A centimeter organ of ossification center and its preparation method and application

Abstract

The application provides a bone nodule organoid and a preparation method and application thereof, the bone nodule organoid comprises 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 a light curing 3D printing technology, the microsphere carrier system is mixed with GelMA hydrogel containing SP to be crosslinked and solidified to form the bone nodule organoid, and the bone nodule organoid is used for in-vivo transplantation to stimulate endogenous bone tissue rapid regeneration;The bone nodule organoid has the ability of two-phase delivery of bioactive factors, different bioactive factors can realize time sequence, differential low dose, slow and continuous release, so that the healing speed and bone repair effect of large-size bone defect are accelerated in a divide-and-rule manner.The application also provides a bioactive factor composition of a bionic bone nodule tissue neurotrophic factor and a physiological dose of bone morphogenetic protein, the composition releases the bone marrow mesenchymal stem cell osteogenic differentiation in the bone nodule organoid according to signal time sequence.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and in particular relates to a mineralization center organoid and a preparation method and application thereof. BACKGROUND

[0002] Bone defect is one of the most common injuries. Since large size bone defects exceed the limit of self-repairing ability, the healing time is long, which easily leads to lifelong disability of patients. Most bone injuries are regenerated in the repair process by forming callus and then remodeling into new bone. Large size, such as critical size, bone defects are difficult to effectively form callus tissue in a short time, thus often leading to delayed healing or non-healing. Metal prosthesis, polymer prosthesis, autologous bone transplantation and allogeneic bone transplantation are considered as the "gold standard" of current clinical treatment. However, these treatment methods face the problems of limited donor, immune rejection, infection and poor bone integration, and are prone to inhomogeneous scar healing or to cause other complications, and eventually lead to secondary surgery. Epidemiological studies show that the prevalence of non-healing bone injury is about 5% in patients with bone injury, and the clinical treatment is still difficult, thus bringing a serious social health burden.

[0003] Biological material prosthesis is the preferred strategy for repairing large size bone defects. However, traditional bone grafts face the problems of not fine internal structure and surface functional modification, and are difficult to fully vascularize and homogenously osteogenesis in vivo, and are difficult to integrate with bone. In recent years, domestic leading research teams based on biomimetic nanohydroxyapatite and micro-nano coating technology make polymer prosthesis materials have similar composition and structural characteristics to natural bone tissue, and have the advantages of promoting bone differentiation and collagen mineralization in vivo. However, synthetic prosthesis materials with high mechanical strength are often difficult to biodegrade after in vivo transplantation, and the potential of internal vascularization and new bone growth is limited, thus it is difficult to integrate with bone. And because the prosthesis has a space occupying effect, the newly formed tissue cannot replace the scaffold material to fill the defect in a short time, leading to difficulty in achieving complete bone regeneration even in a long time.

[0004] Most of the stem cell transplantation-based bone repair treatments 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, and low cell survival rate and local blood supply after in vivo transplantation, it is difficult to form a consistent conclusion on the long-term efficacy of stem cell transplantation, and the potential for further clinical application is limited. At the same time, the heterogeneity of cells expanded in vitro is high, and it is impossible to accurately predict or intervene in their differentiation direction after transplantation, and the potential mechanism of bone repair is still lacking. The strategy of using biomimetic hydrogel to regulate stem cell osteogenic differentiation or pre-culture of in vitro osteogenic differentiation still has challenges in rapid bone healing and structure reconstruction of critical-sized bone defects, which is easy to lead to the formation of disordered and immature bone tissue. In addition, the well-known bone injury treatment methods include adsorbing recombinant human bone morphogenetic protein-2 (BMP-2) in collagen sponge carriers or loading into microspheres to promote the osteogenic differentiation of endogenous stem cells. However, its therapeutic effect is greatly influenced by factors such as ectopic bone formation, activation of osteoclasts, and soft tissue inflammation induced by high doses, as well as the need for additional mechanical stimulation to promote functional bone regeneration. The above studies suggest that it is urgent 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 cell source determination and gene homology, the organoids or microtissues provide a low immunogenicity and sustainable source of tissue replacement for autologous tissue transplantation. In recent years, the concept of bone organoids has gradually been proposed, such as woven bone organoids, callus organoids, trabecular bone organoids, and humanized ossicles, which can be used to study the formation and differentiation mechanism of bone and stimulate tissue regeneration. Bone organoids are based on the directional 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 gradient, and cell-cell connection. The injectable pro-osteogenic microtissue formed by emulsifying stem cells can effectively provide stem cells required for repair; the delivery of frozen gel microspheres containing osteogenic and angiogenic cells can enhance the osteogenic differentiation capacity. In addition, BMP-2 modified demineralized bone matrix particles are beneficial to the repair of bone defects with high mechanical performance requirements. These studies show that the "all-in-one" strategy of loading stem cells and growth factors provides a reference for constructing bone organoids to simulate the process of bone formation. However, the microstructure of bone tissue has a delicate hierarchy and ordered arrangement, and cells are in contact with various cytokines and growth factors, and complex interactions occur with surrounding cells. It is difficult to reproduce in vitro the elements in vivo by simply constructing bone organoids by simulating the structure and composition of mature bone, and the survival time is short after transplantation in vivo.

