Composite growth factor composition capable of promoting proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, method and application

Abstract

The invention provides a composite growth factor composition capable of promoting proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, a method and application. The invention provides the composite growth factor composition by simulating a natural secretion mode of a decalcified bone matrix of the mandible of an SD (Sprague Dawley) rat on the seventh day, the composite growth factor composition comprises BMP-2 (400-600 pg / ml), TGF-beta1 (5-7 ng / ml) and IGF-1 (150-250 ng / ml), and the optimal concentrations are 500 pg / ml, 6 ng / ml and 200 ng / ml respectively. The composition is remarkably superior to a single factor and a high-dosage combination in the aspects of promoting BMSCs proliferation, improving the activity of alkaline phosphatase (ALP) and forming mineralized nodules, meanwhile, the use dosage of growth factors is remarkably reduced, and the risk of side effects such as heterotopic ossification is reduced. The composite growth factor composition has good biocompatibility and safety, and is suitable for the fields of bone tissue engineering, bone defect repair and related regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and tissue engineering, specifically relating to a composite growth factor composition, method and application that can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. Background Technology

[0002] Bone regeneration therapy utilizes stem cells, growth factors, and scaffolds for mechanical support and transplantation to construct a biologically active bone tissue repair system, stimulating bone regeneration capacity. [1] This is considered an alternative therapy for repairing bone defects. Common bone regeneration treatments include biomaterial transplantation, stem cell therapy, and growth factor injections. [2] BMSCs have been proven to be important stem cells in the field of bone regeneration. Studies have shown that their proliferation and differentiation capacity is regulated by a variety of growth factors, mainly including transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), insulin-like growth factor (IGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) [3-7]. Among the many growth factors involved in bone remodeling, BMP has the most prominent role in inducing osteogenicity. It can promote cell proliferation and differentiation, aggregate mesenchymal cells, directionally induce precursor osteoblasts to differentiate into osteoblasts, promote type I collagen synthesis, and increase alkaline phosphatase levels [8-9].

[0003] Currently, BMP-2 has achieved certain therapeutic effects in the clinical treatment of borderline bone defects.

[10] However, due to its short biological half-life and rapid local clearance, this method requires high doses of growth factors (1-12 mg / patient).

[11] Supraphysiological doses of BMP-2 may lead to serious surgical complications, such as heterotopic ossification.

[12] Bone resorption and graft sinking

[13] Bone cyst formation

[14] Inflammation-related complications

[15]

[16] radiculitis

[17] and tumor formation

[18] To maximize the efficacy of BMP-2 and minimize related side effects, the concentration of BMP-2 used should be minimized. One possible approach is to combine it with other growth factors.

[19] .

[0004] Studies have shown that TGF-β1 and IGF-1 play important roles in the coupling pathways of bone remodeling.

[20] TGF-β1 is one of the most abundant cytokines in the bone matrix, and it has the functions of promoting cell proliferation, regulating the cell cycle and inducing apoptosis. At the same time, it can stimulate the secretion and deposition of extracellular matrix.

[21] Furthermore, TGF-β can regulate VEGF expression and strengthen the coupling between angiogenesis and bone formation.

[22] Although TGF-β1 is responsible for recruiting BMSCs to sites of bone resorption, they do not provide signals for osteoblast differentiation.

[23] Conversely, IGF-1 is also one of the most abundant growth factors in the bone matrix, responsible for providing the osteogenic microenvironment required for BMSCs to differentiate into osteoblasts.

[24] It primarily acts on receptors on the surface of osteoblasts, inducing osteoblast proliferation and differentiation, inhibiting apoptosis, promoting the expression of mineralization-related proteins such as osteocalcin, collagen-1, and alkaline phosphatase, and participating in new bone formation.

[25] Furthermore, most studies indicate that the combined use of growth factors is more effective in osteogenic formation than using any single factor alone. However, a consensus has not yet been reached on the specific dosage. Due to safety and cost concerns, controlling the dosage of exogenous growth factors, rationally using growth factors to promote bone regeneration, and avoiding a series of side effects caused by supraphysiological doses are key research issues in the treatment of bone defects. Further research is needed to optimize the dosage and determine long-term safety. It is also necessary to consider using a combination of multiple growth factors in a specific ratio to create a biological agent, which can be administered at a specific time to achieve a truly safe and effective osteogenic effect.

[0005] Bone regeneration therapy utilizes stem cells, growth factors, and scaffolds for mechanical support and transplantation to construct a biologically active bone tissue repair system, stimulating bone regeneration capacity. [1] Bone regeneration is considered an alternative therapy for bone defect repair. Common bone regeneration treatments include biomaterial transplantation, stem cell therapy, and growth factor injection [2]. BMSCs have been proven to be important stem cells in the field of bone regeneration. Studies have shown that their proliferation and differentiation capacity is regulated by a variety of growth factors, including transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), insulin-like growth factor (IGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF).[3]-[7] Among the many growth factors involved in bone remodeling, BMP has the most prominent osteogenic induction effect. It can promote cell proliferation and differentiation, aggregate mesenchymal cells, directionally induce precursor osteoblasts to differentiate into osteoblasts, promote type I collagen synthesis, and increase alkaline phosphatase levels. [8],[9] Currently, BMP-2 has shown some efficacy in the clinical treatment of borderline bone defects.

[10] However, due to its short biological half-life and rapid local clearance, this method requires high doses of growth factors (1-12 mg / patient).

[11] Supraphysiological doses of BMP-2 may lead to serious surgical complications, such as heterotopic ossification.

