Functionalized silk fibroin-based bio-ink, biomimetic cartilage hydrogel and methods of making the same

Abstract

The application belongs to the field of biomaterials and tissue engineering technology, and relates to a functionalized silk fibroin-based bio-ink, a biomimetic cartilage hydrogel and a preparation method thereof. The ink contains an initiator, SFMA, GelMA, PEGDA and OBC, and the ratio of each component is strictly controlled; the ink is uniformly mixed with stem cells, a solidification cross-linking reaction is initiated, and a multiple biomimetic cartilage hydrogel is prepared. The hydrogel includes a multiple network structure hydrogel and OBC and stem cells dispersed in the multiple network structure hydrogel at the same time, and has a compression modulus and a porous structure matched with real cartilage tissue. The functionalized silk fibroin-based bio-ink and the multiple biomimetic cartilage hydrogel provided by the application solve the problems of donor shortage, immune rejection, single biomimetic dimension and unmatched mechanical properties in the prior art, and can realize high-precision printing and efficiently promote cartilage regeneration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials and tissue engineering technology, and relates to a functionalized silk fibroin-based bio-ink, a biomimetic cartilage hydrogel and a preparation method thereof. BACKGROUND

[0002] In human tissues, cartilage plays a crucial role, bearing the key responsibility of physical support and cushioning. However, cartilage is generally deficient in blood vessels and nerves, has a low cell density and limited cell migration ability, which severely limits its self-repairing ability. Once cartilage is damaged, it is highly likely to cause osteoarthritis and other diseases, which seriously affects the quality of life of patients.

[0003] For cartilage defect repair and regeneration, traditional methods include autologous transplantation, allogeneic cartilage transplantation, and tissue engineering scaffolds. However, autologous transplantation has the problem of donor shortage; allogeneic cartilage transplantation can easily cause immune rejection; traditional tissue engineering scaffolds are single in structure and function, and cannot fully match the complex physiological requirements of cartilage, and cannot efficiently prepare personalized scaffolds, making it difficult to meet the efficient and personalized cartilage repair needs.

[0004] Silk fibroin (SF) is a natural polymer material that has attracted much attention in the biomedical field due to its excellent physicochemical properties, especially its excellent biocompatibility and biodegradability. Its raw material is usually derived from cocoon, and after degumming to remove the surface sericin, it can be regenerated into various forms of regenerated silk fibroin (RSF) materials, such as silk fibroin microspheres, fibers, films, hydrogels, and three-dimensional scaffolds.

[0005] For example, document 1 (Nature Communications, 2018, 9:1620.) prepared a high-precision personalized hydrogel scaffold encapsulating living cells by methacrylated silk fibroin bio-ink based on digital light processing 3D printing. Document 2 (Journal of Biomedical Materials Research Part A 2022, 4:884-898.) prepared a silk fibroin-based hydrogel by photocuring method, and verified that the hydrogel can promote the formation of cartilage tissue in vivo by immunohistochemical experiment. However, the silk fibroin-based hydrogels of documents 1-2 do not meet the requirements of cartilage repair materials in terms of physicochemical properties, and the biomimetic dimension is relatively single.

[0006] Document 3 (Bioactive Materials, 2024, 40:541-556.) successfully obtained a silk fibroin hydrogel material with a compression modulus of 2.33 MPa by post-treating methylacrylated silk fibroin with ethanol, which meets the mechanical property requirements of cartilage repair materials. However, the ethanol post-treatment process causes the hydrogel to shrink, losing its porous characteristics and failing to provide the ideal microenvironment conditions required by cells.

[0007] Patent application CN116421780A discloses a silk fibroin and gelatin-based bio-printing ink and its preparation and application. By modifying regenerated silk fibroin with methylacrylated glycidyl ester, a photosensitive silk fibroin (SilGMA) is obtained. It is compounded with methylacrylated gelatin (GelMA), and pigments and photoinitiators are added. A bio-printing ink with excellent biocompatibility, enhanced mechanical properties and rapid photocuring ability is prepared by mixing with cells. With the help of digital light processing bio-printing technology, fine manufacturing of hydrogel scaffold loaded with chondrocytes can be achieved. However, the compression modulus of the hydrogel scaffold is only 90 KPa at most, which is still far from the compression modulus of real cartilage tissue (0.8-2 MPa). Numerous studies have shown that the closer the material properties are to the real tissue, the more conducive it is to the regeneration and repair of the corresponding tissue. This patent application has obvious limitations in this regard.