[0006] Therefore, it is urgent to find a bone nodule organoid to solve the above problems. SUMMARY

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

[0008] A bone nodule organoid comprises a microsphere carrier system, wherein the microsphere carrier system comprises a neuropeptide, a recombinant human bone morphogenetic protein-2, stem cells and a first hydrogel.

[0009] In some ways, the formation of the ossification center is the key basis for the rapid development of bone, and the subsequent bone development is continuously extended around the formed ossification center as the basis to form the final bone tissue. The ossification center organoid provided by the application has similar functions to the ossification center. During the development of the ossification center, the cells in the mesenchymal coagulation develop into chondrocytes and secrete cartilage matrix. The cells around the newly differentiated cartilage form a perichondrium and define the boundary of the developing skeleton. The cells that define the boundary exit the cell cycle and appear hypertrophic, and blood vessels infiltrate the hypertrophic cartilage, and the "primary ossification center" is formed in this area, triggering the differentiation of osteoblasts and eventually forming the bone marrow cavity.

[0010] In some ways, the team of the application found in the previous research (Biomaterials, 2022. High-efficient engineering of osteo-callus organoids for rapid bone regeneration within one month) that the stem cell-loaded hydrogel microsphere unit based on DLP technology 3D printing construction is further assembled into "callus organoids" under the condition of chondrocyte differentiation induction, and shows a high similarity with the transcriptome characteristics in the bone development period, and the state of synchronous activity of cell proliferation and differentiation. Through similarity analysis, the team of the application also found that the bone organoids based on stem cell microspheres are highly similar to the diversified cell composition and cell behavior in the intramembranous ossification of cartilage during bone development, providing a favorable osteogenic microenvironment for the rapid occurrence of new bone. Therefore, on the basis of the previous research, the application provides an ossification center organoid, which is composed of a microsphere carrier system (the above-mentioned stem cell-loaded hydrogel microsphere unit) and a second hydrogel. The microsphere carrier system is made of a first hydrogel, and loads bone marrow mesenchymal stem cells (BMSCs) and one or more bioactive factors. The microsphere carrier system can release the one or more bioactive factors at a low dose, slowly and continuously to promote the osteogenic differentiation of BMSCs. The second hydrogel is added with another one or more bioactive factors, which can release the another one or more bioactive factors at a low dose, slowly and continuously to promote the 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 application is a composite carrier system with the function of double-phase delivery of bioactive factors.

[0011] Further, a second hydrogel is also included, and the microsphere carrier system is cross-linked and solidified with the second hydrogel to form the ossification center organoid.

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

[0013] In some ways, neurogenesis and angiogenesis are necessary processes in the early stages of natural development of bone, and the healing of bone injury is also regulated by a sequence of signals promoting neurogenesis, revascularization and ossification, and the reinnervation of callus precedes angiogenesis and ossification. Early neurogenesis is essential for the normal formation of ossification centers, and signals from nascent sensory nerves can provide osteogenic cues to promote differentiation of stem cells into osteoblast lineage. Studies have found that early innervation / neurogenesis and neuropeptide expression (such as CGRP and NGF) play an important role in enhancing vascularization and osteogenic differentiation in the early stages of bone repair (3 days after injury). However, current bone tissue engineering methods usually focus on optimizing the local delivery of vascular endothelial growth factor (VEGF) and recombinant human bone morphogenetic protein-2 (BMP-2) to achieve low-dose, sustained-release growth factors to enhance the effect of bone regeneration. Although studies have shown that the synergistic function of the two factors can be achieved by changing the release sequence or spatial distribution of the two factors, transplantation of these combined elements still faces limitations such as delayed revascularization in vivo and heterogeneous bone formation caused by critical size defects, and slow healing of bone repair. The team's previous research (Nature Communications, 2022. Msx1 + stem cells recruited by bioactive tissue engineering graft for bone regeneration) found that local transplantation of bioactive hydrogel scaffolds containing neurotrophic factor complexes can specifically expand Msx1 + bone stem cell subpopulations in situ and achieve efficient bone regeneration through the intramembranous ossification pathway. The above studies show that reinnervation and revascularization of bone healing follow a predictable, time-and-space-specific injury response pattern, and the development and repair of bone require corresponding signals to program neurogenesis, angiogenesis and osteogenesis. Therefore, the present application loads multiphasic neurotrophic factors and osteogenic growth factors on osteogenically active tissue-like substrates to simulate the timing process of ossification center formation and construct "ossification center" organoids to stimulate efficient endogenous bone tissue regeneration.

