A mineralized body for bone repair, its preparation method and application

Abstract

This invention discloses a mineralized body for bone repair, its preparation method, and its application. The preparation method includes the following steps: S1, osteogenic-associated cells are cultured in vitro and then subjected to osteogenic induction culture to induce the osteogenic-associated cells to secrete migration bodies and deposit calcium salts; S2, the culture system obtained in step S1 is decellularized to obtain mineralized bodies, wherein the mineralized bodies include the migration bodies and calcium salts. This invention, based on mineralized bodies composed of migration bodies and calcium nodules obtained from osteogenic-associated cells through osteogenic induction culture and decellularization, possesses the characteristics of high interface compatibility and stable binding with materials, and its osteogenic-associated activity remains stable after decellularization. It can induce osteogenic differentiation and promote bone regeneration in vivo, while avoiding the risks associated with live cell transplantation. This provides a novel and efficient cell-free strategy for bone defect repair, which is beneficial for clinical promotion and application.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a mineralized body for bone repair, its preparation method, and its application. Background Technology

[0002] Bone defect repair, especially the repair of large and complex bone defects, has always been a key technical challenge that urgently needs to be addressed in the fields of oral and maxillofacial surgery and implantology. Currently, the mainstream techniques for bone repair in clinical practice mainly rely on autologous bone grafting, allogeneic bone grafting, and artificial bone substitutes. However, each of these three techniques has significant drawbacks: autologous bone sources are limited and there is a risk of donor site damage; allogeneic bone poses risks of immune rejection and disease transmission; and artificial bone materials alone usually lack sufficient osteogenic induction capacity, making it difficult to meet the clinical demand for high-quality bone regeneration.

[0003] To enhance the osteogenic properties of artificial bone materials, existing technologies have proposed various strategies to enhance osteogenic activity, including: (1) introducing growth factors, cytokines, or small molecule osteogenic inducing substances into the material to activate the osteogenic differentiation capacity of host cells. However, this type of technology generally suffers from problems such as poor stability of bioactive substances, difficulty in precisely controlling the release process, strong dose dependence, and potential safety risks, which limit its clinical application; (2) using cell therapy or tissue engineering strategies: directly implanting stem cells or osteogenic-related cells into the bone defect site, or combining them with scaffold materials for transplantation. Although this strategy has shown certain osteogenic effects in experimental studies, it still faces problems such as limited cell sources, complex in vitro expansion, harsh storage and transportation conditions, and high immune and ethical risks, thus limiting its application.

[0004] In recent years, the concept of cell-free therapy has gradually developed, and existing technologies have begun to explore the use of cell secretions and cell-derived microstructures (such as exosomes, matrix vesicles, and extracellular matrix) in bone regeneration and repair research. These technologies mitigate the risks associated with live cell transplantation to some extent, but still generally suffer from complex preparation processes, cumbersome separation and purification steps, insufficient stability and reproducibility of the resulting products, and difficulty in forming stable bonds with bone repair materials. Furthermore, the mechanisms of action of different secretions in osteogenic induction remain unclear, leading to significant differences in their application effects. Other technologies use the induction and separation of stem cell extracellular matrix to endow materials with osteogenic properties, but the resulting products contain a mixture of various components, and the structure that truly exerts its osteogenic effect is not yet clear, hindering targeted regulation and efficient application.

[0005] In summary, current bone defect repair techniques and osteogenic activity enhancement strategies all have insurmountable problems. Developing a bone regeneration enhancement scheme with good osteogenic induction effect, high safety, simple preparation, and stable integration with bone repair materials has become an urgent need for the development of bone defect repair technology in the fields of oral and maxillofacial surgery and implantology. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a mineralized body for bone repair, its preparation method, and its application. The mineralized body and bone repair material loaded with mineralized bodies provided by this invention not only exhibit significant osteogenic effects but also possess simple material sources, convenient preparation methods, and high safety in use.

