Preparation method of polypeptide photo-crosslinking hydrogel material loaded with human dental pulp stem cells

Abstract

The invention belongs to the field of hydrogel preparation, and relates to a preparation method of a polypeptide photo-crosslinking hydrogel material loaded with human dental pulp stem cells, and the method comprises the following steps: uniformly mixing methacrylic acid modified BFP1 polypeptide with a GelMA hydrogel solution and human dental pulp stem cells, and then carrying out photo-crosslinking under the action of a photoinitiator; finally, the polypeptide photo-crosslinking hydrogel material loaded with the human dental pulp stem cells is formed. According to the invention, methacrylic acid modified BFP1 polypeptide and a GelMA hydrogel material are photo-crosslinked for the first time and are combined with human dental pulp stem cells for bone repair, so that the bone repair effect is improved; the obtained polypeptide hydrogel material has excellent stem cell carrying capacity.

Description

Technical Field

[0001] The invention belongs to the field of hydrogel preparation and relates to a method for preparing a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells. Background Art

[0002] Developmental defects, severe periodontitis, apical periodontitis, maxillofacial tumors, and cysts can all lead to maxillofacial bone defects. Large bone defects are difficult to heal on their own. Since traditional bone defect repair has certain risks, how to repair large maxillofacial bone defects and restore their functions remains a difficult point in clinical treatment. The emergence of bone tissue engineering effectively promotes bone regeneration while avoiding the risks of autologous bone and allogeneic bone transplantation. The biological substitutes of bone tissue engineering are composed of seed cells, bioactive factors, and scaffolds, which can restore, maintain, and improve the function of damaged bone tissue, and assist in bone defect repair by delivering exogenous cells and growth factors.

[0003] Some studies have proposed fibroblast growth factor FGF-2, FGF-4, transforming growth factor β1 (TGF-β1) and peptide drugs as alternative osteogenic stimulators. Among these osteogenic factors, peptide drugs have many advantages because their characteristics include low production cost, low incidence of antigen-antibody reaction, and the structure can be easily changed to adapt to the drug target. Finding new peptide drug-related factors is of great significance for improving bone repair effects. Bone morphogenetic protein 7 (BMP-7) is a recognized effective osteogenic induction protein. However, large molecular proteins are easily degraded in the body, and they often denature during the process of fixing them to biological scaffolds, and they have significant immunogenicity. Bone morphogenetic peptide-1 (BFP1) is a peptide sequence of amino acids extracted from the immature region of BMP-7, which has higher osteogenic activity than BMP-7. In addition to good stability, this peptide has a better ability to promote bone formation than BMP-7.

[0004] Bone repair scaffolds are usually loaded with growth factors to stimulate seed cells to proliferate and differentiate into osteoblasts, activate osteoblast function, promote osteoblast activity, and induce bone regeneration. However, the half-life of growth factors in the body is short, which limits their clinical application.

[0005] And because the supply of endogenous cells in the bone defect area is limited, transplantation or replacement materials are needed to deliver exogenous cells to promote bone defect repair. However, the commonly used bone marrow mesenchymal stem cells have limited sources and the acquisition process is relatively traumatic, which limits their clinical application. Summary of the invention

[0006] Purpose of the Invention

[0007] In order to solve the problems existing in the above-mentioned prior art, the present invention aims to provide a method for preparing a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells. The polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells prepared by the method of the present invention can solve the problems of the sudden release of growth factors loaded by hydrogels and the limited supply of endogenous cells in the bone defect area in the prior art.

[0008] Technical solution: A method for preparing a polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells. The methacrylic acid-modified BFP1 polypeptide is evenly mixed with the GelMA hydrogel solution and human dental pulp stem cells, and then photocrosslinked under the action of a photoinitiator to finally form a polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells.

[0009] Furthermore, the density of human dental pulp stem cells in the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells is 1 to 1.5*10 6 Pieces / mL.