[0008] In summary, there is still an urgent need for a simple and easy-to-use functional silk fibroin-based bio-ink preparation method. This method should be able to support and package living cells for printing and achieve high-precision printing, while meeting the needs of cartilage repair materials in structure, physicochemical properties, function and other multidimensional biomimetic requirements, and have high-efficiency cartilage regeneration-promoting effect, to effectively promote the development and progress of cartilage repair treatment technology. SUMMARY

[0009] The purpose of the present application is to solve the problems existing in the prior art and provide a functional silk fibroin-based bio-ink, a biomimetic cartilage hydrogel and a preparation method thereof.

[0010] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0011] The functionalized silk fibroin-based bio-ink contains an initiator (for example, LAP (lithium salt of phenyl-2,4,6-trimethylbenzoyl phosphinic acid) and the like), SFMA, GelMA, PEGDA and OBC, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, and the mass fraction of OBC is 0.3-1.2wt%, and SFMA, GelMA, PEGDA and OBC correspond to methylacrylated silk fibroin, methylacrylated gelatin, polyethylene glycol diacrylate and oxidized bacterial cellulose nanofiber respectively.

[0012] As a preferred technical solution:

[0013] The functionalized silk fibroin-based bio-ink as described above, the molecular weight of SFMA is 70-150kDa, and the grafting rate is 80-95%; the molecular weight of GelMA is 160-220kDa, and the grafting rate is 71-90%; and the molecular weight of PEGDA is 700Da.

[0014] The functionalized silk fibroin-based bio-ink as described above, the mass fraction of the initiator is 0.1-0.4wt%.

[0015] The application further provides a multi-bionic cartilage hydrogel, which comprises a multi-network structure hydrogel and OBC and stem cells (for example, rat bone marrow mesenchymal stem cells (BMSCs) and the like) dispersed in the multi-network structure hydrogel at the same time; the multi-network structure hydrogel is crosslinked by SFMA, GelMA and PEGDA; the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, the mass ratio of GelMA to PEGDA can be adjusted at will, the mass fraction of OBC is 0.3-1.2wt%, and SFMA, GelMA, PEGDA and OBC correspond to methylacrylated silk fibroin, methylacrylated gelatin, polyethylene glycol diacrylate and oxidized bacterial cellulose nanofiber respectively.

[0016] As a preferred technical solution:

[0017] The multi-bionic cartilage hydrogel as described above, the molecular weight of SFMA is 70-150kDa, and the grafting rate is 80-95%; the molecular weight of GelMA is 160-220kDa, and the grafting rate is 71-90%; and the molecular weight of PEGDA is 700Da.

[0018] The multiple biomimetic cartilage hydrogel according to any one of the above has a density of 2*10 5 cells / mL-1*10 6 cells / mL.

[0019] The multiple biomimetic cartilage hydrogel according to any one of the above has a compression modulus of 0.4-0.85 MPa, a pore size of 40-160 μm, and good survival and directional differentiation of stem cells into chondrocytes in the hydrogel.

[0020] The application further provides a method for preparing the multiple biomimetic cartilage hydrogel according to any one of the above, which comprises mixing a functionalized silk fibroin-based bio-ink according to any one of the above with stem cells uniformly, and initiating a solidification cross-linking reaction to obtain the multiple biomimetic cartilage hydrogel.

[0021] As a preferred technical solution,

[0022] The method according to the above initiates the solidification cross-linking reaction by means of ultraviolet light irradiation, and the solidification cross-linking reaction is performed at a temperature of 25-37 ℃ for 20-60 s, and the ultraviolet light intensity is 10-30 mW / cm 2 .

[0023] Principle of the application

[0024] The multiple biomimetic cartilage hydrogel of the application has excellent directional chondrogenic differentiation ability of the stem cells inside, which is due to the following two aspects.

[0025] On one hand, the OBC inside contains a large amount of oxidized carboxyl functional groups, which can promote the chondrogenic differentiation of stem cells.

[0026] On the other hand, the multiple biomimetic cartilage hydrogel of the application is formed by mixing a functionalized silk fibroin-based bio-ink with stem cells uniformly and then initiating a solidification cross-linking reaction.

[0027] The main components of the functionalized silk fibroin-based bio-ink are SFMA, GelMA and PEGDA, which have good biocompatibility and contain carbon-carbon double bonds. The three components can be photo-crosslinked under the joint action of initiator and ultraviolet light to form a multiple crosslinking network, which provides a microenvironment for stem cells. The suitable photo-crosslinking site and shorter printing time avoid damage to the internal wrapped stem cells.

