Matrix-cell co-polymer biological material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of preparation and application of a co-polymer biological material, and discloses a matrix-cell co-polymer biological material, which comprises a matrix-cell co-polymer, and the matrix-cell co-polymer comprises human decellularized alveolar bone matrix microparticles and human dental sac stem cells. According to the matrix-cell copolymer biological material as well as the preparation method and the application thereof, hDABMPs capable of providing a tissue-specific microenvironment and hDFSCs with development potential are utilized to prepare a developmental bionic matrix-cell copolymer biological material, and the developmental bionic matrix-cell copolymer biological regeneration material has good mechanical strength, good mechanical properties, good biocompatibility, good biocompatibility and good biocompatibility. The stem cell polymer carrying the tissue-specific microenvironment and the developmental potential provides key biological signals of a regeneration scaffold and the specific tissue microenvironment for tissue regeneration so as to support and enhance the regeneration capacity of the implanted stem cell polymer. The strategy highlights the synergistic effect of development guidance and microenvironment induction.

Description

Technical Field

[0001] The invention relates to the technical field of preparation and application of copolymer biomaterials, in particular to a matrix-cell copolymer biomaterial and a preparation method and application thereof. Background Art

[0002] With the increasing incidence of trauma, congenital defects and various diseases, the demand for organ regeneration is also growing significantly. To meet this demand, regenerative medicine has become an important field, focusing on the use of innovative methods to repair and replace damaged tissues. Studies have consistently shown that the interaction between specific microenvironments and tissue cells plays a key role in inducing and stabilizing the mesenchymal condensation process, especially in the subsequent morphogenesis. For example, during growth and development, dental mesenchymal stem cells and their epidermal growth factor (ECM) together constitute dental mesenchymal tissue, which differentiates into two sub-condensation regions, namely the dental papilla and the dental follicle tissue, which further develops into tooth-supporting tissues including cementum, periodontal ligament and alveolar bone. However, in the adult stage, tissue repair at the defect can only generate disordered and single tissues, and cannot effectively achieve precise tissue regeneration. Given the temporal dynamic changes of tissues and organs, it is necessary to start from the perspective of "development-guided regeneration" to achieve more precise tissue regeneration to better meet specific regeneration needs, among which regenerative biomaterials include matrix copolymer biomaterials.

[0003] Traditional periodontal tissue regeneration materials only focus on the compounding or modification of materials, ignoring the importance of key biological signals and microenvironment during growth and development, thus failing to achieve more precise tissue regeneration effects. Therefore, it is necessary to develop new periodontal tissue regeneration materials under the principle of "development-guided regeneration". By imitating and reconstructing the developmental condensation process, reconstructing specific tissue microenvironment in adult tissue regeneration to achieve functional tissue, breaking through the bottleneck of existing periodontal regeneration biomaterial research and development. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a matrix-cell copolymer biomaterial and a preparation method and application thereof, which solves the problem that accurate tissue regeneration effects cannot be achieved.

[0005] To achieve the above object, the present invention provides the following technical solution: a matrix-cell copolymer biomaterial, including a matrix-cell copolymer, wherein the matrix-cell copolymer includes human decellularized alveolar bone matrix microparticles and human dental follicle stem cells.

[0006] Preferably, the human dental follicle stem cells are derived from the third molar dental follicle tissue of healthy patients aged 10-25 years, and the human dental follicle stem cells have developmental potential, and the human decellularized alveolar bone matrix microparticles are derived from the alveolar bone around the tooth roots of healthy orthognathic surgery patients aged 18-25 years and without clinical application value.

[0007] Preferably, the human dental follicle stem cells are developmental mesenchymal stem cells remaining in the adult stage of dental follicle tissue, and the human decellularized alveolar bone matrix microparticles are decellularized matrix, and the decellularized matrix mainly contains various ECM components, such as collagen, fibronectin and proteoglycan.

[0008] A method for preparing a matrix-cell copolymer biomaterial comprises the following steps:

[0009] S1: Preparation of human dental follicle stem cell aggregates;

[0010] S2: Prepare polymer induction medium, add fetal bovine serum, GlutaMAXTM-I, penicillin, streptomycin and vitamin C to α-MEM medium, and based on 1L of polymer induction medium, the concentration of GlutaMAXTM-I is 0.5-10mM, the concentration of penicillin is 100U / mL, the concentration of streptomycin is 100g / mL, the concentration of vitamin C is 0-100μg / mL, and the volume concentration of fetal bovine serum is 5-20%, and α-MEM medium is used to make up to 1L;

[0011] S3: The isolated and identified human dental follicle stem cells were placed in an aggregate induction medium for induction culture, and the obtained aggregates were characterized by scanning electron microscopy, H&E staining, Masson staining and immunohistochemical staining;

[0012] S4: Preparation of human acellular alveolar bone matrix microparticles;