[0007] To achieve the above objectives, the present invention first provides a method for preparing mineralized bodies for bone repair, comprising the following steps: S1, after osteoblast-associated cells are cultured in vitro, osteoblast-induced culture is performed to induce the osteoblast-associated cells to secrete migratory bodies and deposit calcium salts; S2, the culture system obtained in step S1 is subjected to decellularization to obtain mineralized bodies, which include the migratory bodies and calcium salts.

[0008] Optionally, the osteogenic induction culture conditions are as follows: cultured for 14 to 21 days in DMEM complete medium containing 40 μg / mL ascorbic acid, 2 mg / mL β-glycerophosphate sodium and 100 nM dexamethasone.

[0009] Optionally, the osteogenic-related cells include at least one of osteoblasts, osteogenic precursor cells, osteogenic precursor stem cells, bone-derived mesenchymal stem cells, or cells that exhibit an osteogenic phenotype after osteogenic induction culture.

[0010] Optionally, the decellularization process includes biological enzyme treatment.

[0011] Optionally, the specific operation of the biological enzyme treatment includes: aspirating the culture medium of the culture system obtained in step S1, washing with PBS, adding 0.25% trypsin-EDTA digestion solution and digesting at 37°C for 30 minutes, aspirating the digestion solution, adding DMEM complete culture medium to stop digestion, and repeatedly pipetting with a pipette.

[0012] Optionally, step S1, before performing the osteogenic induction culture, further includes: seeding the in vitro cultured osteogenic-associated cells onto a biological material.

[0013] Optionally, the mineralized bodies grow in situ on at least a portion of the surface of the biomaterial.

[0014] Optionally, the scaffold includes at least one of a biological scaffold, a titanium implant, and an electrospun membrane.

[0015] In another aspect, the present invention provides a mineralized body for bone repair, wherein the mineralized body for bone repair is obtained by the aforementioned preparation method.

[0016] In another aspect, the present invention provides the application of the aforementioned mineralized bodies in the preparation of bone defect repair materials.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention first provides a mineralized body for bone repair. Specifically, osteogenic-associated cells are induced to form mineralized bodies composed of migratory bodies and calcium nodules through osteogenic induction culture and decellularization treatment. A series of in vitro and in vivo experiments have confirmed that the osteogenic-associated activity of the mineralized body can still be stably maintained after decellularization treatment, thereby playing a role in inducing osteogenic differentiation and promoting bone regeneration.

[0018] Furthermore, a bone repair material loaded with the mineralized bodies is provided. Specifically, the mineralized bodies are grown in situ on the surface of a biomaterial (such as a scaffold), allowing them to form a stable bond with the material interface in a naturally occurring manner. This enhances the interfacial bonding strength and avoids the problems of interfacial instability, easy detachment, inactivation, or uneven spatial distribution of active structures caused by physical adsorption or chemical coupling during the preparation or use of existing technologies. This ensures the stability and repeatability of the osteogenic induction effect of the material.

[0019] The mineralized bodies and bone repair materials loaded with mineralized bodies provided by this invention not only have a significant bone-promoting effect, but also have simple material sources and simple preparation methods. At the same time, the decellularization process avoids the immune and safety issues caused by live cell transplantation, which is conducive to clinical application and promotion. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the preparation process of the mineralized bodies used for bone repair according to the present invention.

[0021] Figure 2 This is a fluorescence imaging image showing the co-localization of calcium salts and migratory bodies in mineralized bodies in Example 1 of the present invention.

[0022] Figure 3 This is a fluorescence imaging image showing the co-localization of calcium salts and migration bodies after Tspan4 siRNA was co-cultured with osteogenic progenitor stem cells in Example 1 of the present invention.

[0023] Figure 4 This is a schematic diagram of the preparation process of the bone repair material loaded with mineralized particles according to the present invention.

[0024] Figure 5 This is a fluorescence imaging image of the bone repair material TSPAN4-GFP in one embodiment of the present invention.