[0028] SFMA is derived from silk fibroin and has excellent biocompatibility. After gelation, it has a three-dimensional network structure and a high water content, which can provide a long-term stable microenvironment for cells. GelMA contains chemical sites that are beneficial to cell adhesion. PEGDA, as a synthetic polymer, is an excellent anti-cell adhesion substance, which has poor adhesion to stem cells. PEGDA has a smaller molecular weight, a higher density of carbon-carbon double bonds per unit mass, a more dense hydrogel network, a higher compression modulus, and a smaller hydrogel pore size. OBC can simultaneously significantly improve the mechanical properties (compression modulus) and pore size of the hydrogel. Adjusting the content of SFMA, GelMA, PEGDA and OBC in the functionalized silk fibroin-based bio-ink can control the mechanical properties, pore size and biological interface properties of the multiple biomimetic cartilage hydrogels.

[0029] Specifically, the total mass fraction of SFMA, GelMA and PEGDA in the functionalized silk fibroin-based bio-ink is 18 wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, and the mass fraction of OBC is 0.3-1.2 wt%. In this way, the mass fractions of SFMA, GelMA, PEGDA and OBC in the multiple biomimetic cartilage hydrogel are all within the appropriate range. If the mass fraction of SFMA is too small, the overall biological activity of the hydrogel will decrease, which may cause inflammation and immune rejection. If the mass fraction of SFMA is too large, the mechanical properties will be insufficient, and the mechanical properties of the cartilage tissue cannot be accurately matched. If the mass fraction of GelMA is too small, the adhesion ability of the hydrogel to the internal stem cells will be insufficient, and the subsequent cell proliferation and differentiation behaviors will be adversely affected. If the mass fraction of GelMA is too large, the adhesion ability of the hydrogel to the internal stem cells will be too strong, and the strong adhesion will make the internal stem cells adhere and spread, which is not conducive to cell aggregation and further directional chondrogenic differentiation. If the mass fraction of PEGDA is too small, the pore size of the hydrogel will be too large, which is not conducive to cell aggregation and further directional chondrogenic differentiation of the internal stem cells. If the mass fraction of PEGDA is too large, the pore size of the hydrogel will be too small, which is not conducive to cell survival and further cell differentiation ability. If the mass fraction of OBC is too small, the mechanical properties will be insufficient, and the mechanical properties of the cartilage tissue cannot be matched. Moreover, the low carboxyl content cannot better induce chondrogenic differentiation of stem cells. If the mass fraction of OBC is too large, too much OBC cannot be uniformly dispersed in the hydrogel, which will cause the hydrogel to be non-uniform as a whole, the physicochemical properties of the hydrogel to be unstable, and thus affect the cell behavior.

[0030] The concentration of the bio-ink of the present application is appropriate and uniformly dispersed, which can ensure high printing accuracy. If the concentration of the bio-ink is too low, the number of photo-crosslinking sites contained is too small, which will result in residual uncured bio-ink, reducing the micro-precision of printing. If the concentration of the bio-ink is too high, the number of photo-crosslinking sites contained is too large, which will easily cause the phenomenon of "over-exposure solidification", resulting in a significant decrease in resolution. If the bio-ink is not uniformly dispersed, the photo-curing process will be unstable, which will also cause a decrease in printing accuracy.

[0031] Advantages:

[0032] (1) The present application uses SFMA, GelMA and other natural polymers as main raw materials, which have excellent biocompatibility and are biodegradable, avoiding the donor limitation of autologous transplantation and the immune rejection risk of allogeneic transplantation, and does not need to rely on additional donor tissues, which is suitable for clinical cartilage repair scenarios.

[0033] (2) The multiple biomimetic cartilage hydrogel of the present application has a porous structure of 40-160 μm, providing a suitable microenvironment for stem cells to survive, solving the problem of hydrogel shrinkage, significant reduction in pore size, or even loss of porosity caused by ethanol post-processing in document 3.

[0034] (3) The compression modulus of the multiple biomimetic cartilage hydrogel of the present application reaches 0.4-0.85 MPa, accurately matching the mechanical requirements of natural cartilage tissue, overcoming the defects of insufficient physicochemical properties of silk fibroin-based hydrogel in documents 1-2 and the large gap between the compression modulus of the material and the real cartilage in patent application CN116421780A.