[0013] S5: Collect human alveolar bone, remove the necrotic periodontal membrane and soft tissue on the surface, rinse it repeatedly with PBS, soak the human alveolar bone in deionized water, use an ultrasonic cleaner to vibrate and clean the human alveolar bone every 30 minutes, soak the human alveolar bone in 17% EDTA for 10 minutes, and then thoroughly wash it with sterile water to remove the residual EDTA. The obtained decellularized alveolar bone matrix is ​​used for scanning electron microscopy observation to evaluate the decellularization effect, and the remaining decellularized alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours;

[0014] S6: Under nitrogen atmosphere, human decellularized alveolar bone matrix microparticles were obtained, and the microparticles were ground at 1200 rpm for 5 min using a medium-throughput tissue grinder, repeated 3-6 times, and sterilized in a high-temperature autoclave for 12 hours. 50 g of human decellularized alveolar bone matrix microparticles were resuspended in sterile PBS (100 mL) containing 100 U / mL penicillin and 100 mg / mL streptomycin, and cultured in a 37°C incubator for 3 days. The sample was filtered with a 70 μm filter and stored at -80°C until needed;

[0015] S7: preparing developmental biomimetic matrix-cell copolymers, placing the above-isolated and identified human dental follicle stem cells and human acellular alveolar bone matrix microparticles in a polymer induction medium for continuous induction culture for 10-12 days to form dense spherical copolymers;

[0016] S8: Place the polymer induction culture medium in a constant temperature incubator, maintain the temperature in the constant temperature incubator at 35°C-37°C, and introduce 5% carbon dioxide into the incubator. Add 2mL of water and 1mL of inorganic salt to the culture medium, and carry out continuous induction culture for 5-6 days to form a dense spherical copolymer.

[0017] and, in vivo application of matrix copolymer biomaterials in alveolar bone defect sites.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The matrix-cell copolymer biomaterial and its preparation method and application, by utilizing hDABMPs that can provide a tissue-specific microenvironment and hDFSCs with developmental potential, a developmental biomimetic matrix-cell copolymer bioregenerative material is prepared. The developmental biomimetic matrix-cell copolymer bioregenerative material has good mechanical strength, carries a tissue-specific microenvironment and stem cell aggregates with developmental potential, has good biological properties, provides a regenerative scaffold for tissue regeneration and key biological signals of a specific tissue microenvironment, to support and enhance the regenerative ability of implanted stem cell aggregates, thereby achieving more efficient and targeted tissue regeneration. This strategy highlights the synergistic effect of developmental guidance and microenvironment induction, and further verifies and expands the concept of "developmentally inspired regeneration".

[0020] 2. The matrix-cell copolymer biomaterial and its preparation method and application. Among the developmental biomimetic matrix-cell copolymer biomaterials, the raw material of human decellularized alveolar bone is simple and easy to obtain, the preparation process is simple, and it has good biosafety. At the same time, the form of microparticles greatly increases the interaction area between the matrix and stem cells, which can be adjusted according to the morphology of the defect site, and the operation of in vivo application is relatively easy. At the same time, the human decellularized alveolar bone matrix microparticles also carry a variety of active substances and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the isolation, culture process and biological characteristics identification of hDFSCs in the present invention;

[0022] Figure 2 This is a schematic diagram of the construction and characterization results of hDFSCA in the present invention;

[0023] Figure 3 The figure is a flow chart of the preparation of hDABMPs and a schematic diagram of the characterization results in the present invention;

[0024] Figure 4 It is a schematic diagram of experimental data for evaluating the in vitro biocompatibility and osteoinductive ability of hDABMPs in the present invention;

[0025] Figure 5 It is a schematic diagram of experimental data for evaluating the osteogenic and periodontal abilities of the developmental biomimetic matrix-cell copolymer (hDFSCA-hDABMPs) of the present invention;

[0026] Figure 6 Schematic diagram of the experimental results of evaluating the effects of hDFSCA, hDABMPs and hDFSCA-hDABMPs in the present invention on periodontal tissue regeneration after treating alveolar bone defects in rats. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] “hDFSCA” stands for “human dental follicle stem cell aggregate”;

[0029] “hDFSCs” stands for “human dental follicle stem cells”;

[0030] “hDABMPs” stands for “human decellularized alveolar bone matrix microparticles”;

[0031] “hAB” stands for “human alveolar bone”;

[0032] “hDABM” stands for “human decellularized alveolar bone matrix”;

[0033] “hDFSCs-hDABMPs” stands for “developmental biomimetic matrix-cell consortium”.

[0034] See also Figure 1-6 , a matrix-cell copolymer biomaterial, including a matrix-cell copolymer, and the matrix-cell copolymer includes human decellularized alveolar bone matrix microparticles and human dental follicle stem cells.

