Preparation method of keratin-based composite bone cartilage

Abstract

The invention relates to the field of tissue engineering biological materials, in particular to a preparation method and application of keratin-based composite osteochondral, and according to the preparation method, a cartilage layer 1, a calcified cartilage layer 2 and a subchondral bone layer 3 are combined into the composite osteochondral. The cartilage layer 1 is keratin-based nanofiber aerogel prepared through an electrostatic spinning method and a foaming technology, the calcified cartilage layer 2 is a keratin nanofiber membrane which is prepared through the electrostatic spinning method and is rich in calcium and phosphorus ions, and the subchondral bone layer 3 is a modified keratin-based hydrogel material prepared through a 3D printing technology. The three layers of structures are combined in sequence to form the keratin-based composite osteochondral bone. The prepared keratin-based scaffold has good biocompatibility, is beneficial to adhesion and proliferation of bone cells, and has the function of promoting repair and regeneration of bone cartilage.

Description

Technical Field

[0001] The present invention relates to the field of tissue engineering biomaterials, and in particular to a composite osteocartilage and a preparation method thereof. Background Art

[0002] Osteochondral injuries caused by orthopedic trauma, disease, and aging are very common orthopedic clinical diseases, and they often occur in the knee, hip, and ankle joints. Osteochondral cartilage is composed of three parts: calcified cartilage, articular cartilage, and subchondral bone. Among them, subchondral bone has a certain self-healing ability, while articular cartilage has a very limited self-regeneration ability due to the lack of blood vessels, lymphatic vessels, and nerves. For osteochondral injuries that are more serious and require surgical treatment, there are currently available treatments such as microfracture, osteochondral transplantation, autologous chondrocyte transplantation / matrix-induced autologous chondrocyte transplantation. These treatments can repair the structure and function of osteochondral cartilage to a certain extent, but they all have limitations in material acquisition.

[0003] The construction of tissue engineering scaffolds with specific spatial structures, using natural or synthetic materials with excellent biocompatibility, offers broad potential for the design and fabrication of biomimetic structures for osteochondral defect repair. Three-phase scaffolds with biomimetic structures can more accurately mimic the physiological structure of osteochondral defects. Keratin, as a natural polymer material, is not only abundant and inexpensive, but also exhibits excellent biocompatibility, biodegradability, and low immune rejection. Keratin and its products have broad development potential and enormous application value in fields such as biomedical materials and sustained-release drug delivery. Natural keratin fibers are widely found in human and animal skin and skin derivatives, but most are directly discarded or incinerated, resulting in resource waste and environmental pollution. Therefore, the development and utilization of discarded natural keratin fibers not only enables the reuse of waste protein resources, but also reduces environmental pollution and has significant economic and social benefits.

[0004] Electrospun materials made from keratin have remarkable structural similarity, scale compatibility, and cellular responsiveness to the lattice structure of the extracellular matrix, leading to widespread interest in nanofiber biomedical materials. However, compared to cartilage tissue, the 3D spatial network structure of conventional nanofiber membranes still needs to be improved. 3D printing technology can produce keratin-based biomaterials with excellent structural controllability, but keratin-based 3D printing materials still have the disadvantage of poor plasticity, which hinders the widespread application of three-phase keratin composite osteochondral materials. Summary of the Invention

[0005] The object of the present invention is to provide a keratin PEO composite bio-nanofiber membrane and a preparation method thereof, so as to solve at least one of the above-mentioned technical problems existing in the prior art.

[0006] The present invention aims to provide a method for preparing a keratin-based composite osteochondral scaffold that promotes osteochondral repair, thereby resolving at least one of the aforementioned technical problems in the prior art. To address the aforementioned technical problems, the present invention provides a method for preparing a keratin-based three-phase structured osteochondral scaffold, comprising the following steps:

[0007] S1. Keratin was extracted from discarded natural hair fibers using a reduction method, and a keratin-based nanofiber membrane was obtained by electrospinning. The keratin-based nanofibers were then subjected to secondary cross-linking and foaming to prepare a cartilage layer.

[0008] S2. keratin was blended with modified nanohydroxyapatite to obtain a keratin / nanohydroxyapatite blended nanofiber membrane by electrospinning, and the nanofiber membrane was then subjected to secondary cross-linking to prepare a calcified cartilage layer;

[0009] S3. Keratin was photocrosslinked and then mixed with photocrosslinked hyaluronic acid, a thickener, and a photoinitiator to prepare a bioink, and the subchondral bone layer was prepared by 3D printing technology;

[0010] S4. The cartilage layer, calcified cartilage layer and subchondral bone layer are combined into a composite osteochondral scaffold by applying biological glue.

[0011] The present invention combines a cartilage layer with a 3D fluffy nanofiber grid structure, a nanofiber calcified cartilage layer with a dense structure and rich in calcium and phosphorus ions, and a subchondral bone layer with a 3D grid structure to form a three-phase osteochondral repair material with a bionic structure. The osteochondral repair material has a bionic structure that matches natural osteocartilage in terms of material structure and composition, can better promote the repair of osteochondral defects, and has broad application prospects in the field of bone repair materials.

[0012] Furthermore, in step S1, the steps of extracting keratin from discarded natural hair fibers by using a reduction method are as follows:

[0013] S11. The natural keratin fibers were opened and cut into 5-10 mm long fiber segments after impurity removal, and then washed with petroleum ether solvent for about 40 min to remove impurities, and then washed with distilled water 4-5 times and dried at room temperature.

