Method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells

Abstract

The invention relates to the field of materials, and particularly provides a method for rapidly generating a mineralized extracellular matrix based on dental pulp stem cells, and the mineralized extracellular matrix can be formed after culturing in a 96-well plate for 7-14 days. And after decellularization treatment, the decellularized osteoclast can be directly used for quantitatively evaluating the bone erosion capability of the osteoclast in a pore plate. According to the method disclosed by the invention, on the premise of ensuring mineralization uniformity and stability, the preparation time of the mineralization matrix is remarkably shortened, and the repeatability and efficiency of experimental operation are improved. The dental pulp stem cell-derived mineralized extracellular matrix provides a substitute material which is natural, low in cost, convenient to operate and wide in application for in-vitro evaluation of osteoclast functions.

Description

Technical Field

[0001] The invention relates to the technical field of materials, in particular to a method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells. Background Art

[0002] Osteoclasts play a vital role in bone development and bone remodeling, and their dysfunction is closely related to a variety of osteoporosis and osteolysis-related diseases. Therefore, it is of great significance to establish a simple and easy-to-use in vitro method to analyze the function of osteoclasts. At present, the commonly used methods for evaluating osteoclast function include traditional bone slice models and the use of artificial mineralized materials as matrices. However, these methods have problems such as high cost, complex operation or unstable materials, which limit their widespread use in scientific research and clinical applications.

[0003] In recent years, the use of natural extracellular matrix (ECM) to evaluate osteoclast function has become an emerging technology. Dental pulp stem cells (DPSCs) are a type of stem cell with high proliferation and mineralization potential, which can form stable mineralized ECM under appropriate culture conditions.

[0004] Chinese patent document CN108998412A discloses a method for preparing mineralized extracellular matrix cell microspheres, and Chinese patent document CN114921404A discloses a method for promoting mineralization of dental pulp stem cells. However, there is no report on a method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells. Summary of the invention

[0005] The purpose of the present invention is to provide a new method based on natural mineralized extracellular matrix (ECM) derived from dental pulp stem cells (DPSCs) for quantitatively evaluating the bone resorption capacity of osteoclasts in vitro, and to provide a natural, low-cost, easy-to-operate and widely used alternative material for the in vitro evaluation of osteoclast function.

[0006] In a first aspect, the present invention provides a method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells, comprising the following steps:

[0007] Primary culture and immortalized mouse dental pulp stem cells (DPSCs) were cultured at 2 × 10 per well. 4 The cells were seeded into 96-well plates at a density of 10 cells / mL. After culturing to 80-90% confluence, the medium was replaced with osteogenic induction medium [α-MEM medium + 10% v / v FBS + 50 µg / mL ascorbic acid (Sigma, A5960) + 10 mM sodium β-glycerophosphate (Sigma, G9422) + 1×10⁻ 8M dexamethasone (Sigma, D4902) + 100 IU / mL penicillin / streptomycin] and the medium was changed every two days; calcified extracellular matrix (ECM) began to form on the 3rd day, and an extracellular matrix (ECM) suitable for etch experiments was formed on the 7th day; at this time, a solution containing 0.5% Triton X-100 and 20 mM ammonium hydroxide was used for decellularization for 5 minutes; washed once with PBS and then washed with ddH 2 O and washed twice, 10 minutes each time; the decellularized extracellular matrix was dried at room temperature and sterilized under ultraviolet light for 2 hours; all operations were performed under sterile conditions; and stored at 4°C.

[0008] Furthermore, the mice are mice of 3 months and above. Still further, the mice are C57 mice of 3 months and above.

[0009] Furthermore, the mouse dental pulp stem cells are primary mouse dental pulp stem cells and immortalized dental pulp stem cells.

[0010] Furthermore, the primary culture and immortalization method of mouse dental pulp stem cells is as follows: the dental pulp is separated from the tooth root, cut into small pieces of about 1 mm³, and then inoculated into a culture dish containing α-MEM medium (HyClone, SH30265.01), 10% v / v fetal bovine serum (Gibco, 12664025) and penicillin / streptomycin (100 IU / mL), and incubated at 37°C, 5% CO 2 Culture at room temperature for 5 days; do not disturb the culture in the first 2 days, which is crucial for the tissue block to attach to the culture dish and for the DPSC cells to migrate out; change the culture medium after 3 days of culture; when the DPSC cells migrate out of the tissue block and expand to form large clonal cell clusters, you can start subculturing; wash the cells three times with preheated PBS (Gibco, 18912014), then add 0.25% trypsin-EDTA to digest for 5 minutes at 37°C; add two volumes of culture medium to terminate the digestion and centrifuge, resuspend the cells to form a single cell suspension, and inoculate them into a new culture dish at a ratio of 1:3. The immortalized mouse dental pulp stem cell line was obtained by infecting the primary cultured mouse dental pulp stem cells with Lenti-SV40T-GFP-Puro immortalization virus for 2 days, selecting, subculturing and amplifying with 1.5µg / mL puromycin. Immortalized DPSCs of passage 5-6 were preserved as cell stocks.

