, or at least 50 % of a bioceramic in the first layer and 0% bioceramic in the second layer.
  • the second layer of the scaffold of the first and third aspects of the invention can comprise a bioceramic, if desired, as the presence of a bioceramic in the second layer will not prevent the formation of a continuous bioceramic gradient throughout the scaffold of the first and third aspects of the invention.
  • the first layer of the scaffold of the first and third aspects of the invention may consist of 100% of a bioceramic and the second layer of the scaffold of the first and third aspects of the invention may comprise up to 10% of a bioceramic.
  • the first layer comprises at least 90 % of a bioceramic and the second layer comprises up to 10% of a bioceramic, such as at least 80 % of a bioceramic in the first layer and up to 10% of a bioceramic in the second layer, at least 70 % of a bioceramic in the first layer and up to 10% of a bioceramic in the second layer, at least 60 % of a bioceramic in the first layer and up to 10% of a bioceramic in the second layer, or at least 50 % of a bioceramic in the first layer and up to 10% of a bioceramic in the second layer.
  • the first layer of the scaffold of the first and third aspects of the invention may consist of 100% of a bioceramic and the second layer of the scaffold of the first and third aspects of the invention may comprise up to 20% of a bioceramic. Accordingly, in some embodiments of the first and third aspects of the invention, the first layer comprises at least 90 % of a bioceramic and the second layer comprises up to 20% of a bioceramic, such as at least 80 % of a bioceramic in the first layer and up to 20% of a bioceramic in the second layer, at least 70 % of a bioceramic in the first layer and up to 20% of a bioceramic in the second layer, at least 60 % of a bioceramic in the first layer and up to 20% of a bioceramic in the second layer, or at least 50 % of a bioceramic in the first layer and up to 20% of a bioceramic in the second layer.
  • the scaffold of the first and third aspects of the invention further comprises a functionalisation layer between the first layer and the second layer.
  • “functionalisation layer” it is meant any layer of material that can be attached to a substrate, for example a bioceramic substrate, and that is capable of undergoing reactions with biomolecules to form covalent linkages.
  • the functionalisation layer may be capable of undergoing reactions with“unactivated biomolecules”, by which it is meant any biomolecule that has not undergone a synthetic transformation, e.g. a functional group transformation.
  • collagen is an unactivated biomolecule that naturally comprises pendant amine groups.
  • the functionalisation layer may be a polymer layer.
  • the polymer may be selected from the group consisting of poly(methacrylate), poly(acrylate),
  • the polymer may be poly(pentafluorophenyl methacrylate), or poly(N- hydroxysuccinimidyl methacrylate).
  • ppPFM plasma polymerised poly(pentafluorophenyl methacrylate)
  • the polymers for use as the functionalisation layer of the first and third aspects of the invention comprise reactive groups capable of undergoing substitution with unactivated biomolecules.
  • collagen comprises amine (-NFh) moieties that can undergo nucleophilic substitution with the pentafluorophenyl or - hydroxysuccinimidyl esters attached to the backbone of methacrylate polymers of a functionalisation layer, forming covalent linkages between the collagen and the functionalisation layer.
  • the functionalisation layer is covalently attached to the bioceramic layer.
  • the functionalisation layer may be coated onto the first layer of the scaffold of the invention.
  • coated it is meant that a material strongly adheres to a substrate, wherein the principal attractive force between the material and the substrate is non-covalent in nature.
  • functionalisation layer may be covalently attached to the collagen hydrogel of the second layer of the scaffold of the invention.
  • the functionalisation layer has a thickness of at least 10 nm, such as at least 20 nm, at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, or at least 100nm, and up to 500nm, such as up to 400nm, up to 300nm, up to 200nm, or up to 150nm.
  • the functionalisation layer has a thickness of from 10-500nm, such as from 20-400nm, from 20-300nm, from 30-200nm, from 30-150nm, from 40-100 nm, from 40-80 nm, or from 40-60nm
  • a continuous gradient of a bioceramic will form through the body of the scaffold.
  • a continuous bioceramic gradient of from 100% to 0 % by weight will form when the scaffold of the first and third aspects of the invention is placed in physiological fluid, such as a gradient of from 100% to 10%, 100% to 20%, 100% to 30%, 90% to 0%, 80% to 0%, 70% to 0%, or 60% to 0%, 90% to 10%, 80% to 20%, 70% to 30%, or 60% to 40% by weight across the scaffold.
  • the first layer consists of bioceramic (i.e. the first layer has a weight percentage of bioceramic of 100%)
  • the first layer has a weight percentage of bioceramic of 100%
  • no significant reduction in the weight percentage of the bioceramic will be observed due to the diffusion of calcium and phosphate ions out of the first layer and into the collagen hydrogel layer, as no other components are present in the bioceramic first layer such that the bioceramic will still be the only component.
  • Some minor impurities may diffuse into the bioceramic layer once the scaffold is placed in physiological fluid, resulting in small decrease of the weight percentage of the bioceramic in the first layer.
  • the bioceramic of the first layer may become more porous as a result of the diffusion of calcium and phosphate ions once the scaffold is placed in physiological fluid.
  • a gradient of the weight ratio of the bioceramic:collagen is formed.
  • the gradient of weight ratio of the bioceramic:collagen formed when the scaffold is placed in physiological fluids is from a maximum of 100:0 to 95:5 to a minimum of 5:95 to 0:100, such as from a maximum of 100:0 to 95:5 to a minimum of 15:85 to 10:90, from a maximum of 100:0 to 95:5 to a minimum of 25:75 to 20:80; from a maximum of 100:0 to 95:5 to a minimum of 45:65 to 30:70; from a maximum of 100:0 to 95:5 to a minimum of 45:55 to 40:60; from a maximum of 90:10 to 85:15 to a minimum of 5:95 to 0:100; from a maximum of 90:10 to 85:15 to a minimum of 15:85 to 10:90; from a maximum of 90:10 to 85:15 to a minimum of 25:75 to 20:80; from a maximum of 90:10 to
  • the gradient may be linear, i.e. for a scaffold with a gradient of a bioceramic across the scaffold from 100% to 0%, there will be an approximately constant rate of change of the weight percentage of a bioceramic from 100% to 0% across the scaffold.