[12] Bone resorption and graft sinking

[13] Bone cyst formation

[14] Inflammation-related complications

[15]

[16] radiculitis

[17] and tumor formation

[18] To maximize the efficacy of BMP-2 and minimize related side effects, the concentration of BMP-2 used should be minimized. One possible approach is to combine it with other growth factors.

[19] .

[0006] Studies have shown that TGF-β1 and IGF-1 play important roles in the coupling pathways of bone remodeling.

[20] TGF-β1 is one of the most abundant cytokines in the bone matrix, and it has the functions of promoting cell proliferation, regulating the cell cycle and inducing apoptosis. At the same time, it can stimulate the secretion and deposition of extracellular matrix.

[21] .

[0007] In addition, TGF-β can regulate VEGF expression and strengthen the coupling between angiogenesis and bone formation.

[22] Although TGF-β1 is responsible for recruiting BMSCs to sites of bone resorption, they do not provide signals for osteoblast differentiation.

[23] Conversely, IGF-1 is also one of the most abundant growth factors in the bone matrix, responsible for providing the osteogenic microenvironment required for BMSCs to differentiate into osteoblasts.

[24] It primarily acts on receptors on the surface of osteoblasts, inducing osteoblast proliferation and differentiation, inhibiting apoptosis, promoting the expression of mineralization-related proteins such as osteocalcin, collagen-1, and alkaline phosphatase, and participating in new bone formation.

[25] Furthermore, most studies indicate that the combined use of growth factors is more effective in osteogenic formation than using any single factor alone. However, a consensus has not yet been reached on the specific dosage. Due to safety and cost concerns, controlling the dosage of exogenous growth factors, rationally using growth factors to promote bone regeneration, and avoiding a series of side effects caused by supraphysiological doses are key research issues in the treatment of bone defects. Further research is needed to optimize the dosage and determine long-term safety. It is also necessary to consider using a combination of multiple growth factors in a specific ratio to create a biological agent, which can be administered at a specific time to achieve a truly safe and effective osteogenic effect. Summary of the Invention

[0008] The purpose of this invention is to provide a complex growth factor composition, method, and application that can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, effectively promoting the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells at a low dose.

[0009] This addresses the problems of high dosage and significant side effects associated with existing single growth factor technologies.

[0010] The technical solution adopted in this invention is:

[0011] This invention provides a complex growth factor composition that can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, the composition comprising the following components:

[0012] Bone morphogenetic protein-2 (BMP-2): concentration 400–600 pg / ml;

[0013] Transforming growth factor-β1 (TGF-β1): concentration 5–7 ng / ml;

[0014] Insulin-like growth factor-1 (IGF-1): concentration 150–250 ng / ml;

[0015] The composition was obtained by simulating the natural secretion pattern of bone matrix on day 7, and its osteogenic induction effect was significantly better than that of single factors and high-dose combinations.

[0016] Preferably, in the composition:

[0017] The concentration of BMP-2 was 500 pg / ml; the concentration of TGF-β1 was 6 ng / ml; and the concentration of IGF-1 was 200 ng / ml.

[0018] Preferably, the composition is derived from decalcified bone matrix extract or artificially formulated using recombinant proteins.

[0019] Preferably, the composition is used to induce the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.

[0020] Secondly, the present invention provides a method for constructing a composite growth factor composition based on the natural secretion pattern of bone matrix, comprising the following steps:

[0021] Preparation of decalcified bone matrix and establishment of growth factor time-release curves;

[0022] The natural secretion concentration on day 7, a critical time window for bone healing, was selected as the baseline for the combination.

[0023] BMP-2, TGF-β1, and IGF-1 were prepared at a ratio of 400–600 pg / ml: 5–7 ng / ml: 150–250 ng / ml.

[0024] Preferably, the concentrations of each factor in the composition are: BMP-2 500 pg / ml, TGF-β1 6 ng / ml, and IGF-1 200 ng / ml.

[0025] Thirdly, the present invention also provides the application of the composite growth factor composition in the preparation of bone tissue engineering materials and in the repair of bone tissue and bone defects, the applications including:

[0026] Used to reduce BMP-2 dosage by ≥90%;

[0027] Simultaneously, it increases ALP activity by ≥50% and calcium nodule formation by ≥25%;

[0028] Significantly reduces the risk of heterotopic ossification.

[0029] Preferably, the composition is used to induce BMSCs mineralization in vitro, and the amount of calcium nodule formation is increased by ≥50% compared with the control group.

[0030] Preferably, the concentration deviation of each component is less than ±10%, the protein purity is ≥95%, and it meets the pharmaceutical grade quality standard.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1) The combined growth factors have a better effect on the proliferation and osteogenic differentiation of BMSCs than the single application of BMP-2, TGF-β1 and IGF-1. The optimal concentrations for the application of single growth factors are: BMP-2 500pg / ml, TGF-β1 6 ng / ml and IGF-1 300ng / ml.

[0033] The composite growth factor constructed from naturally secreted bone matrix at day 7, consisting of 500 pg / ml BMP-2, 6 ng / ml TGF-β1, and 200 ng / ml IGF-1, significantly enhanced the proliferation and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) compared to the composite growth factor constructed from the optimal concentration of a single factor. The difference was statistically significant.

[0034] 2) This application constructs single growth factor groups and compound growth factor groups by referring to the contents of TGF-β1, BMP-2 and IGF-1 naturally secreted by bone matrix, and observes whether the compound growth factor can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) better than the single growth factor. At the same time, the optimal concentration of each growth factor to promote the proliferation and osteogenic differentiation of BMSCs is screened and combined. Attached Figure Description

[0035] Figure 1 The diagram shows the preparation of the decalcified bone matrix of the present invention: (a) collection of mandibles from SD rats; (b) mandibles after removal of soft tissues such as muscles, periosteum, and teeth; (c) demineralized bone matrix particles after crushing, defatting, and demineralization.