[0010] Furthermore, the GelMA hydrogel solution is a freeze-dried GelMA hydrogel material dissolved in a PBS solution at a concentration of 5wt%-10wt%. During the process, a magnetic stirrer is used to heat and stir at a speed of 500-1000 r / min and 50-60° C. for 20-30 min.

[0011] Furthermore, the specific mixing step is as follows: adding methacrylic acid-modified BFP1 polypeptide and photoinitiator to the GelMA hydrogel solution, mixing evenly by blowing and beating with a pipette for 20 to 30 times, and obtaining a polypeptide hydrogel precursor solution for use; taking 3 to 5 generations of human dental pulp stem cells, digesting with trypsin until the cell morphology becomes round and cells begin to float, terminating the digestion with 2 to 3 times the volume of trypsin and a complete culture medium containing 10 to 15% serum, collecting the cell suspension and counting the cell concentration under a microscope, taking the cell suspension containing the required amount of cells after counting, centrifuging at a speed of 800 to 1000 r / min for 5-8 minutes, obtaining a mass of human dental pulp stem cells, removing the supernatant after centrifugation, adding the polypeptide hydrogel precursor solution, blowing and beating with a pipette for 20 to 30 times until the solution begins to become turbid, obtaining a polypeptide hydrogel precursor solution of evenly mixed human dental pulp stem cells, and irradiating with a light source of 360 to 405 nm for 20 to 30 seconds to obtain a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells.

[0012] Furthermore, the concentration of the methacrylic acid-modified BFP1 polypeptide in the polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells is 50-100 μg / mL, and the amount of the photoinitiator added is 0.1-0.5wt% of the polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells, expressed as a percentage by weight of the material.

[0013] Furthermore, the method for extracting human dental pulp stem cells is as follows:

[0014] Healthy and complete young permanent teeth extracted due to impaction or orthodontic reduction were collected. Young permanent teeth were permanent teeth grown by people aged ≤25 years old. The pulp was removed under sterile conditions, and the pulp was cut into pieces with a tissue block size of less than 1 mm. 200-300 μL of a 1:1 mixture of type I and type II collagenase solution was added for digestion for 10-15 minutes, and 1-2 times the volume of the collagenase solution was added to a complete culture medium containing 15%-20% serum to neutralize it. The mixture was centrifuged at a speed of 1800-2000 r / min for 5-8 minutes, and all the supernatant was aspirated, leaving only the tissue block precipitate. Then 500 μL-1 mL of complete culture medium containing 15%-20% serum was added, and the mixture was pipetted 20-30 times with a 2.5 ml plastic pipette until there were no obvious clumps. The mixture was inoculated into a culture dish and cultured in a cell culture dish for 1 to 2 weeks. The cells that crawled out of the tissue block were recorded as cells of the 0th generation, and cells of the 3rd to 5th generations were taken to prepare polypeptide photo-cross-linked hydrogel materials loaded with human dental pulp stem cells.

[0015] Furthermore, the cells of generation 0 have a growth area greater than 4 cm after culturing for 3-4 days. 2 The cells were passaged at 3 to 5 generations and used to prepare polypeptide photo-crosslinked hydrogel materials loaded with human dental pulp stem cells.

[0016] Advantages and effects

[0017] The present invention is the first to photo-crosslink the methacrylic acid-modified BFP1 polypeptide with the GelMA hydrogel material and combine it with human dental pulp stem cells for bone repair. The BFP1 polypeptide can promote the osteogenic differentiation of human dental pulp stem cells and has the effect of promoting angiogenesis, which jointly promotes the function of osteogenesis and improves the bone repair effect; the BFP1 polypeptide is modified with methacrylic acid and then photo-crosslinked with the GelMA hydrogel material and mixed with human dental pulp stem cells to prepare a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells. On the one hand, the BFP1 polypeptide can be released for a long time as the hydrogel material degrades slowly, avoiding the problem of inactivation caused by its short-term burst release, and on the other hand, a better stem cell carrying capacity of the polypeptide hydrogel material is obtained.