  • the rate of change in the weight percentage of the bioceramic per unit distance across a straight line vector across the scaffold will be constant along the length of the vector, from one end point of the gradient to the other.
  • the gradient in bioceramic concentration that forms across the scaffold will be non-linear.
  • the gradient of bioceramic that forms across the scaffold will be exponential.
  • the weight percentage of a bioceramic in the scaffold will approximately exponentially decay from 100% to 0% across the scaffold.
  • the rate of change in the weight percentage of the bioceramic per unit distance across a straight line vector through the scaffold will vary (i.e. increase or decay) exponentially along the length of the vector, from one end point of the gradient to the other.
  • physiological fluids any fluids that are present in the environment where the scaffold of the first and third aspects of the invention might find use, for example, within the articular junction of a mammal.
  • blood plasma or synovial fluid.
  • SBF refers to any solution that simulates the ion concentration of human blood plasma.
  • SBF comprises tris-hydroxymethylaminomethane at a concentration of from 2- 12 g dm -3 , such as 4 - 10 g dm -3 , or 6- 8 g dm -3 ; NaCI at a concentration of from 2- 12 g dm -3 , such as 4 - 10 g dm -3 , or 6- 8 g dm -3 ; NaFICC at a concentration of from 0.5 - 4.5 g dm -3 , such as 1.0 to 3.0 g dm -3 , or 1.5 to 2.5 g dm -3 ; KCI at a concentration of from 0.1 - 0.6 g dm -3 , such as 0.2 - 0.5 g dm -3 , or 0.3 - 0.4 g dm -3 ; Na 2 FIP0 4 ⁇ 12FI2O at a concentration of from 0.01 - 0.12 g dm -3 , such as 0.03 - 0.09
  • the inventors have developed a scaffold for which initiatiation of the dissolution of the bioceramic and formation of a continuous bioceramic gradient is optimum under physiological conditions, i.e. the conditions in which the scaffolds of the invention will find use, e.g. when used in the treatment of OA.
  • physiological conditions i.e. the conditions in which the scaffolds of the invention will find use, e.g. when used in the treatment of OA.
  • the scaffolds of the invention may also undergo spontaneous formation of a continuous bioceramic gradient when placed in other solvents, such as water.
  • articular junction it is meant a joint where at least two bones in an animal meet.
  • bioceramic gradient can also be referred to as
  • the continuous bioceramic gradient that arises in the scaffolds of the first and third aspects of the invention results in a compositional variation and a stiffness gradient across the scaffold which direct differentiation of mesenchymal stem cells.
  • Mesenchymal stem cells in areas of the scaffold with a high concentration of bioceramic (and therefore of high stiffness) will differentiate into osteoblasts (via osteogenic differentiation).
  • Mesenchymal stem cells in parts of the scaffold with a low concentration of bioceramic (and therefore of low stiffness) will differentiate into chondrocytes (via chondrogenic differentiation).
  • the scaffold further comprises mesenchymal stem cells, including osteoblasts and chondrocytes, and combinations thereof.
  • the scaffold comprises a gradient in the concentration of osteoblasts. The concentration of osteoblasts may be highest in the areas of the scaffold with the highest concentration of bioceramic (and therefore the stiffest) and lowest in the areas of the scaffold with the lowest concentration of bioceramic (and therefore the least stiff).
  • the scaffold comprises a gradient in the concentration of chondrocytes.
  • the concentration of chondrocytes may be highest in the areas of the scaffold with the lowest concentration of bioceramic (and therefore the least stiff) and lowest in the areas of the scaffold with the highest concentration of bioceramic (and therefore the stiffest).
  • the scaffold of the invention take a variety of shapes and sizes.
  • the scaffold has a total thickness of at least 25mm, such as at least 30 mm, at least 35mm, at least 40mm, at least 60mm, at least 80mm, or at least 100mm.
  • the scaffold has a total thickness of from 25mm to 200mm, such as from 30mm to 100mm, from 35mm to 80mm, from 40mm to 70mm, or from 50mm to 70mm.
  • the first and second layers of the scaffold have the same thickness. In some embodiments of the invention, the first and second layers of the scaffold have different thicknesses. The thickness of a layer of a scaffold is taken as the distance from the interface with the adjacent layer to the face of the layer opposing the interface.
  • the first layer has thickness of at least 10 mm, such as at least 15 mm, at least 20 mm, at least 30 mm, at least 40 mm, at least 50 mm, or at least 70 mm. In some embodiments of the invention, the first layer has thickness of from 1 mm to 100mm, such as from 5 mm to 80mm, from 10 mm to 70mm, from 20 mm to 60mm, from 30 mm to 50mm, or from 40mm to 60mm. In some embodiments of the invention, the second layer has thickness of at least 10 mm, such as at least 15 mm, at least 20 mm, at least 30 mm, at least 40 mm, at least 50 mm, or at least 70 mm.
  • the second layer has thickness of from 1 mm to 100mm, such as from 5 mm to 80mm, from 10 mm to 70mm, from 20 mm to 60mm, from 30 mm to 50mm, or from 40mm to 60mm.
  • the ratio of the thicknesses of the first layer to the second layer is at least 50:50, such as at least 60:40, at least 70:30, at least 80:20, at least 90:10, or at least 95:05. In some embodiments of the invention, the ratio of the thicknesses of the second layer to the first layer is at least 50:50, such as at least 60:40, at least 70:30, at least 80:20, at least 90:10, or at least 95:05.