[0036] Figure 2 The standard curves for ELISA determination of growth factor content in demineralized bone matrix of SD rat mandibles are as follows: (a) Standard curve for BMP-2 content; (b) Standard curve for TGF-β1 content; (c) Standard curve for IGF-1 content.

[0037] Figure 3 The following figures represent the release levels (ng / g) of growth factors in the decalcified bone matrix of the mandible of SD rats according to the present invention: (a) BMP-2 release levels on days 1, 3, 5, 7, 14, 21, and 28; (b) TGF-β1 release levels on days 1, 3, 5, 7, 14, 21, and 28; and (c) IGF-1 release levels on days 1, 3, 5, 7, 14, 21, and 28.

[0038] Figure 4 Primary culture of rat bone marrow mesenchymal stem cells for the present invention: P0 generation BMSCs cells were basically fused and formed long spindle shapes, with some other cells in suspension. After passage, the cell growth rate was accelerated and the number of suspended miscellaneous cells decreased (10×).

[0039] Figure 5 The flow cytometry results for identifying rat BMSCs of the present invention are as follows: (a) P3 generation BMSCs CD29 detection showed a positive expression rate of 97.82%; (b) P3 generation BMSCs CD44 detection showed a positive expression rate of 94.34%; (c) P3 generation BMSCs CD90 detection showed a positive expression rate of 97.76%; (d) P3 generation BMSCs CD34 detection showed a negative expression rate of 0.74%; (e) P3 generation BMSCs CD45 detection showed a negative expression rate of 0.80%; (f) P3 generation BMSCs CD11b / c detection showed a negative expression rate of 0.96%.

[0040] Figure 6 This is a diagram illustrating the osteogenic induction process of rat BMSCs according to the present invention.

[0041] (a) 21 days after osteogenic induction of BMSCs, a large number of sand-like crystals were observed to precipitate under a microscope;

[0042] (b) Alizarin red staining showed that many calcium nodules stained red (4×);

[0043] (c) After staining with alizarin red, a large area of ​​calcium salts appeared red under a high-power microscope (10×);

[0044] Figure 7 This is a diagram illustrating the adipogenic induction process of rat BMSCs according to the present invention.

[0045] (a) Twenty-one days after adipogenic induction, transparent droplets of varying sizes were observed in the cytoplasm under a microscope;

[0046] (b) After staining with Oil Red O, the transparent droplets were stained red under a low magnification microscope (4×);

[0047] (c) After staining with Oil Red O, the lipid droplets appeared as a deep red beaded pattern under high magnification (10 ×).

[0048] Figure 8 The effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on BMSC proliferation at 1, 3, and 5 days were investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0049] Figure 9 The effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on BMSC proliferation at 1, 3, and 5 days were investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0050] Figure 10 The effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on BMSC proliferation at 1, 3, and 5 days were investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0051] Figure 11 The effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on BMSC proliferation at 1, 3, and 5 days were investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0052] Figure 12 The effects of different concentrations of BMP-2 applied alone on BMSC proliferation at 1, 3, and 5 days were investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0053] Figure 13 The effect of different concentrations of TGF-β1 alone on BMSC proliferation at 1, 3, and 5 days; * p < 0.05; ** p < 0.01, *** p < 0.001; **** p < 0.0001;

[0054] Figure 14 The effect of different concentrations of IGF-1 applied alone on BMSC proliferation at 1, 3, and 5 days was investigated. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0055] Figure 15 The results of the alkaline phosphatase staining experiment of the present invention (4X);

[0056] Figure 16 The effects of BMP-2, TGF-β1, and IGF-1 alone or in combination on ALP activity of BMSCs in compound group 1 of the present invention are shown in the figures: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0057] Figure 17 The effects of BMP-2, TGF-β1, and IGF-1 alone or in combination on ALP activity of BMSCs in compound group 2 of the present invention are shown in the figures: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0058] Figure 18 The effects of BMP-2, TGF-β1, and IGF-1, alone or in combination, on ALP activity of BMSCs in compound group 3 of this invention are shown in the figures: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

[0059] Figure 19 The effects of BMP-2, TGF-β1, and IGF-1 alone or in combination on ALP activity of BMSCs in compound group 4 of the present invention are shown in the figures: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0060] Figure 20The effect of different concentrations of BMP-2 on the ALP activity of BMSCs was investigated in this invention. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001;

[0061] Figure 21 The effect of different concentrations of TGF-β1 on ALP activity of BMSCs was investigated in this invention. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0062] Figure 22 The effect of different concentrations of IGF-1 on ALP activity of BMSCs was investigated in this invention. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001;

[0063] Figure 23 The effect of the composite growth factor of the present invention on the ALP activity of BMSCs is shown in the figures: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0064] Figure 24 The results of alizarin red staining of mineralized nodules in this invention are as follows: (a) Gross view of mineralized nodules in the control group; (b) Gross view of mineralized nodules in compound group 1; (c) Gross view of mineralized nodules in compound group 2; (d) Gross view of mineralized nodules in the optimized group; (e) Microscopic view of mineralized nodules in the control group (4×); (f) Microscopic view of mineralized nodules in compound group 1 (4×); (g) Microscopic view of mineralized nodules in compound group 2 (4×); (h) Microscopic view of mineralized nodules in compound group 5 (4×).

[0065] Figure 25 Quantitative analysis of calcium salt deposition after 21 days of application of different combinations of compound growth factors of the present invention. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Detailed Implementation

[0066] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] 1. Experimental materials

[0068] 1.1 Analysis of variance was used to compare data from different groups in the in vitro cell biology experiments.

