Ossification center organoid and preparation method and application thereof

Abstract

The invention provides an ossification center organoid and a preparation method and application thereof.The ossification center organoid comprises a microsphere carrier system, and the microsphere carrier system is prepared from CGRP, BMP-2, bone marrow mesenchymal stem cells and GelMA-HA-NB hydrogel through the photocuring 3D printing technology; the microsphere carrier system and GelMA hydrogel containing SP are mixed, cross-linked and cured to form an ossification center organ which is used for in-vivo transplantation to stimulate rapid regeneration of endogenous bone tissues; the ossification center organoid has the ability of biphasic delivery of bioactive factors, can enable different bioactive factors to realize sequential, differential, low-dose, slow and sustained release, and accelerates the healing speed and bone repair effect of large-size bone defects in a separate and treatment manner. The invention further provides a bioactive factor composition of the bionic ossification center tissue neurotrophic factor and the physiological dose bone morphogenetic protein. The composition is released in an ossification center organ according to a signal time sequence to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an ossification center organoid and a preparation method and application thereof. Background Art

[0002] Bone defect is one of the most common traumas. Since large-sized bone defects exceed the limit of self-repair ability, the healing time is long and it is easy to cause lifelong disability in patients. Most bone injuries regenerate by forming callus and then remodeling into new bone during the repair process. Large-sized bone defects such as critical-sized bone defects are difficult to effectively form callus tissue in a short period of time, which often leads to delayed healing or non-union. Metal prostheses, polymer prostheses, autologous bone transplants and allogeneic bone transplants are considered to be the "gold standard" for clinical treatment at present. However, these treatments face difficulties such as limited donors, immune rejection, infection and poor bone integration, and are prone to heterogeneous scar healing or other complications, and ultimately lead to secondary surgery. Epidemiological studies have shown that the prevalence of non-union of bone injuries is about 5% in patients with bone injuries. Clinical treatment is still difficult, which has brought serious social health burdens.

[0003] Biomaterial prostheses are the preferred strategy for repairing large-sized bone defects. However, traditional bone grafts face the problems of rough internal structure and surface functional modification. They are difficult to fully vascularize and homogeneously generate bone in the body, and bone integration is difficult. In recent years, domestic leading research teams have made polymer prosthetic materials have similar composition and structural characteristics to natural bone tissue, and have the advantages of promoting bone differentiation and collagen mineralization in vivo based on bionic nanohydroxyapatite and micro-nano coating technology. However, synthetic prosthetic materials with high mechanical strength are often difficult to biodegrade after transplantation in vivo, and the potential for internal vascularization and new bone growth in the structure is limited, so bone integration is difficult. And because the prosthesis has a space-occupying effect, the new tissue cannot replace the scaffold material to fill the defect in a short period of time, making it difficult to achieve complete bone regeneration even for a long time.

[0004] Most bone repair treatments based on stem cell transplantation are still in the early research stage, mainly focusing on the transplantation of mesenchymal stem cells expanded in vitro. Due to the low efficiency of in vitro cell expansion, long culture time, low cell colonization and survival rate after in vivo transplantation, and insufficient local blood supply, it has long been difficult to form a consistent conclusion on the efficacy of stem cell transplantation, and its potential for further clinical application is limited. At the same time, the heterogeneity of cells after in vitro expansion is high, and it is impossible to accurately predict or intervene in their differentiation direction in vivo after transplantation, and there is still a lack of understanding of their potential mechanism of bone repair. The strategy of using biomineralized hydrogels to regulate the osteogenic differentiation of stem cells or pre-culture of osteogenic differentiation in vitro by cutting-edge research teams at home and abroad is still challenging in the rapid bone healing and structural reconstruction of critical-sized bone defects, which can easily lead to the formation of disordered and immature bone tissue. In addition, well-known bone injury treatment methods include adsorbing recombinant human bone morphogenetic protein-2 (BMP-2) on collagen sponge carriers or loading it into microspheres to promote the osteogenic differentiation of endogenous stem cells. However, its therapeutic effect is largely affected by factors such as ectopic bone formation, osteoclast activation and soft tissue inflammation induced by high doses, and requires additional mechanical stimulation to promote functional bone regeneration. The above studies suggest that there is an urgent need to develop a "bionic" bone repair strategy composed of absorbable scaffold materials, potent stem cells and physiological doses of bone-inducing active soluble factors.

[0005] Due to the definite cell source and genetic homology, organoids or microtissues provide a low immunogenicity and sustainable source of tissue alternatives for autologous tissue transplantation. The concept of bone organoids has been gradually proposed in recent years, such as woven bone organoids, callus organoids, trabecular bone organoids, and humanized ossicles, which can be used to study bone formation, differentiation mechanisms and stimulate tissue regeneration. Bone organoids are based on the directed differentiation of stem / progenitor cells and are three-dimensional micro-bone tissues with biomimetic spatial characteristics, self-renewal and self-assembly capabilities. Matrigel and synthetic hydrogels are used to support the assembly of bone organoids to achieve oxygen, metabolic gradients, and cell-to-cell connections. The formation of injectable osteogenic microtissues by emulsifying and incorporating stem cells can effectively provide the stem cells required for repair; the delivery of cryogel microspheres containing osteoblasts and angiogenic cells can enhance osteogenic differentiation ability. In addition, demineralized bone matrix particles modified with BMP-2 are beneficial for the repair of bone defects with high mechanical properties. These studies show that the "all-in-one" strategy of loading stem cells and growth factors provides a reference for the construction of bone organoids by simulating the process of osteogenesis. However, the microstructure of bone tissue has a delicate hierarchy and orderly arrangement, and cells are exposed to a variety of cytokines and growth factors and have complex interactions with surrounding cells. It is difficult to reproduce in vivo elements in vitro by simply mimicking the structure and composition of mature bones to build bone organoids, and the survival time after in vivo transplantation is short.