[0025] Figure 6 For the in vivo efficacy evaluation of the bone repair material loaded with mineralized particles of the present invention, wherein: A represents a schematic diagram of the skull defect regeneration experiment in each group of mice; B represents the microCT reconstruction of mice in each group 8 weeks after surgery; C represents the bone mineral density analysis of the bone defect areas in each group of mice after treatment; D represents the H&E staining analysis of bone tissue in the bone defect area of ​​each group of mice after treatment; E represents the Masson tricolor analysis of bone tissue in the bone defect area of ​​each group of mice after treatment; F represents the OCN and RUNX2 immunofluorescence results of bone tissue in the bone defect area of ​​each group of mice after treatment. Scale bar: 1 mm. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

[0027] Terminology Explanation In this invention, "mineralized bodies" refer to tiny structural units formed and released at the extracellular matrix interface by osteogenic-related cells (such as osteogenic progenitor stem cells) under osteogenic induction culture conditions. These structural units are typically associated with calcium salt deposition, osteogenic protein enrichment, or mineralized matrix formation and can maintain relatively stable biological activity after detaching from the main cell body. The mineralized bodies described in this invention are formed by the co-deposition of migrasomes and calcium nodules (formed by calcium salt accumulation), and their function is to act as carriers of osteogenic-related signals to induce or promote bone formation.

[0028] The term "migratory body" refers to vesicles formed by the breakage of contractile fibers during cell migration. These vesicles are primarily responsible for transmitting signals or clearing residual material after cell migration. Their formation depends on cell migration and contains cytoplasmic components (such as mitochondrial fragments), proteins, and RNA.

[0029] As described in the background section, existing technologies for enhancing the osteogenic induction capacity of artificial bone materials mainly rely on exogenous bioactive factors, live cell transplantation, or the indirect utilization of cell secretions. However, this approach generally suffers from problems such as unstable osteogenic induction signals, non-natural bonding between active structures and material interfaces, and difficulty in maintaining osteogenic activity after decellularization. Therefore, developing a bone regeneration enhancement scheme with good osteogenic induction effect, high safety, simple preparation, and stable integration with bone repair materials has become an urgent need for the development of bone defect repair technologies in the fields of oral and maxillofacial surgery and implantology.

[0030] To achieve the above objectives, this invention has conducted extensive research and analysis, and for the first time discovered that during the in vitro osteogenic induction process, osteogenic progenitor stem cells form and accumulate microstructural units (i.e., mineralized bodies) closely related to the mineralization process in the extracellular space or matrix interface. These mineralized bodies are formed by the co-deposition of migratory bodies and calcium nodules. Through a series of in vitro and in vivo experiments, it has been confirmed that the mineralized bodies obtained by osteogenic progenitor stem cells through osteogenic induction culture and decellularization have the characteristics of originating from the osteogenic progenitor stem cells themselves, forming naturally during the osteogenic process, highly matching and stably binding with the material interface, and maintaining stable osteogenic-related activity after decellularization. They can play a role in inducing osteogenic differentiation and promoting bone regeneration in vivo, providing a novel and efficient cell-free strategy for bone defect repair.

[0031] Based on the above, the present invention first provides a method for preparing mineralized bodies for bone repair, comprising the following steps: S1, after osteoblast-associated cells are cultured in vitro, osteoblast-induced culture is performed to induce the osteoblast-associated cells to secrete migratory bodies and deposit calcium salts; S2, the culture system obtained in step S1 is subjected to decellularization to obtain mineralized bodies, which include the migratory bodies and calcium salts.

[0032] In some embodiments, the osteogenic induction culture conditions are as follows: cultured for 14 to 21 days in DMEM complete medium containing 40 μg / mL ascorbic acid, 2 mg / mL β-glycerophosphate sodium and 100 nM dexamethasone.