[0011] The second aspect of the present invention provides a mineralized extracellular matrix prepared by the method as described above.

[0012] The third aspect of the present invention provides a use of the mineralized extracellular matrix as described above in the in vitro evaluation of osteoclast function.

[0013] Furthermore, the osteoclasts are selected from mouse bone marrow mononuclear cells, rat bone marrow mononuclear cells or human THP-1 cells.

[0014] The present invention has the advantages that:

[0015] 1. The present invention provides a method for rapidly generating a mineralized extracellular matrix by primary cultured dental pulp stem cells, which can be formed after 7-14 days of culture in a 96-well plate. After decellularization, the matrix can be directly used in the well plate to quantitatively evaluate the osteoclast's ability to erode bones. The method of the present invention significantly shortens the preparation time of the mineralized matrix and improves the repeatability and efficiency of the experimental operation while ensuring the uniformity and stability of mineralization.

[0016] 2. Through the method of the present invention, DPSC-derived ECM can not only support the differentiation and maturation of osteoclast precursor cells of different species (such as mouse bone marrow mononuclear cells, rat bone marrow mononuclear cells and human THP-1 cells), but also can conveniently visualize and quantitatively analyze the bone resorption area through Von Kossa staining. In addition, the system is sensitive to cytokines such as RANKL and IFN-γ, and can be used to study the regulatory effects of different molecules on osteoclast activity.

[0017] 3. DPSC-derived mineralized ECM provides a natural, low-cost, easy-to-operate and widely used alternative material for in vitro evaluation of osteoclast function. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : DPSCs were isolated from 3-month-old mice and cultured in primary culture;

[0019] (a, b) Flowchart of dental pulp isolation from 3-month-old mice. The mandibular bone was isolated using forceps and a scalpel (a). The mandibular bone was cut open with a scalpel to isolate the incisor root (b). The pulp was separated from the root (c), cut into small pieces, and incubated at 37°C, 5% CO. 2 (d). The photo was taken under a stereomicroscope. The black dotted line indicates the mandibular midline suture of the mouse. The black arrow indicates the direction of removal of adjacent tissues. The white dotted line indicates the tooth germ. The red dotted line indicates the first molar area. (a'-d') The text describes the detailed operation steps step by step, and a'-d' correspond to ad, respectively.

[0020] Figure 2 :Isolation, expansion and ECM formation of DPSCs;

[0021] (a) Images of primary cultured DPSCs at passage 1. (b) Cell counts of the first three passages of primary cultured DPSCs obtained from the mandibular incisors of 3-month-old C57 mice (n=6). (c) Proliferation capacity of the first three passages of DPSCs was measured using CCK-8 (n=6). (d) Mineralized ECM produced by P1 DPSCs during osteogenic induction for 2, 5, 7, and 14 days, with mineralized ECM shown by Von Kossa staining. Whole-body views of the culture wells are shown in the upper left corner of each image. (e) Quantitative analysis of the area of ​​mineralized ECM in (e) (n=3). (f) Mineralized ECM-forming capacity of the first three passages of DPSCs, with the percentage of mineralized ECM area measured using Image J software. Scale bar = 500 µm.

[0022] Figure 3 : Immortalized DPSCs can produce stable mineralized ECM;

[0023] (a) Primary cultured DPSCs infected with Lenti-SV40T-GFP-Puro virus. (b) Von Kossa staining showed that immortalized DPSCs at passages 5, 10, 15, and 20 can produce stable mineralized ECM. Scale bar = 1000 µm.