[0014] In some ways, recombinant human bone morphogenetic protein-2 (BMP-2) promotes the growth of osteoblasts and chondroblasts by regulating and inducing bone tissue, making undifferentiated mesenchymal stem cells migrate, proliferate and differentiate, thereby accelerating the morphogenesis and cell differentiation of bone and cartilage tissue during the process of bone healing. However, as a recognized factor for promoting the osteogenic differentiation of stem cells, recombinant human bone morphogenetic protein-2 (BMP-2) often causes ectopic ossification due to its large clinical use dose, and low dose is ineffective for bone healing, so its clinical transformation is limited. The present application proves by experiments that neuropeptide VIP, neuropeptide Y (NYP) and calcitonin gene-related peptide (CGRP) can synergistically promote human bone differentiation with physiological dose of recombinant human bone morphogenetic protein-2 (BMP-2).

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

[0016] Further, the second hydrogel comprises neuropeptide substance P; the first hydrogel degrades to release neuropeptides 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 neuropeptide substance P (SP) has the function of promoting the homing of endogenous mesenchymal stem cells (MSCs) to lesions, which in the present application specifically manifests that 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 ways, experiments have shown that the degradation rate of the GelMA hydrogel in vivo is greater than that of the GelMA-HA-NB hydrogel, so that GelMA-HA-NB and GelMA can be prepared into a composite carrier of a biphasic delivery material.

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

[0020] Further, a preparation method of an ossification center organoid comprises the following steps:

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

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

[0023] (3) mixing the microsphere carrier system and the mixture of step (2) and cross-linking and solidifying.

[0024] Further, the final concentration of the neuropeptide added 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] Further, the final concentration of the neuropeptide substance P added in step (2) is 1×10 -8 ~ 1×10 - 7 M; and the volume ratio of the microsphere carrier system to the mixture in step (3) is (4-1):(2-1).

[0026] Further, the use of a bioactive factor composition for preparing a bone nodule organoid for promoting rapid healing of a large-size bone defect, the composition comprising a calcitonin gene-related peptide, a recombinant human bone morphogenetic protein-2, and a neuropeptide substance P.

[0027] Further, the use of a composite carrier system for preparing a bone nodule organoid for promoting rapid healing of a large-size bone defect, the composite carrier system comprising a microsphere carrier system and a second hydrogel, the microsphere carrier system comprising bone marrow mesenchymal stem cells, bioactive factors, and a first hydrogel, and the second hydrogel comprising a neuropeptide substance P.

[0028] In some ways, the process of bone healing and bone development is similar, which is a vascularization and osteogenic differentiation process, first angiogenesis, and then stem cells follow the blood vessels into the ossification center to form osteogenic differentiation, so the first phase of neuropeptide substance P (SP) needs to be released to induce angiogenesis. Experiments have shown that calcitonin gene-related peptide (CGRP) can synergize with physiological doses of recombinant human bone morphogenetic protein-2 (BMP-2) to induce osteogenic differentiation, so CGRP and BMP-2 are used as the second phase of the cytokine combination. The complex carrier system has different degradation rates in the first two days in vivo, and the second hydrogel GelMA releases bioactive factors at a faster rate than the first hydrogel GelMA-HA-NB, so the neuropeptide substance P (SP) carried by the second hydrogel GelMA is released first 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 osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). After two days, the first hydrogel GelMA-HA-NB and the second hydrogel GelMA release bioactive factors at a rate that tends to be consistent, because angiogenesis and osteogenic differentiation occur at the same time. Through the complex 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 time sequence, low dose, slow, and continuous manner for 28 days in vivo.

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

[0030] (1) The present application provides an ossification center organoid, which comprises a microsphere carrier system made of CGRP, BMP-2, bone marrow mesenchymal stem cells, and a first hydrogel by light-cured 3D printing technology, and the microsphere carrier system is mixed and cross-linked with a second hydrogel containing SP to form a complex carrier system for stimulating endogenous bone tissue rapid regeneration.