[0033] In some embodiments, the osteogenic-related cells are osteogenic precursor stem cells, which refer to stem cells or precursor cells derived from bone cortex tissue that have the ability to differentiate into osteoblasts and participate in the bone formation process; in other embodiments, they may also be bone-derived mesenchymal stem cells or cells that exhibit an osteogenic phenotype after osteogenic induction culture.

[0034] In some embodiments, the decellularization method includes enzymatic treatment. Further, the specific steps of the enzymatic treatment include: aspirating the culture medium obtained in step S1, washing with PBS, adding 0.25% trypsin-EDTA digestion solution, digesting at 37°C for 30 minutes, aspirating the digestion solution, adding complete culture medium to stop digestion, and repeatedly pipetting.

[0035] In some embodiments, step S1, prior to the osteogenic induction culture, further includes: seeding in vitro cultured osteogenic-associated cells onto a biomaterial, such that the mineralized bodies are eventually deposited in situ on at least a portion of the surface of the biomaterial through natural growth.

[0036] In some embodiments, the biomaterial is a bioscaffold. It is understood that, compared to using simple mineralized bodies as bone repair materials, bone repair materials formed by growing mineralized bodies on the surface of a scaffold, combined with 3D / 4D printing technology, allow for precise fabrication of the scaffold according to the individual defect shape, achieving the formation of complex structures, improving the matching degree with the defect site, ultimately reducing surgical adjustment time, and improving surgical outcomes and healing quality. Furthermore, compared to existing technologies that load extracellular matrix such as exosomes onto the material surface or interior through physical adsorption, chemical coupling, or embedding, the bone repair material formed by the natural growth and adhesion method of this invention exhibits stronger structural continuity and greater interfacial bonding between the mineralized bodies and the material interface. This avoids the problems of active structures (such as exosomes and other extracellular matrix) easily detaching, becoming inactive, or exhibiting uneven spatial distribution during the preparation or use of existing technologies, thus improving the stability and repeatability of the material's osteogenic induction effect. As an example, the scaffold includes at least one of porous bone scaffolds, bioceramic scaffolds, polymer scaffolds, metal scaffolds, or composite material scaffolds. In porous scaffolds, the mineralized bodies also grow into the inner wall surface of the pores, further improving the bonding strength of the materials. In addition, the high porosity and interconnected channels of porous scaffolds are also conducive to blood vessel ingrowth and cell migration during bone repair, thereby improving the bone regeneration effect.

[0037] In other embodiments, the biomaterial may also be a titanium implant or an electrospun membrane. In practical applications, the mineralized bodies can be inoculated onto the surface of different biomaterials for growth according to the final requirements. This invention does not limit this.

[0038] In another aspect, the present invention provides a mineralized body for bone repair, wherein the mineralized body for bone repair is obtained by the aforementioned preparation method.

[0039] In another aspect, the present invention provides the application of the aforementioned mineralized bodies in the preparation of bone defect repair materials.

[0040] The experimental process and results of the present invention will be described in detail below through specific embodiments and accompanying drawings.

[0041] Unless otherwise specified in the embodiments of this invention, all reagents and consumables used are commercially available products. In the embodiments of this invention, ascorbic acid was purchased from Sigma (product model A4544), β-glycerophosphate was purchased from Sigma (product model G9422), dexamethasone was purchased from Sigma (product model D4902), 0.25% trypsin-EDTA digestion solution was purchased from Gibco (product model C25200), DEME basal medium was purchased from Gibco (product model C11995500BT), and penicillin-streptomycin was purchased from Gibco (product model 15140-122).

[0042] The research on laboratory animals was approved by the Ethics Committee of the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine (Approval No. SH9H-2020-T36-2), and all experimental procedures were conducted in accordance with the policies and ethics of animal research.

