Matrix-cell coacervate biomaterials, methods of making and uses thereof

Abstract

The application relates to the technical field of preparation and application of copolymer biomaterials, and discloses a matrix-cell copolymer biomaterial, which comprises a matrix-cell copolymer, wherein the matrix-cell copolymer comprises human decellularized alveolar bone matrix microparticles and human dental follicle stem cells. The matrix-cell copolymer biomaterial, the preparation method and the application, by using hDABMPs which can provide a tissue-specific microenvironment and hDFSCs which have developmental potential, a developmental biomimetic matrix-cell copolymer biomaterial is prepared, the developmental biomimetic matrix-cell copolymer biomaterial has good mechanical strength, carries a tissue-specific microenvironment and a stem cell aggregate with developmental potential, provides a regeneration scaffold and key biological signals of a specific tissue microenvironment for tissue regeneration, so as to support and enhance the regeneration capacity of the implanted stem cell aggregate. The strategy highlights the synergistic effect of development guidance and microenvironment induction.

Description

Technical Field

[0001] This invention relates to the field of copolymer biomaterial preparation and application technology, specifically to a matrix-cell copolymer biomaterial, its preparation method, and its application. Background Technology

[0002] With the rising incidence of trauma, birth defects, and various diseases, the demand for organ regeneration is also increasing significantly. To meet this demand, regenerative medicine has become a crucial field, focusing on innovative methods to repair and replace damaged tissue. Studies consistently demonstrate that the interaction between specific microenvironments and tissue cells plays a key role in inducing and stabilizing mesenchymal aggregation, particularly during 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-aggregating regions: the dental papilla and dental follicle. The latter further develops into tooth-supporting tissues, including cementum, periodontal ligaments, and alveolar bone. However, in adulthood, tissue repair at the defect site can only generate disordered and monolithic tissue, failing to effectively achieve precise tissue regeneration. Given the temporal dynamics of tissues and organs, it is essential to adopt a "development-guided regeneration" approach to achieve more precise tissue regeneration, thereby better meeting specific regenerative needs. Regenerative biomaterials include matrix copolymer biomaterials.

[0003] Traditional periodontal tissue regeneration materials focus only on the composite or modification of materials, neglecting the importance of key biological signals and the microenvironment during growth and development, thus failing to achieve more precise tissue regeneration effects. Therefore, it is necessary to develop novel periodontal tissue regeneration materials based on the principle of "development-guided regeneration." By mimicking and reconstructing the developmental aggregation process, specific tissue microenvironments can be reconstructed in adult tissue regeneration to achieve functional tissues, breaking through the bottlenecks in the development of existing periodontal regeneration biomaterials. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a matrix-cell copolymer biomaterial, its preparation method, and its application, solving the problem of not being able to achieve precise tissue regeneration effects.

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

[0006] Preferably, the human dental follicle stem cells are derived from the dental follicle tissue of the third molar of a healthy patient 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 roots of healthy orthognathic surgery patients aged 18-25 years who have no clinical application value.

[0007] Preferably, the human dental follicle stem cells are mesenchymal stem cells that remain in the adult stage of dental follicle tissue during development, and the human decellularized alveolar bone matrix microparticles are decellularized matrix, which mainly contains various ECM components, such as collagen, fibronectin and proteoglycans.

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

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

[0010] S2: Prepare polymer induction medium. Add fetal bovine serum, GlutaMAX™-I, penicillin, streptomycin, and vitamin C to α-MEM medium. Based on 1L of polymer induction medium, the concentration of GlutaMAX™-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%. Make up to 1L with α-MEM medium.

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

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

[0013] S5: Collect human alveolar bone, remove necrotic periodontal ligament and soft tissue from the surface, rinse thoroughly with PBS, immerse human alveolar bone in deionized water, clean human alveolar bone with an ultrasonic cleaner every 30 minutes, immerse human alveolar bone in 17% EDTA for 10 minutes, and then rinse thoroughly with sterile water to remove residual EDTA. The obtained decellularized alveolar bone matrix was used for scanning electron microscopy to evaluate the decellularization effect, and the remaining decellularized alveolar bone matrix was transferred to a freeze dryer for sublimation drying for 24 hours.

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

[0015] S7: Prepare a developmental biomimetic matrix-cell copolymer by placing the above-isolated and identified human dental follicle stem cells and human decellularized alveolar bone matrix microparticles in a polymer induction medium and continuously inducing culture for 10-12 days to form a dense spherical copolymer.

