Dental pulp stem cell-derived secretion group, photocuring hydrogel and application of photocuring hydrogel in osteogenic differentiation

Abstract

The invention provides a dental pulp stem cell-derived secretion group, a photocuring hydrogel and application of the photocuring hydrogel in osteogenic differentiation, and belongs to the technical field of biomedicine. According to the extraction method of the dental pulp stem cell-derived secretion group, the dental pulp stem cell-derived secretion group (DPS) can be obtained from dental pulp stem cells through simple operation, the DPS is rich in proliferation and osteogenesis promoting active ingredients, and cell behaviors can be regulated and controlled through the synergistic effect of multiple components. The invention also provides a photocuring hydrogel containing the DPS, and the DPS is combined with a GelMA hydrogel to generate an efficient synergistic induction effect, so that the osteogenic differentiation efficiency of the BMSCs is improved. By integrating the multi-target regulation advantage of the stem cell secretion group and the precise delivery characteristic of the photocuring hydrogel, an innovative strategy is provided for solving the problem of low differentiation efficiency of the BMSCs in bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically designs a secretome derived from dental pulp stem cells and a light-cured hydrogel and their application in osteogenic differentiation. Background Art

[0002] Bone defects are a common and serious clinical problem, caused by a variety of factors such as trauma, infection, or tumor resection. Currently, the insufficient osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs) is a key bottleneck restricting the efficacy of bone regeneration. Existing bone defect repair strategies mainly include the following three types of technologies: growth factor delivery systems (such as BMP-2 loaded hydrogels): although they can stimulate osteogenesis in the short term, they have disadvantages such as sudden factor release (>60% released within 24 hours), dose-dependent side effects (heterotopic ossification), and high costs; direct stem cell transplantation: BMSCs have a low survival rate after transplantation (<30%), and the ischemic microenvironment inhibits their paracrine function; inert bone scaffold materials (such as hydroxyapatite composites): they only provide mechanical support and lack the ability to regulate dynamic biological signals.

[0003] Hydrogels have attracted significant attention in bone tissue engineering due to their three-dimensional network structure, biocompatibility, and customizability. Methacryl-modified gelatin (GelMA) hydrogels, as modified versions of natural gelatin, combine photocrosslinking properties (rapid prototyping and mechanical tunability) with cell adhesion sites, making them ideal bone repair vehicles. However, GelMA hydrogels alone lack sufficient bioactivity to effectively activate the osteogenic differentiation potential of BMSCs. Summary of the Invention

[0004] The present invention provides a secretome derived from dental pulp stem cells and a light-cured hydrogel and their application in osteogenic differentiation. The dental pulp stem cell secretome contains a large number of extracellular vesicles (EVs) and extracellular matrix (ECM), which can promote the osteogenic differentiation ability of BMSCs at bone defect sites and promote the osteogenic potential of BMSCs.

[0005] The present invention provides a method for extracting a secretome derived from dental pulp stem cells, comprising the following steps: culturing dental pulp stem cells to a confluence of 80-90%, transferring the cells to a culture medium free of fetal bovine serum for starvation culture for a period of time, and centrifuging at 3000-4500g to collect a supernatant, wherein the supernatant contains a secretome derived from dental pulp stem cells.

[0006] In a preferred embodiment of the present invention, the dental pulp stem cells include primary dental pulp stem cells extracted from impacted third molars of healthy adults.

[0007] In a preferred embodiment of the present invention, the dental pulp stem cells are cultured in a-MEM containing fetal bovine serum and double antibody and passaged to the 3rd to 5th passage.

[0008] In a preferred embodiment of the present invention, the temperature of the starvation culture is 37° C. and the time is 24 to 48 hours.

[0009] In a preferred embodiment of the present invention, the centrifugation time is 5 to 8 minutes.

[0010] In a preferred embodiment of the present invention, after the centrifugation, the supernatant is concentrated, and the concentration method includes centrifugation, and the centrifugation method includes centrifugation at 5000g for 30 to 60 minutes.

[0011] The present invention also provides a secretome derived from dental pulp stem cells extracted using the above extraction method.

[0012] The present invention also provides a photocurable hydrogel comprising the secretome derived from the dental pulp stem cells.

[0013] The present invention also provides a method for preparing the above-mentioned photocurable hydrogel, comprising the following steps: mixing the dry powder of the secretome derived from the above-mentioned dental pulp stem cells with GelMA and initiator TAP, and stirring to obtain the photocurable hydrogel.

