Shape memory short fiber stiffened hydrogel and application thereof

Abstract

The invention discloses a preparation method and application of shape memory short fiber stiffened hydrogel, and the preparation method comprises the following steps: obtaining a copolymer PLCL nanofiber membrane of poly-L-lactic acid and caprolactone through electrostatic spinning, carrying out stretching shaping and cutting at a certain temperature, dispersing in a low-temperature homogenizer to form shape memory short fibers SMSFs, and carrying out shape memory short fiber reinforcement on the shape memory short fibers SMSFs to obtain the shape memory short fiber hydrogel. The SMSFs are added into the double-network hydrogel, and the shape memory short fiber stiffened hydrogel is obtained by exciting the shape memory effect of the SMSFs. The invention relates to an application of a shape memory short fiber, which is used for preparing shape memory short fiber stiffened hydrogel for promoting osteogenic differentiation and inhibiting osteoclast generation. According to the shape memory short fiber stiffened hydrogel and the application method thereof, the stiffening effect of the shape memory short fiber stiffened hydrogel has the direct regulation and control effect on stem cell osteogenic differentiation and indirectly has the ability of inhibiting osteoclast formation, and a new way is provided for preparing a bone regeneration scaffold for osteoporotic bone defect repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and relates to a preparation method and application of a shape memory short fiber reinforced hydrogel, and particularly relates to a method for promoting osteogenic differentiation and inhibiting osteoclastogenesis of a shape memory short fiber reinforced hydrogel. Background Art

[0002] Osteoporosis is a disease of bone homeostasis imbalance caused by aging and decreased estrogen levels, resulting in increased bone resorption and decreased bone formation. Due to the decrease in bone strength and bone mass, osteoporosis patients are extremely prone to bone defects. Bone tissue engineering combines cells, biological factors, and biomimetic scaffolds to form an analogue of bone tissue in vitro and transplant it to the osteoporosis bone defect site, replacing natural transplanted bone, which can effectively solve the problems of donor shortage and immunogenicity. The design of a biomimetic bone tissue engineering scaffold with clear biological, physical and other functions has guiding significance for the regulation of specific cell behaviors. Therefore, designing an effective bone tissue engineering scaffold for promoting osteogenesis is crucial for breaking bone homeostasis imbalance and treating local osteoporotic bone defects.

[0003] In mechanically sensitive bone tissue, various resident cells latent in the bone tissue can promptly sense the mechanical changes in the surrounding bone tissue and initiate cell biochemical signals. For example, stem cells are affected by the physical signals of the scaffold (such as stiffness, dynamic mechanics, roughness, porosity, pore size, and micropatterns), especially after mechanical stimulation, and initiate the osteogenic differentiation program through the perception of mechanical sensitive receptors on the cell membrane - the classical mechanical signal transduction pathway. Hydrogel is composed of cross-linked water-soluble polymers and is the tissue engineering scaffold that can best mimic the extracellular matrix components of natural tissues. Hydrogels can be used to initiate the bone regeneration in vivo effectively by changing their stiffness and constructing osteogenic differentiated bone tissue engineering implants in vitro. However, the current method for regulating the stiffness of hydrogels mainly relies on increasing the concentrations of polymers, cross-linking agents, and fillers, which will bring problems such as limited biocompatibility and limited level of stiffness improvement.