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

[0017] In addition, the in vivo application of matrix copolymer biomaterials in alveolar bone defects.

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

[0019] 1. This matrix-cell copolymer biomaterial, its preparation method, and its application: By utilizing hDABMPs, which can provide a tissue-specific microenvironment, and hDFSCs, which have developmental potential, a development-inspired matrix-cell copolymer biomaterial was prepared. This development-inspired matrix-cell copolymer bioregenerative material has good mechanical strength and carries a tissue-specific microenvironment and developmental potential stem cell polymer, exhibiting excellent biological properties. It provides key biological signals for tissue regeneration, including a regeneration scaffold and a specific tissue microenvironment, to support and enhance the regenerative capacity of implanted stem cell polymers, thereby achieving more efficient and targeted tissue regeneration. This strategy highlights the synergistic effect of developmental guidance and microenvironment induction, further validating and expanding the concept of "development-inspired regeneration."

[0020] 2. The matrix-cell copolymer biomaterial, its preparation method, and its application: Among the biomimetic matrix-cell copolymer biomaterials, decellularized human alveolar bone is a simple and readily available raw material with a simple preparation process and good biocompatibility. At the same time, the microparticle form greatly increases the interaction area between the matrix and stem cells, allowing for adjustment according to the morphology of the defect site. In vivo application is also relatively easy. Furthermore, the human decellularized alveolar bone matrix microparticles also carry a variety of active substances, showing broad application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the isolation, culture process, and identification of biological characteristics of hDFSCs in this invention;

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

[0023] Figure 3 This is a flowchart of the preparation process and a schematic diagram of the characterization results of hDABMPs in this invention;

[0024] Figure 4 This is a schematic diagram of the in vitro biocompatibility and osteogenic induction capacity evaluation experimental data of hDABMPs in this invention.

[0025] Figure 5 This is a schematic diagram of experimental data on the osteogenic and periodontal capacity assessment of the biomimetic matrix-cell copolymers (hDFSCA-hDABMPs) in this invention.

[0026] Figure 6 This is a schematic diagram of the experimental results evaluating the effects of hDFSCA, hDABMPs, and hDFSCA-hDABMPs on periodontal tissue regeneration after alveolar bone defects in rats in this invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[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 copolymers”.

[0034] Please see Figure 1-6 A matrix-cell copolymer biomaterial comprising a matrix-cell copolymer including 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 dental follicle tissue of the third molar of healthy patients aged 10-25 years, and human dental follicle stem cells have developmental potential; 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 who have no clinical application value.

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

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

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

[0039] S2: Prepare polymer induction medium. Add fetal bovine serum, GlutaMAX™-I, penicillin, streptomycin, and vitamin C to α-MEM medium. Based on 1L of polymer induction medium, the concentration of GlutaMAX™-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%. Make up to 1L with α-MEM medium.

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

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

[0042] S5: Human alveolar bone was collected, and necrotic periodontal ligament and soft tissue were removed from the surface. After repeated rinsing with PBS, the human alveolar bone was immersed in deionized water and cleaned using an ultrasonic cleaner every 30 minutes. The human alveolar bone was then treated with 17% EDTA for 10 minutes, followed by thorough rinsing with sterile water to remove residual EDTA. The resulting decellularized alveolar bone matrix was used for scanning electron microscopy to evaluate the decellularization effect, and the remaining decellularized alveolar bone matrix was transferred to a freeze dryer for sublimation drying for 24 hours.

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

[0044] S7: Prepare a developmental biomimetic matrix-cell copolymer by placing the above-isolated and identified human dental follicle stem cells and human decellularized alveolar bone matrix microparticles in a polymer induction medium and continuously inducing culture for 10-12 days to form a dense spherical copolymer.

[0045] S8: Place the polymer induction medium in a constant temperature incubator, maintain the temperature in the incubator at 35℃-37℃, introduce carbon dioxide with a concentration of 5% into the incubator, and add 2mL of water and 1mL of inorganic salt to the medium. Perform 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 As shown

[0048] The hDFSCs of this invention are extracted from the dental follicle tissue of extracted wisdom teeth, obtained from the third molar of healthy patients aged 10-25 years without significant inflammation.