  • the interfacial area between the first and second layers of the scaffold of the invention is at least 5 mm 2 , such as at least 8 mm 2 , at least 10 mm 2 , at least 20 mm 2 , at least 30 mm 2 , at least 40 mm 2 , or at least 50 mm 2 . In some embodiments, the interfacial area between the first and second layers of the scaffold of the invention is from 5 mm 2 to 100 mm 2 , such as from 8 mm 2 to 80 mm 2 , from 10 mm 2 to 70 mm 2 , from 20 mm 2 to 60 mm 2 , or from 30 mm 2 to 50 mm 2 .
  • the second aspect of the present invention also provides a method of immobilising a collagen hydrogel on a substrate, comprising the functionalising the surface of a substrate with a functionalisation layer, attaching a monolayer of tropocollagen to the functionalisation layer by contacting the functionalisation layer with a first solution comprising tropocollagen, and contacting the tropocollagen monolayer with a second solution comprising tropocollagen to form a collagen hydrogel.
  • the method of the second aspect of the invention allows for the immobilisation of a collagen hydrogel onto a wide variety of surfaces and therefore has a broad applicability.
  • the method of the second aspect of the invention may therefore be used to immobilise collagen hydrogels onto medical devices, for example orthopedic implants.
  • the osseointegration of the medical device is promoted, as the collagen hydrogel can fill any cavities that exist between the implant and the bone to which the implant is attached.
  • the collagen hydrogel thus prevents bacterial colonization and biofilm accumulation around the implant and the associated inflammatory conditions that could occur as a result.
  • the method of the second aspect of the invention can be used to immobilise collagen hydrogels onto dental implants.
  • Plaque accumulation normally occurs between the implant and the surrounding tissue and can cause periimplantitis and ultimate implant rejection. However, by filling the cavities between the implant and the bone in which it is implanted with a collagen hydrogel, plaque accumulation can be prevented.
  • the method of the second aspect of the invention can also be used to make heterogeneous osteochondral scaffolds, such as those of the first and third aspects of the invention. Accordingly, immobilising a collagen hydrogel onto a bioceramic material using the method of the invention enables formation of a heterogeneous scaffold comprising a rigid bioceramic layer covalently attached to collagen hydrogel.
  • the method of the second aspect of the present invention comprises the attachment of a tropocollagen monolayer to the surface of a substrate that has been
  • the substrate comprises a bioceramic wherein the bioceramic has a mean crystalline domain size of 1 to 275 nm, for example 2 to 250 nm, such as 5 to 240 nm, 10 to 230 nm, 50 to 220 nm, 60 to 200 nm, 80 to 210 nm, 100 to 200 nm, or 120 to 180 nm.
  • the method of the second aspect of the invention can therefore be used to prepare scaffolds
  • tropocollagen it is meant a layer of tropocollagen that is covalently attached to the functionalisation layer.
  • functionalising the substrate means introducing reactive groups to the surface of the substrate, in this case reactive groups capable of covalently attaching to unactivated biomolecules.
  • the functionalisation layer in the second aspect of the invention may comprise any material capable of covalently attaching to unactivated biomolecules.
  • a layer comprising functional groups capable of undergoing substitution reactions with an unactivated biomolecule, for example the amine (-NFh) groups attached to collagen.
  • the functionalisation layer can comprise pentafluorophenyl esters, or hydroxysuccinimidyl esters, which will undergo nucleophilic substitution with the amine groups of a biomolecule (e.g. collagen), covalently attaching the biomolecule to the functionalisation layer through an amide bond.
  • the functionalisation layer may be a polymer layer made through polymerizing a monomer such that the resulting polymer is attached to the surface of the substrate.
  • the polymer may be selected from the group consisting of poly(methacrylate), poly(acrylate), poly(methacrylamide), poly(acrylamide), and copolymers thereof, or poly(paracyclophane), or poly(dopamine).
  • the polymers can be obtained by polymerising the respective monomers using methods known in the art.
  • a pentafluorophenyl methacrylate or N-hydroxysuccinimidyl methacrylate monomer is polymerised such that the resulting polymer is attached to the surface of the substrate.
  • methacrylate monomer is polymerised such that the resulting polymer is attached to the surface of the substrate.
  • polymerisation methodologies known to those skilled in the art can be employed to polymerise the respective monomer and obtain a polymer layer attached to the substrate.
  • Such methods include free-radical polymerisation, living anionic polymerisation, and controlled free-radical polymerisation, including atom- transfer radical polymerisation (ATRP), reversible addition-fragmentation atom- transfer polymerisation (RAFT), and photo inverter polymerisation (PIMP)
  • ATRP atom- transfer radical polymerisation
  • RAFT reversible addition-fragmentation atom- transfer polymerisation
  • PIMP photo inverter polymerisation
  • the monomer is polymerised using plasma enhanced chemical vapour deposition (PECVD), initiated chemical vapour deposition (ICVD), or ultra violet chemical vapour deposition (UVCVD).
  • PECVD plasma enhanced chemical vapour deposition
  • the monomer is polymerised using plasma enhanced chemical vapour deposition (PECVD).
  • the polymerization of the monomer results in a functionalisation layer that coats the substrate, wherein the principal attractive form between the coating and the substrate is non-covalent.
  • living anionic polymerization can be used to polymerise a monomer to afford a functionalisation layer that coats the surface of the substrate in such a manner.
  • the functionalisation layer attached to the substrate has a thickness of at least 20 nm, such as at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, or at least 100nm, and up to 500nm, such as up to 400nm, up to 300nm, up to 200nm, or up to 150nm.