[0014] The present invention also provides the use of the above-mentioned photocurable hydrogel in preparing osteogenic differentiation products.

[0015] Beneficial effects: The present invention provides a method for extracting the secretome derived from dental pulp stem cells. The dental pulp stem cell-derived secretome (DPS) can be obtained from dental pulp stem cells through simple operations. The DPS is rich in pro-proliferation and osteogenesis active components, such as extracellular vesicles (EVs), exosomes, soluble proteins and extracellular matrix (ECM), etc., which can regulate cell behavior through the synergistic action of multiple components and avoid the risks of live cell therapy.

[0016] The present invention also provides a photocurable hydrogel (GelDPS) containing the DPS. By combining DPS with GelMA hydrogel, a dual-functional system of "sustained-release carrier + active signal" can be constructed, which solves the problem of rapid degradation of DPS in vivo and lack of spatial localization; at the same time, DPS contains a large number of EVs and ECM, which can provide osteogenic active factors, produce a highly efficient synergistic induction effect, and thus improve the osteogenic differentiation efficiency of BMSCs. The photocuring properties of the GelDPS described in the present invention support instant shaping during surgery and adapt to the needs of repairing irregular bone defects. It is expected to be applied to bone tissue injury sites under clinical conditions, providing potential biologically available for promoting bone tissue injury. By integrating the advantages of multi-target regulation of the stem cell secretome with the precise delivery characteristics of photocurable hydrogels, the present invention provides an innovative strategy for solving the problem of low BMSCs differentiation efficiency in bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow chart of the preparation of GelDPS; Figure 2 Figure 2 is the result of cell colony formation assay, where A is the cell colony image of GelMA group and GelDPS group, and B is the statistical graph of relative cell colony number (fold); Figure 3 Figure 3 is the cell scratch test results of the GelMA group and the GelDPS group. Figure A is the cell scratch test results of the GelMA group and the GelDPS group, and Figure B is the statistical diagram of the relative cell migration width; Figure 4 Figure 1 is the result of Transwell cell migration experiment, in which A is the microscopic examination of GelMA group and GelDPS group under high magnification microscope, and B is the statistical diagram of cell migration number; Figure 5 This is the result of alkaline phosphatase ALP activity detection; Figure 6 This is the result diagram of cell mineralization capacity assessment; Figure 7 Ca 2+ Concentration measurement result graph; Figure 8 Figure 2 is the result of in vivo ectopic bone formation ability test. Figure A is the result of H&E staining, and Figure B is the statistical graph of osteoid formation (multiples). DETAILED DESCRIPTION

[0018] The present invention provides a method for extracting a secretome derived from dental pulp stem cells, comprising the following steps: culturing dental pulp stem cells to a confluence of 80-90%, transferring the cells to a culture medium free of fetal bovine serum for starvation culture for a period of time, and centrifuging at 3000-4500g to collect a supernatant, wherein the supernatant contains a secretome derived from dental pulp stem cells.

[0019] The present invention uses dental pulp stem cells as a host for extraction, and DPS is extracted from the dental pulp stem cells, wherein the dental pulp stem cells of the present invention include primary dental pulp stem cells extracted from impacted third molars of healthy adults. In one embodiment, impacted third molars of healthy adults aged 18 to 25 are selected, and primary dental pulp stem cells are extracted by enzymatic digestion.

[0020] The dental pulp stem cells are cultured and passaged to passages 3 to 5 in α-MEM containing fetal bovine serum (FBS) and a double-streptomycin (PS) solution, wherein the FBS is added at a rate of 10% (v / v) and the PS solution is penicillin and streptomycin, and the PS solution is added at a rate of 1% (v / v). The dental pulp stem cells are passaged in the culture medium and, when the cells reach 80% to 90% confluency, are washed three times with PBS to remove residual FBS and cell debris.

[0021] The cells are then inoculated into FBS-free α-MEM for starvation culture at 37°C for 24-48 hours. In the present invention, the FBS-free α-MEM culture medium is collected after the starvation culture and centrifuged at 3000-4500g for 5-8 minutes to remove residual cells and debris. The supernatant is then centrifuged to achieve concentration, wherein the centrifugation includes passing the supernatant through an Amicon Ultra-15 centrifugal filter device (Millipore, USA) and concentrating it by centrifugation at 5000g for 30 minutes at 4°C. The resulting concentrated supernatant is referred to as the dental pulp stem cell-derived secretome (DPS).