[0049] a. In a laminar flow hood, the dental follicle tissue was separated from the tooth and placed in a culture dish. It was repeatedly rinsed with PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin until no visible bloodstains were found. Then it was transferred to another clean culture dish.

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

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

[0052] d. Termination of digestion: Add an equal volume 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. Resuspension and inoculation: Add 1-2 mL of α-MEM medium to resuspend the tissue fragments, blow them evenly repeatedly, spread them evenly in a culture dish, and incubate them in a constant temperature incubator at 37℃ and CO2 saturation of 5% to allow the tissue blocks to adhere to the wall. Change the medium every three days.

[0055] h. Passage the cells when they reach 80%-90% confluence.

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

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

[0058] 1) Detection of hDFSCs proliferation and clonal capacity

[0059] a. Generate P1 generation hDFSCs in the logarithmic growth phase with a 1×10⁻⁶... 3 Cells were seeded at a density of 10 cm in a culture dish and cultured using standard methods.

[0060] b. After continuous culture for about 2 weeks, wash the cells 3 times with PBS and fix them with 4% PFA for 30 min;

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

[0062] d. Microscopic observation: After taking a macroscopic photograph, 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 isolated hDFSCs cells had the ability to form clonal colonies, and the cells grew in a colony-like manner after low-density seeding. The cells had good proliferation capacity and the colonies were evenly distributed.

[0065] 2) Detection of multi-directional differentiation ability of hDFSCs

[0066] A. Preparation of osteogenic and adipogenic induction solutions

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

[0068] b. Lipid-inducing solution: Weigh 8.943 mg indomethacin into a 1.5 mL EP tube and dissolve it in 250 μL of methanol; weigh 27.78 mg IBMX (3-isobutyl-1-methylxanthine) into a 1.5 mL EP tube and dissolve it in 500 μL of dimethyl sulfoxide; filter both solutions through a 0.22 μM filter, and add the filtrate, 2.5 mL of pre-prepared insulin (1 mg / mL), and 250 μL of dexamethasone (0.391 mg / mL) to 250 mL of α-MEM medium (containing 7% FBS, 100 U / mL penicillin, and 100 g / mL streptomycin) and store at 4°C protected from light.

[0069] B. Osteogenic and adipogenic differentiation capacity testing

[0070] hDFSCs from generation P3 were collected by trypsin digestion and seeded into 6-well plates at a density of 1 × 10⁶. 5 Cells per well were added, and 2 mL of 10% α-MEM medium was added to each well. The cells were then placed in a cell culture incubator. When the cells reached 70%–80% confluence, osteogenic and adipogenic induction differentiation media were added respectively. The osteogenic and adipogenic induction media were changed every 2 days. Alizarin Red staining and Oil Red O staining were performed on 21 days and 14 days of continuous culture.

[0071] The specific staining steps are as follows:

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

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

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

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

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

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

[0078] After being cultured in osteogenic and adipogenic induction media for 21 and 14 days respectively, hDFSCs were stained with Alizarin Red and Oil Red O. Under an inverted optical microscope, dark red mineralized nodules were visible, indicating good osteogenic capacity. Under high magnification, multiple orange-red lipid droplets were visible in the cell cytoplasm, indicating good adipogenic capacity.

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

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

[0081] a. Cell collection: After washing cells twice with PBS, add an appropriate amount of trypsin for digestion for 30-50 seconds, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend in PBS to prepare a single-cell suspension with a cell density of 1×10⁻⁶ cells / cells. 6 cells / mL;

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

[0083] c. Centrifugation and washing: Centrifuge at 1000 rpm for 5 min, discard the supernatant. Wash 3 times with PBS, discarding the supernatant each time.

[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 were positive for CD29, CD44, CD105, and CD166, with positive rates of 98.0%, 92.7%, 98.5%, and 94.2%, respectively. They were also negative for CD34 and CD45. This is consistent with the characteristic expression of hDFSC surface markers.

[0088] 4) Detection of surface markers of hDFSCs by immunofluorescence cell staining

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

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

[0091] b. Antibody incubation: Discard the PFA and wash twice with PBS. Add the primary antibody of the target antibody at the specified dilution and incubate overnight at 4°C. Rinse the sample with PBS, incubate with fluorescent secondary antibody, stain the nucleus with DAPI-containing mounting medium, and mount the slide.

[0092] c. Microscopic observation: The sample is observed using a confocal microscope.