  • the functionalisation layer has a thickness of from 10- 500nm, such as from 20-400nm, from 20-300nm, from 30-200nm, from 30-150nm, from 40-100 nm, from 40-80 nm, or from 40-60nm.
  • the substrate is functionalised with a functionalisation layer using plasma polymerisation, for example PECVD.
  • PECVD plasma polymerisation
  • ppPFM poly(pentafluorophenyl methacrylate) layer
  • Such methods are known in the art, including by Duque et al. ( Biomacromolecules , 2010, 11, 2818-2823; Plasma Process. Polym. 2010, 7, 915-925).
  • Such methods benefit from not requiring initiating species, and allowing precise control of the density of the functional groups of the polymer on the substrate surface through careful control of the energy input provided during the deposition of the functionalisation layer.
  • Example 1 provides a summary of a procedure for the functionalisation of a substrate with poly(pentafluorophenyl methacrylate) using PECVD.
  • the polymerization of the monomer to form the functionalisation layer results in the formation of covalent linkages between the functionalisation layer and the substrate.
  • PPCVD, UVCVD, or ICVD can be used to polymerise a monomer, such as pentafluorophenyl methacrylate, wherein the method results in the formation of covalent linkages between the polymerised poly(pentafluorophenyl methacrylate) layer and the substrate.
  • the linkages that are formed through the reaction of radicals that are created on the surface of the substrate by the plasma, and the monomer can give increased structural integrity to the scaffold.
  • substrates for use in the second aspect of the invention include anything capable of being functionalised with a functionalisation layer for use in the invention.
  • substrates include polyacrylates, polyglycolic/polylactic acid polymers and copolymers, polyhydroxybutyrates, polyesters (such as Dacron(R)), expanded polytetrafluoroethylene (ePTFE), bioactive glass, ceramics (such as
  • the substrate comprises, consists essentially of, or consists of a bioceramic.
  • the substrate is a bioceramic, such as b-trichlorophosphate (b-TCP), a-trichlorophosphate (a-TCP), hydroxyapatite, dicalcium phosphate (DCP), dicalcium phosphate dehydrate (DCPD), tetracalcium phosphate (TTCP),
  • b-TCP b-trichlorophosphate
  • a-TCP a-trichlorophosphate
  • DCP dicalcium phosphate
  • DCPD dicalcium phosphate dehydrate
  • TTCP tetracalcium phosphate
  • the substrate is b-TCP, for example Cerasorb®.
  • the bioceramic is hydroxyapatite, for example bovine hydroxyapatite such as Bio-Oss®, or synthetic hydroxyapatite.
  • the bioceramic is biphasic calcium phosphate (BCP), comprising a mixture of b-TCP and hydroxyapatite, such BCPs including BONIT matrix sterile®, BONIT matrix unsterile®, or Straunmann® .
  • BCP biphasic calcium phosphate
  • the bioceramic is biphasic calcium phosphate (BCP) comprising a-TCP and hydroxyapatite.
  • BCP biphasic calcium phosphate
  • the substrate is selected from bioactive glasses containing silicon dioxide (S1O2), which can firmly attach to living tissue.
  • bioactive glasses include Bioglass(R) (American Biomaterials Corp., USA, 45% silica, 24% calcium oxide (CaO), 24.5% disodium oxide (Na 2 0), and 6% pyrophosphate (P2O5)), Consil(R) (Xeipon Ltd., UK),
  • PerioGlass(R) Block Drug Co., USA
  • Ceravital(R) E. Pfeil & H. Bromer, Germany
  • Corglaes(R) (Giltech Ltd., Ayr, UK) represents another family of bioactive glasses containing pyrophosphate rather than silicon dioxide as a network former. These glasses contain 42-49 mole % of P2O5, the remainder as 10-40 mole % as CaO and Na 2 0.
  • the method of the second aspect of the invention can be used to immobilise collagen hydrogels onto medical devices, such as those made from titanium, silicone, or hydroxyapatite, and taking a variety of forms, including pins, rods, screws, plates, or periodontal implants.
  • substrates that can be used with the method of the second aspect of the invention can be any solid phase that can be modified as discussed herein.
  • Examples include silica, aluminium, cellulose, chitosan, indium tin oxide (ITO), aluminium oxide (AI2O3), magnetite (Fe304), CuO x , hematite (c-Fe203), manganese spiral Ferrite (MnFe 2 0 4 ), magnesium hydroxide (Mg(OH)2), zinc oxide (ZnO), zirconium phosphonate, halloysite, montmorillonite, steel, sapphire, cadmium selenide (CdSe), cadmium sulphide (CdS), gallium Arsenide (GaAs), mica, diamond, plastic, polystyrene, poly(ethyleneterephthalate), polyaniline, poly(cyclopentadiene), polystyrene, poly(vinyl chloride), nylon, poly(divinylbenzene), poly(tetrafluoroethylene), poly(dimethylsiloxane), poly (methylmethacrylate), poly
  • a monolayer of tropocollagen is attached to the functionalisation layer. This is achieved by contacting the functionalised substrate with a first solution comprising tropocollagen. Without being bound by theory, it is thought that once the functionalisation layer has been contacted by the first solution comprising tropocollagen, the amine groups on the tropocollagen covalently react with the functionalisation layer. The substrate is incubated with the first solution comprising tropocollagen to form the tropocollagen monolayer. Once the monolayer has formed, the substrate may be washed to remove any material which is not covalently attached to the functionalisation layer (e.g.
  • the solutions for washing the tropocollagen monolayer include water and phosphate buffered saline.
  • the tropocollagen monolayer is washed with phosphate buffered saline.