[0035] As a preferred technical solution of the present invention: human dental follicle stem cells are derived from the third molar dental follicle tissue of healthy patients aged 10-25 years old, and human dental follicle stem cells have developmental potential, and human decellularized alveolar bone matrix microparticles are derived from the alveolar bone around the tooth roots of healthy orthognathic surgery patients aged 18-25 years old and without clinical application value.

[0036] As a preferred technical solution of the present invention: human dental follicle stem cells are developmental mesenchymal stem cells remaining in the adult stage of dental follicle tissue, human decellularized alveolar bone matrix microparticles are decellularized matrix, and the decellularized matrix mainly contains various ECM components, such as collagen, fibronectin and proteoglycan.

[0037] A method for preparing a matrix-cell copolymer biomaterial comprises the following steps:

[0038] S1: Preparation of human dental follicle stem cell aggregates;

[0039] S2: Prepare polymer induction medium, add fetal bovine serum, GlutaMAXTM-I, penicillin, streptomycin and vitamin C to α-MEM medium, and based on 1L of polymer induction medium, the concentration of GlutaMAXTM-I is 0.5-10mM, the concentration of penicillin is 100U / mL, the concentration of streptomycin is 100g / mL, the concentration of vitamin C is 0-100μg / mL, and the volume concentration of fetal bovine serum is 5-20%, and α-MEM medium is used to make up to 1L;

[0040] S3: The isolated and identified human dental follicle stem cells were placed in an aggregate induction medium for induction culture, and the obtained aggregates were characterized by scanning electron microscopy, H&E staining, Masson staining and immunohistochemical staining;

[0041] S4: Preparation of human acellular alveolar bone matrix microparticles;

[0042] S5: Collect human alveolar bone, remove the necrotic periodontal membrane and soft tissue on the surface, rinse repeatedly with PBS, soak the human alveolar bone in deionized water, and use an ultrasonic cleaner to vibrate and clean the human alveolar bone every 30 minutes. Soak the human alveolar bone in 17% EDTA for 10 minutes, then thoroughly wash with sterile water to remove residual EDTA, and use the obtained decellularized alveolar bone matrix for scanning electron microscopy to evaluate the decellularization effect. The remaining decellularized alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours;

[0043] S6: Under nitrogen atmosphere, human decellularized alveolar bone matrix microparticles were obtained, and the microparticles were ground at 1200 rpm for 5 min using a medium-throughput tissue grinder, repeated 3-6 times, and sterilized in a high-temperature autoclave for 12 hours. 50 g of human decellularized alveolar bone matrix microparticles were resuspended in sterile PBS (100 mL) containing 100 U / mL penicillin and 100 mg / mL streptomycin, and cultured in a 37°C incubator for 3 days. The sample was filtered with a 70 μm filter and stored at -80°C until needed;

[0044] S7: preparing developmental biomimetic matrix-cell copolymers, placing the above-isolated and identified human dental follicle stem cells and human acellular alveolar bone matrix microparticles in a polymer induction medium for continuous induction culture for 10-12 days to form dense spherical copolymers;

[0045] S8: Place the polymer induction culture medium in a constant temperature incubator, maintain the temperature in the constant temperature incubator at 35°C-37°C, and introduce 5% carbon dioxide into the incubator. Add 2mL of water and 1mL of inorganic salt to the culture medium, and carry out continuous induction culture for 5-6 days to form a dense spherical copolymer.

[0046] Example 1: Construction and characterization of hDFSCA

[0047] (1) Cell isolation and culture, such as Figure 1 Shown

[0048] Dental follicle tissue without obvious inflammation was obtained from the third molars of healthy patients aged 10-25 years old, and the hDFSCs of the present invention were extracted from the dental follicle tissue of extracted wisdom teeth.

[0049] a. Separate the dental follicle tissue from the tooth in a clean bench and place it in a culture dish. Rinse it repeatedly with PBS containing 100U / mL penicillin and 100mg / mL streptomycin until no blood is visible, and then transfer it to another clean culture dish.

[0050] b. Use sterile ophthalmic scissors to cut the dental follicle tissue into 1 mm 3The tissue fragments were centrifuged at 800 rpm for 5 min, and the supernatant was removed;

[0051] c. Digestion: Place the centrifuged dental follicle tissue fragments in collagenase I and digest in a humid environment with 5% CO2 at 37°C in the dark for 1.5 hours;

[0052] d. Terminate digestion: Add an equal amount of α-MEM medium containing 10% FBS to terminate digestion;

[0053] e. Centrifugation: Centrifuge at 800 rpm for 5 min and remove the supernatant;

[0054] f. Resuspend and inoculate: add 1-2 mL of α-MEM medium to resuspend the tissue fragments, blow them repeatedly to evenly distribute them in a culture dish, and culture them in a constant temperature incubator at 37°C and 5% CO2 saturation to allow the tissue fragments to adhere to the wall. Change the culture medium every three days;

[0055] h. Cells were passaged when they reached 80%-90% confluence.