[0006] Therefore, it is urgent to find an ossification center organoid to solve the above problems. Summary of the invention

[0007] According to the defects of the prior art, the present invention provides an ossification center organoid and its preparation method and application, the ossification center organoid includes a microsphere carrier system, the microsphere carrier system is made of CGRP, BMP-2, bone marrow mesenchymal stem cells and GelMA-HA-NB hydrogel by light-curing 3D printing technology, and the microsphere carrier system is mixed with GelMA hydrogel containing SP to cross-link and solidify to form an ossification center organoid, which is used for in vivo transplantation to stimulate endogenous regenerative cells to promote rapid regeneration of bone tissue; the ossification center organoid has the ability to deliver bioactive factors in two phases, which can achieve sequential, differential low-dose, slow and continuous release of different bioactive factors, and accelerate the healing speed and bone repair effect of large-sized bone defects in a divide-and-conquer manner. The present invention also provides a bioactive factor composition of biomimetic ossification center tissue neurotrophic factor and physiological dose bone morphogenetic protein, which is released in the ossification center organoid according to the signal sequence to promote the osteogenic differentiation of bone marrow mesenchymal stem cells.

[0008] An ossification center organoid comprises a microsphere carrier system, wherein the microsphere carrier system comprises a neuropeptide, a recombinant human bone morphogenetic protein-2, a stem cell and a first hydrogel.

[0009] In some ways, the formation of an ossification center is the key basis for rapid bone development. Subsequent bone development is based on the formed ossification center and continues to expand and extend to form the final bone tissue. The ossification center organoids provided by the present invention have similar functions to ossification centers. During the development of the ossification center, cells in the mesenchyme condensation develop into chondrocytes and secrete cartilage matrix. The cells around the newly differentiated cartilage form the perichondrium and define the boundaries of the developing bones. The cells that define the boundaries exit the cell cycle and hypertrophy occurs, blood vessels infiltrate the hypertrophic cartilage, and a "primary ossification center" is formed in this area, triggering the differentiation of osteoblasts and eventually forming a bone marrow cavity.

[0010] In some ways, the previous research of the present invention team (Biomaterials, 2022. High-efficient engineering of osteo-callus organoids for rapid bone regeneration within one month) found that the hydrogel microsphere unit loaded with stem cells constructed by 3D printing based on DLP technology was further assembled into "bone callus organoids" under cartilage differentiation induction conditions, and showed transcriptome characteristics highly similar to those during bone development, and a state of synchronous active cell proliferation and differentiation. Through similarity analysis, the present invention team also found that the bone organoids constructed based on stem cell microspheres are highly similar to the diversified cell composition and cell behavior of endochondral ossification during bone development, providing a favorable osteogenic microenvironment for the rapid occurrence of new bone. Therefore, based on the previous research, the present invention provides an ossification center organoid, an ossification center organoid composed of a microsphere carrier system (the hydrogel microsphere unit loaded with stem cells) and a second hydrogel, the microsphere carrier system is made of a first hydrogel, loaded with bone marrow mesenchymal stem cells (BMSCs), one or more bioactive factors, and the microsphere carrier system can release one or more bioactive factors at a low dose, slowly and continuously to promote BMSCs osteogenic differentiation. Another one or more bioactive factors are added to the second hydrogel, which can release another one or more bioactive factors slowly and continuously at low doses to promote osteogenic differentiation of BMSCs. Since the degradation rates of the first hydrogel and the second hydrogel in the body are different, the release rates of the bioactive factors carried by the microsphere carrier system and the bioactive factors carried by the second hydrogel are different. Therefore, the ossification center organoid provided by the present invention is a composite carrier system with the function of biphasic delivery of bioactive factors.

[0011] Furthermore, it also includes a second hydrogel, and the microsphere carrier system and the second hydrogel are cross-linked and solidified to form an ossification center organoid.

[0012] Furthermore, the neuropeptide includes one or more of neuropeptide vasoactive intestinal peptide, neuropeptide Y, and calcitonin gene-related peptide.

[0013] In some ways, neurogenesis and angiogenesis are necessary processes in the early stages of natural bone development. The healing of bone injuries is also regulated by the sequence of signals that promote re-neuralization, revascularization, and ossification. The re-neuralization of callus tissue precedes angiogenesis and ossification. Early neurogenesis is essential for the normal formation of ossification centers. Signals from new sensory nerves can provide osteogenic cues and promote the differentiation of stem cells into osteoblast lineages. Studies have found that early (3 days after injury) neural innervation / occurrence and neuropeptide expression (such as CGRP and NGF) in bone repair play an important role in enhancing angiogenesis and osteogenic differentiation. However, current bone tissue engineering methods usually focus on optimizing the local delivery of angiogenic factors (VEGF) and recombinant human bone morphogenetic protein-2 (BMP-2) to achieve low-dose, sustainable release of growth factors to enhance the effect of promoting bone regeneration. Although studies have shown that the synergistic function of the two factors can be exerted by changing the release order or spatial distribution of the two factors, the transplantation of these combined elements still faces limitations such as delayed in vivo revascularization and heterogeneous bone formation caused by critical size defects, and the healing rate of bone repair is slow. The previous research of the present invention team (Nature Communications, 2022. Msx1 + The results of a study on the local transplantation of bioactive hydrogel scaffolds containing neurotrophic factor complexes showed that Msx1 could be specifically amplified in situ in bone defects. + Skeletal stem cell subsets, and achieve efficient bone regeneration through the endochondral ossification pathway. The above studies have shown that the reinnervation and revascularization of bone healing follow a predictable injury response pattern with temporal and spatial characteristics. Bone development and injury repair require corresponding signals to program neurogenesis, angiogenesis and osteogenesis. Therefore, the present invention uses osteogenic active tissue as a substrate to load multiphasic neurotrophic factors and osteogenic growth factors, simulates the timing process of ossification center formation, and constructs "ossification center" organoids to stimulate efficient endogenous bone tissue regeneration.