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

[0094] (3) Culture, gross and microscopic evaluation, and histological staining of hDFSCs aggregates (hDFSCA), such as... Figure 2 As shown

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

[0096] 2) Polymer induction, gross and microscopic evaluation: P4 generation hDFSCs were cultured in 6-well plates to 80% confluence. The culture medium in the plates was discarded, and sterile PBS was slowly added along the sidewall of the plates for washing, repeated twice. After discarding the PBS, 10 mL of polymer induction medium was added to the plates, and the plates were placed in an incubator for routine culture medium replacement.

[0097] The results are as follows Figure 2 As shown in Figure A, after 12-14 days of continuous culture, a white membranous structure 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 the polymers: The prepared aggregates were washed three times with PBS and then fixed in 2.5% glutaraldehyde at room temperature for 5 min. The polymers were then dehydrated with ethanol solutions of different concentration gradients (60%, 75%, 85%, 95%, and 100%, 5 min each) and dried with hexamethyldisilane. Gold was then deposited onto 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, the hDFSCs in the polymer are arranged in a layered and tightly packed manner, with abundant ECM and tightly connected pseudopodia between the cells.

[0100] 4) Histological staining of the polymers: The prepared polymers were fixed with 4% PFA at room temperature for 24 h. Subsequently, the polymers were dehydrated in a gradient ethanol solution and then embedded in paraffin. 6 μm thick tissue sections of the polymers were prepared for H&E staining, Masson staining, and immunohistochemical staining. Images were acquired using a histological 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. Immunohistochemical staining results ( Figure 2 E) This further demonstrates the positive expression of ECM proteins (such as COL-1) in hDFSCA.

[0102] Example 2: Preparation and Characterization of hDABMPs

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

[0104] Human alveolar bone (hAB) around the tooth roots of healthy orthognathic surgery patients aged 18-25 years that had no clinical application value was collected.

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

[0106] b. Clean hAB using an ultrasonic cleaner that vibrates once every 30 minutes, repeating twice to ensure thorough cleaning.

[0107] c. Immerse hAB in 17% EDTA for 10 minutes, then rinse thoroughly with sterile water to remove residual EDTA.

[0108] d. The obtained decellularized alveolar bone matrix (hDABM) fraction was used for scanning electron microscopy (SEM) to evaluate the decellularization effect. The remaining hDABM was transferred to a freeze dryer for sublimation drying for 24 h. Then, under a nitrogen atmosphere, it was ground using a medium-throughput tissue homogenizer (DHS) at a speed of 1200 rpm for 5 min, 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 100 mL of sterile PBS containing 100 U / mL penicillin and 100 mg / mL streptomycin, and incubate at 37 °C for 3 days.

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

[0112] The results are as follows Figure 3 As shown in Figure A, hAB was taken from the periapical region of the teeth of healthy orthognathic surgery patients. After decellularization, hAB yielded hDABM. After grinding, hDABM was processed into hDABMPs, which are pale yellow powder particles.

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

[0114] 1) SEM observation of hDABM

[0115] The obtained hAB and hDABM samples were dehydrated in a gradient of ethanol solutions (60%, 75%, 85%, 95%, and 100%, 10 min each). Hexamethyldisilane was then added dropwise to the dehydrated samples, which were then dried at room temperature in a fume hood for 8–10 h. Subsequently, gold was deposited on the dried sample surfaces 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 microscopy spectrometer in accordance with the equipment's instructions.

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

[0118] The results are as follows Figure 3 As shown in B-3D, the scanning electron microscopy results after decellularization ( Figure 3 B) indicates that collagen fibers are fully exposed in hDABM. XPS analysis results ( Figure 3 C) indicates 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 groups. These results indicate that hDABMPs have been successfully prepared.

[0119] 2. Evaluate the biocompatibility and osteogenic induction capacity of hDABMPs.

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

[0121] hDFSCs were cultured with hDABMPs in a humid environment of 37°C and 5% CO2, with 4 gh DABMPs uniformly mixed in every 10 mL of standard culture medium. 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 capacity test, such as Figure 4 E, shown in 4F

[0123] hDFSCs were induced with hDABMPs and cultured for 7 days, followed by washing the cells three times with PBS. The cells were then fixed with 4% PFA at room temperature for 30 min, and incubated overnight at 4°C with proportionally diluted antibodies (ALP, RUNX2). After washing three times with PBS, the samples were incubated with fluorescent secondary antibody. The nuclei were stained using a DAPI-containing anti-fluorescence quenching blocking agent. 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 indicate that almost all hDFSCs in the control group and the 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 activity of hDFSCs. Figure 4 A-4B). EdU test results ( Figure 4 C-4D analysis 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 promote the proliferation of hDFSCs. Then, osteogenic-related proteins (OG-related proteins) in hDFSCs were detected using immunofluorescence 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 in vitro assessment of their osteogenic and periodontal capacity.