[0014] S12. The natural keratin fiber is immersed in a dissolving solution containing a reducing agent, a protein denaturant and a surfactant, wherein the solid-liquid ratio of the dissolving solution is 6-12 g / 250 ml; stirred at a temperature of 70-95 ° C for 4-6 hours at a stirrer speed of 160-220 r / min;

[0015] The reducing agent is sodium metabisulfite, dithiothreitol, thioglycolic acid, etc., and the mass percentage concentration of the reducing agent in the dissolving solution is 5%-7%;

[0016] The keratin denaturant is urea, thiourea or lithium bromide, and the mass percentage concentration of the protein denaturant in the dissolving solution is 40-45%;

[0017] The surfactant is sodium lauryl sulfate, and the mass percentage concentration of the surfactant in the dissolving solution is 1-5%;

[0018] S13. The mixture obtained after stirring was filtered through a 100-150 mesh sieve to remove undissolved natural keratin fibers. The filtered keratin salt solution was dialyzed at room temperature using a dialysis bag with a molecular weight cutoff of 8000-14000Da to remove salts and small molecules from the solution. The dialysis time was 36-72h, and distilled water was replaced every 12h.

[0019] S14. The dialyzed keratin solution was centrifuged to remove the precipitate in the dialysate at a centrifugal speed of 5000-6000 rpm for 0.25-0.5 h, and the supernatant was collected;

[0020] S15. The obtained supernatant is concentrated to obtain a keratin concentrated solution having a keratin mass percentage concentration of 10-14%;

[0021] S16. Freeze-dry the concentrated keratin solution for 24-36 hours to obtain keratin powder.

[0022] Furthermore, the natural hair fiber is wool, rabbit hair, human hair, pig hair, cow hair or camel hair.

[0023] Furthermore, the electrospinning method is to prepare nanofibers by passing the spinning solution through a needle-type, bubble-type or disc-type electrospinning device, wherein:

[0024] In the electrospinning method described in step S1, the spinning solution is an aqueous solution of keratin and polyethylene oxide (mass ratio 9:1), with a total mass fraction of 13-17%, and the mass fraction of the crosslinking agent ethylene glycol diglycidyl ether is 5-7% of the solute mass.

[0025] In the electrospinning method in step S2, the electrospinning solution is an aqueous solution of keratin / polyethylene oxide (mass ratio 9:1) with a modified nano-hydroxyapatite addition amount of 5-20%, the mass fraction of keratin and polyethylene oxide in the solution is 13-17%, and the mass fraction of the crosslinker ethylene glycol diglycidyl ether is 5-7% of the solute mass.

[0026] Furthermore, the secondary crosslinking can be oxygen oxidation crosslinking, ultraviolet radiation crosslinking or thermal crosslinking.

[0027] Furthermore, in step S1, the foaming method is to immerse the secondary cross-linked keratin-based nanofiber membrane in a 0.1-0.5M sodium borohydride solution for 15-60 minutes; repeatedly wash it with deionized water and freeze-dry it to obtain the keratin-based nanofiber aerogel.

[0028] Furthermore, in step S2, the method for preparing the sodium hexametaphosphate modified nano-hydroxyapatite powder includes:

[0029] S21. The nano-hydroxyapatite was placed in water and stirred at a speed of 50-100 r / min to disperse the nano-hydroxyapatite to obtain a nano-hydroxyapatite dispersion having a concentration of 10% by mass of the nano-hydroxyapatite, and then a sodium hexametaphosphate solution having a concentration of 10% by mass was prepared;

[0030] S22. Sodium hexametaphosphate solution was added dropwise to the nano-hydroxyapatite solution under constant stirring at 50-100 r / min speed to allow sufficient reaction, and the volume ratio of sodium hexametaphosphate solution to hydroxyapatite dispersion was 12.5:1-7.5:1;

[0031] After the sodium hexametaphosphate solution is added dropwise, stirring is continued for 1 hour to obtain a mixed solution;

[0032] S23. The mixed solution after the reaction was ultrasonically treated for 10 min to uniformly disperse the mixed solution;

[0033] S24. The hydroxyapatite was extracted by adding anhydrous ethanol of the same volume as the mixed solution, and the modified nano-hydroxyapatite was collected by centrifugation at 8000 r / min for 20 min, and then the modified nano-hydroxyapatite was repeatedly washed with deionized water 4-5 times to remove unreacted ions, and centrifuged at 8000 r / min to obtain wet modified nano-hydroxyapatite;

[0034] S25. Freeze-dry the wet modified nano-hydroxyapatite for 24-36 hours to obtain modified nano-hydroxyapatite powder.

[0035] Furthermore, in step S3, the modification method for photocrosslinking keratin is as follows: preparing 50 ml of a keratin solution with a mass percentage concentration of 10-14%, adding 1-6 ml of glycidyl methacrylate dropwise under heating (60° C.) and stirring for 3 hours, and regulating the reaction pH value at about 8.0 with a NaOH solution during the reaction. After the reaction is completed, the resulting solution is dialyzed and freeze-dried to obtain modified keratin powder.

[0036] Furthermore, in step S3, the modification method of the photo-crosslinked modified hyaluronic acid is:

[0037] S31. Prepare 100 ml of hyaluronic acid solution at a concentration of 1%, add 1-3 ml of methacrylic anhydride dropwise with stirring on ice for 24 hours, and control the reaction pH at around 8.0 using NaOH solution during the reaction.

[0038] S32 After the reaction, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed for 36-72h, changing the water every 8h;

[0039] S33. After dialysis, the pH of the resulting solution was adjusted to about 7.4 with NaHCO3 solution;

[0040] S34. Freeze the solution in a -20°C refrigerator overnight, and then freeze-dry for 24-48 hours to obtain a white flocculent product, namely, modified hyaluronic acid.