[0014] In some ways, recombinant human bone morphogenetic protein-2 (BMP-2) regulates and induces bone tissue during bone healing, causing undifferentiated mesenchymal stem cells to migrate, proliferate, and differentiate, thereby promoting the growth of osteoblasts and chondroblasts, and accelerating the morphogenesis and cell differentiation of bone and cartilage tissue. However, recombinant human bone morphogenetic protein-2 (BMP-2), as a recognized factor that promotes osteogenic differentiation of stem cells, often causes heterotopic ossification because its clinical dosage is too high, and low doses are ineffective for bone healing, so its clinical transformation is limited. The present invention has experimentally demonstrated that the neuropeptide vasoactive intestinal peptide (VIP), neuropeptide Y (NYP), and calcitonin gene-related peptide (CGRP) can cooperate with physiological doses of recombinant human bone morphogenetic protein-2 (BMP-2) to promote human bone differentiation.

[0015] Preferably, calcitonin gene-related peptide (CGRP) can significantly synergize with physiological doses of recombinant human bone morphogenetic protein-2 (BMP-2) to promote human bone differentiation.

[0016] Furthermore, the second hydrogel contains neuropeptide substance P; the first hydrogel degrades to release neuropeptide and recombinant human bone morphogenetic protein-2, and the second hydrogel degrades to release neuropeptide substance P.

[0017] In some ways, studies have shown that the neuropeptide substance P (SP) has the function of promoting the homing of endogenous mesenchymal stem cells (MSCs) to the lesion. In the present invention, it is specifically manifested that the neuropeptide substance P (SP) can promote bone marrow mesenchymal stem cells (BMSCs) to express vascular endothelial growth factor (VEGF) and promote the migration of bone marrow mesenchymal stem cells (BMSCs).

[0018] In some embodiments, experiments have shown that the degradation rate of the GelMA hydrogel in vivo is greater than that of the GelMA-HA-NB hydrogel, so GelMA-HA-NB and GelMA can be prepared into a composite carrier for dual-phase delivery of substances.

[0019] Furthermore, the first hydrogel is GelMA-HA-NB, and the second hydrogel is GelMA.

[0020] Furthermore, a method for preparing an ossification center organoid comprises the following steps:

[0021] (1) adding neuropeptides, recombinant human bone morphogenetic protein-2, and stem cells to the first hydrogel, and mixing them evenly to prepare a microsphere carrier system; the neuropeptide comprises one or more of neuropeptide vasoactive intestinal peptide, neuropeptide Y, and calcitonin gene-related peptide;

[0022] (2) adding the neuropeptide substance P to the second hydrogel and mixing evenly;

[0023] (3) The microsphere carrier system and the mixture of step (2) are mixed and then cross-linked and cured.

[0024] Furthermore, the final concentration of the neuropeptide added to the first hydrogel in step (1) is 1×10 -8 ~1×10 -7 M, the final concentration of the recombinant human bone morphogenetic protein-2 is 0.5-5 μg / mL, and the final concentration of the stem cells is 2×10 5 ~2×10 6 cells / mL.

[0025] Furthermore, the final concentration of the neuropeptide substance P added to the second hydrogel in step (2) is 1×10 -8 ~1×10 - 7 M; the volume ratio of the microsphere carrier system and the mixture in step (3) is (4-1): (2-1).

[0026] Furthermore, a bioactive factor composition is used for preparing ossification center organoids that promote rapid healing of large-sized bone defects, wherein the composition includes calcitonin gene-related peptide, recombinant human bone morphogenetic protein-2, and neuropeptide substance P.

[0027] Furthermore, a composite carrier system is used to prepare ossification center organoids that promote rapid healing of large-sized bone defects. The composite carrier system includes a microsphere carrier system and a second hydrogel. The microsphere carrier system includes bone marrow mesenchymal stem cells, bioactive factors and a first hydrogel, and the second hydrogel contains neuropeptide substance P.