[0035] (4) The present application promotes the directional chondrogenic differentiation of stem cells through the synergistic effect of the carboxyl functional groups of OBC and biological interface regulation (GelMA provides suitable adhesion), achieving biomimicry at the functional level.

[0036] (5) The functionalized silk fibroin-based bio-ink of the present application can be quickly cured by ultraviolet light, with high printing precision and a mild curing process, which can effectively encapsulate high-activity stem cells, solving the problems of traditional scaffolds that cannot efficiently prepare personalized structures and are difficult to load living cells, and adapting to the clinical demand for personalized treatment.

[0037] (6) The present application significantly improves the ability of stem cells to differentiate into cartilage through multi-dimensional biomimetic design and the inducing effect of OBC, solving the problem of low chondrogenic gene expression of existing materials, and providing an efficient solution for cartilage defect repair. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The printing model used for printing precision detection;

[0039] Figure 2 The printing result for printing precision detection; wherein (a) corresponds to Example 1, (b) corresponds to Example 4, (c) corresponds to Comparative Example 2, and (d) corresponds to Comparative Example 6;

[0040] Figure 3 The chondrogenic differentiation-related gene expression level comparison chart of the relevant examples and comparative examples in the stem cell chondrogenic differentiation ability detection. DETAILED DESCRIPTION

[0041] The present application will be further described in conjunction with the specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or modifications to the present application after reading the content taught by the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0042] The following are the test methods for the relevant performance indicators in each example and comparative example:

[0043] Grafting rate: 1H-NMR spectrum of the material before and after grafting was determined by nuclear magnetic resonance spectrometer, and the grafting rate was derived according to the reduction degree of lysine peak area. The specific steps are as follows: the freeze-dried powder before and after grafting is dissolved with deuterium reagent, and 1H-NMR test is carried out at a frequency of 400MHz. The integral area of lysine signal peak before grafting is defined as S1, and the integral area of lysine signal peak after grafting is defined as S2. Then the grafting rate is (S1-S2) x 100% / S1, and the stable range is selected by multiple tests.

[0044] Compression modulus: static compression test of hydrogel was carried out by using INSTRON / 5969 electronic universal material testing machine. The specific test method is as follows: adjust the upper and lower clamps so that the clamps just contact with the hydrogel, then compress the hydrogel to rupture at a compression rate of 10mm / min, obtain the stress-strain data of hydrogel at different time points, select the linear fitting region of stress-strain curve in the strain range of 5%-10%, and calculate the compression modulus of the test material.

[0045] Pore size: the pore size of hydrogel was observed by SU8010 scanning electron microscope (SEM). The specific test method is as follows: the hydrogel sample is soaked for 24h to reach swelling equilibrium, then it is brittle fractured after being frozen for 12h and placed in liquid nitrogen, and then it is freeze-dried and gold sprayed for 60-90s (current 10mA). The cross-sectional morphology of the sample is photographed at a test voltage of 10.0kV, and the pore size distribution of the sample is measured and analyzed by using ImageJ software.

[0046] In order to ensure the performance of the substances used in each embodiment and comparative example, the manufacturer information of the substances is disclosed. In addition, the products of other manufacturers meeting the definition of the present application can also be applicable.

[0047] The preparation process of OBC in each of the following embodiments is as follows: first, 100 g of bacterial fiber (manufacturer Hainan Coconut Food Co., Ltd.) is weighed, 200 mL of deionized water is added, and a homogenizer is used to stir to uniformly disperse the bacterial fiber to obtain a bacterial fiber suspension; 0.032 g of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical) and 0.2 g of NaBr (sodium bromide) are taken and added to 20 mL of deionized water, and ultrasonic treatment is performed in a water bath until completely dissolved to prepare a mixed solution; the above mixed solution is added to the bacterial fiber suspension, stirred uniformly, and then 0.5 mol / L NaOH (sodium hydroxide) aqueous solution is used to adjust the pH value of the system to 10-10.5; then 7.8 mL of NaClO (sodium hypochlorite) is added to the reaction system, and 0.5 mol / L NaOH aqueous solution is continuously used to maintain the pH value of the system in the range of 10-10.5, timing is started and reaction is performed for 1-2 h; after the reaction is completed, 0.5 mol / L HCl (hydrochloric acid) aqueous solution is added to adjust the pH value of the system to 7 to terminate the reaction; the mixed solution after reaction is subjected to centrifugal treatment, the precipitate is collected, deionized water is added to wash the precipitate, centrifugation is performed again, and such repeated washing and centrifugation is performed for multiple times until the reaction residues are washed clean, and finally OBC is obtained; the degree of polymerization of the OBC is 2000-6000, the aspect ratio is greater than 100, and the average diameter is 40-60 nm.