[0041] Furthermore, in step S3, the bio-ink uses water as a solvent, the mass fraction of the modified keratin in the ink is 13-17%, the mass fraction of the modified hyaluronic acid is 2-4%, the mass fraction of the thickener is 5-10%, and the mass fraction of the photoinitiator is 0.25-1%;

[0042] The thickener is nanoclay lithium magnesium silicate;

[0043] The photoinitiator is I 2959 or LAP.

[0044] By adopting the above technical solution, the present invention has the following beneficial effects:

[0045] The present invention provides a method for preparing keratin-based composite osteocartilage. In view of the different structural and compositional characteristics of the cartilage layer, calcified cartilage layer and subchondral bone layer in osteocartilage and the good biocompatibility and bioactivity of keratin, a keratin-based nanofiber aerogel prepared by electrospinning and foaming technology is used as the bionic cartilage layer, a nanofiber membrane material rich in calcium and phosphorus ions prepared by electrospinning is used as the bionic calcified cartilage layer, and a hydrogel material prepared by 3D printing technology is used as the bionic subchondral bone layer. By assembling and constructing a three-phase bionic osteocartilage consisting of a cartilage layer, a calcified cartilage layer and a subchondral bone layer from top to bottom, bionic structural simulation of each hierarchical structure of osteocartilage is achieved, and the method has good biocompatibility and tissue repair performance, and has broader application prospects in the field of osteocartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0047] Figure 1 A schematic diagram of a keratin-based composite osteocartilage structure provided in an embodiment of the present invention;

[0048] Figure 2 The surface and cross-sectional electron micrographs of a keratin-based composite osteochondral cartilage layer provided in an embodiment of the present invention are shown. The foaming time is 30 min and the concentration of the NaBH4 solution is 0.3 M.

[0049] Figure 3 This is a graph of live / dead cells detected by Calcein-AM / PI staining solution after different cell culture days of a keratin-based composite osteochondral cartilage layer provided in an embodiment of the present invention;

[0050] Figure 4 This is an infrared spectrum of the cartilage layer of a keratin-based composite osteochondral cartilage and its components provided in an embodiment of the present invention, (A) 4000-400 cm -1 , (B) 1800-400cm -1 , (a) wool keratin nanofiber membrane, (b) human hair keratin / HA / CS nanofiber membrane (cartilage layer), (c) hyaluronic acid (HA), (d) chondroitin sulfate (CS);

[0051] Figure 5 Scanning electron micrographs and fiber diameter distribution diagrams of wool keratin / PEO / nHA nanofiber membranes with different nHA contents in the calcified cartilage layer of a keratin-based composite osteochondral provided in an embodiment of the present invention, (a) 0%, (b) 5%, (c) 10%, (d) 15%, (e) 20%;

[0052] Figure 6 XRD patterns of wool keratin / PEO / nHA nanofiber membranes with different nHA contents in the calcified cartilage layer of a keratin-based composite osteochondral provided in an embodiment of the present invention, (a) 0%, (b) 5%, (c) 10%, (d) 15%, (e) 20%;

[0053] Figure 7ARS staining appearance of wool keratin / PEO / nHA nanofiber membranes with different nHA contents in the calcified cartilage layer of a keratin-based composite osteochondral provided in an embodiment of the present invention after different mineralization times, (a) 0%, (b) 5%, (c) 10%, (d) 15%, (e) 20%;

[0054] Figure 8 A scanning electron micrograph of a wool keratin / PEO / nHA nanofiber membrane having a calcified cartilage layer of a keratin-based composite osteochondral structure with an nHA content of 15% after 28 days of mineralization provided in an embodiment of the present invention;

[0055] Figure 9 Calcein-AM / PI staining assay of live / dead cells after different cell culture days of a wool keratin / PEO / nHA nanofiber membrane with 0% and 15% nHA content in the calcified cartilage layer of a keratin-based composite osteochondral provided in an embodiment of the present invention;

[0056] Figure 10 Scanning electron micrograph of the subchondral bone layer of 3D-printed keratin-based osteocartilage provided in an embodiment of the present invention;

[0057] Figure 11 A graph of live / dead cells detected by Calcein-AM / PI staining of the subchondral bone layer of a keratin-based composite osteochondral provided in an embodiment of the present invention after different cell culture days;

[0058] Figure 12 Micro-CT images of a keratin-based composite osteochondral material provided in an embodiment of the present invention as an osteochondral repair filling material implanted into the osteochondral defect of a rat joint at different times (Control is the group without added material). DETAILED DESCRIPTION

[0059] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0060] The present invention will be further explained below with reference to specific embodiments.

[0061] Example 1

[0062] Reference Figure 1As shown, the keratin-based composite osteocartilage 4 provided in this embodiment is composed of a cartilage layer 1, a calcified cartilage layer 2, and a subchondral bone layer 3. The cartilage layer 1 is prepared by electrospinning keratin nanofiber membranes obtained by extracting keratin from discarded natural hair fibers using a reduction method, followed by secondary cross-linking and foaming of the keratin nanofibers. The calcified cartilage layer 2 is prepared by electrospinning keratin / nanohydroxyapatite blended nanofiber membranes obtained by blending keratin and modified nanohydroxyapatite, followed by secondary cross-linking of the nanofiber membranes. The subchondral bone layer 3 is prepared by 3D printing using a bio-ink prepared by blending photo-crosslinked modified keratin with photo-crosslinked modified hyaluronic acid, a thickener, and a photoinitiator.

[0063] Wherein, keratin is prepared by the following steps:

[0064] ①Open and remove impurities from the wool fibers, then cut them into 5mm long fiber segments. Then wash them with petroleum ether for about 40 minutes to remove impurities. Repeat the washing with distilled water for 4 times and let them dry naturally at room temperature.