[0043] Example 1: Preparation of mineralized bodies for bone repair like Figure 1 As shown, this embodiment provides a method for preparing mineralized bodies for bone repair, including the following steps: Step 1: Obtaining and culturing osteogenic progenitor stem cells Human jawbone cortical fragments were obtained from the buccal and occlusal sides of tissue removed during the extraction of impacted mandibular third molars at the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine. The periosteum and bone marrow tissue were removed by PBS rinsing and mechanical scraping. The fragments were then rinsed with PBS and cut into small pieces for later use. The obtained cortical bone fragments were evenly distributed in culture dishes containing DMEM complete medium (DMEM basal medium containing 10% (v / v) FBS and 1% (v / v) penicillin and streptomycin). After 7 days of culture, osteogenic progenitor stem cells with osteogenic differentiation potential were obtained and cultured in vitro under adherent conditions with the medium changed every 3 days to achieve a stable growth state.

[0044] The purpose of this step is to provide a source of cells with clear osteogenic potential for the subsequent formation of mineralized bodies, thus ensuring the osteogenic properties of the obtained mineralized bodies from the source.

[0045] Step 2: Osteogenic induction and mineralization culture The osteogenic progenitor stem cells obtained above were passaged to P3 and subjected to osteogenic induction medium (DMEM complete medium containing 40 μg / mL ascorbic acid, 2 mg / mL β-glycerophosphate sodium and 100 nM dexamethasone) to induce osteogenic differentiation and mineralization. The osteogenic progenitor stem cells secreted migratory bodies and calcium salts. Obvious mineralization deposition could be observed after 7 days, and stable calcium nodules were formed after 14 to 21 days.

[0046] The purpose of this step is to induce osteogenic progenitor stem cells to gradually form mineralized bodies in the extracellular space, which are associated with calcium salt deposition and mineralization matrix. This allows the mineralized bodies to form naturally during osteoogenesis, rather than being artificially introduced through exogenous means, thereby ensuring their physiological relevance.

[0047] Step 3: Decellularization to obtain mineralized bodies After the formation of mineralized bodies (14 days after osteogenic induction), the culture system was decellularized by aspirating the culture medium, washing three times with PBS, adding 0.25% trypsin-EDTA digestion solution and digesting at 37°C for 30 minutes. After the reaction, the digestion solution was aspirated, and DMEM complete culture medium was added and the main cell structure was removed by pipetting, while retaining the migratory bodies and calcium nodules to obtain the mineralized bodies.

[0048] It is worth mentioning that, such as Figure 2 As shown, in the induced differentiation culture of osteogenic progenitor stem cells, this invention first detected a large amount of calcium salt deposition (ARS fluorescent labeling) and migration body co-localization (GFP fluorescent labeling of TSPAN4 protein). Furthermore, during the decellularization process to eliminate the risks associated with live cells, this invention, through comparison, found that different treatment techniques and digestion times significantly affected the retention rate of migration bodies in the final mineralized bodies, ultimately resulting in significant differences in osteopromoting effects. Specifically, compared to incomplete mineralized bodies with removed or destroyed migration body structures, mineralized bodies retaining migration bodies exhibit superior osteopromoting effects.

[0049] TSPAN4 protein is essential for the formation of migration bodies. This example also used Tspan4 siRNA co-cultured with osteogenic progenitor stem cells. Results showed that... (See...) Figure 3 As shown, compared with the control group (si-Con), the experimental group (si-Tspan4) showed a significant decrease in the content of migration bodies (WGA fluorescent label) and the amount of calcium nodule deposition (ARS fluorescent label), indicating that migration bodies are beneficial to promoting the formation and deposition of calcium salts and play an important role in the osteogenic effect of the mineralized bodies.

[0050] Therefore, the decellularization process in step 3 of this invention needs to remove cells while preserving the migratory bodies as much as possible, avoiding the removal or destruction of the migratory bodies. The degree of decellularization can be complete removal or basic removal of the main cell structure, but the principle followed is not to damage the structure of the migratory bodies or affect their retention. After screening and optimization, comparing single or combined methods of various conventional decellularization methods such as physical-mechanical methods, chemical methods, freeze-drying methods, and enzymatic hydrolysis, this embodiment ultimately adopts a combination of biological enzyme treatment and mechanical pipetting for decellularization. This method achieves a high decellularization rate while minimizing damage to the migratory bodies, resulting in greater retention of migratory bodies in the mineralized bodies.