  • the concentration of tropocollagen in the first solution is about 0.1 mg mL ⁇ 1 . In other embodiments of the invention, the concentration is greater than 0.1 mg mL 1 , for example 0.2 mg mL ⁇ 1 ,
  • the concentration is lower than 0.1 mg mL ⁇ 1 , for example 0.08 mg mL ⁇ 1 , 0.05 mg mL ⁇ 1 , 0.02 mg mL ⁇ 1 , 0.01 mg mL ⁇ 1 , or 0.001 mg mL ⁇ 1 .
  • the concentration of tropocollagen in the first solution is from 0.001 - 1 mg mL ⁇ 1 , such as from 0.01 - 0.5 mg mL ⁇ 1 , from 0.05 - 0.3 mg mL ⁇ 1 , from 0.08 - 0.2 mg mL ⁇ 1 , or from 0.09 - 0.15 mg mL ⁇ 1 .
  • the solvent is water.
  • Other solvents capable of dissolving collagen can be employed, such as acetic acid (or other organic acids such as methanoic, propanonic, butanoic, benzoic, lactic, citric, tartaric, or gallic acid) solutions in water, water, or acetone.
  • acetic acid or other organic acids such as methanoic, propanonic, butanoic, benzoic, lactic, citric, tartaric, or gallic acid
  • the solvent is phosphate buffered saline (PBS).
  • the organic acid may be at a concentration of at least 0.001 M, such as 0.005M, 0.01 M, 0.02M, 0.05M, 0.1 M, 0.2M, 0.3M, 0.4M, 0.5M, or at least 1 M.
  • the organic acid is at a concentration of at from 0.0001 - 5M, such as from 0.0001 - 2M, 0.0005 - 3M, 0.001 - 2.5M, 0.002 - 1 M, 0.005 - 0.8M, 0.008 - 0.5M, or from 0.01 - 0.3M.
  • the attachment of the tropocollagen monolayer can be performed under
  • a tropocollagen molecule comprises three left-handed procollagens that join to form a right-handed triple helical structure.
  • Procollagen is a three-dimensional stranded structure comprising the amino acids glycine and proline as its principle components.
  • the tropocollagen of the first solution comprising tropocollagen for use in the invention is Type I.
  • the tropocollagen is Type III.
  • the tropocollagen is a mixture of Type I and Type III, for example Fibricol®, available from Advanced Biohydrogel, USA, and comprising a high monomer content.
  • Fibricol® available from Advanced Biohydrogel, USA
  • By“monomer” of collagen it is referred to a collagen polypeptide chain (e.g. tropocollagen) that has not self-assembled to form higher-order structures, such as fibrils. It will be understood that the terms
  • tropocollagen and“collagen monomer” are used interchangeably.
  • “high monomer content” it is meant that at least 50% of the collagen in the solution is tropocollagen, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the collagen in the solution is tropocollagen.
  • the tropocollagen monolayer that is formed in step c) in the method of the second aspect of the invention comprises tropocollagen, i.e. collagen monomers that have not self-assembled into higher-order structures such as fibrils.
  • the tropocollagen monolayer can therefore act as an anchoring point for the attachment and self- assembly of further tropocollagen to form higher-order structures, such as collagen fibrils.
  • the collagen hydrogel layer can be formed by contacting the substrate comprising the
  • the tropocollagen monolayer directs the self-assembly of the tropocollagen in the second solution, facilitating the formation of higher-order fibrillar structures.
  • the tropocollagen monolayer covalently attached to the surface of the substrate comprises collagen monomers and is not already self-assembled into higher order structures.
  • the tropocollagen monolayer can therefore act as an anchoring point for the self-assembly of the tropocollagen in the second solution when it is contacted with the tropocollagen monolayer.
  • substrates can be functionalized with collagen hydrogels to afford functionalized substrates that have mechanical integrity between the substrate and the collagen hydrogel layers.
  • the mechanical integrity results from the nature of the interactions between the respective layers, particularly the covalent interactions between the functionalisation layer and the tropocollagen monolayer, the non-covalent interactions between the collagen monomers resulting from the self-assembly of tropocollagen, and the covalent interactions between the functionalisation layer and the substrate.
  • the method of the second aspect of the invention can provide a heterogeneous scaffold with
  • the tropocollagen used (e.g. Type I, Type II, Type III, or combination thereof) in the first and second solutions comprising tropocollagen is the same.
  • the tropocollagen used (e.g. Type I, Type II, Type III, or combination thereof) in the first and second solutions comprising tropocollagen is different.
  • the tropocollagen used in the first and second solutions comprising tropocollagen is Fibricol®, available from Advanced BioMatrix.
  • the density of the collagen hydrogel is determined by the density of the functional groups of the functionalisation layer.
  • the method further comprises the step of seeding the scaffold with mesenchymal stem cells.
  • the concentration of tropocollagen in the second solution is about 4.0 mg mL ⁇ 1 . In some embodiments of the second aspect of the invention, the concentration is greater than 4.0 mg mL ⁇ 1 , for example greater than 5.0 mg mL ⁇ 1 , greater than 6.0 mg mL ⁇ 1 , greater than 8.0 mg mL ⁇ 1 , greater than 10.0 mg mL ⁇ 1 , or greater than 15.0 mg mL ⁇ 1 . In some
  • the concentration is lower than 4.0 mg mL ⁇ 1 , for example lower than 2.0 mg mL ⁇ 1 , lower than 1 .0 mg mL ⁇ 1 , lower than 0.5 mg mL ⁇ 1 , lower than 0.2 mg mL ⁇ 1 , or lower than 0.1 mg mL ⁇ 1 .
  • the concentration of tropocollagen in the second solution is from 0.01 - 20 mg mL ⁇ 1 , such as from 0.1 - 10 mg mL ⁇ 1 , 0.5 - 9 mg mL ⁇ 1 , from 1 .0 - 8.0 mg mL ⁇ 1 , from 2.0 - 6.0 mg mL ⁇ 1 , or from 3.0 - 5.0 mg mL ⁇ 1 .