[0056] The results are as follows Figure 1 As shown in A, after 2-3 days of continuous culture, hDFSCs emerged from the periphery of the dental follicle tissue block with a characteristic fibroblast morphology, and these cells maintained the mesenchymal cell morphology during the entire subculture process.

[0057] (2) Cell function identification, such as Figure 1 Shown

[0058] 1) Detection of proliferation and cloning ability of hDFSCs

[0059] a. P1 hDFSCs in the logarithmic growth phase were cultured at 1×10 3 The cells were seeded at a density of 10 cells in a 10 cm culture dish for routine culture;

[0060] b. After continuous culture for about 2 weeks, the cells were washed three times with PBS and fixed with 4% PFA for 30 min;

[0061] c. After 20-40 minutes of crystal violet staining, discard the staining solution and wash twice with PBS;

[0062] d. Observation under the microscope: After taking a macroscopic photo, observe the morphology of individual clones under an inverted microscope.

[0063] The results are as follows Figure 1 As shown in B.

[0064] Crystal violet staining results showed that the hDFSCs cells isolated had the ability to form clone colonies, and the cells grew in a colony-like manner after being seeded at a low density. The cells had good proliferation ability and uniform colony distribution.

[0065] 2) Detection of multidirectional differentiation ability of hDFSCs

[0066] A. Preparation of osteogenic and adipogenic induction fluid,

[0067] a. Osteogenic induction solution: weigh 0.765 g of sodium β-glycerophosphate into a 15 mL centrifuge tube, pipette 10 mL of α-MEM medium (containing 7% FBS, 100 U / mL penicillin, and 100 g / mL streptomycin by volume) into the centrifuge tube, mix well to fully dissolve the sodium β-glycerophosphate, filter with a 0.22 μm filter, add the filtrate, 0.417 mL of VC (30 mg / mL) and 50 μL of dexamethasone (0.02 mg / mL) prepared in advance into 240 mL of α-MEM medium, and store at 4°C in the dark;

[0068] b. Adipogenesis induction solution: weigh 8.943 mg of indomethacin into a 1.5 mL EP tube, add 250 μL of methanol to dissolve; weigh 27.78 mg of IBMX (3-isobutyl-1-methylxanthine) into a 1.5 mL EP tube, add 500 μL of dimethyl sulfoxide to dissolve; filter the above two liquids with a 0.22 μM filter, add the filtrate, 2.5 mL of insulin (1 mg / mL) and 250 μL of dexamethasone (0.391 mg / mL) prepared in advance into 250 mL of α-MEM medium (containing 7% FBS, 100 U / mL penicillin, and 100 g / mL streptomycin by volume), and store at 4°C in the dark.

[0069] B. Osteogenic and adipogenic differentiation ability detection

[0070] hDFSCs at the P3 generation were collected by trypsin digestion and seeded into 6-well culture plates at a seeding density of 1 × 10 5 2 mL of 10% α-MEM medium was added to each well and the cells were cultured in a cell culture incubator. When the cells grew to 70% to 80% confluence, osteogenic and adipogenic differentiation medium were added respectively. The osteogenic and adipogenic induction medium were replaced every 2 days. Alizarin red staining and Oil Red O staining were performed on the 21st and 14th days of continuous culture.

[0071] The specific staining steps are as follows:

[0072] a. Washing and fixation: remove the original culture medium, wash twice with PBS, add 2 mL of 4% PFA fixative to each well and fix for 30 minutes on a shaker at room temperature;

[0073] b. Washing: remove the fixative and wash twice with PBS;

[0074] c. Staining: Add 2 mL of 1% Alizarin Red stain or 2 mL of Oil Red O stain to each well and stain for 30 minutes at room temperature;

[0075] d. Washing: Remove the staining solution and wash thoroughly with PBS until the staining solution is completely washed away;

[0076] e. Observe the formation of calcium nodules and fat droplets under an inverted optical microscope.

[0077] The results are as follows Figure 1 C and 1D.

[0078] hDFSCs were cultured in osteogenic and adipogenic induction medium for 21 days and 14 days respectively, and then stained with Alizarin Red and Oil Red O. Dark red mineralized nodules were visible under an inverted optical microscope, indicating good osteogenic ability. Multiple orange-red fat droplets were visible in the cell cytoplasm under a high-power microscope, indicating good adipogenic ability.

[0079] 3) Flow cytometry identification of surface markers of hDFSCs

[0080] hDFSCsP2 cells in the logarithmic growth phase were taken and the surface markers of hDFSCs were detected by flow cytometry. The antibodies of the detected indicators were: positive indicators CD29, CD44, CD105, CD166, and negative indicators CD34, CD45.