[0004] Osteoblasts and osteoclasts are in a balanced state in normal bone tissue, mainly relying on the signal communication between their respective paracrine factors. For example: osteoblasts secrete receptor activator of nuclear factor κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), interleukin-6 (IL-6), chemokine CX3C ligand 1 (CX3CL1), monocyte chemoattractant protein (MCP-1), etc., to regulate the attachment, proliferation, differentiation, and maturation of osteoclasts on the bone surface to promote bone resorption. Osteoblasts can also secrete osteoprotegerin (OPG), prostaglandin E2 (PGE 2)、IL-18, IL-33 and other active factors inhibit the bone resorption behavior of osteoclasts. Conversely, osteoclasts also secrete active factors that positively and negatively regulate the behavior of osteoblasts. Under specific stimuli, the types and contents of active factors produced by osteoblasts and osteoclasts will change, which depends on which signal pathways for the synthesis of biochemical factors are activated by the stimuli given to the cells from the outside world. Summary of the Invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the existing methods for regulating the stiffness of hydrogels mainly rely on increasing the concentrations of polymers, cross-linking agents and fillers, which will bring problems such as limited biocompatibility, limited level of stiffness improvement and affecting injectability, and cannot stimulate the growth of osteoblasts nor inhibit osteoclasts. Therefore, the present invention provides a preparation method and application of a shape memory short fiber-reinforced hydrogel, whose stiffening effect has a direct regulatory effect on the osteogenic differentiation of stem cells and an indirect inhibitory ability on osteoclast formation, providing a new method for the preparation of bone regeneration scaffolds for the repair of osteoporotic bone defects.

[0006] To achieve the above object, the present invention provides a preparation method of a shape memory short fiber-reinforced hydrogel. A PLCL nanofiber membrane is obtained by electrospinning, stretched and shaped at a certain temperature, cut into pieces, and then dispersed into shape memory short fibers (SMSFs) by a low-temperature homogenizer;

[0007] The SMSFs are added to a double-network hydrogel, and a shape memory short fiber-reinforced hydrogel is obtained by activating the shape memory effect of the SMSFs;

[0008] Among them, the double network includes a first network and a second network. The first network is a Schiff base reaction between an aldehyde-functionalized polymer and adipic dihydrazide; the second network is a photocrosslinking reaction between a methacrylated polymer and a diacrylated polymer under the irradiation of an initiator, blue light / ultraviolet light.

[0009] Further, obtaining a PLCL nanofiber membrane by electrospinning specifically includes: weighing 12 wt% of PLCL and 0.5 wt% of polyethylene oxide (PEO), dissolving them in 10 mL of hexafluoroisopropanol, and stirring overnight, where the LA:CL of PLCL is 90:10 mol:mol;

[0010] Preparing a PLCL electrospun fiber membrane under the conditions of a flow rate of 0.5 mL / h, an electric field strength of 5 - 10 kV, a receiving distance of 10 - 15 cm, a roller rotation speed of 1000 rpm, room temperature and a humidity of 40 - 60%.

[0011] Furthermore, the stretching and shaping are carried out at a certain temperature. After being cut into pieces, they are dispersed into shape memory short fibers (SMSFs) by a low-temperature homogenizer, which specifically includes: cutting the prepared fiber membrane into rectangles, clamping it on a shaping fixture, stretching and shaping it at 40 °C, and then quickly fixing the fiber membrane at 0 °C; cutting the shaped PLCL fiber membrane into fiber fragments, placing them in cold water at 0 - 4 °C, and dispersing them for 10 minutes under high-speed shearing at 20,000 rpm by the homogenizer to obtain SMSFs.

[0012] Another preferred embodiment of the present invention provides an application of a shape memory short fiber-rigidified hydrogel for promoting osteogenic differentiation and inhibiting osteoclastogenesis.

[0013] Another preferred embodiment of the present invention provides a method for promoting osteogenic differentiation and inhibiting osteoclastogenesis of a shape memory short fiber-rigidified hydrogel, including the following steps:

[0014] Preparing shape memory short fibers (SMSFs);

[0015] Introducing a certain amount of SMSFs into the precursor solution of the double-network hydrogel, forming the first-stage stiffness N1 through Schiff base cross-linking, forming the double-network hydrogel with the second-stage stiffness N2 after ultraviolet or blue light irradiation, and then activating the shape memory effect (SME) of SMSFs to enhance the stiffness of the double-network hydrogel to form the third-stage stiffness N3;

[0016] The hydrogel with the N3 stiffness promotes the osteogenic differentiation of stem cells, and the conditioned medium (CM) after promoting osteogenic differentiation inhibits the osteoclastic differentiation of macrophages.

[0017] Furthermore, the dosage of SMSFs is 0.5 - 2%.