  • the solvent is water.
  • the solvent is phosphate buffered saline (PBS).
  • acetic acid solution in water or other organic acid solutions such as methanoic,
  • the organic acid may be at a concentration of at least 0.001 M, such as at least 0.005M, 0.01 M, 0.02M, 0.05M, 0.1 M, 0.2M, 0.3M, 0.4M, 0.5M, or at least 1 M.
  • the organic acid is at a concentration of at from 0.0001 -5M, such as from 0.0001 -2M, 0.0005-3M, 0.001 - 2.5M, 0.002-1 M, 0.005-0.8M, 0.008-0.5M, or from 0.01 -0.3M.
  • the attachment of the tropocollagen monolayer can be performed under
  • physiological conditions it is meant any conditions that occur where collagen is found in nature. Such conditions include a temperature of from 20 to 40 °C, such as 22 to 40 °C, 25 to 40 °C, 30 to 40 °C, 32 to 38 °C, or 35 to 37 °C, and at a pH of from 6-8.
  • the attachment of the tropocollagen monolayer can be performed using the method of the invention at about pH 7 and about 37 °C.
  • the substrate In some embodiments of the second aspect of the invention, the substrate
  • tropocollagen monolayer is contacted with the second solution comprising tropocollagen for 40 minutes to allow for self-assembly of the collagen hydrogel.
  • the tropocollagen monolayer is contacted with the second solution comprising tropocollagen for at least 40 minutes, such as at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, or at least 48 hours.
  • hydroxyapatite may be conducted under physiological conditions, for example at about 37 °C and about pH 7.
  • contacting is conducted at a temperature greater than about 37 °C, for example greater than 38 °C, greater than 39 °C, greater than 40 °C, greater than 41 °C, greater than 45 °C, or greater than 50 °C.
  • contacting is conducted at a temperature lower than 37 °C, for example lower than 36 °C, lower than 35 °C, lower than 34 °C, lower than 32 °C, lower than 30 °C, lower than 28 °C, lower than 26 °C, or lower than 24 °C.
  • a temperature lower than 37 °C for example lower than 36 °C, lower than 35 °C, lower than 34 °C, lower than 32 °C, lower than 30 °C, lower than 28 °C, lower than 26 °C, or lower than 24 °C.
  • contacting is conducted at a temperature from 24 °C - 50 °C, such as from 26 °C - 45 °C, from 28 °C - 41 °C, from 30 °C - 39 °C, from 32 °C - 38 °C, from 35 °C - 38 °C, or from 36 °C - 37 °C,
  • the second solution comprising collagen may contain additional components.
  • Said additional components include polysaccharides, such as glycosaminoglycans
  • GAGs are long unbranched polysaccharides consisting of a repeating disaccharide unit, wherein one of the units is an amino sugar.
  • GAGs for use in the second aspect of the invention include heparin, heparin sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and hyaluronic acid.
  • Other polysaccharides that can be incorporated into the second solution comprising collagen include dextran, heparan, alginate, agarose, carrageenan, amylopectin, amylose, glycogen, starch, cellulose, chitin, chitosan and various sulfated
  • the GAG is added to the second solution comprising collagen to assist with the self-assembly of the collagen hydrogel (for example, chondroitin sulfate, hyaluronic acid, and combinations thereof).
  • the second solution comprising collagen further comprises chondroitin sulfate, hyaluronic acid, and combinations thereof.
  • chondroitin sulfate, hyaluronic acid helps recreates the cartilage extracellular matrix in the scaffold of the invention, as they constitute two of its main components.
  • chondroitin sulfate and hyaluronic acid assist in the self-assembly of the collagen hydrogel and promote the differentiation of mesenchymal stems cells into either osteoblasts or chondrocytes.
  • Additional components that may be incorporated into the second solution comprising collagen include DNA, RNA, low molecular weight nucleic acids, proteins, polypeptides, peptides, hormones, growth factors, cytokines, metabolites and cells.
  • the collagen hydrogel of the second layer of the scaffold of the invention forms from self-assembly to the collagen monomers into higher order structures, including fibrils, and therefore does not require crosslinking in order to stabilize the hydrogel and/or to impart structural integrity to the scaffold.
  • the collagen hydrogel can be optionally cross-linked.
  • the method further comprises the step of crosslinking the collagen hydrogel, e.g., through the addition of a suitable crosslinking agent.
  • Suitable crosslinking methods include agents include dehydrothermal (DHT) crosslinking and addition of a chemical cross-linking agent.
  • Suitable chemical crosslinking agents include are known to those skilled in the art and include 1 - ethyl- 3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) or glutaraldehyde.
  • the collagen hydrogel is cross-linked using DHT and EDAC cross-linkers.
  • Figure 1 is a schematic diagram of stainless steel vertical plasma reactor and its electrical components: (1 ) monomer feed, (2) gases feed, (3) cylindrical chamber, (4) holder sample, (5) pirani gauge, (6) matching box and electrical system, (7) cold trap, and (8) chemical trap.
  • Figure 8. XRD Spectra of Cerasorb® samples. Cerasorb® 50-150 pm (B) Cerasorb 150-500 pm (C) Cerasorb 1000-2000 pm.
  • Bonitmatrix® sterile Figure 10. XRD Spectra of (A) Straumann® samples (B) BioOss® samples and (C) Hydroxyapatite sample.
  • Example I Procedure to obtain a plasma polymerized PFM layer onto a desired substrate by plasma enhanced chemical vapor deposition
  • Example 1 relates to a method of functionalizing the surface of a substrate with a functionalisation layer of plasma polymerized poly(pentafluorophenyl methacrylate) (ppPFM) by polymerizing a pentafluorophenyl methacrylate (PFM) via plasma polymerization in the presence of a substrate.