[0065] ② Immerse the cleaned and dried wool fibers in a solution containing 5% sodium metabisulfite, 40% urea, and 1% sodium lauryl sulfate, with a solid-to-liquid ratio of 6g / 250ml; stir at 70°C for 6 hours at a stirrer speed of 160r / min;

[0066] ③ Filter the mixture obtained after stirring through a 100-mesh sieve to remove undissolved wool fibers. Dialyze the filtered keratin salt solution using a dialysis bag with a molecular weight cutoff of 8000-14000Da at room temperature to remove salt and small molecules in the solution. The dialysis time is 36 hours, and the distilled water is replaced every 12 hours.

[0067] ④ Centrifuge the dialyzed keratin solution to remove the precipitate in the dialysate at a speed of 5000 pm for 0.25 h, and then collect the supernatant;

[0068] ⑤ Concentrating the obtained supernatant to obtain a keratin concentrated solution with a keratin mass percentage concentration of 10%;

[0069] ⑥ Freeze-dry the concentrated keratin solution for 24 hours to obtain keratin powder.

[0070] The cartilage layer 1 is prepared by the following steps:

[0071] ① Prepare an aqueous solution of wool keratin and polyethylene oxide (mass ratio 9:1) with a 3% addition of chondroitin sulfate / hyaluronic acid, with a total mass fraction of 13% and a mass fraction of ethylene glycol diglycidyl ether as the crosslinker of 5% of the solute mass;

[0072] ② Preparation of wool keratin-based nanofibers by needle electrospinning;

[0073] ③ Placing the obtained wool keratin-based nanofibers in oxygen for 7 days for secondary crosslinking to obtain water-insoluble nanofibers;

[0074] ④ Immerse the secondary cross-linked wool keratin-based nanofiber membrane in a 0.3M sodium borohydride solution for 30 minutes; wash it repeatedly with deionized water and freeze-dry it to obtain a wool keratin-based nanofiber cartilage layer. The surface and cross-sectional scanning electron microscopy images of the obtained wool keratin-based nanofiber cartilage layer are shown in Figure 4. Figure 2 As shown, the cartilage layer was cultured for different days and the live / dead cells were detected by Calcein-AM / PI staining. Figure 3 As shown, the infrared spectra of the cartilage layer and its components of human hair keratin-based composite osteochondral are as follows Figure 4 As shown.

[0075] The calcified cartilage layer 2 is prepared by the following steps:

[0076] ① Modification of nano-hydroxyapatite, the steps are as follows:

[0077] a. The nano-hydroxyapatite was placed in water and stirred at a speed of 50r / min to disperse the nano-hydroxyapatite to obtain a nano-hydroxyapatite dispersion having a concentration of 10% by mass of the nano-hydroxyapatite, and then a sodium hexametaphosphate solution having a concentration of 10% by mass was configured;

[0078] b. sodium hexametaphosphate solution was added dropwise to the nano-hydroxyapatite solution under constant stirring at a speed of 50 r / min to allow for sufficient reaction, and the volume ratio of sodium hexametaphosphate solution to hydroxyapatite dispersion was 12.5: 1; stirring was continued for 1 hour after completion of the dropwise addition of the sodium hexametaphosphate solution to obtain a mixed solution;

[0079] c. The mixed solution after the reaction was ultrasonically treated for 10 min to uniformly disperse the mixed solution;

[0080] d. The hydroxyapatite was extracted by adding the same volume of anhydrous ethanol as the mixed solution, and the modified nano-hydroxyapatite was collected by centrifugation at 8000 r / min for 20 min, and then the modified nano-hydroxyapatite was repeatedly washed with deionized water four times to remove unreacted ions, and the modified nano-hydroxyapatite was centrifuged at 8000 r / min to obtain a wet modified nano-hydroxyapatite;

[0081] e. freeze-drying the wet modified nano-hydroxyapatite for 24 hours to obtain modified nano-hydroxyapatite powder.

[0082] ② Prepare an aqueous solution of keratin / polyethylene oxide (mass ratio 9:1) with a modified nano-hydroxyapatite addition amount of 15%, the mass fraction of keratin and polyethylene oxide in the solution is 13%, and the mass fraction of the cross-linking agent ethylene glycol diglycidyl ether is 5% of the solute mass;

[0083] ③Preparation of wool keratin-based nanofibers containing nanohydroxyapatite by needle electrospinning;

[0084] ④ The obtained wool keratin-based nanofibers were placed in oxygen for 7 days for secondary crosslinking to obtain a non-water-soluble wool keratin-based nanofiber calcified cartilage layer with a dense structure. The scanning electron microscopy images and fiber diameter distribution of the wool keratin / PEO / nHA nanofiber membranes with different hydroxyapatite addition amounts in the wool keratin-based nanofiber calcified cartilage layer are shown in Figure 4. Figure 5 As shown; XRD pattern as Figure 6 As shown; ARS staining appearance after different mineralization time is shown Figure 7 As shown, the scanning electron microscope image after 28 days of mineralization is as follows Figure 8 As shown in the figure, the live / dead cells were detected by Calcein-AM / PI staining after different cell culture days. Figure 9 shown.

[0085] The subchondral bone layer 3 is prepared by the following steps:

[0086] ① Photocrosslinking modification of wool keratin: prepare 50 ml of keratin solution with a mass percentage concentration of 10%, add 1 ml of glycidyl methacrylate dropwise under heating (60°C) and stirring for 3 hours. During the reaction, the reaction pH value is adjusted to about 8.0 with NaOH solution. After the reaction is completed, the resulting solution is dialyzed and freeze-dried to obtain modified keratin powder.

[0087] ②Photocrosslinking modification of hyaluronic acid:

[0088] a. Prepare 100 ml of hyaluronic acid solution at a concentration of 1%, add 1 ml of methacrylic anhydride dropwise under stirring on an ice bath and react for 24 hours. During the reaction, use NaOH solution to control the reaction pH at around 8.0.