[0051] This embodiment ultimately yielded mineralized bodies derived from osteogenic progenitor stem cells and possessing osteogenic-related characteristics, providing a foundation for subsequent applications.

[0052] Example 2: Preparation of bone repair materials and evaluation of their in vivo bone repair effects (I) Preparation of bone repair materials This embodiment, based on Embodiment 1, further applies the mineralized bodies to the functional construction of bone repair materials. The preparation process is as follows: Figure 4 As shown.

[0053] Step 1: Obtaining and culturing osteogenic progenitor stem cells For specific instructions, please refer to step 1 in Example 1, which will not be repeated here.

[0054] Step 2: Osteogenic induction and mineralization culture The osteogenic progenitor stem cells obtained above were passaged to P3, then seeded on the surface of a BCP scaffold and cultured in osteogenic induction medium (DMEM complete medium containing 40 μg / mL ascorbic acid, 2 mg / mL β-glycerophosphate sodium and 100 nM dexamethasone) to allow them to enter the osteogenic differentiation and mineralization stage. The osteogenic progenitor stem cells secreted migratory bodies and calcium salts, and obvious mineralization deposition could be observed after 7 days.

[0055] Step 3: Decellularization to obtain bone repair material with surface-loaded mineralized bodies After mineralization bodies formed (14 days after osteogenic induction), the culture system was decellularized by aspirating the culture medium, washing three times with PBS, adding 0.25% trypsin-EDTA digestion solution, and digesting at 37°C for 30 minutes. After the reaction, the digestion solution was aspirated, and DMEM complete culture medium was added. The main cell structure was removed by pipetting, retaining the migratory bodies and calcium nodules. Figure 5 The results showed that many migration bodies remained on the scaffold even after decellularization. Simultaneously, the mineralized bodies and their bonding state with the scaffold interface were preserved, resulting in a bone repair material with surface-loaded mineralized bodies.

[0056] In this embodiment, the mineralized bodies are naturally formed and bonded to the surface of the scaffold material, which improves the interfacial bonding strength between the mineralized bodies and the scaffold and avoids the material structure instability problem caused by physical adsorption or chemical coupling in the prior art. By removing the main cells through decellularization treatment and retaining the mineralized bodies attached to the scaffold surface, a cell-free bone repair material with osteogenic induction function is finally obtained, which can be used to regulate host cell behavior and promote bone defect repair.

[0057] (II) Evaluation of in vivo bone repair efficacy The repair material with surface-loaded mineralized bodies obtained above from human osteogenic progenitor stem cells was applied to a mouse model of bone defects to evaluate the in vivo therapeutic effect and immune safety of the material of the present invention.

[0058] To establish a clinically relevant in vivo model of critical-sized bone injury, skull defects (4 mm in diameter) were induced in 10-week-old male C57BL / 6 mice (n=6). Specifically, under isoflurane inhalation anesthesia, a midline scalp incision and periosteal dissection were performed, followed by the creation of bilateral defects in the central parietal bone using a trephine drill. The aforementioned bone repair material loaded with mineralized particles (…) Figure 6 The text describes the implantation of mig-BCP into a mouse model of skull defects, using an unmodified BCP scaffold as a control. Figure 6 (A) After skin suturing, buprenorphine was injected every 12 hours for 48 hours. Eight weeks postoperatively, bone regeneration was analyzed by micro-computed tomography (CT) and histomorphometry. After decalcification, bone tissue sections were sectioned and stained for histological analysis.