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

[0032] (3) The osteogenic center organoid provided by the application is a composite carrier system made of two different hydrogels, the two different hydrogels have different degradation rates in vivo, have the ability of biphasic delivery of bioactive factors, and can realize time-sequential low-dose, slow and continuous release of different bioactive factors for 28 days to enhance the effect of promoting bone regeneration. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0035] Figure 1 A-B is the ALP staining result and quantitative evaluation result of stem cells added with different neuropeptides in Example 2;

[0036] Figure 1 C-F is the ALP and RUNX2 expression content of BMSCs at 7 days and 14 days after osteogenic induction in Example 2;

[0037] Figure 1 G-H is the ALP staining and alizarin red staining absorbance value at 14 days in Example 2;

[0038] Figure 1 I-J is the ALP staining and alizarin red staining section picture at 14 days in Example 2;

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

[0040] Figure 2 B is the live and dead staining result of the microsphere carrier system of Example 2 at 1 day, 4 days, 7 days and 14 days;

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

[0042] Figure 2 D is the Bright Field detection result of the microsphere carrier system of Example 2 at 1 day of culture;

[0043] Figure 2 E is the ALP staining result of the microsphere carrier system of Example 2 at 7 days of culture;

[0044] Figure 2 F is the alizarin red staining result of the microsphere carrier system of Example 2 at 14 days of culture;

[0045] Figure 2G-H are the results of fluorescence staining of Collagen I, Phalloidin and DAPI of the microsphere carrier system of Example 2 on the 14th day of culture;

[0046] Figure 3 A-B are the results of the determination of the degradation rate of the two hydrogels in Example 3 in rats subcutaneously;

[0047] Figure 3 C is the image of the subcutaneous tissue of the two groups of rats in Example 3 at 1st, 2nd and 4th weeks respectively;

[0048] Figure 3 D is the flow chart of the construction of the biphasic composite carrier system in Example 3;

[0049] Figure 3 E-F are the results of the detection of the ability of the biphasic composite carrier system in Example 3 to release bioactive factors;

[0050] Figure 4 C is the situation of the implanted solidified carrier system in the bone defect in Example 4;

[0051] Figure 4 D is the different carrier system groups in Example 4;

[0052] Figure 4 E-F are the results of BV / TV of each group at 1st and 2nd months after the operation in Example 4;

[0053] Figure 4 G-H are the images of the stained sections of the bone tissue of each group at 1st and 2nd months after the operation in Example 4;

[0054] Figure 5 a-b are the images of the stained sections of the bone tissue of each group at 1st and 2nd months after the operation in Example 4;

[0055] Figure 5 c-d are the results of Safranin-O staining of the osteogenic center organ group two (OCO-2) in Example 4 at 1st and 2nd months after the operation;

[0056] Figure 5 e-f are the fluorescence staining images of the expression amounts of OCN and NGFR of the osteogenic center organ group two (OCO-2) in Example 4 at 1st and 2nd months after the operation;

[0057] Fig. g-i is the diagram of the bone healing process;

[0058] Fig. j is the fluorescence staining image of the expression amounts of CGRP and βIII tubulin of the different carrier systems in Example 4 at 2nd month after the operation;

[0059] Figure K is a fluorescence staining image of CD31 and COLX expression of different carrier systems in Example 4 at the second month after surgery.

[0060] Figure L is a fluorescence staining image of RUNX2 expression of different carrier systems in Example 4 at the second month after surgery. DETAILED DESCRIPTION

[0061] The application will be further described in conjunction with the accompanying drawings and examples, it should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0062] Example 1, preparation of a mineralization center organoid

[0063] 1. A method for preparing a mineralization center organoid, the steps are as follows:

[0064] (1) Preparation of microsphere carrier system: add BMSCs (rat bone marrow primary extraction) to 50 mg / ml GelMA-HA-NB hydrogel ink (purchased from Sigma-Aldrich, gelatin type V900863; methacrylic anhydride type 276685) to a final concentration of 2 x 10 6 cells / ml, add CGRP (purchased from Abeam, type Rat CGRP peptide ab47101) to a final concentration of 10 -7 M, add BMP-2 (purchased from Peprotech, type Human / Mouse / Rat BMP-2 Recombinant Protein, Gibco Catalog #120-02C) to a final concentration of 5 μg / ml, mix well, and prepare the microsphere carrier system by light-cured 3D printing technology, as shown in Figure 4 A;

[0065] (2) Mix the above-mentioned microsphere carrier system with 100 mg / ml GelMA (purchased from Sigma-Aldrich, gelatin type V900863) loaded with 10 -7 M SP (purchased from MCE, type Substance P MCE Cat. No.: HY-P0201) hydrogel according to the volume ratio of 2:1, as shown in Figure 4 B;

[0066] (3) Preparation of ossification center organoid precursor: 20 μl of the mixture prepared in step (2) is taken and cross-linked and solidified using UV light to form a biphasic composite carrier system with a diameter of 5 mm and a thickness of 1 mm (for example, in the case of a rat bone defect model). It should be noted that the shape of the bone defect model of the animal model is regular, so it can be pre-shaped and implanted into the bone defect model. In actual applications, the shape of the bone defect may be irregular, and the mixture of step (2) can be injected into the bone defect position first, and then cross-linked and solidified by UV light.