[0089] b. After the reaction, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed for 36h, changing the water every 8h;

[0090] c. After dialysis, the pH of the resulting solution was adjusted to about 7.4 with NaHCO3 solution;

[0091] d. The solution was placed in a -20°C refrigerator and frozen overnight, followed by freeze-drying for 24 hours to obtain white flocculent modified hyaluronic acid.

[0092] ③ Using water as the solvent, prepare a 3D printing bio-ink with a modified keratin mass fraction of 13%, a modified hyaluronic acid mass fraction of 2%, a nanoclay lithium magnesium silicate mass fraction of 5%, and a I 2959 mass fraction of 0.25%;

[0093] ④ Prepare the subchondral bone layer through 3D printing and UV cross-linking.

[0094] Preparation of composite osteochondral scaffold 4

[0095] The cartilage layer 1, calcified cartilage layer 2 and subchondral bone layer 3 were assembled by applying biological glue in a dot-coating manner to prepare a wool keratin-based biomimetic composite osteochondral scaffold. Micro-CT images of the wool keratin-based composite osteochondral scaffold implanted as an osteochondral repair filling material into the rat joint osteochondral defect at different times are shown in the figure. Figure 12 shown.

[0096] Example 2

[0097] Reference Figure 1 As shown, the keratin-based composite osteocartilage 4 provided in this embodiment is composed of a cartilage layer 1, a calcified cartilage layer 2, and a subchondral bone layer 3. The cartilage layer 1 is prepared by electrospinning keratin nanofiber membranes obtained by extracting keratin from discarded natural hair fibers using a reduction method, followed by secondary cross-linking and foaming of the keratin nanofibers. The calcified cartilage layer 2 is prepared by electrospinning keratin / nanohydroxyapatite blended nanofiber membranes obtained by blending keratin and modified nanohydroxyapatite, followed by secondary cross-linking of the nanofiber membranes. The subchondral bone layer 3 is prepared by 3D printing using a bio-ink prepared by blending photo-crosslinked modified keratin with photo-crosslinked modified hyaluronic acid, a thickener, and a photoinitiator.

[0098] Wherein, keratin is prepared by the following steps:

[0099] ① After opening and removing impurities from the human hair, cut it into 10mm long fiber segments, then wash it with petroleum ether solvent for about 40 minutes to remove impurities, repeatedly rinse it with distilled water for 5 times, and then dry it naturally at room temperature;

[0100] ② Immerse the cleaned and dried wool fibers in a solution containing 7% dithiothreitol, 45% thiourea, and 5% sodium lauryl sulfate, with a solid-to-liquid ratio of 10 g / 250 ml; stir at 95°C for 4 hours at a stirrer speed of 220 rpm.

[0101] ③ Filter the mixture obtained after stirring through a 150-mesh sieve to remove undissolved human hair. Dialyze the filtered keratin salt solution at room temperature using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove salt and small molecules in the solution. The dialysis time is 72 hours, and the distilled water is replaced every 12 hours.

[0102] ④ Centrifuge the dialyzed keratin solution to remove the precipitate in the dialysate at a speed of 6000 pm for 0.5 h, and then collect the supernatant;

[0103] ⑤ Concentrating the obtained supernatant to obtain a keratin concentrated solution with a keratin mass percentage concentration of 14%;

[0104] ⑥ Freeze-dry the keratin concentrated solution for 36 hours to obtain keratin powder.

[0105] The cartilage layer 1 is prepared by the following steps:

[0106] ① Prepare an aqueous solution of wool keratin and polyethylene oxide (mass ratio 9:1) with a 10% addition of chondroitin sulfate / hyaluronic acid, with a total mass fraction of 17% and a mass fraction of ethylene glycol diglycidyl ether as the crosslinker of 7% of the solute mass;

[0107] ②Preparation of human hair keratin-based nanofibers by bubble electrospinning;

[0108] ③ The obtained human hair keratin-based nanofibers were placed in an oven at 100°C for 2 hours for secondary crosslinking to obtain water-insoluble nanofibers;

[0109] ④ The secondary cross-linked wool keratin-based nanofiber membrane was immersed in a 0.1 M sodium borohydride solution for 60 minutes; it was repeatedly washed with deionized water and freeze-dried to obtain a human hair keratin-based nanofiber cartilage layer.

[0110] The calcified cartilage layer 2 is prepared by the following steps:

[0111] ① Modification of nano-hydroxyapatite, the steps are as follows:

[0112] a. The nano-hydroxyapatite was placed in water and stirred at a speed of 100 r / min to disperse the nano-hydroxyapatite to obtain a nano-hydroxyapatite dispersion having a concentration of 10% by mass of the nano-hydroxyapatite, and then a sodium hexametaphosphate solution having a concentration of 10% by mass was configured;

[0113] b. sodium hexametaphosphate solution was added dropwise to the nano-hydroxyapatite solution under constant stirring at 100 r / min speed to allow for sufficient reaction, and the volume ratio of sodium hexametaphosphate solution to hydroxyapatite dispersion was 7.5: 1; stirring was continued for 1 hour after completion of the dropwise addition of sodium hexametaphosphate solution to obtain a mixed solution;

[0114] c. The mixed solution after the reaction was ultrasonically treated for 10 min to uniformly disperse the mixed solution;

[0115] d. The hydroxyapatite was extracted by adding the same volume of anhydrous ethanol as the mixed solution, and the modified nano-hydroxyapatite was collected by centrifugation at 8000 r / min for 20 min, and then the modified nano-hydroxyapatite was repeatedly washed with deionized water 5 times to remove unreacted ions, and the modified nano-hydroxyapatite was centrifuged at 8000 r / min to obtain a wet modified nano-hydroxyapatite;

[0116] e. freeze-drying the wet modified nano-hydroxyapatite for 36 hours to obtain modified nano-hydroxyapatite powder.