[0081] a. Collect cells: Wash the cells twice with PBS, add appropriate amount of trypsin to digest for 30-50 seconds, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add PBS to resuspend to make a single cell suspension, the cell density is 1×10 6 Pieces / mL;

[0082] b. Antibody incubation: Take 200 μL of cell suspension into 6 1.5 mL EP tubes, add target antibodies in different proportions to each EP tube, and incubate at 4°C in the dark for 30-60 min;

[0083] c. Centrifugal washing: Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash with PBS 3 times and discard the supernatant;

[0084] d. Resuspend: Collect cells and resuspend in 200 μL PBS;

[0085] e. On-machine testing.

[0086] The results are as follows Figure 1 As shown in E.

[0087] Flow cytometry results showed that the isolated hDFSCs cells showed positive expression of CD29, CD44, CD105 and CD166, with positive rates reaching 98.0%, 92.7%, 98.5% and 94.2% respectively. At the same time, CD34 and CD45 were negatively expressed, which was consistent with the characteristic expression of hDFSCs surface markers.

[0088] 4) Immunofluorescence cell staining to detect surface markers of hDFSCs

[0089] P3 generation hDFSCs were cultured in 24-well plates, and the surface markers of hDFSCs were detected by immunofluorescence cell staining. The antibodies of the detected indicators were: positive indicators STRO-1, Vimentin, Nestin, and negative indicator CK14.

[0090] a. Washing and fixation: When the cells reach 80%-90% confluence, discard the culture medium and rinse with PBS, then fix the cells with 4% PFA for 30 minutes at room temperature;

[0091] b. Antibody incubation: After discarding PFA and washing twice with PBS, add the target antibody at the specified dilution and incubate at 4°C overnight. Rinse the sample with PBS, incubate with fluorescent secondary antibody, and stain the nucleus with DAPI-containing mounting medium and seal the slide;

[0092] c. Observation under microscope: Use a confocal microscope to observe the samples.

[0093] The results are as follows Figure 1 As shown in F. The results of immunofluorescence cell staining showed that the hDFSCs cells isolated showed positive expression of STRO-1, Vimentin and Nestin, and negative expression of CK14, which was consistent with the characteristic expression of hDFSCs surface markers.

[0094] (3) Cultivation, gross, microscopic evaluation, and histological staining of hDFSCs aggregates (hDFSCA), such as Figure 2 Shown

[0095] 1) Preparation of polymer induction medium: Add 25 mL of fetal bovine serum, 2.5 mL of 100×GlutaMAXTM-I, 2.5 mL of 100×penicillin-streptomycin and 500 μL of 3 mg / mL VC solution to 220 mL of α-MEM medium.

[0096] 2) Aggregate induction, gross and microscopic evaluation: hDFSCs at P4 were cultured in a 6-well plate until 80% confluence. The culture medium in the dish was discarded, and sterile PBS was slowly added along the side of the dish for washing, and repeated twice. After discarding PBS, 10 mL of aggregate induction medium was added to the dish and placed in an incubator for regular medium replacement.

[0097] The results are as follows Figure 2 As shown in A, after 12-14 days of continuous culture, white membrane-like structures can be easily observed, and the aggregates gradually thicken over time. hDFSCA can be scraped off the culture plate using a cell scraper.

[0098] 3) Scanning electron microscopy observation of polymers: The prepared aggregates were rinsed three times with PBS and then fixed in 2.5% glutaraldehyde at room temperature for 5 minutes. Then, the polymers were dehydrated with ethanol solutions of different concentration gradients (60%, 75%, 85%, 95% and 100%, 5 minutes each), and then dried with hexamethyldisilane. Then, gold was plated on the samples using an ion sputtering device, and the morphology of the polymers was observed and photographed under a scanning electron microscope.

[0099] The results are as follows Figure 2 As shown in B, hDFSCs in the aggregate are tightly arranged in layers, with abundant ECM and tightly connected pseudopodia between cells.

[0100] 4) Histological staining of polymers: The prepared polymers were fixed with 4% PFA at room temperature for 24 h. Afterwards, the polymers were dehydrated in a gradient ethanol solution and then embedded in paraffin. 6 μm thick polymer tissue sections were prepared for H&E staining, Masson staining, and immunohistochemical staining. Images were collected using a tissue morphology analysis workstation.

[0101] The results are as follows Figure 2 As shown in C-2E, H&E staining ( Figure 2 C) and Masson staining ( Figure 2 D) confirmed the presence of abundant cells and dense ECM in hDFSCA. Figure 2 E) Further evidence of the positive expression of ECM proteins (such as COL-1) in hDFSCA.

[0102] Example 2: Preparation and characterization of hDABMPs

[0103] 1. Preparation method of hDABMPs, such as Figure 3 As shown in A

[0104] Human alveolar bones (hAB) of no clinical value surrounding the tooth roots were collected from healthy orthognathic surgery patients aged 18-25 years.

[0105] a. First, remove the necrotic periodontal membrane and soft tissue, rinse them repeatedly with PBS, and then soak the hAB in deionized water.