[0022] The present invention also provides a secretome derived from dental pulp stem cells extracted using the above extraction method.

[0023] The DPS of the present invention contains a large number of EVs and ECM, which can provide osteogenic active factors and improve the osteogenic differentiation efficiency of BMSCs. After obtaining the DPS, the present invention can also dry the DPS to obtain a powder. For example, in one embodiment, the obtained DPS is freeze-dried using a vacuum freeze dryer. The DPS extraction method of the present invention is simple, and only requires concentrating and freeze-drying the cell culture supernatant, which is low in cost. The secretome freeze-dried powder obtained by the present invention can be stored at -80°C for 12 months without loss of activity, avoiding the storage and transportation problems of living cell preparations.

[0024] The present invention also provides a photocurable hydrogel comprising the secretome derived from the dental pulp stem cells.

[0025] The photocuring properties of GelDPS described in the present invention support instant shaping during surgery and adapt to the needs of repairing irregular bone defects. It is expected to be applied to bone tissue damage sites under clinical conditions and provide a potentially available biological gel preparation for promoting bone tissue damage.

[0026] The present invention also provides a method for preparing the above-mentioned photocurable hydrogel, comprising the following steps: mixing the dry powder of the secretome derived from the above-mentioned dental pulp stem cells with GelMA and initiator TAP, and stirring to obtain the photocurable hydrogel.

[0027] In the present invention, freeze-dried DPS powder is added to a methacrylated gel solution (containing GelMA and initiator TAP) in a sterile operating chamber. The solution is then stirred in a magnetic stirrer at less than 500 rpm at room temperature for 30 minutes to thoroughly mix the mixture. This yields a dental pulp stem cell secretory group photocurable hydrogel (GelDPS). This hydrogel is liquid at room temperature and exhibits excellent fluidity, making it easy to perform common cell culture operations such as plating. In the present invention, curing and shaping the fluid liquid requires only 10-30 seconds of irradiation using a 405 nm ultraviolet (UV) light source to achieve gelation. The mass ratio of DPS powder to methacrylated gel is 100 μg DPS powder / mL GelMA solution.

[0028] The present invention also provides the use of the above-mentioned photocurable hydrogel in preparing osteogenic differentiation products.

[0029] The GelDPS of the present invention can significantly promote BMSCs colony formation, that is, promote BMSCs cell proliferation; GelDPS significantly promotes BMSCs cell migration and chemotaxis, and GelDPS promotes the early osteogenic differentiation and mineralization ability of BMSCs cells, and the GelDPS can promote the in vivo osteogenic ability of BMSCs.

[0030] To further illustrate the present invention, the dental pulp stem cell-derived secretome and photocurable hydrogel provided by the present invention and their applications in osteogenic differentiation are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 according to Figure 1 The following process is used to prepare the dental pulp stem cell secretome photocurable hydrogel: Dental pulp stem cell-derived secretome (DPSCs secretome, DPS) extraction method: Impacted third molars from healthy adults aged 18 to 25 years were selected. Primary dental pulp stem cells were extracted using an enzymatic digestion method and cultured in α-MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) for passages 3 to 5. When cells reached 80% to 90% confluence in a 10 cm dish, they were washed three times with PBS to remove residual FBS and cell debris. The cells were then placed in 10 ml of α-MEM without FBS and incubated at 37°C for 24 hours. After incubation, the conditioned medium was collected and centrifuged at 3000–4500 g for 5–8 minutes to remove residual cells and debris. The supernatant was concentrated by centrifugation at 5000 g for 30 minutes at 4°C using an Amicon Ultra-15 centrifugal filter device (Millipore, USA). The resulting concentrated supernatant is referred to as the dental pulp stem cell-derived secretome (DPS). The obtained DPS was freeze-dried using a vacuum freeze dryer and then used for later use.

[0032] Preparation of dental pulp stem cell secretory structure photocurable hydrogel (GelDPS): Lyophilized DPS powder was added to a pre-prepared methacrylated gel (containing GelMA and initiator TAP) solution in a sterile operating chamber to a concentration of 100 μg DPS powder / mL GelMA solution. The mixture was stirred thoroughly in a magnetic stirrer at less than 500 rpm for 30 minutes at room temperature to obtain the dental pulp stem cell secretory structure photocurable hydrogel (GelDPS). Irradiation with a 405 nm UV light source for 10–30 seconds resulted in gelation.