[0117] ② Prepare an aqueous solution of keratin / polyethylene oxide (mass ratio 9:1) with a modified nano-hydroxyapatite addition amount of 5%, wherein the mass fraction of keratin and polyethylene oxide in the solution is 17%, and the mass fraction of the cross-linking agent ethylene glycol diglycidyl ether is 7% of the solute mass;

[0118] ③Preparation of human hair keratin-based nanofibers containing nanohydroxyapatite by bubble electrospinning;

[0119] ④ The obtained wool keratin-based nanofibers were placed in an oven at 100°C for 2 hours for secondary cross-linking to obtain a non-water-soluble human hair keratin-based nanofiber calcified cartilage layer with a dense structure.

[0120] The subchondral bone layer 3 is prepared by the following steps:

[0121] ① Photocrosslinking modification of human hair keratin: 50 ml of a keratin solution with a mass percentage concentration of 14% was prepared, and 6 ml of glycidyl methacrylate was added dropwise under heating (60°C) and stirring for 3 h. During the reaction, the pH value of the reaction was adjusted to approximately 8.0 with a NaOH solution. After the reaction, the resulting solution was dialyzed and freeze-dried to obtain modified keratin powder.

[0122] ②Photocrosslinking modification of hyaluronic acid:

[0123] a. Prepare 100 ml of hyaluronic acid solution at a concentration of 1%, add 3 ml of methacrylic anhydride dropwise under stirring on an ice bath and react for 24 hours. During the reaction, use NaOH solution to control the reaction pH at around 8.0.

[0124] b After the reaction, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed for 72h, changing the water every 8h;

[0125] c. After dialysis, the pH of the resulting solution was adjusted to about 7.4 with NaHCO3 solution;

[0126] d. The solution was placed in a -20°C refrigerator and frozen overnight, followed by freeze-drying for 48 hours to obtain white flocculent modified hyaluronic acid.

[0127] ③ Using water as the solvent, prepare a 3D printing bio-ink with a modified keratin mass fraction of 17%, a modified hyaluronic acid mass fraction of 4%, a nanoclay lithium magnesium silicate mass fraction of 10%, and a LAP mass fraction of 1%;

[0128] ④ Prepare the subchondral bone layer through 3D printing and UV cross-linking.

[0129] Preparation of composite osteochondral scaffold 4

[0130] The cartilage layer 1, the calcified cartilage layer 2 and the subchondral bone layer 3 were assembled by using biological glue in a spot coating manner to prepare a wool keratin-based biomimetic composite osteochondral scaffold.

[0131] Example 3

[0132] Reference Figure 1 As shown, the keratin-based composite osteocartilage 4 provided in this embodiment is composed of a cartilage layer 1, a calcified cartilage layer 2, and a subchondral bone layer 3. The cartilage layer 1 is prepared by electrospinning keratin nanofiber membranes obtained by extracting keratin from discarded natural hair fibers using a reduction method, followed by secondary cross-linking and foaming of the keratin nanofibers. The calcified cartilage layer 2 is prepared by electrospinning keratin / nanohydroxyapatite blended nanofiber membranes obtained by blending keratin and modified nanohydroxyapatite, followed by secondary cross-linking of the nanofiber membranes. The subchondral bone layer 3 is prepared by 3D printing using a bio-ink prepared by blending photo-crosslinked modified keratin with photo-crosslinked modified hyaluronic acid, a thickener, and a photoinitiator.

[0133] Wherein, keratin is prepared by the following steps:

[0134] ① After opening and removing impurities from the rabbit hair, cut it into 8mm long fiber segments, then wash it with petroleum ether solvent for about 40 minutes to remove impurities, repeatedly wash it with distilled water for 5 times, and then dry it naturally at room temperature;

[0135] ② Immerse the cleaned and dried wool fibers in a solution containing 6% thioglycolic acid, 42% lithium bromide, and 3% sodium lauryl sulfate, with a solid-to-liquid ratio of 6g / 250ml; stir at 80°C for 5 hours at a stirrer speed of 200r / min;

[0136] ③ Filter the mixture obtained after stirring through a 120-mesh sieve to remove undissolved human hair. Dialyze the filtered keratin salt solution at room temperature using a dialysis bag with a molecular weight cutoff of 8000-14000 Da to remove salt and small molecules in the solution. The dialysis time is 72 hours, and the distilled water is replaced every 12 hours.

[0137] ④ Centrifuge the dialyzed keratin solution to remove the precipitate in the dialysate at a speed of 6000 pm for 0.4 h, and then collect the supernatant;

[0138] ⑤ Concentrating the obtained supernatant to obtain a keratin concentrated solution with a keratin mass percentage concentration of 12%;

[0139] ⑥ Freeze-dry the keratin concentrated solution for 36 hours to obtain keratin powder.

[0140] The cartilage layer 1 is prepared by the following steps:

[0141] ① Prepare an aqueous solution of wool keratin and polyethylene oxide (mass ratio 9:1) with a total mass fraction of 15% and a crosslinker of ethylene glycol diglycidyl ether of 7% of the solute mass;

[0142] ②Preparation of human hair keratin-based nanofibers by bubble electrospinning;

[0143] ③ Placing the obtained human hair keratin-based nanofibers in a UV generator for 2 hours for secondary crosslinking to obtain water-insoluble nanofibers;

[0144] ④ Immerse the secondary cross-linked wool keratin-based nanofiber membrane in a 0.5 M sodium borohydride solution for 15 minutes; repeatedly wash with deionized water and freeze-dry to obtain a human hair keratin-based nanofiber cartilage layer.