[0106] b. Use an ultrasonic cleaner to clean the hAB by vibrating it once every 30 minutes. Repeat twice to ensure thorough cleaning.

[0107] c. Soak hAB in 17% EDTA for 10 min, then wash thoroughly with sterile water to remove residual EDTA.

[0108] d. The obtained decellularized alveolar bone matrix (hDABM) was partially observed by scanning electron microscopy (SEM) to evaluate the decellularization effect, and the remaining hDABM was transferred to a freeze dryer for sublimation drying for 24 hours. Then, under a nitrogen atmosphere, a medium-throughput tissue grinder (DHS) was used to grind at a speed of 1200 rpm for 5 minutes, and repeated 3-6 times if necessary to obtain hDABMPs.

[0109] e. Sterilization: To ensure the sterility of hDABMPs, sterilize them in a high-temperature autoclave for 12 hours.

[0110] f. Resuspend 50 g of hDABMPs in sterile PBS (100 mL) containing 100 U / mL penicillin and 100 mg / mL streptomycin and culture in a 37°C incubator for 3 days.

[0111] g. Filter the sample through a 70 μm filter and store at -80°C until needed.

[0112] The results are as follows Figure 3 As shown in A, hAB was obtained from the root of a healthy orthognathic surgery patient. hDABM was obtained after decellularization of hAB. After grinding, hDABM was processed into hDABMPs, which appeared as light yellow powder particles.

[0113] (2) Characterization of hDABM and hDABMPs, such as Figure 3 B-3D

[0114] 1) SEM observation of hDABM

[0115] The obtained hAB and hDABM samples were dehydrated in a gradient ethanol solution (60%, 75%, 85%, 95% and 100%, 10 min each), hexamethyldisilane was added dropwise on the dehydrated samples, and the samples were placed in a fume hood and dried at room temperature for 8-10 h. Subsequently, gold was plated on the surface of the dried samples using an ion sputtering device, and the morphology of hAB and hDABM was observed and photographed under SEM.

[0116] 2) X-ray photoelectron spectroscopy analysis: The chemical composition of hDABM was analyzed using an X-ray scanning electron microscope spectrometer according to the operating instructions of the equipment.

[0117] 3) The chemical properties of hDABMPs were analyzed using Fourier transform infrared spectrometer (Shimadzu, Japan).

[0118] The results are as follows Figure 3 As shown in B-3D, after decellularization, the scanning electron microscopy results ( Figure 3 B) shows that collagen fibers are fully exposed in hDABM. XPS analysis results ( Figure 3 C) showed that in addition to basic chemical elements such as calcium and phosphorus, hDABM also contains carbon, oxygen, magnesium and sodium. FTIR analysis results ( Figure 3 D) shows that both hDABMPs and hydroxyapatite (HAP) contain organic groups such as carbonate and phosphate. These results indicate that hDABMPs have been successfully prepared.

[0119] 2. Evaluation of the biocompatibility and osteoinductive capacity of hDABMPs

[0120] (1) Biocompatibility testing, such as Figure 4 Shown in AD

[0121] hDFSCs were cultured with hDABMPs by mixing 4 hDABMPs per 10 mL of regular culture medium in a humidified atmosphere at 37°C and 5% CO2. Cell viability was assessed using the Calcein / PI Cell Viability and Cytotoxicity Assay Kit according to the manufacturer's instructions. Cell proliferation was assessed using the kFlour488-EdU Cell Proliferation Assay Kit (EdU).

[0122] (2) Osteogenic induction ability test, such as Figure 4 E, shown in 4F

[0123] After inducing hDFSCs with hDABMPs and culturing for 7 days, the cells were washed 3 times with PBS. Then they were fixed with 4% PFA at room temperature for 30 minutes, and antibodies (ALP, RUNX2) diluted in proportion were added and incubated overnight at 4°C. After being washed three times with PBS, the samples were incubated with fluorescent secondary antibodies. The cell nuclei were stained with an anti-fluorescence quenching blocking agent containing DAPI. Finally, the cells were observed and photographed under a confocal microscope. The relative fluorescence intensity was analyzed using ImageJ software.

[0124] The results are as follows Figure 4 As shown in A-4F, the live / dead staining results showed that almost all hDFSCs in the control group and hDFSCs-hDABMPs group expressed green fluorescence at 1, 3, and 5 days of culture ( Figure 4A), while induction with hDABMPs had no significant effect on the viability of hDFSCs ( Figure 4 A-4B). EdU detection results ( Figure 4 C-4D) showed that after 48 hours of culture, the proportion of EdU-positive cells in the hDABMPs-induced group was significantly higher than that in the control group, indicating that hDABMPs are beneficial to the proliferation of hDFSCs. Then, the osteogenic-related proteins in hDFSCs were detected by immunofluorescence cell staining ( Figure 4 E-4F). After 7 days of co-culture, the expression levels of ALP and RUNX2 were significantly increased in the hDABMPs-induced group, confirming the osteogenic induction ability of hDABMPs. These findings indicate that hDABMPs have good biocompatibility and can significantly promote the proliferation and osteogenic differentiation of hDFSCs.