[0018] GelMA hydrogel is loaded with peptide drugs BFP1 and human dental pulp stem cells at the same time. The methacrylic acid-modified BFP1 peptide is covalently bonded to the GelMA hydrogel under the action of a photoinitiator, allowing the peptide to be released slowly and for a long time as the GelMA hydrogel degrades, avoiding the problem of inactivation caused by short-term burst release. At the same time, the obtained peptide hydrogel material has excellent stem cell carrying capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. The protection scope of the present invention is not limited to the following description.

[0020] Figure 1 This is a surface scanning electron micrograph of a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells;

[0021] Figure 2 Live / Dead staining results of human dental pulp stem cells in hydrogels of the peptide photocrosslinking group, peptide physical mixing group and peptide-free control group (a: Live / Dead staining results in hydrogel materials on day 1; b: Live / Dead staining results in hydrogel materials on day 7);

[0022] Figure 3 For Figure 2 A view showing the fluorescence intensity statistics of Live / Dead staining results of human dental pulp stem cells in the peptide photocrosslinking group, peptide physical mixing group, and peptide-free control group hydrogels;

[0023] Figure 4 The results of cytoskeleton staining of human dental pulp stem cells after culturing in the peptide photo-crosslinking group, the peptide physical mixing group and the peptide-free control group hydrogels for 1 day and 5 days respectively; Figure 5 ALP staining results of human dental pulp stem cells induced into osteogenesis in the peptide photocrosslinking group, peptide physical mixing group and peptide-free control group hydrogels after 14 days; Figure 6 Expression levels of osteogenesis-related genes (a: RUNX2; b: COL-1; c: OCN; d: ALP) in human dental pulp stem cells after 14 days of osteogenesis in peptide photocrosslinking group, peptide physical mixing group and peptide-free control group hydrogels. DETAILED DESCRIPTION

[0024] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0026] The preparation method of polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells comprises: methacrylic acid-modified BFP1 polypeptide is uniformly mixed with GelMA hydrogel solution and human dental pulp stem cells, and then photo-crosslinked occurs under the action of a photoinitiator, and finally a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells is formed. The density of human dental pulp stem cells carried in the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells is 1 to 1.5*10 6 The GelMA hydrogel solution is a lyophilized GelMA hydrogel material dissolved in a PBS solution at a concentration of 5wt%-10wt%. During the process, a magnetic stirrer is used to heat and stir at a speed of 500-1000 r / min and 50-60° C. for 20-30 minutes.

[0027] The specific mixing steps are as follows: adding methacrylic acid-modified BFP1 polypeptide and photoinitiator to the GelMA hydrogel solution, mixing by pipetting 20 to 30 times to obtain a polypeptide hydrogel precursor solution for use; taking 3 to 5 generations of human dental pulp stem cells, digesting with trypsin until the cell morphology becomes round and cells begin to float, terminating the digestion with a complete culture medium containing 10 to 15% serum in a volume of 2 to 3 times that of trypsin, collecting the cell suspension and counting the cell concentration under a microscope, taking the cell suspension containing the required amount of cells after counting, centrifuging at a speed of 800 to 1000 r / min for 5 to 8 minutes to obtain a mass of human dental pulp stem cells, removing the supernatant after centrifugation, adding the polypeptide hydrogel precursor solution, blowing with a pipette 20 to 30 times until the solution begins to become turbid, obtaining a polypeptide hydrogel precursor solution uniformly mixed with human dental pulp stem cells, and irradiating with a light source of 360 to 405 nm for 20 to 30 seconds to obtain a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells.

[0028] Specifically, the concentration of methacrylic acid-modified BFP1 polypeptide in the polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells is 50-100 μg / mL, and the amount of photoinitiator added is 0.1-0.5wt% of the polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells, expressed as a percentage by weight of the material.