[0033] Example 2 The effect of GelDPS prepared in Example 1 on the proliferation ability of BMSCs was detected by cell colony formation assay: The experiment was divided into two groups: GelMA group and GelDPS group.

[0034] ① Prepare GelDPS at a concentration of 100 ng DPS / mL GelMA, take 5 ml of the prepared GelDPS and spread it on a 10 cm cell culture dish, and solidify it by cross-linking under a 405 nm UV light source for 30 seconds.

[0035] ② Take BMSCs cells (in the logarithmic growth phase) at approximately 80%-90% confluency, digest and centrifuge, resuspend the cells, and count the cells. Add 1000 cells to each well and culture in a CO2 incubator.

[0036] ③ After 1 week of culture, when colonies are visible to the naked eye, remove the culture dish, discard the culture medium, and add 5 mL of methanol to fix for 30 minutes.

[0037] ④Discard the methanol and add 5 mL of 0.1% crystal violet to each well for staining for 3 min. Then wash off the crystal violet, take pictures with a digital camera, and count the colonies under a light microscope.

[0038] The test results are as follows Figure 2 As shown, GelDPS significantly promoted BMSCs colony formation, that is, promoted BMSCs cell proliferation.

[0039] Example 3 (1) Cell scratch test: The experimental groups were GelMA group and GelDPS group.

[0040] ① Prepare GelDPS at a concentration of 100 ng DPS / mL GelMA. Take 2 ml of the prepared GelDPS and spread it in a 6-well plate. Crosslink it under a 405 nm UV light source for 30 seconds to solidify it.

[0041] ② Take 80% to 90% confluency green fluorescent protein gene GFP-labeled BMSCs (in the logarithmic growth phase), digest and centrifuge, resuspend the cells and count the cells. 4 Cells were seeded into 6-well culture plates and cultured in a 37°C, 5% CO2 incubator for 24 h.

[0042] ③ On the second day, use a 200 μL pipette tip to scratch the cells along a ruler, rinse the cells three times with PBS, remove the scratched cells, and add serum-free culture medium.

[0043] ④ Take pictures under a fluorescence microscope at 0h and 24h time points, and calculate the cell migration rate.

[0044] The test results are as follows Figure 3 As shown in the figure, GelDPS significantly promoted the migration and motility of BMSCs.

[0045] (2) Transwell cell migration assay: The experimental groups were GelMA group and GelDPS group.

[0046] ① BMSCs with a confluency of about 80% were starved and cultured in serum-free medium for 24 h, digested and centrifuged, and washed 1-2 times with PBS. Cells were resuspended in serum-free medium containing BSA and the cell density was adjusted to 5×10 5 pcs / ml.

[0047] ② Prepare GelDPS at a concentration of 100 ng DPS / mL GelMA. Place 500 μL of the prepared GelDPS in the lower chamber of a well plate. Crosslink under a 405 nm UV light source for 30 seconds to solidify. Add 600 μL of culture medium containing 10% fetal bovine serum.

[0048] ③ Take 100 μL of the cell suspension and add it to the Transwell chamber, evenly distributing the cell suspension on the top of the Transwell chamber. Incubate the cells in a 37°C, 5% CO2 incubator for 24 hours.

[0049] ④ Carefully remove the Transwell chamber, aspirate the culture medium in the upper chamber, and gently wipe the cells in the upper chamber with a cotton swab. Then, add 600 μL of 4% paraformaldehyde to a new 24-well plate and place the chamber in it for fixation for 20-30 minutes.

[0050] ⑤ After aspirating the fixative from the upper chamber, transfer the chamber to a well pre-filled with approximately 800 μL of 0.1%–0.2% crystal violet dye. Allow the cells to stain for 15–30 minutes. Next, gently wipe the upper side of the chamber with a cotton swab to remove any unbound crystal violet, allowing for subsequent cell counting during microscopic examination.

[0051] ⑥ Gently rinse with clean water several times. After removing the chamber, drain the liquid in the upper chamber and wipe the cells on the membrane surface at the bottom of the upper chamber with a wet cotton swab. Next, carefully use tweezers to peel off the membrane, ensuring that the bottom side is facing up, and wait for an appropriate time for it to air dry.