[0018] Furthermore, the precursor solution of the double-network hydrogel includes aldehyde-functionalized sodium alginate, methacrylated gelatin, polyethylene glycol diacrylate, adipic dihydrazide, and a photoinitiator.

[0019] Furthermore, the stem cells are bone marrow mesenchymal stem cells.

[0020] Furthermore, the macrophages include RAW264.7 macrophages.

[0021] Furthermore, the osteogenic differentiation CM is a mixture formed by the paracrine factors produced by the osteogenic differentiation of bone marrow mesenchymal stem cells and the culture medium.

[0022] Technical effects

[0023] In the present invention, PLCL is processed into SMSFs by methods such as electrospinning, shearing, and homogenization. The preparation process is simple, easy to operate, and has stable batches, which is conducive to large-scale preparation.

[0024] The use of SMSFs in SMEs of the present invention can effectively improve the stiffness of hydrogels, which is a new way to enhance stiffness; the double-network hydrogel precursor solution containing SMSFs has the ability to minimally invasively inject and fill bone defects.

[0025] The SMSFs-rigidified hydrogels of the present invention can directly promote the osteogenic differentiation of bone marrow mesenchymal stem cells and indirectly inhibit the ability of osteoclast differentiation.

[0026] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, features and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a microscopic observation of SMSFs (pseudo-color processing) of a preferred embodiment of the present invention;

[0028] Figure 2 are SEM images (A) and pore size quantification (B) of hydrogels with different stiffnesses;

[0029] Figure 3 are compression stress-strain curves (A) and Young's moduli (B) of hydrogels with different stiffnesses;

[0030] Figure 4 is the rheological test of hydrogels with different stiffnesses;

[0031] Figure 5 is the promotion of osteogenic differentiation of bone marrow mesenchymal stem cells by SMSFs-rigidified hydrogels: (A) cell spreading; (B) cell proliferation; (C-D) alkaline phosphatase (ALP) staining and quantification; (E-F) alizarin red staining and quantification; (G) osteogenic-related gene expression;

[0032] Figure 6 is the expression of mechanics-related genes for the promotion of osteogenic differentiation of bone marrow mesenchymal stem cells by SMSFs-rigidified hydrogels;

[0033] Figure 7 is the promotion of osteogenic differentiation by SMSFs-rigidified hydrogels and indirect inhibition of osteoclastogenesis: (A-B) tartrate-resistant acid phosphatase (TRAP) staining and quantification; (C) osteoclastogenesis-related gene expression. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] In the following description, specific details such as specific internal programs and technologies are presented for the purpose of illustration rather than limitation, in order to thoroughly understand the embodiments of the present invention. However, those skilled in the art should understand that the present invention can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to avoid unnecessary details from hindering the description of the present invention.

[0036] Synthesis of raw materials for preparing hydrogels:

[0037] Dissolve 2 g of sodium alginate in 100 mL of deionized water and dissolve it evenly. Dissolve 1.08 g of sodium periodate in 20 mL of deionized water, add it to the sodium alginate solution under light-shielded conditions, stir at room temperature for 2 h, then add 1.6 mL of ethylene glycol to terminate the reaction, and stir for 1 h. Finally, dialyze the reaction solution with a dialysis bag with a molecular weight of 0.8 - 1.2 kDa for 3 days and freeze-dry for 3 days to obtain aldehyde-functionalized sodium alginate.

[0038] Dissolve 4 g of gelatin in 30 mL of carbonate buffer (0.075 M Na 2 CO 3 and 0.175 M NaHCO 3 ), heat to 60 °C to completely dissolve, then add 0.2 mL of methacrylic anhydride, react at 50 °C for 3 h, finally add 150 mL of deionized water to terminate the reaction, dialyze with a dialysis bag with a molecular weight of 0.8 - 1.2 kDa for 3 days, and freeze-dry for 3 days to obtain methacryloylated gelatin.