[0029] The extraction method of human dental pulp stem cells is as follows:

[0030] Healthy and complete young permanent teeth extracted due to impaction or orthodontic reduction were collected. Young permanent teeth were permanent teeth grown by people aged ≤25 years old. The pulp was removed under sterile conditions, and the pulp was cut into pieces with a tissue block size of less than 1 mm. 200-300 μL of a 1:1 mixture of type I and type II collagenase solution was added for digestion for 10-15 minutes, and 1-2 times the volume of the collagenase solution was added to a complete culture medium containing 15%-20% serum to neutralize it. The mixture was centrifuged at a speed of 1800-2000 r / min for 5-8 minutes, and all the supernatant was aspirated, leaving only the tissue block precipitate. Then 500 μL-1 mL of complete culture medium containing 15%-20% serum was added, and the mixture was pipetted 20-30 times with a 2.5 ml plastic pipette until there were no obvious clumps. The mixture was inoculated into a culture dish and cultured in a cell culture dish for 1 to 2 weeks. The cells that crawled out of the tissue block were recorded as cells of the 0th generation, and cells of the 3rd to 5th generations were taken to prepare polypeptide photo-cross-linked hydrogel materials loaded with human dental pulp stem cells.

[0031] Specifically, the cells of generation 0 grew to an area greater than 4 cm after 3-4 days of culture. 2 The cells were passaged at 3 to 5 generations and used to prepare polypeptide photo-crosslinked hydrogel materials loaded with human dental pulp stem cells.

[0032] Example

[0033] This embodiment provides a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells, and the preparation method thereof includes:

[0034] Extraction of human dental pulp stem cells:

[0035] Healthy and complete young permanent teeth extracted due to impaction or orthodontic reduction were collected. Young permanent teeth were permanent teeth grown by people aged ≤25 years old. The dental pulp was removed under sterile conditions, and the pulp was cut into pieces until the size of the tissue block was less than 1 mm. 300 μL of 1:1 mixed type I and type II collagenase was added for digestion for 10-15 minutes, and the mixture was centrifuged at 1800-2000 r / min for 5 minutes. 300 μL of supernatant was removed, and only the tissue block precipitate was retained. Then, complete culture medium containing 20% ​​serum was added, and the mixture was pipetted 20-30 times with a 2.5 ml plastic pipette until there were no obvious clumps. The mixture was inoculated into a culture dish and cultured in a cell culture dish for 1 to 2 weeks. The cells that crawled out of the tissue block were recorded as generation 0, and cells of generation 3-5 were used to prepare polypeptide photo-cross-linked hydrogel materials loaded with human dental pulp stem cells.

[0036] Preparation of polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells:

[0037] Weigh 10 g of gelatin and dissolve it in 100 mL of distilled water. After dissolving at 60°C, add 6 mL of methacrylic anhydride and react at room temperature for 8 h. Dialyze against distilled water for 3 to 5 days (molecular weight cutoff: 3500). Centrifuge at 12000 rpm for 5 min, filter with neutral filter paper, and freeze-dry the filtrate to obtain freeze-dried GelMA hydrogel material.

[0038] Weigh 1 mg of methacrylic acid-modified BFP1 polypeptide, dissolve it in 1 mL of PBS solution to prepare a solution with a polypeptide concentration of 1 μg / μL, weigh 0.1 g of freeze-dried GelMA hydrogel material, dissolve it in 1 mL of PBS solution to prepare a GelMA hydrogel solution with a weight percentage of 10 wt%, take 100 μL of polypeptide solution and add it to 0.5 mL of 10 wt% GelMA hydrogel solution, take 1 mg of photoinitiator and add it to the GelMA hydrogel solution, take 400 μL of PBS solution and add it to the above solution, mix well, and obtain 1 mL of polypeptide hydrogel precursor solution, wherein the polypeptide concentration is 100 μg / mL; in terms of weight percentage, the GelMA concentration is 5 wt%, and the amount of photoinitiator added is 0.1 wt%; take 3 to 5 generations of human dental pulp stem cells, digest them with trypsin until the cell morphology becomes round and cells begin to float , terminate the digestion with 1.5 to 2 times the volume of complete culture medium containing 10% serum, collect the cell suspension and count the cell concentration under a microscope, take the cell suspension containing the required amount of cells after counting, centrifuge at a speed of 800 to 1000 r / min for 5 minutes to obtain a mass of human dental pulp stem cells, remove the transparent supernatant solution after centrifugation, add the polypeptide hydrogel precursor solution, pipette 20 to 30 times until the solution begins to become turbid, obtain a polypeptide hydrogel precursor solution with uniformly mixed cells, and irradiate with a light source of 360 to 405 nm for 20 to 30 seconds to obtain a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells.