  • ppPFM plasma polymerized poly(pentafluorophenyl methacrylate)
  • the reactor consists of a stainless steel chamber (diameter, 25.5 cm; length, 41.6 cm) vertical plate reactor.
  • the ground electrode is the reactor chamber, and the radio frequency (RF) electrode is an aluminum plate, which is used to hold the samples for polymerization. Additionally, the RF electrode is connected to a RF pulse generator (13.56 MHz) via a matching box. Gases and monomers are supplied via a standard manifold where gas fluxes can be adjusted with a tree of needle valves.
  • FIG. 1 depicts the reactor equipment used to functionalize the substrates. Briefly, the samples were placed on the aluminum plate placed in the middle of the reactor chamber. The monomer, PFM (Apollo Scientific Ltd., Stockport, U.K.), is introduced inside the reactor at nearly constant pressure around 0.02-0.04 mbar.
  • Example II Attachment of a tropocollagen monolayer onto a PFM functionalised substrate
  • Example II relates to a method of attaching a tropocollagen monolayer to the surface of a substrate functionalized with a functionalisation layer of ppPFM.
  • the pp-PFM-functionalised substrate is placed in a vial and an excess of a tropocollagen solution (Fibricol®, Advanced BioMatrix) (0.1 mg/ml in milliQ water) is added to ensure that the functionalized surface of the substrate is covered by the solution.
  • the substrate is then incubated overnight at 37 °C with agitation (using an orbital shaker) to obtain the tropocollagen monolayer on the polymeric layer.
  • the substrate is then removed from the solution and rinsed with phosphate buffered saline (PBS) to remove any non-covalently attached collagen molecules and the displaced pentafluorophenol groups.
  • PBS phosphate buffered saline
  • Example III Collagen hydrogel self-assembly onto a collagen-ppPFM-functionalised substrate
  • Example III related to a method of forming a collagen hydrogel on a substrate functionalized with a functionalisation layer and a monolayer of tropocollagen covalently attached to the tropocollagen monolayer.
  • a first aqueous solution is prepared comprising Type I tropocollagen (Fibricol®, Advanced Biohydrogel) (4 mg mL 1 ) and 0.01 N HCI.
  • the first solution comprises additional components as outlined in Table 1.
  • Table 1 Protocol for 3D hydrogel production from Fibricol®
  • a second aqueous solution comprising hyaluronic acid (0.15 mg mL 1 ) and chondroitin sulfate (0.15 mg mL ⁇ 1 ) is prepared by dissolving hyaluronic acid (Bioiberica, S.A.U) and chondroitin sulfate (Bioiberica, S.A.U) in water. The solution is then filtered through a 0.22 pm filter.
  • Equal volume s of the two solutions are mixed together and the resulting solution added to the substrate functionalized with the functionalisation layer and the tropocollagen monolayer.
  • the hydrogel forming solution and substrate are then incubated at about 37 °C for about 45 minutes to allow the self-assembly of hydrogels.
  • each of the bioceramic samples were compacted into a disk and immersed at 37 °C in simulated body fluid (SBF).
  • composition of SBF is provided below (Table 3):
  • composition of SBF effectively mimics the salt concentration present in blood plasma (Table 4):
  • Table 4 Comparison of the standardized ions concentration between blood plasma and SBF.
  • the bioceramics for use in the invention can recrystallize from solution after initial dissolution into the SBF phase.
  • the data demonstrate the dissolution and recrystallization kinetics of several bioceramics for use with the invention.
  • the data further demonstrate how incorporation of said bioceramics into a scaffold of the invention will facilitate the formation of a continuous bioceramic gradient across the scaffold, once the scaffold is inserted into a physiological fluid.
  • Example V Probing the effect of covalently linking the collagen and bioceramic layers
  • QCM-D technology can be used to study protein adsorption onto different surfaces in order to evaluate the ability of a determinate material to promote protein adsorption. This technique was used to evaluate collagen adsorption onto different substrates and investigate the influence of different parameters over the adsorption process.
  • QCM- D technique changes in frequency (f) and dissipation (D) of an oscillating sensor are measure, as mass adsorbs onto the sensor, a frequency shift can be observed and the dissipation parameter provides information about the viscoelastic properties of the adsorbed layer.
  • Softer layers lead to an increase of the oscillation damping, which is transed as an increase of dissipation.
  • Figure 3 illustrates the behavioral differences between a rigid and a soft layer.
  • a non-treated stainless-steel sensor ( Figure 6A) has been taken as reference to evaluate the collagen adsorption that takes place onto modified substrates. Adsorption takes place once the protein solution is flown through the QCM-D chamber, as a decrease in sensor’s oscillation frequency can be appreciated. The subsequent PBS wash leads to the detachment of a little amount of protein, corresponding to those molecules that are not interacting with the substrate. Flowever, the final water wash causes an increase of frequency which almost reaches the baseline value. The flow of water through the QCM-D chamber causes a change in ionic strength and the detachment of a great amount of protein molecules, as their interaction with stainless steel is purely electrostatic 52 .
  • the mineral surface of the sensor could lead to ionic interactions with the collagen molecules, which will also enhance the whole adsorption process.
  • a hydroxyapatite substrate compared to stainless steel, would promote collagen adsorption under the studied conditions.
  • the PBS wash does not alter the amount of adsorbed molecules, reflecting the existence of specific electrostatic interactions between the protein molecules themselves and with the substrate.
  • the final water wash results in a significant increase of the frequency signal due to the detachment of a fraction of protein molecules.
  • the interface between the bone-like platform and the collagen hydrogel can be disrupted in response to ionic strength fluctuations in the surrounding medium.