[0024] Figure 4 :DPSC-derived ECM supports normal differentiation and maturation of osteoclasts;

[0025] (a, b) Mouse BMMs were cultured on polystyrene for 7 days in culture medium and osteoclast induction (OCI) medium. Differentiated osteoclasts were detected by TRAP staining (a) and F-actin staining (b). Mature osteoclasts are TRAP positive, multinucleated, and have characteristic F-actin rings. (c, d) Mouse BMMs were cultured on DPSC-derived ECM for 7 days in culture medium and OCI medium. Differentiated osteoclasts were detected by TRAP staining (c) and F-actin staining (d). F-actin was visualized using rhodamine phalloidin (red), and nuclei were stained with DAPI (blue). CM, BMM culture medium; OCI, osteoclast induction medium. Scale bar = 500 µm.

[0026] Figure 5 : DPSC-derived ECM is compatible with the bone resorption function of osteoclasts from different species;

[0027] (ac) Mouse BMMs (a), rat BMMs (b), and human THP-1 cells (c) were seeded on DPSC-derived ECM and cultured in BMM medium and osteoclast induction (OCI) medium for 14 days, respectively. White areas indicate areas of osteoclast-induced bone resorption. Mineralized ECM was visualized by Von Kossa staining. Scale bar = 500 µm.

[0028] Figure 6 : DPSC-derived ECM for evaluating osteoclast bone resorption capacity;

[0029] (a, b) Effects of RANKL and IFN-γ at different concentrations on osteoclast differentiation and bone resorption of mouse bone marrow mononuclear cells. Resorption pits were shown by Von Kossa staining. (c, d) Quantitative analysis of the resorption area in (a) and (b) showed that RANKL and IFN-γ exhibited dose-dependent promoting and inhibiting effects on bone resorption, respectively. *, p<0.05, **, p<0.01. Scale bar = 500 µm. DETAILED DESCRIPTION

[0030] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0031] Example:

[0032] 1. Experimental Materials and Methods

[0033] 1. Primary Culture of DPSCs

[0034] A 3-month-old mouse was euthanized and completely immersed in 75% ethanol for 3 minutes. Then, the mandible was separated using forceps and a scalpel, and the surrounding tissue was thoroughly removed with gauze. The mandible was cut open with a scalpel to separate the incisor root, and the pulp was separated from the root, cut into small pieces (about 1 mm³), and then inoculated into a culture dish containing α-MEM medium (HyClone, SH30265.01), 10% fetal bovine serum (Gibco, 12664025) and penicillin / streptomycin (P / S, 100 IU / mL) at 37°C, 5% CO 2Culture at room temperature for 5 days. Do not disturb the culture during the first 2 days, which is critical for the tissue block to attach to the culture dish and for the DPSC cells to migrate out. Change the culture medium after 3 days of culture. When the DPSC cells migrate out of the tissue block and expand to form large clonal cell clusters, passaging can begin. The cells are washed three times with pre-warmed PBS (Gibco, 18912014) and then digested with 0.25% trypsin-EDTA at 37°C for 5 minutes. After adding two volumes of culture medium to terminate the digestion, centrifuge, resuspend the cells into a single cell suspension, and seed them into a new culture dish at a ratio of 1:3. Please note that over-digestion will affect the proliferation and differentiation ability of DPSC. DPSC can be passaged at least 5 times, but its proliferation and differentiation ability will decrease with the increase of passage number. It is recommended to use cells before the third passage.

[0035] 2. Immortalization of DPSCs

[0036] Lenti-SV40T-GFP-Puro immortalization virus (1×10 4 TU / ml), after culturing for 48 hours, the selection medium (basal medium plus 1.5µg / mL puromycin (Gibico, A1113803) was replaced every 2 days, and the cells were subcultured after reaching 90% confluence. The 5th-6th generation immortalized DPSCs were cryopreserved in liquid nitrogen as cell stocks.

[0037] 3. Formation of DPSC-derived mineralized extracellular matrix

[0038] DPSC cells were cultured at 2 × 10 per well. 4 The cells were seeded into 96-well plates at a density of 10 cells / mL. After culturing to 80-90% confluence, the medium was replaced with osteogenic induction medium [α-MEM + 10% FBS + 50 µg / mL ascorbic acid (Sigma, A5960) + 10 mM β-glycerophosphate (Sigma, G9422) + 1×10⁻ 8 M dexamethasone (Sigma, D4902) + P / S] and the medium was changed every two days. Calcified ECM began to form on day 3, and ECM suitable for scarring experiments was formed on day 7. At this time, decellularization was performed using a solution containing 0.5% Triton X-100 and 20 mM ammonium hydroxide for 5 minutes. Wash once with PBS and then wash with ddH 2 O wash twice, 10 minutes each time. ECM air-dried and sterilized under UV for 2 hours. All operations were performed under sterile conditions. This ECM can be stored at 4 ° C for a long time.