[0039] Test case 1

[0040] This test case uses cell experiments to explore the cytotoxicity of hydrogel samples in the peptide photocrosslinking group, the peptide physical mixing group, and the peptide-free control group. The specific test methods are as follows:

[0041] After culturing three hydrogel materials, namely, polypeptide-free hydrogel material loaded with human dental pulp stem cells, polypeptide physically mixed hydrogel material loaded with human dental pulp stem cells, and polypeptide photocrosslinked hydrogel material loaded with human dental pulp stem cells for 1 day and 5 days respectively, each hydrogel material was washed 1-2 times with PBS solution at room temperature. Then, calcein (AM) and propidium iodide (PI) were added to the detection buffer at a ratio of 1:1000 to prepare a Live / Dead solution, and the hydrogel complex (1 ml for each 24-well plate) was added and incubated at 37°C for 30 minutes. Fluorescence images of live cells and dead cells were taken under excitation at wavelengths of 490nm and 545nm using a confocal microscope. The images were then merged using image J, and the ratio of live cells to dead cells was calculated ( Figure 3 ).

[0042] Live / Dead staining results are as follows Figure 2 As shown in the figure, the cells in the non-peptide control group, the peptide physical mixing group, and the peptide photocrosslinking group all survived well, with the vast majority being live cells (green fluorescence), while the number of dead cells (red fluorescence) was small. On the 7th day, there was very little red fluorescence in the dead cell view, which has been marked with arrows; the statistical results of the Live / Dead staining fluorescence intensity are shown in the figure. Figure 3 As shown, on the first day, the proportion of living cells in the three hydrogels reached more than 80%, and on the seventh day, the proportion of living cells in the three hydrogels reached more than 99%, indicating that the polypeptide hydrogel materials in the polypeptide physical mixing group and the polypeptide photocrosslinking group have good biocompatibility.

[0043] Test case 2

[0044] This experimental case uses a cytoskeleton staining test to explore the cell growth status of hydrogel samples in the peptide photocrosslinking group, the peptide physical mixing group, and the peptide-free control group. The specific experimental methods are as follows:

[0045] After culturing three hydrogel materials, namely, a polypeptide-free hydrogel material loaded with human dental pulp stem cells, a polypeptide physically mixed hydrogel material loaded with human dental pulp stem cells, and a cross-linked polypeptide photo-cross-linked hydrogel material loaded with human dental pulp stem cells for 1 day and 7 days, each hydrogel material was washed twice with PBS solution, fixed with a 3.7% formaldehyde solution prepared with PBS solution for 20 minutes at room temperature; then washed 2-4 times with a PBS solution containing 0.1% Triton X-100, 5 minutes each time. Then, Actin-Tracker Red was diluted with a PBS solution containing 3% BSA and 0.1% TritonX-100 at a ratio of 1:100, and the diluted solution was the staining working solution. The staining working solution was added to the fixed hydrogel complex at a ratio of 300 μl per well, and incubated at room temperature in the dark for 30 minutes. Then, the PBS solution containing 0.1% Triton X-100 was washed 3 times, 5 minutes each time. Then, the cells were incubated with DAPI stain at room temperature in the dark for 5 minutes, and then washed 3 times with PBS solution containing 0.1% Triton X-100, each time for 5 minutes. Finally, a confocal microscope was used to capture fluorescence images under excitation at a wavelength of 581 nm.