[0052] ⑦ Transfer the membrane to a glass slide and seal with neutral gum. Observe and photograph under a high-power microscope, count the purple-colored positive cells, and then tally the results.

[0053] The test results are as follows Figure 4 As shown, the number of cells migrating through the Transwell pores in the GelDPS group was significantly greater than that in the GelMA group, and GelDPS significantly promoted the chemotactic ability of BMSCs cells to migrate.

[0054] Example 4 The early osteogenic differentiation ability of cells was assessed by alkaline phosphatase (ALP) activity detection. The cells were divided into GelMA group and GelDPS group.

[0055] ① Prepare GelDPS at a concentration of 100 ng DPS / mL GelMA. Take 1 ml of the prepared GelDPS and spread it in a 6-well plate. Crosslink it under a 405 nm UV light source for 30 seconds to solidify it.

[0056] ②Add 1.5mL of 2×10 5 The cells were cultured in a 37°C incubator for 24 hours.

[0057] ③ Replace the osteogenic differentiation induction medium and induce in a 37°C incubator for 72 hours.

[0058] ④ Take the 6-well plate that has been induced for 3 days of osteogenic differentiation, discard the osteogenic differentiation induction medium, wash the cell growth surface twice with PBS, and discard the residual PBS liquid; add 600 μL of ALP extraction and lysis solution, and place it in a 37°C environment for incubation for 15 minutes.

[0059] ⑤ Use a clean cell scraper to gently scrape the cells from each well, transfer the lysate suspension into a 1.5 mL EP tube, centrifuge at 14,000 rpm at 4°C for 10 min, and transfer the supernatant to a new EP tube. This is the extracted ALP protein sample solution.

[0060] ⑥ Take a new 96-well plate, add 50 μL of ALP reaction substrate solution to each well, continue to add 50 μL of ALP buffer to each well, tap to mix, add 10 μL of ALP protein sample solution to the detection well, place in a constant temperature environment at 37°C, and incubate for 15 minutes.

[0061] ⑦ Take out the 96-well test plate and observe with the naked eye until a clear light yellow color change appears in the test wells. Then, add 110 μL of 0.5 N NaOH solution to each reaction well to terminate the reaction.

[0062] ⑧Select a microplate reader with a wavelength of 405 nm to measure the corresponding absorbance value (OD value).

[0063] The test results are as follows Figure 5 As shown in the figure, the intracellular alkaline phosphatase activity in the GelDPS group was higher than that in the GelMA group, and GelDPS promoted the early osteogenic differentiation ability of BMSCs cells.

[0064] Example 5 The mineralization capacity of cells was assessed by Alizarin red staining of mineralized nodules.The cells were divided into GelMA group and GelDPS group.

[0065] ① Prepare GelDPS at a concentration of 100 ng DPS / mL GelMA. Take 1 ml of the prepared GelDPS and spread it in a 6-well plate. Crosslink it under a 405 nm UV light source for 30 seconds to solidify it.

[0066] ②Add 1.5 ml of 2×10 5 The cells were cultured in a 37°C incubator for 24 hours.

[0067] ③ Replace the osteogenic differentiation induction medium and induce in a 37°C incubator for 14 days.

[0068] ④ Take the 6-well plate that has been induced for 14 days, discard the osteogenic differentiation induction medium, wash the cell growth surface twice with PBS, and discard the residual PBS liquid; add 2 mL of 70% ethanol to each well, place in a constant temperature environment at 4℃, and fix for 1 hour.

[0069] ⑤ Take out the 6-well plate and equilibrate it to room temperature, discard the 70% ethanol fixative in the wells, and wash twice with ultrapure water.

[0070] ⑥ Add 2 mL of 40 mM Alizarin Red Solution (ARS, pH = 4.2) to each well and stain at room temperature. Once a distinct bright red color is observed, discard the ARS staining solution immediately.

[0071] ⑦ Wash with ultrapure water 5 times, place on a horizontal shaker at 100 rpm, and continue to shake gently for 15 minutes, trying to remove non-specific staining.

[0072] ⑧The scanner acquires images in transmission mode.

[0073] The test results are as follows Figure 6 As shown in the figure, the mineralization ability of cells in the GelDPS group was higher than that in the GelMA group.

[0074] Example 6 Calcium ions (Ca 2+ ) concentration determination ① Take the 6-well culture plate after ARS staining in Example 4, add 10% w / v CPC, and incubate at 37°C for 30 minutes to completely dissolve the ARS stain.