[0039] 4. Primary Culture of Bone Marrow Mononuclear Cells

[0040] The mice were euthanized and thoroughly soaked in 75% ethanol for 3 minutes. The femur and tibia of each mouse were separated, ensuring that the muscles and epiphysis on the bones were completely removed with sterile gauze. The cleaned bones were placed in 10 mL of buffer (PBS + 2% FBS + P / S), the two ends of the bones were cut off, and the bone marrow was flushed out with a 5 mL syringe. Subsequently, the buffer containing the bone marrow was centrifuged at 300g for 10 minutes. The supernatant was discarded, and 5 mL of 1× red blood cell lysis buffer (eBioscience, 00-4300-54) was added and incubated at room temperature for 5 minutes. After adding 20 mL of PBS, the buffer was filtered through a 70μm cell strainer and centrifuged at 300g for 10 minutes. The cell pellet was resuspended in culture medium (α-MEM + 10% FBS + P / S), and the cells of each mouse were seeded in a 10cm culture dish and cultured for 24 hours. The non-adherent cells are bone marrow-derived mononuclear cells (BMMs).

[0041] 5. Osteoclast Differentiation

[0042] After primary culture in BMM, the non-adherent cell supernatant was collected and centrifuged at 300 g for 5 minutes at room temperature. After resuspending the cell pellet, the cells were counted by trypan blue staining. After counting, the cells were inoculated in BMM adherence medium (α-MEM +10% FBS + 30 ng / mL M-CSF + P / S) at a concentration of 0.5~1×10 6 The cells were cultured at a density of 10 cells / cm² for 24 hours to promote adhesion. The BMM medium was then replaced with osteoclast induction (OCI) medium [α-MEM + 10% FBS + 30 ng / mL M-CSF (R&D, 416-ML-010) + 100 ng / mL RANKL (R&D, 462-TR-010) + P / S], and the OCI medium was changed every 2 days.

[0043] 6. THP-1 cell culture and differentiation

[0044] THP-1 cells were cultured in RPMI-1640 medium (HyClone, SH30027) containing 10% FBS and cultured at 5 × 10 4 THP-1 monocytes were seeded in 96-well plates at a density of 100 cells / well. THP-1 monocytes were differentiated into adherent macrophages after 48 hours of stimulation with 100 ng / mL phorbol-12-myristate-13-acetate (PMA, Sigma, 524400). Adherent THP-1 cells were then cultured with OCI medium, and the medium was changed every two days.

[0045] 7. TRAP and Actin Ring Staining

[0046] After being collected and washed, osteoclasts were fixed with 4% paraformaldehyde solution for 15 minutes at room temperature. TRAP staining was performed using an acid phosphatase activity detection kit (Sigma, 387A) according to the provided protocol. For actin ring staining, cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. Subsequently, cells were washed three times with PBS and stained with rhodamine phalloidin (Invitrogen, R451) and DAPI (Sigma, D9542) dyes for 30 minutes. After washing with PBS, cells were observed using an inverted fluorescence microscope.

[0047] 8. Von Kossa dyeing

[0048] The cells were fixed with 4% paraformaldehyde solution for 15 min at room temperature and then washed with ddH 2 O and washed three times. Then, 5% silver nitrate solution was added to the culture plate and incubated for 30 minutes, followed by ddHO. 2 Wash three times with ddHO for 5 minutes each. 2 Prepare 1% pyrogallic acid solution (Sangon, A606880) in ddHO and add to the 96-well plate. Incubate at room temperature for 2 minutes. 2 O wash three times, air dry and take pictures with a microscope or camera.

[0049] 9. Statistics

[0050] The density of ECM and the rate of osteoclast uptake were calculated using Image J. Images that required quantification were first converted to grayscale. Pixels with grayscale values ​​between 0 and 50 were defined as mineralized areas for area measurements. For statistical analysis, at least three biological replicates were used for each group of samples. Statistical significance was determined using an unpaired t-test with a significance level of p < 0.05. All statistical analyses were performed in GraphPad Prism version 9.1.1 software (GraphPad Software, Inc., La Jolla, CA, USA).