[0069] 1.2 Time and Location The experiment was completed in the laboratory of Yunnan Provincial Institute of Stomatology from March to December 2023.

[0070] 1.3 Experimental Animals

[0071] This experiment was approved by the Animal Experiment Ethics Review Committee of Kunming Medical University (Approval No.: Kmmu20220875). 36 healthy SD rats (age: 3-6 months, average weight 250±110g) were provided by the Animal Experiment Center of Kunming Medical University, with half being male and half female.

[0072] 1.4 Main Experimental Reagents and Instruments: BMP-2, TGF-β1, IGF-1 ELISA kits (Origene, USA), rat bone marrow mesenchymal stem cell complete culture medium (Oricell, China), fetal bovine serum (Evacell, Australia), low-glucose DMEM medium (Gibco, USA), penicillin-streptomycin antibiotics (Gibco, USA), phosphate buffer (Liji Biotechnology, China), trypsin (Gibco, USA), BMP-2 (Peprotech, USA), TGF-β1 (Peprotech, USA), IGF-1 (Peprotech, USA), alkaline phosphatase activity assay kit (Beyotime, China), alkaline phosphatase staining kit (Beyotime, China), 4% paraformaldehyde (Biosharp, China), osteogenic induction differentiation medium (Haixing Biotechnology, China), adipogenic... Differentiation induction medium (Haixing Biotechnology, China), Alizarin Red (Haixing Biotechnology, China), Saturated Oil Red O staining solution (Solepro, China), 4% paraformaldehyde (Solepro, China), CCK-8 kit (Glpbio, China), Cetylpyridine chloride hydrate (Solepro, China), L-ascorbic acid (Sigma, USA), Sodium β-glycerophosphate (Sigma, USA), Dexamethasone (Sigma, USA), CD45, CD29, CD90 flow cytometry antibodies (Bio-legend, USA), CD34, cd44, CD11b / c flow cytometry antibodies (Oricel, USA), High-throughput tissue homogenizer (Sinzhi, China), Cell incubator (Leica, Germany), Inverted phase contrast microscope (Leica, Germany), Carbon dioxide cell incubator (Leica, Germany)

[0073] 2 Experimental Methods

[0074] Preparation and growth factor detection of decalcified bone matrix extract from the mandible of SD rats.

[0075] Thirty-six SD rats were euthanized by cervical dislocation. Their mandibles were harvested, and attached muscles, periosteum, and other soft tissues, as well as teeth, were removed. The mandibles were washed thoroughly with pure water and prepared into 1×1cm specimens. 2Bone blocks were dehydrated in anhydrous ethanol for 2 hours, the anhydrous ethanol was discarded, and the bone was soaked in 0.1 mol / L sodium hydroxide solution for 36 hours to defatted. The 0.1 mol / L sodium hydroxide solution was discarded, and the bone was rinsed with plenty of pure water until the pH was neutral. The bone was then air-dried in a clean bench, aliquoted into 5 ml centrifuge tubes, and frozen at -20°C for 4 hours and -80°C for 4 hours. The bone was then ground in liquid nitrogen using a high-throughput tissue homogenizer (1200 rpm / min, 1 min, repeated 3-5 times) to achieve a particle size of 500-1000 μm. The bone was then dynamically decalcified with 0.6 mol / L HCl (solid-to-liquid ratio: 1 g / 30 ml) for 48 hours. The bone was then dehydrated, rinsed thoroughly with pure water, and soaked in pure water overnight to ensure that the hydrochloric acid was completely removed until the pH was neutral.

[0076] Soak in anhydrous ethanol for 2 hours, air-dry the bone particles overnight in a clean bench, weigh, dispense, and sterilize by gamma irradiation (unit: 15kGy). Figure 1 (As shown).

[0077] Extraction solutions were prepared according to ISO 10993 requirements: Decalcified bone particles from the mandibles of SD rats, dispensed into EP tubes, were added to DMEM culture medium at a powder-to-liquid ratio of 1g to 5ml. Extraction was performed in a cell culture incubator for 1, 3, 5, 7, 14, 21, and 28 days. Culture medium was collected at the corresponding time points and stored at -80℃ for later analysis. The levels of decalcified BMP-2, TGF-β1, and IGF-1 in the mandibles of SD rats were detected using an ELISA kit according to the kit instructions. All experimental results are presented as mean ± standard deviation. The mean ± standard deviation (±s) indicates that the growth factor release curve was plotted by statistically analyzing the data using Graph Pad Prism software.

[0078] Isolation, culture and identification of bone marrow mesenchymal stem cells from 2 SD rats;

[0079] Three- to four-week-old SD rats were euthanized by cervical dislocation. The bilateral femurs and tibias were separated, washed three times in PBS solution containing 10% penicillin and antibiotics, and the metaphysis was removed. The bone marrow cavity was flushed with DMEM complete culture medium until it turned white. The flushing fluid was collected, centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded, and the rat bone marrow mesenchymal stem cells were resuspended in 4 ml of rat bone marrow mesenchymal stem cell complete culture medium and seeded at a height of 25 cm. 2 Cells were placed in cell culture flasks and cultured in a cell culture incubator. The medium was completely changed after 24 hours, and then changed every three days thereafter. When the cells covered 85-90% of the bottom of the flask, they were passaged at a ratio of 1:2. BMSCs were identified by detecting the surface marker CD molecules and their osteogenic and adipogenic differentiation abilities.

[0080] 3. Compare the effects of compound growth factors and single growth factors on BMSC proliferation and osteogenic differentiation.