[0046] DAPI / Actin-Tracker Red-594 fluorescence staining results are as follows Figure 4 As shown in the figure, the cells loaded in the peptide physical mixing group and the peptide photocrosslinking group showed obvious F-actin stress fibers after 24 hours. The cells loaded in the peptide photocrosslinking group had more F-actin stress fibers, while the cells loaded in the peptide physical mixing group were round and had significantly fewer stress fibers. After 5 days of 3D culture, the number of cells under the microscope in the peptide photocrosslinking group was more and a denser cell network was formed. On the one hand, it may be because BFP1 can promote cell adhesion, and the crosslinking method of peptide and hydrogel prolongs the contact time between peptide and cell. On the other hand, it may be because the peptide modified with methacrylic acid forms a covalent bond with GelMA to form a carbon-carbon double bond, resulting in less crosslinking between hydrogels, changing the pores of the hydrogel, which is more conducive to cell adhesion and growth. Figure 4 It can be shown that human dental pulp stem cells can adhere and proliferate better in GelMA-BFP1 hydrogel.

[0047] Test case 3

[0048] This experimental case uses ALP staining test to explore the osteogenesis induction effect of human dental pulp stem cells in the peptide photocrosslinking group, peptide physical mixing group and peptide-free control group hydrogel samples. The specific experimental methods are as follows:

[0049] After 14 days of osteoinduction, the three hydrogel materials, namely, the polypeptide-free hydrogel material loaded with human dental pulp stem cells, the polypeptide physically mixed hydrogel material loaded with human dental pulp stem cells, and the cross-linked polypeptide photo-cross-linked hydrogel material loaded with human dental pulp stem cells, were discarded the original culture medium, washed three times with PBS solution, and then the dye solution was prepared with the BCIP / NBT alkaline phosphatase color development kit. After adding the dye solution and incubating for half an hour, the sample was washed with ultrapure water, the staining reaction was terminated, and the results were observed.

[0050] The results of ALP staining were Figure 5 As shown in the figure, after 14 days of inducing human dental pulp stem cells into osteogenesis, the ALP staining color of the hydrogel material with added polypeptides was darker than that of the hydrogel material without polypeptides, indicating that BFP1 can enhance the activity of alkaline phosphatase in human dental pulp stem cells. The ALP staining color of the polypeptide photocrosslinking group was darker than that of the polypeptide physical mixing group, indicating that the fixation of BFP1 by photocrosslinking can have a better effect of inducing hDPSCs into osteogenesis.

[0051] Test case 4

[0052] This experimental case uses cell experiments to explore the osteogenesis-promoting effects of hydrogel samples in the peptide photocrosslinking group, the peptide physical mixing group, and the peptide-free control group. The specific experimental methods are as follows:

[0053] After 14 days of osteoinduction, three hydrogel materials, namely, polypeptide-free hydrogel material loaded with human dental pulp stem cells, polypeptide physically mixed hydrogel material loaded with human dental pulp stem cells, and cross-linked polypeptide photo-cross-linked hydrogel material loaded with human dental pulp stem cells, were dissolved with hydrogel dissolving agent and centrifuged to obtain cell aggregates. RNA in the cells was extracted with RNAiso Plus, and after reverse transcription, the expression levels of COL-1, RUNX2, OCN, and ALP genes were detected by qPCR.

[0054] qPCR test results Figure 6 As shown, at 14 days, the expression levels of RUNX2, COL-1, ALP and OCN of hDPSCs in the polypeptide photocrosslinking group were higher than those in the polypeptide physical mixing group, P<0.05 (n=5), indicating that the polypeptide photocrosslinking group had a better osteogenesis induction effect than the physical mixing group.