[0051] 2. Experimental Results

[0052] 1. Mouse DPSCs are easy to isolate and can be rapidly expanded

[0053] Mouse incisors provide a good source of DPSCs due to their easy isolation. In the present invention, 3-month-old C57 mice were used as a source of DPSCs. Although DPSCs isolated from younger mice are easier to obtain and have stronger proliferation ability, their mineralization and differentiation ability is not as good as DPSCs from adult mice. Therefore, the present invention recommends using mice 3 months and older as donors. For detailed methods for isolating dental pulp from mouse incisors, see the Materials and Methods section ( Figure 1 ). It should be noted that the present invention does not recommend culturing dental pulp after digestion into single cells, but rather directly culturing small tissue blocks of about 1 mm³. This method can obtain a larger number of DPSC cells with better proliferation ability, which may be due to the sensitivity of primary cells to the digestion process. According to the separation scheme of the present invention, a 3-month-old C57 mouse can obtain about 4×10 5 Primary DPSC cells (P0 generation). These cells exhibit a flat fibroblast-like morphology ( Figure 2 a). After rapid expansion and three generations of passage, about 6×10 6 cells ( Figure 2 b). Similar to other mesenchymal stem cells, the proliferation ability of DPSC cells gradually decreases with the increase of passage number, especially in P3 cells. Figure 2 c). This may be due to cell damage that occurred during the passaging process. Based on these results, the present invention recommends using adult mice as a source of DPSCs and using DPSCs within the first three passages.

[0054] 2. DPSCs rapidly form a uniform mineralized extracellular matrix

[0055] To verify the ability of DPSCs to form mineralized ECM, P1 DPSCs were cultured at 2×10 4 Cells were seeded in 96-well plates at a density of 10 cells / well. When the cells reached 80-90% confluence, osteogenic induction medium was added. Von Kossa staining and quantitative analysis showed that a significant amount of ECM began to form on the second day of osteogenic differentiation, and the mineralized ECM formation reached 80% on the fifth day, and was almost completely mineralized on the seventh day ( Figure 2 d, e). In addition, the present invention also tested the in vitro mineralization ability of DPSCs of different passages. The results showed that on the 7th day of differentiation, P1, P2 and P3 cells were able to form mature mineralized ECM ( Figure 2 f). Similar to proliferation capacity, mineralization capacity also decreases with increasing passage number. However, this does not prevent the use of cells from the first three passages to produce mineralized ECM ( Figure 2f). After DPSCs formed a fully mineralized extracellular matrix, the cell cultures were treated with 0.5% Triton X-100 and 20 mM NH 4 Cl treatment to remove cells. After washing, air drying and UV sterilization, they can be used for osteoclast bone resorption experiments. These results show that primary cultured DPSCs from adult mice are an ideal source for obtaining in vitro calcified ECM in a short period of time.

[0056] 3. Immortalized DPSCs

[0057] In order to obtain a stable source of mineralized cells, the present invention uses SV40T immortalization virus and puromycin screening to successfully establish an immortalized DPSC cell line that can be stably passaged. After 20 rounds of passage, the cell line can still maintain a stable ability to form extracellular mineralized matrix, and can be used as a source of germplasm cells for the production of mineralized extracellular matrix to meet the needs of large-scale culture.

[0058] 4. DPSC-derived ECM is suitable for osteoclast differentiation

[0059] To evaluate whether DPSC-derived ECM is suitable for osteoclast culture, the present invention inoculated primary mouse bone marrow mononuclear cells (BMMs) on extracellular matrix for differentiation induction and compared with cells inoculated on polystyrene under the same conditions. TRAP staining results showed that after 7 days of induction in OCI medium, similar to polystyrene, DPSC-derived ECM was able to support the differentiation of mature osteoclasts, as manifested by TRAP-positive staining and multinuclear morphology. In addition, compared with polystyrene, ECM supported a larger number of mature osteoclasts, whose morphology was closer to osteoclasts differentiated on bone slices. In addition, phalloidin staining showed that osteoclasts differentiated on ECM formed typical F-actin rings, which are necessary structures for osteoclast bone resorption. No TRAP-positive cells or F-actin rings were observed in the BMM basal culture medium on polystyrene or ECM ( Figure 4 ad). These results suggest that DPSC-derived ECM is suitable for osteoclast differentiation and bone resorption.

[0060] 5. Bone resorption pits of different osteoclasts on ECM

[0061] To verify the applicability of ECM on the differentiation and resorption functions of different osteoclast precursor cells, different cell types including primary mouse bone marrow mononuclear cells, primary rat bone marrow mononuclear cells and human THP-1 cells (human leukemic mononuclear cells) were used in the present invention. The cells were cultured in culture medium and OCI induction medium for two weeks, and Von Kossa staining showed that all three cell types exhibited osteoclast differentiation and resorption capabilities ( Figure 5The above results indicate that DPSCs-derived ECM is compatible with the differentiation of pre-osteoclasts from different species and types.