[0067] Example 2, Effect of neuropeptides and neuropeptide-BMP-2 combination on BMSCs osteogenic differentiation

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

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

[0070] During the osteogenic induction culture of BMSCs, the following seven groups of substances shown in Table 1 were added separately. The addition ratio is referred to Example 1. VIP, NYP, and CGRP were added in the culture medium to a final concentration of 10 -7 M, and BMP-2 was added in 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 the osteogenic induction culture of BMSCs, the above seven groups were subjected to ALP staining and quantitative evaluation, respectively, with BMSCs without the addition of bioactive components as a control. The results of ALP staining and quantitative evaluation. The results show that, as shown in A-B, compared with the first, second, and third groups, the addition of CGRP alone is more likely to induce the osteogenic differentiation of BMSCs than the addition of VIP and NPY. The osteogenic differentiation of the fifth, sixth, and seventh groups of BMSCs is better than that of the first, second, third, and fourth groups, indicating that the combination of neuropeptides and BMP-2 can synergistically promote the osteogenic differentiation of BMSCs. Figure 1

[0074] ​ALP staining and ALP quantitative evaluation, alizarin red staining and RUNX2 quantitative evaluation were performed on the 7th day and the 14th day after the osteogenic induction of BMSCs. The expression content of ALP and RUNX2 can evaluate the osteogenic differentiation of BMSCs cells. The results show that the osteogenic differentiation effect of the cells in the seventh group adding CGRP+BMP-2 is significantly better than that of the cells in the fifth group adding VIP+BMP-2 and the cells in the sixth group adding NYP+BMP-2; on the seventh day, the ALP expression content of the cells in the seventh group adding CGRP+BMP-2 is 25.6% and 31.7% higher than that of the cells in the fifth group adding VIP+BMP-2 and the cells in the sixth group adding NYP+BMP-2 respectively, and the RUNX2 expression content of the cells in the seventh group adding CGRP+BMP-2 is 40.6% and 50% higher than that of the cells in the fifth group adding VIP+BMP-2 and the cells in the sixth group adding NYP+BMP-2 respectively; on the fourteenth day, the ALP expression content of the cells in the seventh group adding CGRP+BMP-2 is 37.4% and 45.6% higher than that of the cells in the fifth group adding VIP+BMP-2 and the cells in the sixth group adding NYP+BMP-2 respectively, and the RUNX2 expression content of the cells in the seventh group adding CGRP+BMP-2 is 20.8% and 33.1% higher than that of the cells in the fifth group adding VIP+BMP-2 and the cells in the sixth group adding NYP+BMP-2 respectively. Comparing the results of the third group, the fourth group and the seventh group, from the ALP staining and alizarin red staining sections and the absorbance values on the fourteenth day, as shown in Figs. Figure 1 I-J、 Figure 1 G-H, compared with the cells in the third group adding CGRP alone or the cells in the fourth group adding BMP-2 alone, the absorbance value of the cells in the seventh group adding CGRP+BMP-2 is higher, and the staining depth is deeper than the other two groups, indicating that the content of the expressed osteogenic differentiation related proteins is more. From the ALP quantitative evaluation and the RUNX2 quantitative evaluation on the seventh day and the fourteenth day, as shown in Figs. Figure 1 C-F, whether on the seventh day or on the fourteenth day, the cells in the seventh group adding CGRP+BMP-2 express the most content of ALP and RUNX2, which is significantly higher than the other two groups. In summary, it is proved that the composition of CGRP+BMP-2 added to the BMSCs cells in the seventh group has stronger osteogenic differentiation ability and more calcium nodule generation.

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

[0076] 2, The influence of the microsphere carrier system prepared by the combination of CGRP and BMP-2 on the osteogenic differentiation of BMSCs

[0077] In this embodiment, different microsphere carrier systems were prepared for cell culture according to step (1) of embodiment 1, and the preparation process is as shown in Fig. Figure 2A, three groups of microsphere carrier systems were prepared as follows:

[0078] The first group: GelMA-HA-NB hydrogel ink and BMSCs were made into a microsphere carrier system by light-cured 3D printing technology, and this group was used as a control;

[0079] The second group: GelMA-HA-NB hydrogel ink, BMSCs and BMP-2 were made into a microsphere carrier system by light-cured 3D printing technology;

[0080] The third group: GelMA-HA-NB hydrogel ink, BMSCs, CGRP and BMP-2 were made into a microsphere carrier system by light-cured 3D printing technology.