[0081] 3.1 Experimental Grouping

[0082] Single growth factor groups: BMP-2 (200, 300, 400, 500) pg / ml; TGF-β1 (6, 8, 9, 10) ng / ml; IGF-1 (50, 100, 200, 300) ng / ml;

[0083] Complex growth factor group:

[0084] Combined group 1 (day 3): BMP-2 400pg / ml + TGF-β1 9ng / ml + IGF-1 300ng / ml;

[0085] Group 2 (Day 7): BMP-2 500 pg / ml + TGF-β1 6 ng / ml + IGF-1 200 ng / ml;

[0086] Group 3 (Day 14): BMP-2 200pg / ml + TGF-β1 10ng / ml + IGF-1 100ng / ml;

[0087] Group 4 (Day 21): BMP-2 300pg / ml + TGF-β1 8ng / ml + IGF-1 50ng / ml;

[0088] Compound group 5 (optimized group): BMP-2 (500 pg / ml), TGFβ1 (6 ng / ml), IGF-1 (300 ng / ml);

[0089] The control group contained no growth factors, while the blank group was supplemented with only complete culture medium.

[0090] 3.2 CCK-8 Experiment: Fourth-generation bone marrow mesenchymal stem cells were seeded at 2000 cells / well in 96-well plates. Each plate of cells was randomly divided into a blank group, BMP-2 group, TGF-β group, IGF-1 group, and a combination group, with 5 replicates per group. After culturing for 1, 3, and 5 days, one culture plate was taken, the culture medium was discarded, and the operation was performed according to the CCK-8 kit (Glpbio, China). The absorbance of each well was measured at 450 nm using an ELISA reader. The cell growth curve was plotted with the culture time on the x-axis and the average absorbance on the y-axis.

[0091] 3.3 Alkaline phosphatase staining of fourth-generation bone marrow mesenchymal stem cells at 5×10⁻⁶ 4Cells were seeded per well in 12-well plates. Each plate of cells was randomly divided into a blank group, a BMP-2 group, a TGF-β group, an IGF-1 group, and a composite group, with 3 replicates per group. After 7 days of culture, the culture medium was discarded. The alkaline phosphatase staining kit (Beyotime) was used to perform the following procedures: an appropriate amount of BCIP / NBT staining working solution was added to each well and the cells were incubated at room temperature in the dark for 30 minutes. After 30 minutes, the BCIP / NBT staining working solution was removed, and the cells were washed 3 times with PBS. The cells were then photographed and observed under a microscope.

[0092] 3.4 Alkaline phosphatase activity detection

[0093] Third-generation bone marrow mesenchymal stem cells at 2×10 4 Cells were seeded per well in 24-well plates. Each plate of cells was randomly divided into a blank group, a BMP-2 group, a TGF-β group, an IGF-1 group, and a combination group, with 3 replicates per group. After 7 days of culture, the culture medium was discarded. The alkaline phosphatase kit (alkaline phosphatase detection kit, Beyotime) was used to perform the operation. The absorbance of each group of cells was measured at 490 nm using a microplate reader. Each group was measured 3 times and the average value was taken. The alkaline phosphatase activity (King's units) of each group was calculated.

[0094] 4. The results of the alizarin red staining CCK-8 cell proliferation experiment and ALP activity detection showed that compound group 1 (day 3), compound group 2 (day 7) and compound group 5 (optimized group) significantly promoted cell proliferation and increased ALP activity expression. In order to reduce the experimental workload, compound group 1 (day 3), compound group 2 (day 7) and compound group 5 (optimized group) were selected for the alizarin red staining experiment.

[0095] (1) BMSCs were divided into experimental groups at a rate of 1×10 5 Cells were seeded per well into 6-well plates, with 3 replicates per group. After culturing at 37°C for 21 days, the osteogenic induction medium was discarded. 2 ml of PBS was added to each well, and the cells were washed three times. After fixation with 4% paraformaldehyde for 30 min, the paraformaldehyde was discarded, and the cells were gently washed three times with PBS. Alizarin Red staining solution (Solepro, China) was added to each well and stained for 5 min. The Alizarin Red staining solution was discarded, and the cells were washed three times with PBS. The cells were observed and photographed under an inverted phase-contrast microscope.

[0096] (2) Add 2% hexadecylpyridine chloride (Solepro, China) to dissolve the complex solution formed by alizarin red and calcium ions. Perform the decolorization reaction at room temperature for 1 hour, and then use an ELISA reader to detect the absorbance value at 562 nm. Repeat the experiment 3 times.

[0097] 5. Statistical Analysis: The experimental data were analyzed using Graph Pad Prism software. One-way ANOVA was performed to examine the differences in the proliferation and osteogenic differentiation capacity of BMSCs induced by different growth factors. P < 0.05 was considered statistically significant.

[0098] Conclusion Summary:

[0099] 1. Determination of growth factor content in decalcified bone matrix of mandible in SD rats

[0100] A polynomial standard curve was generated based on the results of the standard dilution, BMP-2 group R 2 =1, TGF-β1 group R 2 =0.9992, IGF-1 group R 2 =0.9997, the closer the R-value is to 1, the better the linear fit of the standard curve, and the stronger the correlation between the experimental data and the fitted line. Calculate the corresponding growth factor concentration (e.g., ...) based on the standard curve. Figure 2 (As shown). The content of growth factors in the extract was measured at 1, 3, 5, 7, 14, 21, and 28 days.

[0101] The results showed that BMP-2 release decreased slowly from day 1 to 28, with a significant decrease from day 7 to 14, and then stabilized after day 14. The highest BMP-2 release levels were on day 1 and day 5, at 0.1±0.02 and 0.1±0.09 ng / g demineralized bone matrix, respectively. TGF-β1 release exhibited a wave-like pattern from day 1 to 28, with a low level on day 1, increasing on day 3 and then continuously decreasing until reaching a peak of approximately 1.98±0.56 ng / g on day 14. After day 14, the release level continued to decrease significantly. IGF-1 release showed a continuous but slow decreasing trend from day 1 to 28, with a peak release level of approximately 91.62±22.51 ng / g on day 1. The IGF-1 level decreased slowly over time, reaching a low of 2.41±2.03 ng / g on day 28. Figure 3 (As shown in Table 1).