[0039] Example 1

[0040] This example describes the preparation method of SMSFs, including the following steps:

[0041] (1) Preparation of PLCL 90 / 10 electrospun fibers

[0042] Weigh 12 wt% of PLCL (LA:CL = 90:10 mol:mol), 0.5 wt% of PEO and dissolve them in 10 mL of hexafluoroisopropanol, and stir overnight. Prepare a PLCL electrospun fiber membrane under the conditions of a flow rate of 0.5 mL / h, an electric field strength of 5 - 10 kV, a receiving distance of 10 - 15 cm, a roller rotation speed of 1000 rpm, room temperature, and a humidity of 40 - 60%.

[0043] (2) Preparation of SMSFs

[0044] Cut the prepared fiber membrane into rectangles, clamp it on the shaping fixture, stretch and shape it at 40 °C, and then quickly fix the fiber membrane at 0 °C. Cut the shaped PLCL fiber membrane into fiber fragments, place them in cold water at 0-4 °C, and disperse them under high-speed (20,000 rpm) shearing by a homogenizer for 10 min to obtain SMSFs (as Figure 1 shown). Cut the unshaped PLCL fiber membrane into fiber fragments, place them in cold water at 0-4 °C, and disperse them under high-speed (20,000 rpm) shearing by a homogenizer for 10 min to obtain non-shape memory short fibers, named P0.

[0045] Example 2

[0046] This example describes a method for preparing a double-network hydrogel, including the following steps:

[0047] Weigh 0.6 g of aldehyde-functionalized sodium alginate, 0.3 g of methacrylated gelatin, 0.5 mL of polyethylene glycol diacrylate, and 0.02 g of LAP photoinitiator and dissolve them in 10 mL of PBS (pH = 7.4). Stir the mixed solution evenly to form Solution 1; weigh a certain amount of adipic dihydrazide and prepare a 4 wt% adipic dihydrazide solution to form Solution 2. After mixing according to the volume ratio of Solution 1: Solution 2 of 10:1, Schiff base cross-linking occurs in 5 min to form the first-layer network cross-linked hydrogel; then irradiate it with blue light for 1 min to prepare the second-layer network cross-linked hydrogel.

[0048] Among them, the double network refers to a hydrogel formed by two layers of networks. The first layer of network is formed by cross-linking aldehyde-functionalized sodium alginate and adipic dihydrazide; the second layer of network is formed by photo-cross-linking methacrylated gelatin and polyethylene glycol diacrylate.

[0049] Example 3

[0050] This example describes a method for preparing hydrogels with different stiffnesses based on SMSFs and double networks, including the following steps:

[0051] Weigh 0.6 g of aldehyde-functionalized sodium alginate, 0.3 g of methacrylated gelatin, 0.5 mL of polyethylene glycol diacrylate, and 0.02 g of LAP photoinitiator, and dissolve them in 10 mL of PBS (pH = 7.4). Stir the mixed solution evenly to form Solution 1; weigh 0.1 g of the SMSFs prepared in Example 1 and P0 non-shape memory short fibers respectively, and disperse them evenly to form Solution 2; stir Solution 1 evenly with 1 mL of Solution 3 (4 wt% adipic dihydrazide), and Schiff base crosslinking occurs in 5 min to prepare a first-stage stiffness hydrogel N1; then irradiate N1 with blue light for 1 min to prepare a second-stage stiffness hydrogel N2; after mixing Solutions 1, 2, and 3 together, let it stand for 5 min, then irradiate with blue light for 1 min, and finally heat the hydrogel in a water bath to 40 °C and maintain it for 10 min to obtain a hydrogel with enhanced shape memory effect and a hydrogel reinforced with non-shape memory short fibers, named N3 and P0-N3 respectively.

[0052] The microscopic morphology (scanning electron microscopy) of the hydrogels N1, N2, N3, and P0-N3 prepared in this example shows that as the stiffness increases, the pores of the hydrogel decrease, N1 (240.40 ± 40.39 μm) > N2 (218.93 ± 40.90 μm) > P0-N3 (208.90 ± 48.21 μm) > N3 (124.79 ± 18.56 μm) (as Figure 2 shown). The Young's moduli are ((N1: 9.27 ± 0.77 kPa, N2: 26.49 ± 3.48 kPa, N3: 53.40 ± 3.40 kPa, P0-N3: 34.35 ± 3.31 kPa) (as Figure 3 shown). The results of rheological tests (as Figure 4 shown) show that for the N3 group compared with the P0-N3 group, as the temperature increases (from 25 - 50 °C), the storage modulus gradually increases.