[0028] In some ways, the process of bone healing is similar to that of bone development, which is a sequential process of vascularization and osteogenic differentiation. First, angiogenesis occurs, and then stem cells follow the blood vessels into the ossification center to differentiate into osteoblasts. Therefore, it is necessary to release the first phase neuropeptide substance P (SP) to induce angiogenesis. Experiments have shown that calcitonin gene-related peptide (CGRP) can cooperate with physiological doses of recombinant human bone morphogenetic protein-2 (BMP-2) to induce osteogenic differentiation. Therefore, CGRP and BMP-2 are used together as a combination of cytokines released in the second phase. The degradation rate of the composite carrier system in the first two days in the body is different, and the degradation rate of the second hydrogel GelMA releasing the bioactive factors is greater than that of the first hydrogel GelMA-HA-NB, so the neuropeptide substance P (SP) carried by the second hydrogel GelMA is first released to induce bone marrow mesenchymal stem cells (BMSCs) to express vascular endothelial growth factor (VEGF) to promote angiogenesis, and then the calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) carried by the first hydrogel GelMA-HA-NB are released to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). After two days, the degradation rates of the first hydrogel GelMA-HA-NB and the second hydrogel GelMA releasing the bioactive factors tend to be consistent, because angiogenesis and osteogenic differentiation are carried out simultaneously at this time. Through the composite carrier system, the neuropeptide substance P (SP), calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) can be released in a sequential low dose, slowly and continuously in the body for 28 days.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention provides an ossification center organoid, including a microsphere carrier system, wherein the microsphere carrier system is made of CGRP, BMP-2, bone marrow mesenchymal stem cells and a first hydrogel by light-curing 3D printing technology, and the microsphere carrier system is mixed with a second hydrogel containing SP to form a composite carrier system for stimulating rapid regeneration of endogenous bone tissue;

[0031] (2) The present invention provides a bioactive factor composition, including calcitonin gene-related peptide, recombinant human bone morphogenetic protein-2 and neuropeptide substance P, wherein the neuropeptide substance P can promote bone marrow mesenchymal stem cells to express vascular endothelial growth factor to accelerate angiogenesis, and the calcitonin gene-related peptide and recombinant human bone morphogenetic protein-2 can cooperate with physiological doses to promote osteogenic differentiation of bone marrow mesenchymal stem cells;

[0032] (3) The ossification center organoid provided by the present invention is a composite carrier system made of two different hydrogels. The two different hydrogels have different degradation rates in the body and have the ability to deliver bioactive factors in two phases. Different bioactive factors can be released in a sequential, low-dose, slow, and sustained manner for up to 28 days to enhance the effect of promoting bone regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 4 A is the preparation process of the microsphere carrier system in Example 1;

[0034] Figure 4 B is the preparation process of step (2) of Example 1;

[0035] Figure 1 AB are the ALP staining results and quantitative evaluation results of stem cells with different neuropeptides added in Example 2;

[0036] Figure 1 CF is the expression levels of ALP and RUNX2 on the 7th and 14th days after osteogenic induction of 2BMSCs;

[0037] Figure 1 GH is the absorbance value of ALP staining and alizarin red staining on day 14 of implementation 2;

[0038] Figure 1 IJ is the ALP staining and alizarin red staining section image of Example 2 on day 14;

[0039] Figure 2 A is the preparation process of the microsphere carrier system in Example 2;

[0040] Figure 2 B is the live-dead staining results of the microsphere carrier system in Example 2 on days 1, 4, 7, and 14;

[0041] Figure 2 C is the cell number detection result of the microsphere carrier system in Example 2 on days 1, 4, 7, and 14;

[0042] Figure 2 D is the Bright Field test result of the microsphere carrier system in Example 2 on the first day of culture;

[0043] Figure 2 E is the ALP staining result of the microsphere carrier system in Example 2 on the 7th day of culture;

[0044] Figure 2 F is the alizarin red staining result of the microsphere carrier system in Example 2 on the 14th day of culture;

[0045] Figure 2GH is the fluorescence staining result of Collagen I, Phalloidin and DAPI of the microsphere carrier system in Example 2 on the 14th day of culture;

[0046] Figure 3 AB are the results of the degradation rate determination of the two hydrogels under rat skin in Example 3;

[0047] Figure 3 C is the observation image of the subcutaneous tissues of the two groups of rats made into slices at the 1st, 2nd and 4th week in Example 3;

[0048] Figure 3 D is a flow chart of the construction of the dual-phase composite carrier system in Example 3;

[0049] Figure 3 EF is the test result of the ability of the two-phase composite carrier system to release bioactive factors in Example 3;

[0050] Figure 4 C is the carrier system of Example 4 after bone defect implantation and curing;

[0051] Figure 4 D is the different vector system groups of Example 4;

[0052] Figure 4 EF is the BV / TV results of each group at 1 month and 2 months after surgery in Example 4;

[0053] Figure 4 GH is the first image of the observation of the stained sections of the bone tissues of each group at the 1st and 2nd month after surgery in Example 4;

[0054] Figure 5 ab are the second images of the bone tissues of each group made into stained sections for observation at the 1st and 2nd month after surgery in Example 4;

[0055] Figure 5 cd are the results of Safranin-O staining of ossification center organoid group 2 (OCO-2) in Example 4 at the first and second months after surgery;

[0056] Figure 5 ef is the fluorescence staining diagram of OCN and NGFR expression in the ossification center organoid group 2 (OCO-2) in Example 4 at the first and second months after surgery;

[0057] Figure gi is a diagram of the bone healing process;

[0058] Figure j is a fluorescence staining diagram of the expression of CGRP and βⅢtubulin in different vector systems in Example 4 at the second month after surgery;

[0059] Figure k is a fluorescence staining diagram of the expression of CD31 and COLX in different vector systems in Example 4 at the second month after surgery;

[0060] FIG1 is a fluorescence staining diagram of RUNX2 expression in different vector systems of Example 4 at the second month after surgery. DETAILED DESCRIPTION