[0075] ② Collect the CPC solution into a clean EP tube, dilute it 10 times with ultrapure water, and take out 200 μL to add to a 96-well plate.

[0076] ③ Measure the OD value with a microplate reader at a wavelength of 562 nm.

[0077] ④ Collect the cell membranes in the secondary wells of each culture plate using RIPA lysis buffer, extract the total protein, and determine the protein concentration using the BCA method.

[0078] ⑤Calculate Ca using ARS standard curve 2+ The relative concentration of Ca 2+ Calculate the Ca of the sample using a concentration curve 2+ concentration.

[0079] The test results are as follows Figure 7 As shown, the intracellular calcium ion concentration in the GelDPS group was higher than that in the GelMA group.

[0080] Example 7 The ectopic osteogenic ability of BMSCs in vivo was detected by subcutaneous implantation in nude mice. The experimental groups were GelMA group and GelDPS group.

[0081] ① Prepare GelDPS at a ratio of 100 ng DPS / mL GelMA. Disperse hydroxyapatite-β-tricalcium phosphate (HA-TCP) powder in the GelMA solution at a ratio of 20% (w / w). 6 BMSCs cells were cross-linked with 100 μL of a mixture of GelDPS and HA-TCP under a 405 nm UV light source for 30 seconds to solidify and form a cylindrical gel.

[0082] ② BALB / c nude mice (male, weighing 20-25 g) aged 6-8 weeks were selected and randomly divided into groups of 6 mice each. They were adaptively raised for 1 week.

[0083] ③ Anesthesia was performed by intraperitoneal injection of sodium pentobarbital (50 mg / kg), the back was shaved, and the surgical area was disinfected alternately with iodine-alcohol.

[0084] ④ Make a 5mm incision on each side along the midline of the back, and bluntly separate the subcutaneous tissue to form a cavity; after implanting the material, suture the incision with 5-0 absorbable sutures.

[0085] ⑤ After surgery, the animals were housed in a single cage and given free access to food. The wound healing and activity status were observed daily. Penicillin (50,000 units / kg) was injected 3 days after surgery to prevent infection.

[0086] ⑥ After 8 weeks of implantation, the animals were killed and the samples were fixed with 4% paraformaldehyde for 48 hours. After 4 weeks of EDTA decalcification, they were embedded in paraffin and sectioned (5 μm).

[0087] ⑦ Perform H&E staining on the sections; The test results are as follows Figure 8 As shown in the figure, the pink eosinophilic osteoid formation in the GelDPS group was significantly more than that in the GelMA group, demonstrating that GelDPS promoted the osteogenic ability of BMSCs in vivo.

[0088] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for extracting a secretome derived from dental pulp stem cells, characterized in that: The method comprises the following steps: culturing dental pulp stem cells to a confluence of 80-90%, transferring the cells to a culture medium without fetal bovine serum for starvation culture for a period of time, and centrifuging at 3000-4500g to collect the supernatant, wherein the supernatant contains a secretome derived from dental pulp stem cells.

2. The extraction method according to claim 1, wherein The dental pulp stem cells include primary dental pulp stem cells extracted from impacted third molars of healthy adults.

3. The extraction method according to claim 1 or 2, characterized in that The method comprises culturing the dental pulp stem cells in a-MEM containing fetal bovine serum and double antibodies and passaged to the 3rd to 5th generation.

4. The extraction method according to claim 1, wherein The starvation culture temperature is 37° C. and the time is 24 to 48 hours.

5. The extraction method according to claim 1, wherein The centrifugation time is 5 to 8 minutes.

6. The extraction method according to claim 1 or 5, characterized in that After the centrifugation, the supernatant is concentrated. The concentration method includes centrifugation, and the centrifugation method includes centrifugation at 5000g for 30 to 60 minutes.

7. The secretome derived from dental pulp stem cells extracted using the extraction method according to claims 1 to 6.

8. A photocurable hydrogel comprising the secretome derived from dental pulp stem cells according to claim 7.

9. The method for preparing the light-curable hydrogel according to claim 8, characterized in that: The method comprises the following steps: mixing the dry powder of the secretome derived from dental pulp stem cells according to claim 7 with GelMA and initiator TAP, and stirring to obtain the light-cured hydrogel.

10. Use of the light-curable hydrogel according to claim 8 in preparing osteogenic differentiation products.

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