[0048] Example 1

[0049] A preparation method of a multi-bionic cartilage hydrogel, and the specific steps are as follows:

[0050] (1) Preparation of materials;

[0051] SFMA: molecular weight of 70-100 kDa, grafting rate of 80-85%;

[0052] GelMA: molecular weight of 160-220 kDa, grafting rate of 71-90%, manufacturer Huaxi Sengen (Shanghai) Biotechnology Co., Ltd., product number R201B2;

[0053] PEGDA: molecular weight of 700 Da, manufacturer Sigma-Aldrich Co., product number 455008;

[0054] Water;

[0055] LAP: manufacturer Shanghai Yuan Ye Biotechnology Co., Ltd., product number Y43995-5g;

[0056] OBC;

[0057] Stem cells: rat BMSCs, manufacturer Shanghai Yuchun Biotechnology Co., Ltd., product number ER0113;

[0058] (2) Preparation of functionalized silk fibroin-based bio-ink;

[0059] After dissolving SFMA, GelMA and PEGDA in water, adding LAP after filtration, and then mixing the filtrate with OBC by stirring, a functionalized silk fibroin-based bio-ink is obtained;

[0060] In the functionalized silk fibroin-based bio-ink, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, the mass ratio of GelMA to PEGDA is 1.5:7.5, the mass fraction of OBC is 0.9wt%, and the mass fraction of LAP is 0.2wt%;

[0061] (3) Preparation of multi-bionic cartilage hydrogel;

[0062] After mixing the functionalized silk fibroin-based bio-ink with stem cells uniformly, under the irradiation of ultraviolet light with a strength of 20mW / cm 2 at 25℃, the cross-linking reaction is cured for 40s to obtain a multi-bionic cartilage hydrogel.

[0063] In the finally obtained multi-bionic cartilage hydrogel, the density of stem cells is 1×10 6 cells / mL, the compression modulus of the multi-bionic cartilage hydrogel is 0.8MPa, and the pore size is 100-120μm.

[0064] Example 2

[0065] A preparation method of a multi-bionic cartilage hydrogel, the specific steps are as follows:

[0066] (1) Preparation of materials;

[0067] SFMA: molecular weight of 100-150kDa, grafting rate of 80-85%;

[0068] GelMA: molecular weight of 160-220kDa, grafting rate of 71-90%, manufacturer: Huaxia Siyin (Shanghai) Biotechnology Co., Ltd., product number: R201B2;

[0069] PEGDA: molecular weight of 700Da, manufacturer: Sigma-Aldrich Company, product number: 455008;

[0070] Water;

[0071] LAP: manufacturer: Shanghai Yuanye Biotechnology Co., Ltd., product number: Y43995-5g;

[0072] OBC;

[0073] Stem cells: rat BMSCs, manufacturer Shanghai Yunchun Biotechnology Co., Ltd., item number ER0113;

[0074] (2) Preparation of functional silk fibroin-based bio-ink;

[0075] After dissolving SFMA, GelMA and PEGDA in water, adding LAP after filtration, and then stirring and mixing the filtrate with OBC, a functional silk fibroin-based bio-ink is obtained;

[0076] In the functional silk fibroin-based bio-ink, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, the mass ratio of GelMA to PEGDA is 1:8, the mass fraction of OBC is 0.3wt%, and the mass fraction of LAP is 0.1wt%;

[0077] (3) Preparation of multi-bionic cartilage hydrogel;

[0078] After mixing the functional silk fibroin-based bio-ink with stem cells uniformly, under the irradiation of ultraviolet light with an intensity of 10mW / cm 2 at 25℃, the cross-linking reaction is cured for 20s to obtain a multi-bionic cartilage hydrogel.

[0079] In the finally obtained multi-bionic cartilage hydrogel, the density of stem cells is 2×10 5 cells / mL; the compression modulus of the multi-bionic cartilage hydrogel is 0.4MPa, and the pore size is 40-60μm.