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

[0127] P4 generation hDFSCs were seeded into 6-well plates and cultured using 10 mL of standard medium containing 4 g HDABMPs. When the cell density reached 80% confluence, the original medium was replaced with polymer induction medium, followed by a standard medium change. It is important to note that when changing the medium, the new medium should be gently added along the edge of the culture dish to avoid displacing the hDABMPs.

[0128] The results are as follows Figure 5 As shown in Figure B, pale yellow membrane structures appeared around hDABMPs after 7 days of cultivation. Over time, these membrane structures gradually curled and thickened, eventually forming a dense spherical copolymer on days 10-12 of cultivation.

[0129] (2) In vitro osteogenic and periodontal capacity assessment of hDFSCA-hDABMPs, such as Figure 5 As shown in C

[0130] Total RNA from the prepared hDFSCA-hDABMPs was extracted using Trizol reagent and converted to cDNA using the PrimeScript™ RT kit. Subsequently, quantitative PCR was performed using a PCR system (and...). Premix ExTaq TM II. qRT-PCR was performed. The relative expression levels of each gene were obtained by normalizing them to GAPDH expression levels.

[0131] The results are as follows Figure 5 As shown in Figure C, qRT-PCR results revealed that, compared to hDFSCA, hDFSCA-hDABMPs exhibited higher expression levels of osteogenic markers such as ALP, RUNX2, COL-1, and Osteopontin (OPN), as well as periodontal markers such as Laminin-β1 and Periodin. This indicates that hDFSCA-hDABMPs possess better osteogenic and periodontal formation capabilities than hDFSCA.

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

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

[0134] The aforementioned biomimetic matrix-cell copolymer was implanted into the alveolar bone defect site in rats. Four weeks post-surgery, mandibular bone samples were extracted from the rats and fixed in 4% PFA for 2 hours. The mandibular bone samples were scanned using a Quantum GX2 micro-CT imaging system at a resolution of 8 μm, voltage of 80 kV, and current of 80 μA. After three-dimensional image reconstruction, the original alveolar bone defect area was defined as the region of interest to acquire periodontal bone data, and parameters such as bone volume / total volume (BV / TV), trabecular bone thickness (Tb.Th), and trabecular bone number (Tb.N) were calculated.

[0135] (2) Histology and osteogenic staining of the mandible, such as Figure 6 As shown in C-6F

[0136] Rat mandibular bone samples were collected after scanning and decalcified with 17% EDTA for one month. After paraffin embedding, 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 five images were randomly selected for quantification using ImageJ.

[0137] For immunofluorescence staining on paraffin sections, the sections were first subjected to routine dewaxing and antigen retrieval. The sections were penetrated with 1% Triton X-100 for 5 min, then blocked with goat serum at room temperature for 30 min. Diluted antibody (RUNX2) was then added to the sections, and the sections were incubated overnight at 4°C in a humidified chamber. Excess primary antibody was washed away with PBS, followed by incubation with secondary antibody at room temperature for 1.5 h. Finally, images were acquired and analyzed using confocal microscopy and ImageJ software.

[0138] The results are as follows Figure 6 As shown, representative Micro-CT 3D reconstructed images ( Figure 6 A) shows the tissue regeneration status of the alveolar bone defect area in all surgical groups. Micro-CT quantitative analysis ( Figure 6 B) showed that, compared with the control group, the transplant group had increased BV / TV, Tb.Th, and Tb.N. Furthermore, among all experimental groups, the hDFSCA-hDABMPs group had the highest BV / TV, Tb.Th, and Tb.N values. These results indicate that the hDFSCA-hDABMPs group produced the highest amount of new bone. In addition, Figure 6 CE showed that both the control group and the hDFSCA transplantation group had very little alveolar bone formation in the bone defect area, with most of it filled by connective tissue. However, the hDABMPs and hDFSCA-hDABMPs transplantation groups showed increased alveolar bone formation. The hDFSCA-hDABMPs group exhibited abundant new bone formation, characterized by a continuous, smooth surface and bone density comparable to natural bone. Figure 6 C (top and middle). Additionally, such as Figure 6 As shown in C (bottom) and F, immunofluorescence staining results indicated that the proportion of RUNX2+ cells in the alveolar bone was higher in the hDABMPs group and the hDFSCA-hDABMPs group compared to the other two groups. The proportion was highest in the hDFSCA-hDABMPs group. These findings support the superior regenerative capacity of hDFSCA-hDABMPs for periodontal tissues, thus confirming the regenerative potential of developing biomimetic cell-matrix co-aggregates.