[0081] The above three groups of microsphere carrier systems were subjected to cell culture, and the Bright Field of the three groups of microsphere carrier systems was detected on the first day of culture, as shown in Figure 2 D, the same initial number of cells were encapsulated in the three groups of microsphere carrier systems. The number of cells in each group was detected on the 1st, 4th, 7th and 14th day, as shown in Figure 2 C, indicating that the number of cells in the third group was higher than that in the second group from the 1st to the 14th day. Live and dead staining was performed on the 1st, 4th, 7th and 14th day, as shown in Figure 2 B, the cells in the three groups of microsphere carrier systems grew well, and by the 14th day of culture, the cells in the three groups almost covered the sphere unit. ALP staining was performed on the 7th day, as shown in Figure 2 E, indicating that the expression level of the third group of cells was the highest. Alizarin red staining was performed on the 14th day, as shown in Figure 2 F, indicating that 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 fluorescent staining were performed on the 14th day, and the results of the three staining were combined, as shown in Figure 2 G-H, compared with the first and second groups, the expression level of collagen I in the third group was the highest.

[0082] In summary, the composition containing CGRP and BMP-2 in the third group of microsphere carrier system significantly improved the expression level of cells in cell culture compared with the first and second groups, further proving that the neuropeptide CGRP can promote the osteogenic differentiation of BMSCs in combination with physiological dose of BMP-2.

[0083] Example 3, degradation rate of different hydrogel carriers

[0084] GelMA and GelMA-HA-NB two kinds of hydrogel materials were prepared, and the preparation process referred to the preparation method of one osteogenic center organoid in Example 1, steps (2) and (3), and the specific steps were as follows: GelMA and GelMA-HA-NB in Example 1 were configured into 100 mg / ml hydrogel solution, respectively, 20 μl of GelMA and GelMA-HA-NB hydrogel solution was sucked, and UV light was used for crosslinking and solidification to form a columnar hydrogel with a diameter of 5 mm and a thickness of 1 mm (taking the rat bone defect model as an example).

[0085] The above two kinds of columnar hydrogels were respectively implanted subcutaneously in rats to detect their biological safety and in vivo differential degradation rate. The size of the two kinds of hydrogels was measured at 1, 2, 4 weeks to evaluate the degradation rate, as shown in Figure 3 A-B, the degradation rate of GelMA in vivo was significantly faster than that of GelMA-HA-NB, and at each time point, the degradation degree of GelMA was higher than that of GelMA-HA-NB, and at 4 weeks after subcutaneous transplantation, GelMA had been basically completely degraded, while GelMA-HA-NB was delayed degradation. The subcutaneous tissues of the two groups of rats were respectively made into sections and dyed at 1, 2, 4 weeks to detect the biological safety of the two kinds of hydrogels, as shown in Figure 3 C, the subcutaneous tissues of the two groups of rats at each time point were normal, indicating that the two kinds of hydrogels had high biological safety and were harmless to the tissues after degradation.

[0086] The difference in the in vivo degradation rate of GelMA and GelMA-HA-NB indicated that they could be used to construct a composite delivery system for differential release of cytokines. The construction process was as shown in Figure 3 D, and the preparation steps referred to Example 1, and the specific steps were as follows:

[0087] (1) Mode protein A was added to 50 mg / ml GelMA-HA-NB hydrogel solution to a final concentration of 10 -7 M, and after mixing uniformly, a microsphere carrier system carrying mode protein A was prepared by photocuring 3D printing technology;

[0088] (2) The above microsphere carrier system was mixed with 100 mg / ml GelMA loaded with 10 -7 M mode protein B according to the volume ratio of 2:1;

[0089] (3) 20 μl of the mixture prepared in step (2) was sucked, and UV light was used for crosslinking and solidification to form a biphasic 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 biphasic composite carrier system to carry and release bioactive factors was detected, and the experimental results were as shown in Figure 3As shown in E-F, the release rate of pattern protein B is faster than that of pattern protein A in the first 2 days, and there is a differential release effect. After 2 days, both A and B are slowly and continuously released, and the release time can be up to 28 days.