[0102] Based on the key time points of bone injury healing

[26] The study included the following phases: inflammation (days 1-5), cartilaginous callus formation (days 5-14), bony callus formation (days 14-21), and remodeling phase (days 21-35). We will refer to the release of growth factors from the mandibular DBM of 5g SD rats at four time points (days 3, 7, 14, and 21) (as shown in Table 2) and set corresponding concentrations to study their effects on BMSCs proliferation and osteogenic differentiation (as shown in Table 3).

[0103] Table 1. Growth factor release from demineralized bone matrix in the mandible of SD rats (ng / g) ±s, n=6)

[0104]

[0105] Table 2. Growth factor release from demineralized bone matrix in the mandible of 5g SD rats at 3, 7, 14, and 21 days. ±s,n=6)

[0106]

[0107] Table 3. List of growth factor concentrations used in subsequent studies

[0108]

[0109] 2. Morphology of Rat Bone Marrow Mesenchymal Stem Cells: After 2-3 days of primary cell culture, most cells are spindle-shaped or triangular, with a small number being round. Cell proliferation is rapid; after 3-5 days, most cells are spindle-shaped, with obvious cell aggregation and radial arrangement. A small number of suspended cells and cell debris are visible in the culture medium. With medium changes and increased culture time, cell debris and suspended cells gradually decrease. After passage, cells grow rapidly, exhibiting long spindle-shaped or irregular shapes (e.g., ...). Figure 4 (As shown).

[0110] 3. BMSCs Marker Detection: Flow cytometry was used to detect surface markers CD29, CD44, CD90, CD34, CD45, and CD11b / c in BMSCs. The expression of CD34, CD45, and CD11b / c was very low, at 0.74%, 0.80%, and 0.96%, respectively; while CD29, CD44, and CD90 were highly expressed, at 97.82%, 94.34%, and 97.76%, respectively. (e.g.) Figure 5 (As shown).

[0111] 4. Osteoblastic Differentiation Potential Detection: After 21 days of in vitro osteoblastic differentiation induction culture medium, P4 generation cells exhibited multilayered growth, with irregular, dense mineralized nodules forming at their aggregation sites. Alizarin Red staining revealed the formation of orange-red or red mineralized nodules, indicating that BMSCs possess the ability to differentiate into osteoblasts (e.g., Figure 6 (As shown).

[0112] 5. Assay for adipogenic differentiation potential: After 21 days of in vitro adipogenic differentiation induction culture medium, P3 generation cells showed varying numbers and sizes of clear droplets in the cytoplasm. Saturated Oil Red O staining revealed these droplets to be a bright, deep red, indicating that BMSCs possess the ability to differentiate into adipocytes (e.g., ...). Figure 7(As shown).

[0113] 6. The effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on BMSC proliferation were detected using the CCK8 assay. The levels of BMP-2, TGF-β1, and IGF-1 in the demineralized bone matrix of the mandible of SD rats on days 3, 7, 14, and 21 were used to determine the concentrations of growth factors. The effects of applying growth factors alone or in combination on BMSC proliferation were examined. The results showed that, compared with any single growth factor, the combined growth factors significantly promoted BMSC proliferation (as shown in Table 4-7). Figure 8-11 (As shown in Table 8-10) When used alone, the optimal concentrations for BMP-2 are 500 pg / ml, TGF-β1 is 6 ng / ml, and IGF-1 is 300 ng / ml. Figure 12-14 (As shown).

[0114] Table 4 Compound Group 1: Effects of different concentrations ofBMP-2, TGF-β 1 and IGF-1 on BMSCs proliferation alone or in combination( ±s,n=3)

[0115]

[0116] Notes: Control vs BMP-2 a Control vs TGF-β1 b Control vs IGF-1 c ;ControlVS Compound growth factor d BMP-2 vs TGF-β1 e BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor g TGF-β1 vs IGF-1 h ;TGF-β1 VS Compound growth factor i ;IGF-1 VS Compound growth factor j ;

[0117] Table 5 Compound Group 2: Effects of different concentrations ofBMP-2, TGF-β 1 and IGF-1 on BMSCs proliferation alone or in combination( ±s,n=3)

[0118]

[0119] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;Control VS Compound growth factor d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor i ;IGF-1 VS Compound growth factor j ;

[0120] Table 6 Compound Group 3: Effects of different concentrations ofBMP-2, TGF-β 1 and IGF-1 on BMSCs proliferation alone or in combination( ±s,n=3)

[0121]

[0122] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;ControlVS Compound growth factor d ;BMP-2 VS TGF-β1e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor i ;IGF-1 VS Compound growth factor j ;

[0123] Table 7 Compound Group 4: Effects of different concentrations of BMP-2, TGF-β 1 and IGF-1 on BMSCs proliferation alone or in combination( ±s,n=3)

[0124]

[0125] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;ControlVS Compound growth factor d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor i ;IGF-1 VS Compound growth factor j ;

[0126] Table 8 Effects of different concentrations of BMP-2 on BMSCsproliferation( ±s,n=3)

[0127]