[0061] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0062] Example 1. Preparation of an ossification center organoid

[0063] 1. A method for preparing an ossification center organoid, comprising the following steps:

[0064] (1) Preparation of microsphere carrier system: BMSCs (primary extract from rat bone marrow) were added to 50 mg / ml GelMA-HA-NB hydrogel (purchased from Sigma-Aldrich, gelatin model V900863; methacrylic anhydride model 276685) ink to a final concentration of 2×10 6 cells / ml, and CGRP (purchased from Abcam, model Rat CGRP peptide ab47101) was added to a final concentration of 10 -7 M, add BMP-2 (purchased from Peprotech, model Human / Mouse / Rat BMP-2 Recombinant Protein, Gibco Catalog #120-02C) to a final concentration of 5 μg / ml, mixed evenly, and made into a microsphere carrier system by light-curing 3D printing technology, such as Figure 4 As shown in A;

[0065] (2) The above microsphere carrier system was loaded with 100 mg / ml GelMA (purchased from Sigma-Aldrich, gelatin model V900863) for 10 -7 MSP (purchased from MCE, model Substance P MCE Cat. No.: HY-P0201) hydrogel was mixed at a volume ratio of 2:1. Figure 4 As shown in B;

[0066] (3) Preparation of ossification center organoid precursors: 20 μl of the mixture prepared in step (2) was taken and cross-linked and cured by UV light to form a two-phase composite carrier system with a diameter of 5 mm and a thickness of 1 mm (taking the rat bone defect model as an example). It should be noted that the shape of the bone defect model of the animal model is regular, so the shape can be prefabricated and then implanted into the bone defect model. In actual applications, the shape of the bone defect may be irregular. The mixture of step (2) can be first injected into the bone defect position and then cross-linked and cured by UV light.

[0067] Example 2 Effects of neuropeptides and neuropeptide and BMP-2 combinations on osteogenic differentiation of BMSCs

[0068] 1. Effects of neuropeptides and neuropeptide and BMP-2 combination on osteogenic differentiation of BMSCs

[0069] The osteogenic differentiation of endogenous stem cells requires contact with BMP-2 and multiple neuropeptides. This example explores the effect of neuropeptides synergistically with physiological doses of BMP-2 on the osteogenic differentiation of BMSCs. Neuropeptides include neuropeptide vasoactive intestinal peptide (VIP), neuropeptide Y (NYP), and calcitonin gene-related peptide (CGRP).

[0070] During the osteogenic induction culture of BMSCs, seven groups of substances were added separately as shown in Table 1. The addition ratios were as shown in Example 1. VIP, NYP and CGRP were added to the culture medium to a final concentration of 10 -7 M, BMP-2 was added to the culture medium to a final concentration of 5 μg / ml.

[0071] Table 1. Different BMSCs osteogenic induction culture additives

[0072]

[0073] On the 1st, 3rd and 5th days of BMSCs osteogenic induction culture, the above seven groups were subjected to ALP staining and quantitative evaluation, and BMSCs without bioactive ingredients were used as controls. The results of ALP staining and quantitative evaluation showed that compared with the results of the first, second and third groups, Figure 1 As shown in AB, it is shown that when neuropeptides are added alone, CGRP is more likely to induce osteogenic differentiation of BMSCs than VIP and NPY. The osteogenic differentiation of BMSCs in the fifth, sixth, and seventh groups is better than that in the first, second, third, and fourth groups, indicating that the combination of neuropeptides and BMP-2 can synergistically promote the osteogenic differentiation of BMSCs.

[0074] ALP staining and ALP quantitative evaluation, Alizarin red staining and RUNX2 quantitative evaluation were performed on the 7th and 14th days after BMSCs osteogenic induction. The expression levels of ALP and RUNX2 can evaluate the osteogenic differentiation of BMSCs cells. The results showed that the osteogenic differentiation effect of the cells in the seventh group with CGRP+BMP-2 was significantly better than that of the cells in the fifth group with VIP+BMP-2 and the cells in the sixth group with NYP+BMP-2; on the seventh day, the ALP expression level of the cells in the seventh group with CGRP+BMP-2 was 25.6% and 31.7% higher than that of the cells in the fifth group with VIP+BMP-2 and the cells in the sixth group with NYP+BMP-2, respectively, and the RUNX2 expression level of the cells in the seventh group with CGRP+BMP-2 was 25.6% and 31.7% higher than that of the cells in the fifth group with VIP+BMP-2 and the cells in the sixth group with NYP+BMP-2, respectively. -2 cells, and the sixth group of cells with NYP+BMP-2 were 40.6% and 50% higher, respectively; on the fourteenth day, the ALP expression content of the seventh group of cells with CGRP+BMP-2 was 37.4% and 45.6% higher than that of the fifth group of cells with VIP+BMP-2, and the sixth group of cells with NYP+BMP-2, respectively; the RUNX2 expression content of the seventh group of cells with CGRP+BMP-2 was 20.8% and 33.1% higher than that of the fifth group of cells with VIP+BMP-2, and the sixth group of cells with NYP+BMP-2, respectively. Comparing the results of the third, fourth, and seventh groups, from the perspective of ALP staining and alizarin red staining sections and absorbance values ​​on the fourteenth day, as shown in Figure 1 IJ Figure 1 As shown in GH, compared with the third group of cells with CGRP alone or the fourth group of cells with BMP-2 alone, the seventh group with CGRP+BMP-2 had a higher absorbance value and a deeper staining depth than the other two groups, indicating that more osteogenic differentiation-related proteins were expressed. From the quantitative evaluation of ALP and RUNX2 on the seventh and fourteenth days, as shown in Figure 1 As shown in CF, whether on the seventh day or the fourteenth day, the seventh group of cells with CGRP+BMP-2 expressed the highest levels of ALP and RUNX2, which were significantly higher than those in the other two groups. In summary, the BMSCs cells in the seventh group with the addition of the combination of CGRP+BMP-2 had stronger osteogenic differentiation ability and more calcium nodules.