[0062] 6. Quantitative analysis of osteoclast bone resorption capacity based on DPSCs-ECM

[0063] To further evaluate the quantitative performance of DPSC-derived ECM, the present invention quantitatively analyzed the ability of cytokines to regulate osteoclast bone resorption. RANKL is a cytokine belonging to the tumor necrosis factor (TNF) family. It triggers the expression of transcription factors required for osteoclast differentiation by activating signaling pathways, thereby playing a key role in osteoclast differentiation. IFN-γ directly inhibits osteoclast differentiation by reducing c-Fos and NFATc1 expression or increasing TRAF6 degradation, and can also indirectly inhibit osteoclast differentiation by stimulating the osteoclast production inhibitory factor OPG. The present invention tested the effects of different concentrations of IFN-γ and RANKL on osteoclast differentiation and bone resorption in mouse BMMs. Von Kossa staining and quantitative analysis of the resorption area showed that with the increase of RANKL concentration (0, 5, 25 and 100 ng / mL), the bone resorption area gradually increased ( Figure 6 a, c). In contrast, IFN-γ effectively inhibited the bone resorption area from 20% to 2% with increasing IFN-γ concentrations (0, 0.1, 0.5, and 1 ng / mL). Figure 6 b, d). These results indicate that DPSC-derived ECM can be used to functionally validate the bone resorption capacity of osteoclasts.

[0064] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells, characterized in that: The following steps are involved: Primary culture and immortalized mouse dental pulp stem cells were cultured at 2 × 10 per well. 4 The cells were inoculated into a 96-well plate at a density of 10 cells / mL. After culturing to 80-90% confluence, the medium was replaced with osteogenic induction medium, which was α-MEM medium + 10% v / v FBS + 50 µg / mL ascorbic acid + 10 mM sodium β-glycerophosphate + 1×10⁻ 8 M dexamethasone + 100 IU / mL penicillin / streptomycin, and the culture medium was changed every two days; calcified extracellular matrix began to form from the 3rd day, and an extracellular matrix suitable for etch experiments was formed on the 7th day; at this time, decellularization was performed with a solution containing 0.5% Triton X-100 and 20 mM ammonium hydroxide for 5 minutes; washed once with PBS and then twice with ddH2O for 10 minutes each; the decellularized extracellular matrix was dried at room temperature and sterilized under ultraviolet light for 2 hours; all operations were performed under sterile conditions; stored at 4°C.

2. The method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells according to claim 1, characterized in that: The mice are mice aged 3 months or older.

3. The method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells according to claim 1, characterized in that: The mice are C57 mice of 3 months and above.

4. The method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells according to claim 1, characterized in that: The mouse dental pulp stem cells are primary mouse dental pulp stem cells and immortalized dental pulp stem cells.

5. The method for rapidly generating mineralized extracellular matrix based on dental pulp stem cells according to claim 1, characterized in that: The primary culture and immortalization method of mouse dental pulp stem cells is as follows: the dental pulp is separated from the tooth root, cut into small pieces of about 1 mm³, and then inoculated into a culture dish containing α-MEM culture, 10% v / v fetal bovine serum and 100 IU / mL penicillin / streptomycin, and incubated at 37°C, 5% Culture under CO2 for 5 days; do not disturb the culture within the first 2 days; change the culture medium after 3 days of culture; start passaging when dental pulp stem cells migrate out of the tissue block and expand to form large clonal cell clusters; wash the cells three times with preheated PBS, then add 0.25% trypsin-EDTA to digest for 5 minutes at 37°C; add two volumes of culture medium to terminate digestion and centrifuge, resuspend the cells to form a single cell suspension, and inoculate them into a new culture dish at a ratio of 1:3; the immortalized mouse dental pulp stem cell line was obtained by infecting primary cultured mouse dental pulp stem cells with Lenti-SV40T-GFP-Puro immortalization virus for 2 days, followed by selection, passaging and amplification with 1.5µg / mL puromycin; immortalized DPSCs of passage 5-6 were preserved as cell stocks.

6. A mineralized extracellular matrix prepared by the method according to any one of claims 1 to 5.

7. Use of the mineralized extracellular matrix according to claim 6 in the in vitro assessment of osteoclast function.

8. The use according to claim 7, characterized in that: The osteoclasts are selected from mouse bone marrow mononuclear cells, rat bone marrow mononuclear cells or human THP-1 cells.

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