[0125] 3. Preparation of developmental biomimetic matrix-cell copolymer (hDFSCA-hDABMPs) biomaterials and evaluation of their osteogenic and periodontal abilities in vitro

[0126] (1) Preparation of hDFSCA-hDABMPs, such as Figure 5 A, as shown in 5B

[0127] hDFSCs at P4 were seeded into 6-well plates and cultured with 10 mL of conventional culture medium containing 4 hDABMPs. When the cell density reached 80% confluence, the original culture medium was replaced with aggregate induction medium and conventional culture medium was replaced. It should be noted that when replacing the culture medium, new culture medium should be gently added along the edge of the culture dish to avoid displacement of hDABMPs.

[0128] The results are as follows Figure 5 As shown in B, after 7 days of culture, pale yellow membrane structures appeared around hDABMPs. Over time, these membrane structures gradually curled and thickened, and finally formed dense spherical copolymers on the 10th to 12th day of culture.

[0129] (2) Evaluation of the in vitro osteogenic and periodontal abilities of hDFSCA-hDABMPs, e.g. Figure 5 C

[0130] The total RNA of hDFSCA-hDABMPs prepared above was extracted using Trizol reagent and converted into cDNA using PrimeScriptTMRT kit. Subsequently, quantitative PCR system (and Premix ExTaq TM II was used for qRT-PCR. The relative expression level of each gene was obtained by normalizing it with the expression level of GAPDH.

[0131] The results are as follows Figure 5 As shown in C, qRT-PCR results showed that the expression levels of osteogenic markers such as ALP, RUNX2, COL-1 and Osteopontin (OPN) and periodontal markers such as Laminin-β1 and Periostin were higher in hDFSCA-hDABMPs compared with hDFSCA. This indicates that hDFSCA-hDABMPs have better osteogenic and periodontal formation abilities than hDFSCA.

[0132] 4. Good tissue remodeling effect was observed after implantation of biomimetic matrix-cell copolymer biomaterial into alveolar bone defect in rats

[0133] (1) Micro-CT detection, such as Figure 5 A. Figure 6 As shown in A and B

[0134] The above-mentioned developmental biomimetic matrix-cell copolymer was implanted into the alveolar bone defect of rats. Mandibular bone samples were extracted from rats 4 weeks after surgery and fixed in 4% PFA for 2 hours. The mandibular bone samples were scanned using the Quantum GX2micro-CT imaging system with a resolution of 8μm, a voltage of 80kV, and a current of 80μA. After scanning and three-dimensional image reconstruction, the original alveolar bone defect area was selected as the region of interest to obtain periodontal bone data, and the parameters such as bone volume / total volume (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) were calculated.

[0135] (2) Mandibular histology and osteogenic staining, such as Figure 6 C-6F

[0136] The rat mandibular samples were collected after scanning and decalcified with 17% EDTA for 1 month. After embedding with paraffin, 6 μm thick paraffin sections were prepared. H&E staining and Masson staining were performed according to the manufacturer's instructions. Images were obtained under a microscope, and 5 images were randomly selected to quantify the results using ImageJ.

[0137] When performing immunofluorescence staining on paraffin sections, the sections were first subjected to routine dewaxing and antigen retrieval. The sections were permeabilized with 1% TritonX-100 for 5 minutes and then blocked with goat serum for 30 minutes at room temperature. The diluted antibody (RUNX2) was then added to the sections and incubated overnight at 4°C in a humidified chamber. Excess primary antibodies were washed off with PBS and then incubated with secondary antibodies for 1.5 hours at room temperature. Finally, images were collected and analyzed using a confocal microscope and ImageJ software.

[0138] The results are as follows Figure 6 Representative Micro-CT 3D reconstruction images ( Figure 6 A) shows the tissue regeneration in the alveolar bone defect area of ​​all surgical groups. Micro-CT quantitative analysis ( Figure 6 B) shows that BV / TV, Tb.Th, and Tb.N increased in the transplanted group compared to the control group. In addition, the hDFSCA-hDABMPs group had the highest BV / TV, Tb.Th, and Tb.N values ​​among all experimental groups. These results indicate that the hDFSCA-hDABMPs group produced the highest amount of new bone. In addition, Figure 6 CE showed that the alveolar bone formation in the bone defect area was very small in the control group and hDFSCA transplantation group, and most of it was filled with connective tissue, while the alveolar bone formation in the hDABMPs and hDFSCA-hDABMPs transplantation groups increased. The hDFSCA-hDABMPs group showed a large amount of new bone, which was characterized by a continuous and smooth surface and a bone density comparable to that of natural bone ( Figure 6 C top and middle). In addition, Figure 6 As shown in C (bottom) and F, the results of immunofluorescence staining showed that the proportion of RUNX2+ cells in the alveolar bone was higher in the hDABMPs group and the hDFSCA-hDABMPs group compared with the other two groups. The hDFSCA-hDABMPs group had the highest proportion. These findings support the excellent regenerative ability of hDFSCA-hDABMPs for periodontal tissue, thus confirming the regenerative potential of developmental biomimetic cell-matrix coaggregates.