[0091] From the above experimental results, it can be seen that the double complex carrier system has different hydrolysis rates of the two hydrogels in the first two days, so the release rates of the active substances carried by the two hydrogels in the first two days are different. Because the processes of bone healing and bone development are similar, they are a time sequence process of vascularization and osteogenic differentiation. First, angiogenesis occurs, and then stem cells follow the blood vessels into the ossification center to differentiate into bone differentiation. Neuropeptide substance P (SP) induces bone marrow mesenchymal stem cells (BMSCs) to express vascular endothelial growth factor (VEGF) to promote angiogenesis, so the first phase neuropeptide substance P (SP) needs to be released first. It is verified by Example 2 that the degradation and release of calcitonin gene-related peptide (CGRP) can synergize with a physiological dose of recombinant human bone morphogenetic protein-2 (BMP-2) to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), 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 release rates of the first and second hydrogels tend to be consistent, because angiogenesis and osteogenic differentiation occur at the same time. In summary, neuropeptide substance P (SP) is added to GelMA hydrogel as the first phase 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 release. The neuropeptide substance P (SP), calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) can be released in the body in a time sequence, slow and continuous manner for 28 days.

[0092] Example 4, Effect of Different Microcarrier Systems on Bone Defect Healing

[0093] As shown in Figure 5 g-i, for the process of bone healing, the bone defect site is first innervated, then vascularized and ossified.

[0094] To explore the effect of different microcarrier systems on bone defect healing, as shown in Figure 4 D, five groups of carrier systems were set up, and the preparation process was referred to Example 1:

[0095] The first group: blank group (Blank / Defect), without any bioactive factors and microcarriers;

[0096] The second group: blank microcarrier group (Vehicle), blank microcarriers 2:1 mixed with GelMA hydrogel were prepared from GelMA-HA-NB;

[0097] Group 3: Hybrid cell and active factor mixture, using GelMA hydrogel 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 one of the ossification center organoid preparation methods in Example 1, but without SP in the GelMA hydrogel;

[0099] Group 5: Ossification center organoid group 2 (OCO-2), prepared according to one of the ossification center organoid preparation methods in Example 1.

[0100] The above five carrier systems were implanted into the bone defect sites of rats, such as Figure 4 Figure C shows the bone defect implantation system, examining the bone defect healing status of each group at different time points. Bone volume and fraction (BV / TV) were measured in all five groups at 1 and 2 months post-surgery. Figure 4 As shown in EF, the BV / TV value of ossification center organoid group II (OCO-2) was significantly higher than that of the other four groups, indicating that bone synthesis metabolism of ossification center organoid group II (OCO-2) is greater than catabolism metabolism, resulting in increased bone mass and promoting rapid healing of bone defects. Bone tissue sections from each group were prepared and stained for observation at 1 month and 2 months postoperatively. Figure 4 GH, Figure 5As shown in a-b, at 1 month after surgery, the new bone of the blank microcarrier group (Vehicle) was mainly generated along the surface of the microcarrier, showing an outside-in bone formation pattern, while the microcarriers of the second bone nodule center organ group (OCO-2) generated new bone on the surface and inside, showing an inside-out and outside bone formation pattern, and the bone healing of the remaining groups was not as good as that of the second bone nodule center organ group (OCO-2). At 2 months after surgery, the spherical new bone of the blank microcarrier group (Vehicle) still showed an outside bone formation and a central non-bone formation pattern, while the second bone nodule center organ group (OCO-2) had a large amount of bone formation on the surface of the defect and inside, and the bone healing of the remaining groups was not as good as that of the second bone nodule center organ group (OCO-2). Compared the bone healing of the second bone nodule center organ group (OCO-2) and the first bone nodule center organ group (OCO-1) and the cell and active factor mixed group (Hybrid), the bone formation content of the second bone nodule center organ group (OCO-2) was much higher than that of the first bone nodule center organ group (OCO-1), and the bone healing of the cell and active factor mixed group (Hybrid) was better than that of the first bone nodule center organ group (OCO-1), which on the one hand indicated that the addition of SP accelerated the speed of CGRP and BMP-2 promoting BMSCs osteogenic differentiation, and on the other hand indicated that the two-phase delivery of bioactive factors could further promote the osteogenic differentiation of BMSCs compared with the single-phase delivery of bioactive factors. Because bone healing is a process of blood vessel formation first, and then stem cells follow the blood vessels into the bone nodule center to form bone differentiation, therefore, the first phase of neuroactive substance P (SP) induces blood vessel formation, and the second phase of release of calcitonin gene-related peptide (CGRP) and recombinant human bone morphogenetic protein-2 (BMP-2) play a role in bone nodule center organ group two (OCO-2) have better bone healing effect.