[0145] The calcified cartilage layer 2 is prepared by the following steps:

[0146] ① Modification of nano-hydroxyapatite, the steps are as follows:

[0147] a. The nano-hydroxyapatite was placed in water and stirred at a speed of 80r / min to disperse the nano-hydroxyapatite to obtain a nano-hydroxyapatite dispersion having a concentration of 10% by mass of the nano-hydroxyapatite, and then a sodium hexametaphosphate solution having a concentration of 10% by mass was configured;

[0148] b. sodium hexametaphosphate solution was added dropwise to the nano-hydroxyapatite solution under constant stirring at 80 r / min speed to allow for sufficient reaction, and the volume ratio of sodium hexametaphosphate solution to hydroxyapatite dispersion was 8: 1; stirring was continued for 1 hour after completion of the dropwise addition of sodium hexametaphosphate solution to obtain a mixed solution;

[0149] c. The mixed solution after the reaction was ultrasonically treated for 10 min to uniformly disperse the mixed solution;

[0150] d. The hydroxyapatite was extracted by adding the same volume of anhydrous ethanol as the mixed solution, and the modified nano-hydroxyapatite was collected by centrifugation at 8000 r / min for 20 min, and then the modified nano-hydroxyapatite was repeatedly washed with deionized water 5 times to remove unreacted ions, and the modified nano-hydroxyapatite was centrifuged at 8000 r / min to obtain a wet modified nano-hydroxyapatite;

[0151] e. freeze-drying the wet modified nano-hydroxyapatite for 36 hours to obtain modified nano-hydroxyapatite powder.

[0152] ② Prepare an aqueous solution of keratin / polyethylene oxide (mass ratio 9:1) with a modified nano-hydroxyapatite addition amount of 20%, the mass fraction of keratin and polyethylene oxide in the solution is 15%, and the mass fraction of the cross-linking agent ethylene glycol diglycidyl ether is 6% of the solute mass;

[0153] ③Prepare hair keratin-based nanofibers containing nanohydroxyapatite using bubble electrospinning;

[0154] ④ The obtained wool keratin-based nanofibers were placed in an ultraviolet light box for 2 hours for secondary cross-linking to obtain a non-water-soluble rabbit hair keratin-based nanofiber calcified cartilage layer with a dense structure.

[0155] The subchondral bone layer 3 is prepared by the following steps:

[0156] ① Photocrosslinking modification of human hair keratin: 50 ml of a keratin solution with a mass percentage concentration of 12% was prepared, and 4 ml of glycidyl methacrylate was added dropwise under heating (60°C) and stirring for 3 h. During the reaction, the pH value of the reaction was adjusted to approximately 8.0 with a NaOH solution. After the reaction, the resulting solution was dialyzed and freeze-dried to obtain modified keratin powder.

[0157] ②Photocrosslinking modification of hyaluronic acid:

[0158] a. Prepare 100 ml of hyaluronic acid solution at a concentration of 1%, add 2 ml of methacrylic anhydride dropwise under stirring on an ice bath and react for 24 hours. During the reaction, use NaOH solution to control the reaction pH at around 8.0.

[0159] b After the reaction, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed for 72h, changing the water every 8h;

[0160] c. After dialysis, the pH of the resulting solution was adjusted to about 7.4 with NaHCO3 solution;

[0161] d. The solution was placed in a -20°C refrigerator and frozen overnight, followed by freeze-drying for 48 hours to obtain white flocculent modified hyaluronic acid.

[0162] ③ Using water as the solvent, prepare a 3D printing bio-ink with a modified keratin mass fraction of 15%, a modified hyaluronic acid mass fraction of 3%, a nanoclay lithium magnesium silicate mass fraction of 7%, and a LAP mass fraction of 0.5%;

[0163] ④ Prepare the subchondral bone layer through 3D printing and UV cross-linking.

[0164] Preparation of composite osteochondral scaffold 4:

[0165] The cartilage layer 1, the calcified cartilage layer 2 and the subchondral bone layer 3 were assembled by using biological glue in a spot coating manner to prepare a wool keratin-based biomimetic composite osteochondral scaffold.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing keratin-based composite osteochondrocytes, characterized in that: The following steps are involved: S1. Keratin was extracted from discarded natural hair fibers using a reduction method, and a keratin / chondroitin sulfate / hyaluronic acid composite nanofiber membrane was obtained by electrospinning. The keratin-based nanofibers were then subjected to secondary cross-linking and foaming to prepare a cartilage layer. S2. keratin was blended with modified nanohydroxyapatite to obtain a keratin / nanohydroxyapatite blended nanofiber membrane by electrospinning, and the nanofiber membrane was then subjected to secondary cross-linking to prepare a calcified cartilage layer; S3. Keratin was photocrosslinked and then mixed with photocrosslinked hyaluronic acid, a thickener, and a photoinitiator to prepare a bioink, and the subchondral bone layer was prepared by 3D printing technology; S4. The cartilage layer, calcified cartilage layer and subchondral bone layer are combined into a composite osteochondral scaffold by applying biological glue.

2. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: The steps of extracting keratin from discarded natural hair fibers by the reduction method are as follows: S11. The natural keratin fibers were opened and cut into 5-10 mm long fiber segments after impurity removal, and then washed with petroleum ether solvent for about 40 min to remove impurities, and then washed with distilled water 4-5 times and dried at room temperature. S12. The natural keratin fibers were immersed in a dissolving solution containing a reducing agent, a protein denaturant and a surfactant, the solid-liquid ratio of the dissolving solution was 6-12g / 250ml; stirred at a temperature of 70-95°C for 4-6h, the stirrer speed was 160-220r / min; The reducing agent is sodium metabisulfite, dithiothreitol, thioglycolic acid, etc., and the mass percentage concentration of the reducing agent in the dissolving solution is 5%-7%; The keratin denaturant is urea, thiourea or lithium bromide, and the mass percentage concentration of the protein denaturant in the dissolving solution is 40-45%; The surfactant is sodium lauryl sulfate, and the mass percentage concentration of the surfactant in the dissolving solution is 1-5%; S13. The mixture obtained after stirring was filtered through a 100-150 mesh sieve to remove undissolved natural keratin fibers. The filtered keratin salt solution was dialyzed at room temperature using a dialysis bag with a molecular weight cutoff of 8000-14000Da to remove salts and small molecules from the solution. The dialysis time was 36-72h, and distilled water was replaced every 12h. S14. The dialyzed keratin solution was centrifuged to remove the precipitate in the dialysate at a centrifugal speed of 5000-6000 rpm for 0.25-0.5 h, and the supernatant was then collected; S15. The obtained supernatant is concentrated to obtain a keratin concentrated solution having a keratin mass percentage concentration of 10-14%; S16. Freeze-dry the concentrated keratin solution for 24-36 hours to obtain keratin powder.

3. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: The natural hair fibers are wool, rabbit hair, human hair, pig hair, cow hair or camel hair.

4. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: The electrospinning method is to prepare nanofibers by passing the spinning solution through a needle-type, bubble-type or disc-type electrospinning device, wherein: In the electrospinning method described in step S1, the spinning solution is an aqueous solution of keratin and polyethylene oxide (mass ratio 9:1) with a chondroitin sulfate / hyaluronic acid addition amount of 3-10%, with a total mass fraction of 13-17%, and the mass fraction of the crosslinker ethylene glycol diglycidyl ether is 5-7% of the solute mass. In the electrospinning method in step S2, the electrospinning solution is an aqueous solution of keratin / polyethylene oxide (mass ratio 9:1) with a modified nano-hydroxyapatite addition amount of 5-20%, the mass fraction of keratin and polyethylene oxide in the solution is 13-17%, and the mass fraction of the crosslinker ethylene glycol diglycidyl ether is 5-7% of the solute mass.

5. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: The secondary crosslinking can be oxygen oxidation crosslinking, ultraviolet radiation crosslinking or thermal crosslinking.

6. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: In step S1, the foaming method is: immersing the secondary cross-linked keratin-based nanofiber membrane in a 0.1-0.5M sodium borohydride solution for 15-60 minutes; repeatedly washing with deionized water and freeze-drying to obtain a keratin-based nanofiber aerogel.

7. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: In step S2, the method for preparing the sodium hexametaphosphate modified nano-hydroxyapatite powder comprises: S21: placing nanohydroxyapatite in water and stirring at a speed of 50-100 r / min to disperse the nanohydroxyapatite to obtain a nanohydroxyapatite dispersion with a mass percentage concentration of 10%; and then preparing a sodium hexametaphosphate solution with a mass percentage concentration of 10%; S22. The sodium hexametaphosphate solution was added dropwise to the nano-hydroxyapatite solution under continuous stirring at a speed of 50-100 r / min to allow for sufficient reaction. The volume ratio of the sodium hexametaphosphate solution to the hydroxyapatite dispersion was 12.5:1-7.5:1; stirring was continued for 1 hour after the addition of the sodium hexametaphosphate solution to obtain a mixed solution. S23. The mixed solution after the reaction was ultrasonically treated for 10 min to uniformly disperse the mixed solution; S24. The hydroxyapatite was extracted by adding anhydrous ethanol of the same volume as the mixed solution, and the modified nano-hydroxyapatite was collected by centrifugation at 8000 r / min for 20 min, and then the modified nano-hydroxyapatite was repeatedly washed with deionized water 4-5 times to remove unreacted ions, and centrifuged at 8000 r / min to obtain wet modified nano-hydroxyapatite; S25. Freeze-dry the wet modified nano-hydroxyapatite for 24-36 hours to obtain modified nano-hydroxyapatite powder.

8. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: In step S3, the photocrosslinking modification method for keratin comprises: preparing 50 ml of a keratin solution with a mass percentage concentration of 10-14%, adding 1-6 ml of glycidyl methacrylate dropwise under heating (60° C.) and stirring for 3 hours, adjusting the reaction pH to about 8.0 with a NaOH solution during the reaction, and after the reaction, dialyzing and freeze-drying the resulting solution to obtain modified keratin powder.

9. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: In step S3, the modification method of the photo-crosslinked modified hyaluronic acid is: S31. Prepare 100 ml of hyaluronic acid solution at a concentration of 1%, add 1-3 ml of methacrylic anhydride dropwise with stirring on ice for 24 hours, and control the reaction pH at around 8.0 using NaOH solution during the reaction. S32 After the reaction, the solution was poured into a dialysis bag with a molecular weight cutoff of 8000-14000Da and dialyzed for 36-72h, changing the water every 8h; S33. After dialysis, the pH of the resulting solution was adjusted to about 7.4 with NaHCO3 solution; S34. Freeze the solution in a -20°C refrigerator overnight, and then freeze-dry for 24-48 hours to obtain a white flocculent product, namely, modified hyaluronic acid.

10. The method for preparing keratin-based composite osteochondral according to claim 1, wherein: In step S3, the bio-ink uses water as a solvent, the mass fraction of modified keratin in the ink is 13-17%, the mass fraction of modified hyaluronic acid is 2-4%, the mass fraction of thickener is 5-10%, and the mass fraction of photoinitiator is 0.25-1%; The thickener is nanoclay lithium magnesium silicate; The photoinitiator is I 2959 or LAP.

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