[0055] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the embodiments of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the embodiments here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells, characterized in that: The methacrylic acid-modified BFP1 polypeptide was uniformly mixed with the GelMA hydrogel solution and human dental pulp stem cells, and then photo-crosslinked under the action of a photoinitiator, ultimately forming a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells.

2. The method for preparing the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells according to claim 1, characterized in that: The density of human dental pulp stem cells carried in the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells is 1 to 1.5*10 6 Pieces / mL.

3. The method for preparing the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells according to claim 1, characterized in that: The GelMA hydrogel solution is a freeze-dried GelMA hydrogel material dissolved in a PBS solution at a concentration of 5wt%-10wt%. During the process, a magnetic stirrer is used to heat and stir at a speed of 500-1000r / min and 50-60°C for 20-30min.

4. The method for preparing the polypeptide photo-crosslinked hydrogel loaded with human dental pulp stem cells according to claim 2, characterized in that: The specific mixing steps are: Add methacrylic acid-modified BFP1 polypeptide and photoinitiator to the GelMA hydrogel solution, blow and beat with a pipette for 20 to 30 times to mix evenly, and obtain a polypeptide hydrogel precursor solution for standby use; take 3 to 5 generations of human dental pulp stem cells, digest with trypsin until the cell morphology becomes round and cells begin to float, terminate the digestion with 2 to 3 times the volume of trypsin and a complete culture medium containing 10 to 15% serum, collect the cell suspension, and count the cell concentration under a microscope, and after counting, take the cell suspension containing the required amount of cells, centrifuge at a speed of 800 to 1000 r / min for 5 to 8 minutes to obtain a mass of human dental pulp stem cells, remove the supernatant after centrifugation, add the polypeptide hydrogel precursor solution, blow and beat with a pipette for 20 to 30 times until the solution begins to become turbid, and obtain a polypeptide hydrogel precursor solution uniformly mixed with human dental pulp stem cells, and irradiate with a light source of 360 to 405 nm for 20 to 30 seconds to obtain a polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells.

5. The method for preparing the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells according to claim 1, characterized in that: The concentration of the methacrylic acid-modified BFP1 polypeptide in the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells is 50-100 μg / mL, and the amount of the photoinitiator added is 0.1-0.5wt% of the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells, expressed as a percentage by weight of the material.

6. The method for preparing the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells according to claim 4, characterized in that: The extraction method of human dental pulp stem cells is as follows: Healthy and complete young permanent teeth extracted due to impaction or orthodontic reduction were collected. Young permanent teeth were permanent teeth grown by people aged ≤25 years old. The pulp was removed under sterile conditions, and the pulp was cut into pieces with a tissue block size of less than 1 mm. 200-300 μL of a 1:1 mixture of type I and type II collagenase solution was added for digestion for 10-15 minutes, and 1-2 times the volume of the collagenase solution was added to a complete culture medium containing 15%-20% serum to neutralize it. The mixture was centrifuged at a speed of 1800-2000 r / min for 5-8 minutes, and all the supernatant was aspirated, leaving only the tissue block precipitate. Then 500 μL-1 mL of complete culture medium containing 15%-20% serum was added, and the mixture was pipetted 20-30 times with a 2.5 ml plastic pipette until there were no obvious clumps. The mixture was inoculated into a culture dish and cultured in a cell culture dish for 1 to 2 weeks. The cells that crawled out of the tissue block were recorded as cells of the 0th generation, and cells of the 3rd to 5th generations were taken to prepare polypeptide photo-cross-linked hydrogel materials loaded with human dental pulp stem cells.

7. The method for preparing the polypeptide photo-crosslinked hydrogel material loaded with human dental pulp stem cells according to claim 6, characterized in that: The cells of the 0th generation grew to an area greater than 4 cm after 3-4 days of culture. 2 The cells were passaged at 3 to 5 generations and used to prepare polypeptide photo-crosslinked hydrogel materials loaded with human dental pulp stem cells.

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