[0080] Example 3

[0081] A preparation method of a multi-bionic cartilage hydrogel, the specific steps are as follows:

[0082] (1) Preparation of materials;

[0083] SFMA: molecular weight 100-150kDa, grafting rate 90-95%;

[0084] GelMA: molecular weight 160-220kDa, grafting rate 71-90%, manufacturer Huaxia Siyin (Shanghai) Biotechnology Co., Ltd., item number R201B2;

[0085] PEGDA: molecular weight 700Da, manufacturer Sigma-Aldrich Co., item number 455008;

[0086] Water;

[0087] LAP: manufacturer Shanghai Yuanye Biotechnology Co., Ltd., item number Y43995-5g;

[0088] OBC;

[0089] Stem cells: Rat BMSCs, manufactured by Shanghai Yuchun Biotechnology Co., Ltd., catalog number ER0113;

[0090] (2) Preparation of functionalized silk fibroin-based bio-ink;

[0091] After dissolving SFMA, GelMA and PEGDA in water, LAP was added and the mixture was filtered. The filtrate was then stirred and mixed with OBC to obtain functionalized silk fibroin-based bio-ink.

[0092] In the functionalized silk fibroin-based bio-ink, the total mass fraction of SFMA, GelMA, and PEGDA is 18 wt%, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, the mass ratio of GelMA to PEGDA is 2:7, the mass fraction of OBC is 0.6 wt%, and the mass fraction of LAP is 0.2 wt%.

[0093] (3) Preparation of multiple biomimetic cartilage hydrogels;

[0094] After uniformly mixing functionalized silk fibroin-based bio-ink with stem cells, at an intensity of 25 mW / cm 2 Under ultraviolet light irradiation, the cross-linking reaction is cured at 37°C for 60 seconds to obtain a multilayer biomimetic cartilage hydrogel.

[0095] In the final multi-layered biomimetic cartilage hydrogel, the density of stem cells was 2 × 10⁻⁶. 5 The multiple biomimetic cartilage hydrogel has a compression modulus of 0.6 MPa and a pore size of 60-80 μm.

[0096] Example 4

[0097] A method for preparing a multi-layered biomimetic cartilage hydrogel, the specific steps of which are as follows:

[0098] (1) Preparation of materials;

[0099] SFMA: Molecular weight 100-150kDa, grafting rate 90-95%;

[0100] GelMA: Molecular weight 160-220kDa, grafting rate 71-90%, manufacturer Huaxia Siyin (Shanghai) Biotechnology Co., Ltd., product number R201B2;

[0101] PEGDA: Molecular weight 700 Da, manufacturer Sigma-Aldrich, catalog number 455008;

[0102] water;

[0103] LAP: Vendor Shanghai Yuanye Biotechnology Co., Ltd., Item No. Y43995-5g;

[0104] OBC;

[0105] Stem cells: Rat BMSCs, vendor Shanghai Yuchun Biotechnology Co., Ltd., item No. ER0113;

[0106] (2) Preparation of functional silk fibroin-based bio-ink;

[0107] After dissolving SFMA, GelMA and PEGDA in water, add LAP after filtration, then mix the filtrate with OBC by stirring to obtain functional silk fibroin-based bio-ink;

[0108] In the functional silk fibroin-based bio-ink, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, the mass ratio of GelMA to PEGDA is 3:6, the mass fraction of OBC is 1.2wt%, and the mass fraction of LAP is 0.4wt%;

[0109] (3) Preparation of multi-bionic cartilage hydrogel;

[0110] After mixing the functional silk fibroin-based bio-ink with stem cells evenly, irradiate under ultraviolet light with intensity of 30mW / cm 2 at 37℃ for 60s to solidify and cross-link, a multi-bionic cartilage hydrogel is obtained.

[0111] In the final multi-bionic cartilage hydrogel, the density of stem cells is 1×10 6 cells / mL; the compression modulus of the multi-bionic cartilage hydrogel is 0.85MPa, and the pore size is 140-160μm.

[0112] Comparative Example 1

[0113] A method for preparing a hydrogel, and the difference from Example 1 is only that in step (2), the addition amount of SFMA, GelMA and PEGDA is adjusted, so that the total mass fraction of SFMA, GelMA and PEGDA in the product of step (2) is 20wt%.

[0114] The pore size of the final hydrogel is 20-30μm.

[0115] Compared with Example 1, the pore size of the hydrogel in Comparative Example 1 is significantly smaller, which is not conducive to cell survival, because the total mass fraction of SFMA, GelMA and PEGDA is too high, the polymer molecules are more closely aggregated during photo-crosslinking, and the network structure formed is more dense, resulting in a smaller pore size.

[0116] Comparative Example 2

[0117] A method for preparing a hydrogel, and the difference from Example 1 is only that in step (2), the addition amount of SFMA, GelMA and PEGDA is adjusted, so that the total mass fraction of SFMA, GelMA and PEGDA in the product of step (2) is 15wt%.