[0075] It is proved that the neuropeptide CGRP can promote osteogenic differentiation synergistically with physiological doses of BMP-2.

[0076] 2. Effect of the microsphere carrier system made of CGRP and BMP-2 combination on osteogenic differentiation of BMSCs

[0077] This example refers to step (1) of Example 1 to prepare different microsphere carrier systems for cell culture. The preparation process is as follows: Figure 2As shown in A, three groups of microsphere carrier systems were prepared, respectively as follows:

[0078] Group 1: GelMA-HA-NB hydrogel ink and BMSCs were made into a microsphere carrier system by photocuring 3D printing technology, and this group served as a control;

[0079] Group 2: GelMA-HA-NB hydrogel ink was combined with BMSCs and BMP-2 to form a microsphere carrier system through photocuring 3D printing technology;

[0080] The third group: GelMA-HA-NB hydrogel ink was combined with BMSCs, CGRP, and BMP-2 to form a microsphere carrier system through photocuring 3D printing technology.

[0081] The three groups of microsphere carrier systems were cultured for cells, and the Bright Field of the three groups of microsphere carrier systems was detected on the first day of culture. Figure 2 As shown in D, cells with the same initial cell number were encapsulated in three groups of microsphere carrier systems. The cell number of each group was detected on days 1, 4, 7, and 14. Figure 2 As shown in C, the number of cells in the third group was higher than that in the second group from day 1 to day 14. Live-dead staining was performed on days 1, 4, 7, and 14. Figure 2 As shown in B, the cells in the three groups of microsphere carrier systems grew well. On the 14th day of culture, the three groups of cells almost grew full in the sphere unit. ALP staining was performed on the 7th day. Figure 2 As shown in E, it shows that the expression level of the third group of cells is the highest. Alizarin red staining was performed on the 14th day. Figure 2 As shown in F, the third group of cells produced a large number of mineralized nodules compared with the first and second groups of cells. Collagen I, Phalloidin, and DAPI fluorescence staining were performed on day 14, and the three staining results were combined, as shown in Figure 2 As shown in GH, compared with the first and second groups, the third group had the highest expression level of type I collagen.

[0082] In summary, the third group of microsphere carrier systems containing a combination of CGRP and BMP-2 showed significantly higher cell expression levels in cell culture than those in the first and second groups, further proving that the neuropeptide CGRP can synergize with physiological doses of BMP-2 to promote the osteogenic differentiation of BMSCs.

[0083] Example 3: Study on the degradation rate of different hydrogel carriers

[0084] Two hydrogel materials, GelMA and GelMA-HA-NB, were prepared. The preparation process referred to steps (2) and (3) of the method for preparing an ossification center organoid in Example 1. The specific steps were as follows: GelMA and GelMA-HA-NB in ​​Example 1 were respectively configured into 100 mg / ml hydrogel solutions, 20 μl of GelMA and GelMA-HA-NB hydrogel solutions were respectively absorbed, and cross-linked and cured using UV light to form a columnar hydrogel with a diameter of 5 mm and a thickness of 1 mm (taking a rat bone defect model as an example).

[0085] The above two columnar hydrogels were implanted under the skin of rats to test their biosafety and differential degradation rates in vivo. The sizes of the two hydrogels were measured at 1, 2, and 4 weeks to evaluate the degradation rate. Figure 3 As shown in AB, the degradation rate of GelMA in vivo is significantly faster than that of GelMA-HA-NB. At each time point tested, the degradation degree of GelMA is higher than that of the latter. In addition, at the 4th week after subcutaneous transplantation, GelMA has been almost completely degraded, while GelMA-HA-NB has delayed degradation. The subcutaneous tissues of the two groups of rats were sliced ​​and stained at the 1st, 2nd and 4th week to test the biosafety of the two hydrogels. Figure 3 As shown in C, the subcutaneous tissues of the two groups of rats were normal at each time point, indicating that the two hydrogels have high biosafety and are harmless to tissues after degradation.

[0086] The difference in the degradation rates of GelMA and GelMA-HA-NB in ​​vivo indicates that they can be used to construct a composite delivery system for differential release of cytokines. Figure 3 D, the preparation steps refer to Example 1, and the specific steps are as follows:

[0087] (1) Add model protein A to 50 mg / ml GelMA-HA-NB hydrogel solution to a final concentration of 10 -7 M, after being mixed evenly, a microsphere carrier system carrying model protein A was prepared by photocuring 3D printing technology;

[0088] (2) The above microsphere carrier system was loaded with 100 mg / ml GelMA. -7 The hydrogel of M-mode protein B was mixed at a volume ratio of 2:1;

[0089] (3) 20 μl of the mixture prepared in step (2) was taken and cross-linked and cured using UV light to form a two-phase composite carrier system with a diameter of 5 mm and a thickness of 1 mm (taking the rat bone defect model as an example).