[0139] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

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

Claims

1. A matrix-cell coacervate biomaterial, characterized in that, The matrix-cell coagulation body comprises human acellular alveolar bone matrix microgranules and human dental follicle stem cells.

2. The matrix-cell coacervate biomaterial of claim 1, wherein: The human dental follicle stem cells are derived from the dental follicle tissue of the third molar of a healthy patient aged 10-25 years, and have developmental potential, and the human acellular alveolar bone matrix microgranules are derived from the alveolar bone around the tooth root of a healthy orthognathic surgery patient aged 18-25 years and have no clinical application value.

3. The matrix-cell coacervate biomaterial of claim 2, wherein: The human dental follicle stem cells are mesenchymal stem cells remaining in the adult stage during development in the dental follicle tissue, and the human acellular alveolar bone matrix microgranules are acellular matrices.

4. A method of producing a matrix-cell coacervate biomaterial as claimed in claim 3, wherein, The method comprises the following steps: S1: preparing a human dental follicle stem cell aggregate, preparing an aggregate induction medium, and placing the separated and identified human dental follicle stem cells in the aggregate induction medium for induction culture; the obtained aggregate is characterized by scanning electron microscopy, H&E staining, Masson staining and immunohistochemical staining; S2: collecting human alveolar bone, removing the necrotic periodontal membrane and soft tissue on the surface, repeatedly rinsing with PBS, and then soaking the human alveolar bone in deionized water; the human alveolar bone is cleaned by using an ultrasonic cleaner to vibrate once every 30 min; the human alveolar bone is treated in 17% EDTA for 10 min, and then washed thoroughly with sterile water to remove residual EDTA; the obtained acellular alveolar bone matrix is used for scanning electron microscopy observation to evaluate the decellularization effect; the remaining acellular alveolar bone matrix is transferred to a freeze dryer for sublimation drying for 24 h under a nitrogen atmosphere, and human acellular alveolar bone matrix microgranules are obtained; S3: preparing a development biomimetic matrix-cell coagulation body, placing the separated and identified human dental follicle stem cells and human acellular alveolar bone matrix microgranules in the aggregate induction medium, placing the aggregate induction medium in a constant temperature incubator, keeping the temperature in the constant temperature incubator at 35-37℃, and introducing carbon dioxide with a concentration of 5% into the incubator, and adding 2 mL of water and 1 mL of inorganic salt into the culture medium for continuous induction culture for 5-6 days, so as to form a dense spherical coagulation.

5. The method for preparing a matrix-cell copolymer biomaterial according to claim 4, characterized in that: In the step S2, 25 mL of fetal bovine serum, 2.5 mL of 100X GlutaMAXTM-I, 2.5 mL of 100X penicillin-streptomycin and 500 μL of 3 mg / mL vitamin C solution are added into the α-MEM culture medium, and the concentration of GlutaMAXTM-I is 0.5-10 mM, the concentration of penicillin is 100 U / mL, the concentration of streptomycin is 100 g / mL, the concentration of vitamin C is 0-100 μg / mL, and the volume concentration of fetal bovine serum is 5-20%, and the α-MEM culture medium is supplemented to 1 L.

6. The method for preparing a matrix-cell copolymer biomaterial according to claim 5, characterized in that: Step S6 specifically comprises: using a medium flux tissue grinding crusher to grind at a speed of 1200 revolutions / min for 5 min, repeating 3-6 times, sterilizing for 12 hours using a high-temperature high-pressure pot, resuspending 50 grams of human decellularized alveolar bone matrix microgranules in 100 mL of sterile PBS 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 for use.

7. Use of a matrix-cell co-culture biomaterial, characterized in that: The use of a matrix-cell co-culture biomaterial as claimed in claim 3 in the preparation of alveolar bone defect materials.

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