[0128] Notes:Control VS BMP-2 200pg / ml a ;Control VS BMP-2 300pg / ml b ;ControlVS BMP-2 400pg / ml c ;Control VS BMP-2 500pg / ml d ;BMP-2 200pg / ml VS BMP-2 300pg / ml e ;BMP-2 200pg / m VS BMP-2 400pg / ml f ;BMP-2 200pg / ml VS BMP-2 500pg / ml g ;BMP-2 300pg / ml VS BMP-2 400pg / ml h ;BMP-2 300pg / ml VS BMP-2 500pg / ml i ;BMP-2 400pg / ml VS BMP-2 500pg / ml j ;

[0129] Table 9 Effects of different concentrations of TGF-β1 on BMSCsproliferation( ±s,n=3)

[0130]

[0131] Notes:Control VS TGF-β1 6ng / ml a ;Control VS TGF-β1 8ng / ml b ;Control VSTGF-β1 9ng / ml c ;Control VS TGF-β1 10ng / ml d ;TGF-β1 6ng / ml VS TGF-β1 8ng / ml e ;BMP-2 VS TGF-β1 9ng / ml f ;BMP-2 VS TGF-β1 10ng / ml g ;TGF-β1 8ng / ml VS TGF-β19ng / ml h ;TGF-β1 8ng / ml VS TGF-β1 10ng / ml i;TGF-β19ng / ml VS TGF-β1 10ng / ml j ;

[0132] Table 10 Effects of different concentrations of IGF-1 on BMSCsproliferation ( ±s,n=3)

[0133]

[0134] Notes: Control VS IGF-1 50ng / ml a Control VS IGF-1 100ng / ml b ControlVS IGF-1 200ng / ml c Control VS IGF-1 300ng / ml d ; IGF-1 50ng / ml VS IGF-1100ng / ml e ;IGF-1 5ng / ml VS IGF-1 200ng / ml f ;IGF-1 50ng / ml VS IGF-1 300ng / ml g ; IGF-1 100ng / ml VS IGF-1 200ng / ml h ; IGF-1 100ng / ml VS IGF-1 30ng / ml i ;IGF-1 200ng / ml VS IGF-1 300ng / ml j ;

[0135] 7. ALP staining method to detect the effects of different concentrations of BMP-2, TGF-β1, and IGF-1 alone or in combination on osteogenic differentiation of BMSCs. Compared with the control group, rat BMSCs were cultured in osteogenic induction medium containing different concentrations of BMP-2, TGF-β1, and IGF-1. After alkaline phosphatase staining, both the experimental and control groups showed blue expression, but the blue staining degree of the experimental group was greater than that of the control group. In the single growth factor experimental group, the cytoplasm color was deep blue when the BMP-2 concentration was 500 pg / ml, the TGF-β1 concentration was 6 ng / ml, and the IGF-1 concentration was 200 ng / ml, and the color was deeper than that of other concentration groups. Compared with the single growth factor group, the compound growth factor experimental group showed a deeper blue staining degree in the cytoplasm, indicating that the compound group produced more ALP (e.g., Figure 15(As shown).

[0136] 8. The ALP activity assay was used to detect the effects of different concentrations of BMP-2, TGF-β1, and IGF-1, alone or in combination, on osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Based on the levels of BMP-2, TGF-β1, and IGF-1 in demineralized bone matrix of the mandible of SD rats on days 3, 7, 14, and 21, growth factor concentrations were used to construct composite growth factor groups, named composite group 1, composite group 2, composite group 3, and composite group 4, respectively. ALP activity was measured by co-culturing BMSCs with growth factors alone or in combination at the four time points for 7 days. The results showed that compared with any single growth factor, the composite growth factor group most significantly promoted the proliferation of BMSCs (e.g., ...). Figure 16-19 (As shown in Table 11-14) When using growth factors alone, the optimal concentrations are: BMP-2 500 pg / ml, TGF-β1 6 ng / ml, and IGF-1 300 ng / ml, and an optimized group of compound growth factors was constructed based on this. (e.g.) Figure 20-22 (As shown in Table 15-17).

[0137] Table 11 Compound Group 1: Different concentrations of BMP-2, TGF-β 1and IGF-1 alone or in combination Effect of ALP activity on BMSCs( ±s,n=3)

[0138]

[0139] Notes: Control vs BMP-2 a Control vs TGF-β1 b Control vs IGF-1 c ;ControlVS Compound growth factor 1 d BMP-2 vs TGF-β1 e BMP-2 VS IGF-1 f ;BMP-2 VS Compound growth factor 1 g TGF-β1 vs IGF-1 h ;TGF-β1 VS Compound growth factor1 i ; IGF-1 VS Compound growth factor 1j ;

[0140] Table 14 Compound Group 4: Different concentrations of BMP-2, TGF-β 1and IGF-1 alone or in combination Effect of ALP activity on BMSCs( ±s,n=3)

[0141]

[0142] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;ControlVS Compound growth factor 1 d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor 1 g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor1 i ; IGF-1 VS Compound growth factor 1 j ;

[0143] Table 12 Compound Group 2: Different concentrations of BMP-2, TGF-β1 and IGF-1 alone or in combination Effect of ALP activity on BMSCs( ±s,n=3)

[0144]

[0145] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c;ControlVS Compound growth factor 1 d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor 1 g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor1 i; IGF-1 VS Compound growth factor 1 j ;

[0146] Table 13 Compound Group 3: Different concentrations of BMP-2, TGF-β1 and IGF-1 alone or in combination Effect of ALP activity on BMSCs( ±s,n=3)

[0147]

[0148] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;ControlVS Compound growth factor 1 d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor 1 g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor1 i ; IGF-1 VS Compound growth factor 1 j ;

[0149] Table 14 Compound Group 4: Different concentrations of BMP-2, TGF-β 1and IGF-1 alone or in combination Effect of ALP activity on BMSCs( ±s,n=3)

[0150]