[0090] The ability of the above-mentioned two-phase composite carrier system to carry and release bioactive factors was tested. The experimental results are as follows Figure 3As shown in Figure EF, in the first two days of release, the release rate of model protein B was faster than that of model protein A, and there was a differential release effect. After two days, both A and B began to be released slowly and continuously, and the release time could be as long as 28 days.

[0091] From the above experimental results, it can be seen that the dual composite carrier system has different hydrolysis rates in the first two days of the two hydrogels, so the release rate of active substances carried by different hydrogels in the first two days in the body is different. Because the process of bone healing and bone development is similar, it is a sequential process of vascularization and osteogenic differentiation. First, angiogenesis occurs, and then stem cells follow the blood vessels to enter the ossification center for osteogenic differentiation. Neuropeptide substance P (SP) induces bone marrow mesenchymal stem cells (BMSCs) to express vascular endothelial growth factor (VEGF) to promote angiogenesis, so it is necessary to release the first phase neuropeptide substance P (SP) first. Example 2 verifies that the degradation and release of calcitonin gene-related peptide (CGRP) can promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in coordination with physiological doses of recombinant human bone morphogenetic protein-2 (BMP-2), so calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) are released as the second phase. After two days, the rate of degradation and release of bioactive factors of the first hydrogel and the second hydrogel tends to be consistent, because angiogenesis and osteogenic differentiation are carried out simultaneously at this time. In summary, the neuropeptide substance P (SP) is added to GelMA hydrogel as the first phase of release, and calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) are added to GelMA-HA-NB hydrogel as the second phase and released. The neuropeptide substance P (SP), calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) can be released sequentially, slowly and continuously in the body for 28 days.

[0092] Example 4: Effects of different microcarrier systems on bone defect healing

[0093] like Figure 5 As shown in gi, this is the process of bone healing, in which the bone defect site is first neuralized, then vascularized and ossified.

[0094] To investigate the effects of different microcarrier systems on bone defect healing, Figure 4 As shown in D, five groups of carrier systems are set up, and the preparation process refers to Example 1:

[0095] Group 1: Blank group (Blank / Defect), without any bioactive factors and microcarriers;

[0096] Group 2: blank microcarrier group (Vehicle), blank microcarriers were made with GelMA-HA-NB mixed with GelMA hydrogel in a ratio of 2:1;

[0097] Group 3: Hybrid group, in which GelMA hydrogel was used to mix SP, CGRP, BMP-2 and BMSCs (single-phase carrier system);

[0098] Group 4: Ossification center organoid group 1 (OCO-1), prepared according to the method for preparing an ossification center organoid in Example 1, but the GelMA hydrogel does not contain SP;

[0099] The fifth group: Ossification center organoid group 2 (OCO-2), was prepared according to the ossification center organoid preparation method in Example 1.

[0100] The above five carrier systems were implanted into the bone defect of rats. Figure 4 C shows the situation of implanting the carrier system into the bone defect, and the bone defect healing of each group at each time point was detected. The bone volume and score (BV / TV) of the five groups were detected at the first and second months after surgery. Figure 4 As shown in EF, the BV / TV value of ossification center organoid group 2 (OCO-2) was significantly higher than that of the other four groups, indicating that the bone anabolism of ossification center organoid group 2 (OCO-2) was greater than the catabolism, and the increase in bone mass can promote the rapid healing of bone defects. At the first and second months after surgery, the bone tissues of each group were sliced ​​and stained for observation. Figure 4 GH, Figure 5As shown in ab, at 1 month after surgery, the new bone at the edge of the defect in the blank microcarrier group (Vehicle) was mainly generated along the surface of the microcarrier, showing an ossification pattern from outside to inside, while the microcarriers of the ossification center organoid group II (OCO-2) generated new bone on both the surface and inside, showing a pattern of simultaneous ossification inside and outside, and the bone healing of the remaining groups was not as good as that of the ossification center organoid group II (OCO-2). At 2 months after surgery, the spherical new bone at the edge of the blank microcarrier group (Vehicle) still showed the form of external ossification while the center had not yet ossified, while the ossification center organoid group II (OCO-2) had connected new bone on the surface of the defect and a large amount of bone formation inside, and the bone healing of the remaining groups was not as good as that of the ossification center organoid group II (OCO-2). Comparing the bone healing conditions of the cell and active factor mixed group (Hybrid), ossification center organoid group 1 (OCO-1) and ossification center organoid group 2 (OCO-2), the bone generation content of ossification center organoid group 2 (OCO-2) was much higher than that of ossification center organoid group 1 (OCO-1), and the bone healing of the cell and active factor mixed group (Hybrid) was better than that of ossification center organoid group 1 (OCO-1). On the one hand, it shows that the addition of SP accelerates the speed of CGRP and BMP-2 in promoting the osteogenic differentiation of BMSCs. On the other hand, it shows that the dual-phase delivery of bioactive factors can further promote the osteogenic differentiation of BMSCs compared with the single-phase delivery of bioactive factors. Because bone healing is a process in which angiogenesis occurs first and then stem cells follow the blood vessels into the ossification center to undergo osteogenic differentiation, the ossification center organoid group 2 (OCO-2), which has a better bone healing effect, has a first phase in which the neuropeptide substance P (SP) induces angiogenesis and a second phase in which calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) are released to induce osteogenic differentiation.