[0118] The compression modulus of the finally prepared hydrogel is 0.36MPa.

[0119] Compared with Example 1, the compression modulus of the hydrogel is obviously reduced, because the total mass fraction of SFMA, GelMA and PEGDA is too low, the cross-linked network formed is too sparse, and the network support is insufficient, resulting in the reduction of the compression modulus.

[0120] Comparative Example 3

[0121] A method for preparing a hydrogel, and the difference from Example 2 is only that in step (2), the addition amount of PEGDA is adjusted, so that the mass ratio of GelMA to PEGDA in the product of step (2) is 0.5:8.5.

[0122] The pore size of the finally prepared hydrogel is 20-30μm.

[0123] Compared with Example 2, the pore size of the hydrogel is obviously smaller, which is not conducive to the survival of cells, because the content of PEGDA in the ink is too high, the molecular weight of PEGDA is smaller, the density of carbon-carbon double bonds contained in unit mass is higher, and the hydrogel network formed is more dense, and the increase of the content of PEGDA leads to the densification of the hydrogel network, thereby causing the reduction of the pore size.

[0124] Comparative Example 4

[0125] A method for preparing a hydrogel, and the difference from Example 4 is only that in step (2), the addition amount of PEGDA is adjusted, so that the mass ratio of GelMA to PEGDA in the product of step (2) is 3.5:5.5.

[0126] The pore size of the finally prepared hydrogel is 170-180μm.

[0127] Compared with Example 4, the pore size of the hydrogel is obviously larger, which is not conducive to the growth of cell aggregation, because the content of GelMA in the ink is too high, the density of carbon-carbon double bonds contained in unit mass is lower, the hydrogel network formed is more sparse, and the pore size of the hydrogel is larger.

[0128] Comparative Example 5

[0129] A preparation method of a hydrogel, and the difference between Example 2 is only that the amount of OBC added in step (2) is adjusted, so that the mass fraction of OBC in the product of step (2) is 0.2wt%.

[0130] The compression modulus of the finally prepared hydrogel is 0.31MPa.

[0131] Compared with Comparative Example 5 and Example 2, the compression modulus of the hydrogel is obviously reduced, because when the content of OBC is insufficient, it cannot effectively enhance the supporting force of the cross-linked network, so the compression modulus is obviously reduced.

[0132] Comparative Example 6

[0133] A preparation method of a hydrogel, and the difference between Example 4 is only that the amount of OBC added in step (2) is adjusted, so that the mass fraction of OBC in the product of step (2) is 1.5wt%.

[0134] In order to explore the influence of the functional silk fibroin-based bio-ink formula on the printing precision of the hydrogel and the chondrogenic differentiation ability of the stem cells, the present application verifies through the following experiments:

[0135] (1) Printing precision detection;

[0136] The printing model (containing microneedle array structure) as shown in Figure 1 is designed, and the hydrogels of Comparative Example 2, Comparative Example 6, Example 1 and Example 4 are printed respectively, and the matching degree of the printed hydrogel and the model, the integrity of the microstructure are observed and compared, and the printing precision is evaluated;

[0137] The results are shown in Figure 2 (a)-(d), from Figure 2 it can be seen that the printed hydrogels of Example 1 and Example 4 have higher matching degree with the printing model, higher resolution of complex model and more complete microstructure compared with Comparative Example 2 and Comparative Example 6, which indicates that reasonable formula can guarantee higher printing precision, and the formula design of Comparative Example 2 and Comparative Example 6 will destroy the uniformity of the cross-linked network and reduce the printing precision; among them, the smaller density of carbon-carbon double bond in Comparative Example 2 affects the printing precision; in Comparative Example 6, the content of OBC is too high, which is difficult to disperse uniformly in the hydrogel, destroys the uniformity of the cross-linked network, and reduces the printing precision;

[0138] (2) Chondrogenic differentiation ability detection of stem cells;

[0139] Hydrogels prepared in Comparative Examples 4, 6, 1, and 4 were cultured for 7 days, and then chondrogenic differentiation was assessed using RT-PCR. The hydrogels were crushed with liquid nitrogen, and the fragments were transferred to centrifuge tubes. mRNA was extracted using chloroform, isopropanol, and anhydrous ethanol, and reverse transcribed to obtain cDNA templates. Real-Time PCR reaction solution and primers were used, with ACAN as the chondrogenic characteristic gene and GAPDH as the internal reference gene. The results were obtained by 2... -ΔΔCT Relative quantitative analysis of gene expression levels;