[0059] The results showed that 8 weeks post-surgery, the experimental group (mig-BCP) mice exhibited significantly enhanced new bone formation, resulting in a reduction in defect area, while the control group (BCP) mice showed only minimal osteogenic activity at the defect margins. Figure 6 (B). Although the trabecular thickness remained comparable between the two groups, the experimental group mice showed higher bone mineral density, volume percentage, and trabecular number. Figure 6 (C). Compared with the control group mice, which showed minimal bone regeneration and vascular infiltration at the periphery of the bone defect area, the experimental group mice showed more extensive vascular infiltration and new bone formation in the central region of the bone defect. Figure 6 In addition, the expression of osteogenic markers OCN and RUNX2 was slightly elevated around the defect in the control group, but significantly elevated in the newly formed bone tissue in the experimental group. Figure 6 (FG).

[0060] The above experimental results show that the repair material with surface-loaded mineralized bodies prepared from human osteogenic progenitor stem cells provided by this invention can provide stable osteogenic-related microstructural signals in mice with bone defects, thereby regulating the bone regeneration process under cell-free conditions, successfully promoting the formation of vascularized bone, and avoiding the immune and safety issues caused by live cell transplantation.

[0061] In summary, this invention, based on mineralized bodies composed of migratory bodies and calcium nodules obtained from osteogenic progenitor stem cells through osteogenic induction culture and decellularization, possesses the characteristics of originating from the osteogenic progenitor stem cells themselves, naturally forming during osteogenic processes, highly matching and stably binding with material interfaces, and maintaining stable osteogenic-related activities after decellularization. It can induce osteogenic differentiation and promote bone regeneration in vivo, while avoiding the risks associated with live cell transplantation, providing a novel and efficient cell-free strategy for bone defect repair.

[0062] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for preparing mineralized bodies for bone repair, characterized in that, Includes the following steps: S1, after osteoblast-associated cells are cultured in vitro, osteoblast-induced culture is performed to induce the osteoblast-associated cells to secrete migratory bodies and deposit calcium salts; S2, the culture system obtained in step S1 is subjected to decellularization to obtain mineralized bodies, which include the migratory bodies and calcium salts.

2. The method for preparing mineralized bodies for bone repair as described in claim 1, characterized in that, The osteogenic induction culture conditions were as follows: cultured in DMEM complete medium containing 40 μg / mL ascorbic acid, 2 mg / mL β-glycerophosphate sodium and 100 nM dexamethasone for 14 to 21 days.

3. The method for preparing mineralized bodies for bone repair as described in claim 1, characterized in that, The osteogenic-related cells include at least one of osteoblasts, osteogenic progenitor cells, osteogenic progenitor stem cells, bone-derived mesenchymal stem cells, or cells that exhibit an osteogenic phenotype after osteogenic induction culture.

4. The method for preparing mineralized bodies for bone repair as described in claim 1, characterized in that, The decellularization process includes biological enzyme treatment.

5. The method for preparing mineralized bodies for bone repair as described in claim 4, characterized in that, The specific operation of the biological enzyme treatment includes: aspirating the culture medium of the culture system obtained in step S1, washing with PBS, adding 0.25% trypsin-EDTA digestion solution, digesting at 37°C for 30 minutes, aspirating the digestion solution, adding DMEM complete culture medium to stop digestion, and repeatedly pipetting with a pipette.

6. The method for preparing mineralized bodies for bone repair as described in claim 1, characterized in that, In step S1, before performing the osteogenic induction culture, the method further includes: seeding osteogenic-associated cells cultured in vitro onto a biological material.

7. The method for preparing mineralized bodies for bone repair as described in claim 6, characterized in that, The mineralized bodies grow in situ on at least a portion of the surface of the biomaterial.

8. The method for preparing mineralized bodies for bone repair as described in claim 6, characterized in that, The biomaterials include at least one of the following: bioscaffolds, titanium implants, and electrospun membranes.

9. A mineralized body for bone repair, characterized in that, The mineralized bodies used for bone repair are obtained by the preparation method described in any one of claims 1-8.

10. The application of the mineralized bodies according to claim 9 in the preparation of bone defect repair materials.

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