[0139] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0140] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A matrix-cell copolymer biomaterial, characterized in that: The invention comprises a matrix-cell copolymer, wherein the matrix-cell copolymer comprises human decellularized alveolar bone matrix microparticles and human dental follicle stem cells.

2. The matrix-cell copolymer biomaterial according to claim 1, characterized in that: The human dental follicle stem cells are derived from the third molar dental follicle tissue of healthy patients aged 10-25 years, and the human dental follicle stem cells have developmental potential. The human decellularized alveolar bone matrix microparticles are derived from the alveolar bone around the tooth root of healthy orthognathic surgery patients aged 18-25 years and without clinical application value.

3. The matrix-cell copolymer biomaterial according to claim 2, characterized in that: The human dental follicle stem cells are mesenchymal stem cells remaining in the adult stage of the dental follicle tissue during development, and the human decellularized alveolar bone matrix microparticles are decellularized matrix, and the decellularized matrix mainly contains ECM components, collagen, fibronectin and proteoglycan.

4. A method for preparing a matrix-cell copolymer biomaterial, characterized in that: Using a matrix-cell copolymer biomaterial as claimed in claim 3, comprising the following steps: S1: Preparation of human dental follicle stem cell aggregates; S2: Preparation of aggregate induction medium; S3: The isolated and identified human dental follicle stem cells were placed in an aggregate induction medium for induction culture, and the obtained aggregates were characterized by scanning electron microscopy, H&E staining, Masson staining and immunohistochemical staining; S4: Preparation of human acellular alveolar bone matrix microparticles; S5: Collect human alveolar bone, remove the necrotic periodontal membrane and soft tissue on the surface, rinse it repeatedly with PBS, soak the human alveolar bone in deionized water, use an ultrasonic cleaner to vibrate and clean the human alveolar bone every 30 minutes, soak the human alveolar bone in 17% EDTA for 10 minutes, and then thoroughly wash it with sterile water to remove the residual EDTA. The obtained decellularized alveolar bone matrix is ​​used for scanning electron microscopy observation to evaluate the decellularization effect, and the remaining decellularized alveolar bone matrix is ​​transferred to a freeze dryer for sublimation drying for 24 hours; S6: and obtaining human acellular alveolar bone matrix microparticles under a nitrogen atmosphere; S7: preparing a developmental biomimetic matrix-cell copolymer, placing the human dental follicle stem cells and human acellular alveolar bone matrix microparticles separated and identified above in a polymer induction culture medium; S8: Place the polymer induction culture medium in a constant temperature incubator, maintain the temperature in the constant temperature incubator at 35°C-37°C, and introduce 5% carbon dioxide into the incubator. Add 2mL of water and 1mL of inorganic salt to the culture medium, and carry out continuous induction culture for 5-6 days to form a dense spherical copolymer.

5. The method for preparing a matrix-cell copolymer biomaterial according to claim 4, characterized in that: Step S2 specifically includes: adding 25mL of fetal bovine serum, 2.5mL of 100XGlutaMAXTM-I, 2.5mL of 100× penicillin-streptomycin and 500μL of 3mg / mL vitamin C solution to the α-MEM culture medium, and based on 1L of polymer induction culture medium, the concentration of GlutaMAXTM-I is 0.5-10mM, the concentration of penicillin is 100U / mL, the concentration of streptomycin is 100g / mL, the concentration of vitamin C is 0-100μg / mL, the volume concentration of fetal bovine serum is 5-20%, and 1L is supplemented with α-MEM culture medium.

6. The method for preparing a matrix-cell copolymer biomaterial according to claim 5, characterized in that: Step S6 specifically includes: grinding at 1200 rpm for 5 min using a medium throughput tissue grinder, repeating 3-6 times, sterilizing using a high temperature autoclave for 12 hours, resuspending 50 g of human acellular alveolar bone matrix microparticles in sterile PBS (100 mL) containing 100 U / mL penicillin and 100 mg / mL streptomycin, and culturing in a 37°C incubator for 3 days, filtering the sample with a 70 μm filter, and storing at -80°C until needed.

7. An application of a matrix-cell copolymer biomaterial, characterized in that: A matrix-cell copolymer biomaterial as claimed in claim 3 is used in vivo in an alveolar bone defect.

Images