  • scaffolds comprising a bioceramic and collagen, wherein the bioceramic is not covalently attached to the collagen hydrogel, have a risk of hydrogel leakage from the bioceramic platform once placed into the body.
  • PFM layers can be used as active functionalisation layers for different biomaterials. PFM is reactive towards collagen-based hydrogels and will form covalent bonds with both the bone-like platform and the collagen membrane cover.
  • QCM-D technique to evaluate collagen adsorption onto a PFM- functionalised stainless-steel sensor ( Figure 6C). As PFM contains active sites, a high mass adsorption per surface unit is expected.
  • protein adsorption should be more homogeneous, as the active points of the polymeric layer throughout the sensor surface will act as nucleation points.
  • collagen will be covalently attached to the sensor surface, the ionic strength change caused by the final water wash does not cause protein detachment.
  • the amount of adsorbed mass is significantly higher than that observed for ether the HA sensor or the non-treated stainless sensor according to the Sauerbrey relation.
  • the PFM layer causes a frequency shift around 300 Hz for the seventh overtone which represents more than twice the value obtained for the hydroxyapatite sensor.
  • the PBS wash does not alter the amount of adsorbed molecules on the PFM functionalised sensor.
  • the presence of a covalent interaction between the PFM layer and the collagen molecules is confirmed by the observation that the final water wash produces a lower frequency shift compared with both the non-treated stainless-steel sensor and the HA sensor.
  • the collagen molecules are retained on the surface of the PFM layer. Only a small fraction of the protein molecules were electrostatically interacting with the PFM layer sensor and were removed by the change of ionic strength.
  • a method for immobilising a collagen hydrogel on a substrate comprising the steps of: a. Functionalising a surface of the substrate with a functionalisation layer; b. Attaching a monolayer of tropocollagen to the functionalisation layer by contacting the functionalisation layer with a first solution comprising tropocollagen;
  • step a) comprises the step of polymerising a monomer such that the resulting polymer is attached to the surface of the substrate.
  • step b) comprises the step of covalently attaching the tropocollagen to the functionalisation layer to form the tropocollagen monolayer.
  • the substrate is a bioceramic selected from the group consisting of b-TCP, bovine hydroxyapatite, synthetic hydroxyapatite, biphasic calcium phosphate (BCP) comprising a mixture of b-TCP and hydroxyapatite, biphasic calcium phosphate (BCP) comprising a mixture of a-TCP and hydroxyapatite, or a combination thereof.
  • BCP biphasic calcium phosphate
  • BCP biphasic calcium phosphate
  • a scaffold comprising a first layer and a second layer adjacent to the first layer, wherein the first layer comprises a bioceramic and the second layer comprises a collagen hydrogel, wherein the bioceramic has a mean crystalline domain size of 1 to 275 nm.
  • the scaffold of embodiments 7 to 10 wherein the second layer comprises less than 16% by weight of a bioceramic.
  • a scaffold comprising a first layer, a second layer, and a functionalisation layer between the first layer and the second layer, wherein the first layer comprises a bioceramic and the second layer comprises a collagen hydrogel, wherein the functionalisation layer is attached to a surface of the first layer, and wherein the second layer is covalently attached to the functionlisation layer.

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Abstract

L'invention concerne un procédé d'immobilisation d'un hydrogel de collagène sur un substrat, par exemple un dispositif médical. L'invention concerne également un échafaudage destiné à être utilisé dans le traitement de l'arthrose, l'échafaudage comprenant une couche de biocéramique et une couche d'hydrogel de collagène, l'échafaudage formant un gradient de biocéramique continu lorsqu'il est placé dans des conditions physiologiques. L'invention concerne également un procédé de fabrication correspondant. Enfin, l'invention concerne un échafaudage comprenant une couche de biocéramique, une couche d'hydrogel de collagène et une couche de fonctionnalisation fixée à la fois au substrat et aux couches d'hydrogel de collagène. L'invention concerne également un procédé de fabrication correspondant.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115304775A (zh) * 2022-06-27 2022-11-08 天津大学 一种改性透明质酸修饰的胶原蛋白基质的制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232198A1 (en) 2002-02-21 2003-12-18 Encelle, Inc. Immobilized bioactive hydrogel matrices as surface coatings
WO2006092718A2 (fr) 2005-03-04 2006-09-08 Fin-Ceramica Faenza S.P.A. Substitut cartilagineux et osteochondrial comprenant une structure multicouche et son utilisation
WO2010084481A1 (fr) 2009-01-23 2010-07-29 Royal College Of Surgeons In Ireland Échafaudage stratifié adapté à la réparation ostéochondrale
US8110242B2 (en) 2006-03-24 2012-02-07 Zimmer, Inc. Methods of preparing hydrogel coatings

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004071546A1 (fr) * 2003-02-14 2004-08-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procede de production d'une couche de collagene

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232198A1 (en) 2002-02-21 2003-12-18 Encelle, Inc. Immobilized bioactive hydrogel matrices as surface coatings
WO2006092718A2 (fr) 2005-03-04 2006-09-08 Fin-Ceramica Faenza S.P.A. Substitut cartilagineux et osteochondrial comprenant une structure multicouche et son utilisation
US8110242B2 (en) 2006-03-24 2012-02-07 Zimmer, Inc. Methods of preparing hydrogel coatings
WO2010084481A1 (fr) 2009-01-23 2010-07-29 Royal College Of Surgeons In Ireland Échafaudage stratifié adapté à la réparation ostéochondrale

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DUQUE ET AL., BIOMACROMOLECULES, vol. 11, 2010, pages 2818 - 2823
PLASMA PROCESS. POLYM., vol. 7, 2010, pages 915 - 925

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115304775A (zh) * 2022-06-27 2022-11-08 天津大学 一种改性透明质酸修饰的胶原蛋白基质的制备方法

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