[0140] The results are as follows Figure 3 As shown, from Figure 3 As can be seen, after stem cells were cultured in hydrogels in Examples 1 and 4, the expression level of the chondrogenic differentiation-related gene (ACAN) was significantly better than that in Comparative Examples 4 and 6. This indicates that a reasonable formulation can enable stem cells to survive well and differentiate into chondrocytes. However, an imbalance in the proportion of each component in the formulation can reduce the chondrogenic differentiation ability of stem cells by affecting the pore size of the hydrogel, cell adhesion, and physicochemical stability. In Comparative Example 4, the excessive GelMA content led to an excessively strong adhesion of the hydrogel to the internal stem cells. This strong adhesion caused the internal stem cells to adhere and spread, which was not conducive to cell aggregation and thus hindered chondrogenic differentiation. In Comparative Example 6, the excessive OBC content could not be evenly dispersed in the hydrogel, resulting in unstable physicochemical properties of the overall hydrogel, which affected cell behavior and further reduced the chondrogenic differentiation ability of the internal stem cells.

Claims

1. A functionalized silk fibroin-based bio-ink, characterized in that, The initiator, SFMA, GelMA, PEGDA and OBC are contained, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, and the mass fraction of OBC is 0.3-1.2wt%, and SFMA, GelMA, PEGDA and OBC correspond to methacrylated silk fibroin, methacrylated gelatin, polyethylene glycol diacrylate and oxidized bacterial cellulose nanofiber respectively.

2. The functionalized silk fibroin-based bioink according to claim 1, wherein, The molecular weight of SFMA is 70-150kDa, and the grafting rate is 80-95%; the molecular weight of GelMA is 160-220kDa, and the grafting rate is 71-90%; and the molecular weight of PEGDA is 700Da.

3. The functionalized silk fibroin-based bio-ink according to claim 1, wherein, The mass fraction of the initiator is 0.1-0.4wt%.

4. A multiplexed biomimetic cartilage hydrogel, characterized in that, The multiple network structure hydrogel and OBC and stem cells dispersed in the multiple network structure hydrogel at the same time are included; the multiple network structure hydrogel is crosslinked by SFMA, GelMA and PEGDA; the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, and the mass fraction of OBC is 0.3-1.2wt%, and SFMA, GelMA, PEGDA and OBC correspond to methacrylated silk fibroin, methacrylated gelatin, polyethylene glycol diacrylate and oxidized bacterial cellulose nanofiber respectively.

5. The multiphasic biomimetic cartilage hydrogel of claim 4, wherein, The molecular weight of SFMA is 70-150kDa, and the grafting rate is 80-95%; the molecular weight of GelMA is 160-220kDa, and the grafting rate is 71-90%; and the molecular weight of PEGDA is 700Da.

6. The multiphasic biomimetic cartilage hydrogel of claim 4, wherein, In the multiple biomimetic cartilage hydrogels, the density of stem cells was 2 x 10 5 cells / mL-1 x 10 6 cells / mL.

7. The multiphasic biomimetic cartilage hydrogel of claim 4, wherein, The compression modulus of the multiple biomimetic cartilage hydrogel is 0.4-0.85MPa, and the pore size is 40-160μm.

8. A method of preparing a multiphasic biomimetic cartilage hydrogel according to claim 4, wherein, After the functionalized silk fibroin-based bio-ink according to any one of claims 1-3 is mixed with stem cells uniformly, a solidification crosslinking reaction is initiated, and the multiple biomimetic cartilage hydrogel is obtained. The initiator, SFMA, GelMA, PEGDA and OBC are contained, the total mass fraction of SFMA, GelMA and PEGDA is 18wt%, the mass ratio of GelMA to PEGDA is 1:8-3:6, the mass ratio of SFMA to the total mass of GelMA and PEGDA is 1:1, and the mass fraction of OBC is 0.3-1.2wt%, and SFMA, GelMA, PEGDA and OBC correspond to methacrylated silk fibroin, methacrylated gelatin, polyethylene glycol diacrylate and oxidized bacterial cellulose nanofiber respectively.

9. The method of claim 8, wherein, The initiation of the curing cross-linking reaction is performed by irradiation of ultraviolet light, the curing cross-linking reaction is performed at a temperature of 25-37℃ for 20-60s, and the intensity of the ultraviolet light is 10-30mW / cm 2 .

Images