[0101] In the second month after surgery, fluorescence staining was performed on the first, second, third, and fifth groups to compare the bone healing conditions of each group. Figure 5 As shown in Figure j, DAPI was used to fix and stain living cells, CGRP was stained green by CollagenⅠ, and βⅢtubulin was stained red by Phalloidin. βⅢtubulin is an important component of the cytoskeleton. The results showed that only the fifth group (OCO-2) secreted CGRP and produced more βⅢtubulin, while the other groups only produced a small amount of βⅢtubulin, indicating that the BMSCs of the fifth group (OCO-2) differentiated into more osteoblasts. Figure 5As shown in k, DAPI is used to fix and stain living cells, CD31 is green after staining with CollagenⅠ and is used to detect angiogenesis, and COLX is red after staining with Phalloidin and is mainly present in rat serum and plasma. The results show that only the fifth group (OCO-2) generated a large number of blood vessels, while the other groups only generated a small amount of COLX and did not generate a large number of blood vessels. Figure 5 As shown in Figure 1, DAPI was used to fix and stain living cells, and RUNX2 was green after staining with Collagen I. RUNX2 plays a key role in osteoblast differentiation and bone morphogenesis. The results showed that only the fifth group (OCO-2) produced a large amount of RUNX2, while the other groups only produced a small amount of RUNX2, indicating that the fifth group (OCO-2) secreted more RUNX2 to promote bone healing during bone healing. In summary, the ossification center organoids of the fifth group (OCO-2) have stronger bone healing ability and faster healing speed.

[0102] Safranin-O staining was performed on the ossification center organoid group 2 (OCO-2) at 1 month and 2 months after surgery. The staining results are shown in Figure 5 As shown in cd, it can be seen that a large amount of cartilage tissue has been formed in the first month, and a large amount of bone tissue has been formed in the second month. Fluorescence staining was performed on the ossification center organoid group 2 (OCO-2) at the first and second months after surgery. DAPI was used to fix and stain living cells. OCN was stained with CollagenⅠ fluorescent dye to appear green. OCN is a protein secreted by osteoblasts. NGFR was stained with Phalloidin fluorescent dye to appear red. NGFR plays an important role in nerve growth and development. The staining results are shown in Figure 5 As shown in Figures ef, it can be seen that in the first month, a small amount of NGFR and OCN have been generated, indicating that nerves and bone cells have been generated. In the second month, more NGFR and OCN are generated, indicating that a large number of nerves and bone cells have been generated, indicating that bone healing is good.

[0103] In summary, SP can assist CGRP and BMP-2 in promoting the osteogenic differentiation of BMSCs, and the ossification center organoids with dual-phase delivery of bioactive factors can further promote the rapid healing of bone defects. It also proves that the ossification center organoids provided by the present invention have the ability to first neuralize, then vascularize and ossify the bone defect site.

[0104] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. An ossification center organoid, characterized in that The invention comprises a microsphere carrier system, wherein the microsphere carrier system comprises neuropeptide, recombinant human bone morphogenetic protein-2, stem cells and a first hydrogel.

2. The ossification center organoid according to claim 1, characterized in that It also includes a second hydrogel, and the microsphere carrier system is cross-linked and solidified with the second hydrogel to form an ossification center organoid.

3. The ossification center organoid according to claim 1, wherein The neuropeptide comprises one or more of neuropeptide vasoactive intestinal peptide, neuropeptide Y and calcitonin gene-related peptide.

4. The ossification center organoid according to claim 2, characterized in that The second hydrogel contains neuropeptide substance P; the first hydrogel degrades to release neuropeptide and recombinant human bone morphogenetic protein-2, and the second hydrogel degrades to release neuropeptide substance P.

5. The ossification center organoid according to claim 3, characterized in that The first hydrogel is GelMA-HA-NB, and the second hydrogel is GelMA.

6. A method for preparing ossification center organoids, characterized in that: The following steps are involved: (1) adding neuropeptides, recombinant human bone morphogenetic protein-2, and stem cells to the first hydrogel, and mixing them evenly to prepare a microsphere carrier system; the neuropeptide comprises one or more of neuropeptide vasoactive intestinal peptide, neuropeptide Y, and calcitonin gene-related peptide; (2) adding the neuropeptide substance P to the second hydrogel and mixing evenly; (3) The microsphere carrier system and the mixture of step (2) are mixed and then cross-linked and cured.

7. The preparation method according to claim 6, characterized in that: The final concentration of the neuropeptide added to the first hydrogel in step (1) is 1×10 -8 ~1×10 -7 M, the final concentration of the recombinant human bone morphogenetic protein-2 is 0.5-5 μg / mL, and the final concentration of the stem cells is 2×10 5 ~2×10 6 cells / mL.

8. The preparation method according to claim 7, characterized in that: The final concentration of the neuropeptide substance P added to the second hydrogel in step (2) is 1×10 -8 ~1×10 -7 M; the volume ratio of the microsphere carrier system and the mixture in step (3) is (4-1): (2-1).

9. Use of a bioactive factor composition for preparing ossification center organoids that promote rapid healing of large-sized bone defects, characterized in that: The composition comprises calcitonin gene-related peptide, recombinant human bone morphogenetic protein-2 and neuropeptide substance P.

10. Use of a composite carrier system for preparing ossification center organoids that promote rapid healing of large-sized bone defects, characterized in that: The composite carrier system comprises a microsphere carrier system and a second hydrogel. The microsphere carrier system comprises bone marrow mesenchymal stem cells, bioactive factors and a first hydrogel. The second hydrogel comprises a neuropeptide substance P.

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