[0101] At the second month after surgery, the first, second, third, and fifth groups were compared by fluorescence staining to compare the bone healing of each group. As shown in Figure 5 j, DAPI was used for fixed staining of living cells, CGRP was stained with Collagen I and showed green, and βΙΙΙ tubulin was stained with Phalloidin and showed red. βΙΙΙ tubulin is an important component of the cytoskeleton, and the results show that only the fifth group (OCO-2) secretes CGRP and generates more βΙΙΙ tubulin, while the other groups only generate a small amount of βΙΙΙ tubulin, indicating that the BMSCs of the fifth group (OCO-2) differentiate to form more osteocytes. As shown in Figure 5As shown in k, DAPI is used for fixing and staining living cells, CD31 is stained by Collagen I and appears green, which is used for detecting the angiogenesis, COLX is stained by Phalloidin and appears red, which mainly exists in rat serum and plasma, and the results show that only the fifth group (OCO-2) generates a large amount of blood vessels, while other groups only generate a small amount of COLX and do not generate a large amount of blood vessels. As shown in Figure 5 As shown in l, DAPI is used for fixing and staining living cells, RUNX2 is stained by Collagen I and appears green, and RUNX2 plays a key role in the process of osteoblast differentiation and bone morphogenesis, and the results show that only the fifth group (OCO-2) generates a large amount of RUNX2, while other groups only generate a small amount of RUNX2, which indicates that the fifth group (OCO-2) secretes more RUNX2 to promote bone healing in the process of bone healing. In summary, the bone healing ability of the osteogenic center organoids of the fifth group (OCO-2) is stronger, and the healing speed is faster.

[0102] The osteogenic center organoids group two (OCO-2) is subjected to Safranin-O staining at the first month and the second month after the operation, and the staining results are as shown in Figure 5 As shown in c-d, it can be seen that a large amount of cartilage tissue is formed in the first month, and a large amount of bone tissue is formed in the second month. The osteogenic center organoids group two (OCO-2) is subjected to fluorescence staining at the first month and the second month after the operation, DAPI is used for fixing and staining living cells, OCN is stained by Collagen I fluorescent dye and appears green, OCN is a protein secreted by osteoblasts, and NGFR is stained by Phalloidin fluorescent dye and appears red, and NGFR plays an important role in the process of nerve growth and development, and the staining results are as shown in Figure 5 As shown in e-f, it can be seen that a small amount of NGFR and OCN is generated in the first month, indicating that a small amount of nerve and bone cells is generated, and a large amount of NGFR and OCN is generated in the second month, indicating that a large amount of nerve and bone cells is generated, which indicates that the bone healing is good.

[0103] In summary, SP can assist CGRP and BMP-2 in promoting the osteogenic differentiation of BMSCs, and the osteogenic center organoids for biphasic delivery of bioactive factors can further promote the rapid healing of bone defects, and it is also proved that the osteogenic center organoids provided by the application have the ability to neuralize, vascularize and ossify the bone defect site.

[0104] Although the present application is disclosed as above, the present application 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 application, and therefore the protection scope of the present application should be subject to the scope defined by the claims.

Claims

1. An ossification center organoid, characterized in that, The composition comprises a microsphere carrier system and a second hydrogel, the microsphere carrier system comprises a neuropeptide, recombinant human bone morphogenetic protein-2, stem cells and a first hydrogel; the microsphere carrier system is cross-linked and solidified with the second hydrogel to form an ossification center organoid; the second hydrogel comprises a neuropeptide substance P; the first hydrogel degrades to release the neuropeptide and the recombinant human bone morphogenetic protein-2, and the second hydrogel degrades to release the neuropeptide substance P; the first hydrogel is GelMA-HA-NB, and the second hydrogel is GelMA.

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

3. A method of preparing an ossification center organoid, characterized by, The method comprises the following steps: (1) adding a neuropeptide, recombinant human bone morphogenetic protein-2 and stem cells to a first hydrogel, and mixing uniformly to prepare a microsphere carrier system; the neuropeptide comprises one or more of a neuropeptide intestinal vasoactive peptide, a neuropeptide Y and a calcitonin gene-related peptide; (2) adding a neuropeptide substance P to a second hydrogel and mixing uniformly; (3) mixing the microsphere carrier system and the mixture of step (2) uniformly, and cross-linking and solidifying.

4. The production method according to claim 3, wherein The final concentration of the neuropeptide added in step (1) is 1 x 10 -8 ~1 x 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 x 10 5 ~2 x 10 6 cells / mL.

5. The production method according to claim 4, wherein The final concentration of the neuropeptide substance P added in step (2) is 1 x 10 -8 ~ 1 x 10 -7 The volume ratio of the microsphere carrier system and the mixture in step (3) is (4-1):(2-1).

6. Use of a bioactive factor composition for the preparation of an ossification center organoid for promoting rapid healing of large size bone defects, characterized in that, The composition comprises a calcitonin gene-related peptide, recombinant human bone morphogenetic protein-2 and a neuropeptide substance P.

7. Use of a composite carrier system for the preparation of an ossification center organoid that promotes rapid healing of large size 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, a bioactive factor and a first hydrogel, and the second hydrogel comprises a neuropeptide substance P.

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