[0053] Among them, the decrease in the pores of the hydrogel is caused by the shrinkage of the shape memory short fibers, and it is also a phenomenon reflecting the increase in stiffness; the Young's modulus is a direct measure parameter of the stiffness, and the larger the value, the greater the stiffness; and the storage modulus is another detection method for the change in stiffness, and the larger the storage modulus, the greater the stiffness.

[0054] Experimental Example 1

[0055] This experimental example describes the ability of hydrogels with different stiffnesses to promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro:

[0056] The raw materials for preparing hydrogels with different stiffnesses in Example 3, oxidized sodium alginate and methacrylated gelatin, were soaked in 90% alcohol for 3 h and then air-dried for later use, obtaining solid A. The SMSFs were sterilized by ultraviolet irradiation for 12 h to form solid B. Polyethylene glycol acrylate and LAP were dissolved in PBS and filtered through a 0.22-μm filter membrane for sterilization to form solution A. Adipic dihydrazide was dissolved in deionized water to form solution B. Solid A and B were dissolved in solution A, and then mixed with solution B, and immediately added to a 24-well cell culture plate. The subsequent cross-linking process was the same as that in Example 3. The cross-linked toxic substances in the hydrogel were refreshed with the culture medium.

[0057] 2×10 4 mesenchymal stem cells per milliliter were seeded on the surface of the hydrogel. After culturing for 1 day, the cell spreading ability was observed; after adding osteogenic induction medium for differentiation for 7 days, the ability of promoting osteogenic differentiation was detected by ALP, alizarin red staining and RT-PCR. The results showed (as Figure 5 shown) that compared with N2 and P0-N3, the increase in stiffness of the N3 group was beneficial to cell spreading and proliferation. Through ALP staining and quantification of early osteogenic markers, it was found that the increase in stiffness significantly promoted the osteogenic differentiation of stem cells; in the detection of mid- and late-stage osteogenic markers, it was found that more calcium nodules were deposited in the N3 group; through RT-PCR detection of genes related to mechanical promotion of osteogenic differentiation, it was found that the osteogenic differentiation markers COL I, Runx 2, OPG and OCN genes were all up-regulated, and further detection of genes such as Vinculin, Rho A, Rock 2 and YAP in the mechanical signaling pathway showed that they were all significantly up-regulated in the N3 group (as Figure 6 shown). These results indicate that the SMSFs-rigidified hydrogel may promote osteogenic differentiation through a series of signaling pathways of focal adhesion proteins (FAs)-Rho A / ROCK-YAP nuclear translocation.

[0058] Experimental Example 2

[0059] After collecting the conditioned media of the hydrogels with different stiffnesses for osteogenic induction and differentiation of mesenchymal stem cells for 7 days in Experimental Example 1, they were added in a ratio of 2:8 to the macrophage medium and mixed with 100 ng / mL RANKL osteoclast differentiation inducer. After 5 days of differentiation, the effect on osteoclast differentiation ability was detected by TRAP and RT-PCR. The results are as Figure 7As shown, the paracrine factors of the hydrogel with enhanced shape memory effect that promote osteogenic differentiation have a significantly stronger ability to inhibit the expression of TRAP compared to the paracrine factors of the other two groups. Further, through gene detection, it was found that the genes of the osteoclast differentiation-related markers CTSK, SRC, and TRAP were all downregulated. At the same time, the genes of the upstream key factor TRAF 6 and the osteoclast differentiation transcription factor NFATc 1 of osteoclast differentiation were also found to be downregulated, indicating that the enhanced osteogenic differentiation ability caused by the increased stiffness has an indirect inhibitory effect on osteoclast differentiation, and it also shows that the osteogenesis-related paracrine factors regulate the osteogenesis / osteoclast balance by inhibiting the RANKL / RANK / OPG signaling pathway.

[0060] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the protection scope determined by the claims.

Claims

1. A method for preparing a shape memory short fiber rigid hydrogel, characterized in that: The PLCL nanofiber membrane of poly (L-lactic acid) and caprolactone copolymer was obtained by electrospinning, stretched and shaped at a certain temperature, cut into pieces, and dispersed into shape memory short fibers SMSFs in a low-temperature homogenizer. SMSFs were added into the double network hydrogel, and the shape memory short fiber rigid hydrogel was obtained by stimulating the shape memory effect of SMSFs. Among them, the double network includes a first layer network and a second layer network. The first layer network is a Schiff base reaction between the aldehyde-modified polymer and adipic acid dihydrazide; the second layer network is a photo-crosslinking reaction between the methacrylated polymer and the bisacrylated polymer under the irradiation of an initiator, blue light / ultraviolet light.

2. The method for preparing a shape memory staple fiber according to claim 1, characterized in that: The PLCL nanofiber membrane was obtained by electrospinning, specifically comprising: weighing 12 wt% PLCL and 0.5 wt% g polyethylene oxide PEO and dissolving them in 10 mL hexafluoroisopropanol, stirring overnight, wherein LA:CL of PLCL=90:10 mol:mol; The PLCL electrospun fiber membrane was prepared under the conditions of flow rate 0.5 mL / h, electric field strength 5-10 kV, acceptance distance 10-15 cm, drum speed 1000 rpm, room temperature and 40-60% humidity.

3. The method for preparing a shape memory staple fiber according to claim 2, characterized in that: The fiber membrane is stretched and shaped at a certain temperature, shredded, and dispersed into shape memory short fibers SMSFs in a low-temperature homogenizer, specifically comprising: cutting the prepared fiber membrane into a rectangle, clamping it on a shaping fixture, stretching and shaping it at 40°C, and then quickly fixing the fiber membrane at 0°C; cutting the shaped PLCL fiber membrane into fiber fragments, placing them in cold water at 0-4°C, and dispersing them for 10 minutes under high-speed shearing of 20,000 rpm in a homogenizer to obtain SMSFs.

4. An application of a shape memory short fiber rigidified hydrogel, characterized in that: Used to promote bone differentiation and inhibit osteoclastogenesis.

5. A method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using shape memory short fiber rigidified hydrogel, characterized in that: The following steps are involved: Preparation of shape memory short fibers SMSFs; A certain amount of SMSFs is introduced into the double network hydrogel precursor solution, and the first-level stiffness N1 is formed after Schiff base crosslinking. After ultraviolet light or blue light irradiation, the double network hydrogel is formed to form the second-level stiffness N2. Then, the shape memory effect SME of SMSFs is stimulated to enhance the stiffness of the double network hydrogel to form the third-level stiffness N3. The hydrogel with N3 stiffness promoted the osteogenic differentiation of stem cells, and the conditioned medium CM after promoting osteogenic differentiation inhibited the osteoclast differentiation of macrophages.

6. The method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using a shape memory short fiber rigidified hydrogel according to claim 5, characterized in that: The dosage of SMSFs is 0.5-2%.

7. The method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using a shape memory short fiber rigidified hydrogel according to claim 5, characterized in that: The double network hydrogel precursor solution includes aldehyde-modified sodium alginate, methacrylated gelatin, polyethylene glycol diacrylate, adipic acid dihydrazide and a photoinitiator.

8. The method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using a shape memory short fiber rigidified hydrogel according to claim 5, characterized in that: The stem cells are bone marrow mesenchymal stem cells.

9. The method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using a shape memory short fiber rigidified hydrogel according to claim 5, characterized in that: The macrophages include RAW264.7 macrophages.

10. The method for promoting osteodifferentiation and inhibiting osteoclastogenesis by using a shape memory short fiber rigidified hydrogel according to claim 5, characterized in that: Osteogenic differentiation CM is a mixture of paracrine factors produced by osteogenic differentiation of bone marrow mesenchymal stem cells and culture medium.

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