[0151] Notes:Control VS BMP-2 a ;Control VS TGF-β1 b ;Control VS IGF-1 c ;ControlVS Compound growth factor 1 d ;BMP-2 VS TGF-β1 e ;BMP-2 VS IGF-1 f ;BMP-2 VSCompound growth factor 1 g ;TGF-β1 VS IGF-1 h ;TGF-β1 VS Compound growth factor1 i ; IGF-1 VS Compound growth factor 1 j ;

[0152] Table 15 Effects of Different Concentrations of BMP-2 on ALPActivity of BMSCs( ±s,n=3)

[0153]

[0154] Notes:Control VS BMP-2 200pg / ml a ;Control VS BMP-2 300pg / ml b ;ControlVS BMP-2 400pg / ml c ;Control VS BMP-2 500pg / ml d ;BMP-2 200pg / ml VS BMP-2 300pg / ml e;BMP-2 200pg / m VS BMP-2 400pg / ml f ;BMP-2 200pg / ml VS BMP-2 500pg / ml g ;BMP-2 300pg / ml VS BMP-2 400pg / ml h ;BMP-2 300pg / ml VS BMP-2 500pg / ml i ;BMP-2 400pg / ml VS BMP-2 500pg / ml j ;

[0155] Table 16 Effects of Different Concentrations of TGF-β1 on ALPActivity of BMSCs( ±s,n=3)

[0156]

[0157] Notes:Control VS TGF-β1 6ng / ml a ;Control VS TGF-β1 8ng / ml b ;Control VSTGF-β1 9ng / ml c ;Control VS TGF-β1 10ng / ml d ;TGF-β1 6ng / ml VS TGF-β1 8ng / ml e ;BMP-2 VS TGF-β1 9ng / ml f ;BMP-2 VS TGF-β1 10ng / mlr g ;TGF-β1 8ng / ml VS TGF-β19ng / ml h ;TGF-β1 8ng / ml VS TGF-β1 10ng / ml i ;TGF-β1 9ng / ml VS TGF-β1 10ng / ml j

[0158] Table17 Effects of Different Concentrations of IGF-1 on ALP Activityof BMSCs( ±s,n=3)

[0159]

[0160] Notes: Control VS IGF-1 50ng / ml a Control VS IGF-1 100ng / ml b ControlVS IGF-1 200ng / ml c ; Control VS IGF-1 300ng / ml d ; IGF-1 50ng / ml VS IGF-1100ng / ml e ;IGF-1 50ng / ml VS IGF-1 200ng / ml f ; IGF-1 50ng / ml VS IGF-1 300ng / ml g ; IGF-1 100ng / ml VS IGF-1 200ng / ml h ; IGF-1 100ng / ml VS IGF-1 300ng / ml i ; IGF-1 200ng / ml VS IGF-1 300ng / ml j ;

[0161] The composite growth factors, based on the natural content of demineralized bone matrix in the mandible of SD rats at days 3, 7, 14, and 21, and the optimized growth factor groups selected in the experiment, were designated as: Composite Group 1 (day 3), Composite Group 2 (day 7), Composite Group 3 (day 14), Composite Group 4 (day 21), and Composite Group 5 (optimized group). The ability of different composite growth factors to promote osteogenic differentiation of BMSCs was detected using the ALP activity assay. The results showed that compared with the control group, the expression of ALP activity was significantly increased in all composite growth factor groups, with the most significant increases observed in Composite Groups 1, 2, and 5 (p<0.001). ALP activity also increased in Composite Group 3 with a statistically significant difference (p<0.01). ALP activity also increased in Composite Group 4, but the difference was not statistically significant (p>0.5) (as shown in Table 18). Figure 23 (As shown).

[0162] Table 18 Effects of Different Combination Growth Factors on ALPActivity of BMSCs( ±s,n=3)

[0163]

[0164] Notes:Control VS Compound Growth factor 1 a ;Control VS CompoundGrowth factor 2 b ;Control VS Compound Growth factor 3 c ;Control VS CompoundGrowth factor 4 d ;Control VS Compound Growth factor 5 e ; Compound Growthfactor1 VS Compound Growth factor 2 f ;Compound Growth factor1 VS CompoundGrowth factor 3 g ;Compound Growth factor1 VS Compound Growth factor 4 h ;Compound Growth factor1 VS Compound Growth factor 5 i ;Compound Growth factor2 VS Compound Growth factor 3 j ;Compound Growth factor2 VS Compound Growthfactor 4 k ;Compound Growth factor2 VS Compound Growth factor 5 l ;CompoundGrowth factor 3 VS Compound Growth factor 4 m ;Compound Growth factor 3 VSCompound Growth factor 5 n ;Compound Growth factor4 VS Compound Growth factor 5 o ;

[0165] 9. Alizarin Red Staining and Semi-Quantitative Detection of the Effects of Combined Application of BMP-2, TGF-β1, and IGF-1 on the Mineralization Capacity of BMSCs Based on the results of CCK8 and ALP activity assays, Alizarin Red staining experiments were performed on compound group 1 (day 3), compound group 2 (day 7), and compound group 5 (screening and optimization group). The Alizarin Red staining results showed that after 21 days of osteogenic induction by adding different concentrations of the compound growth factors, obvious red deposits were observed in all groups. In contrast, the control group showed less red deposits and sparser calcification, indicating that BMSCs exhibited greater calcification and higher osteogenic activity under the intervention of the compound growth factors. In summary, the Alizarin Red staining results showed that compound group 2 (day 7), composed of BMP-2 at 500 pg / ml, TGF-β1 at 6 ng / ml, and IGF-1 at 200 ng / ml, showed a significant increase in mineralized deposits and demonstrated a better ability to promote osteogenic differentiation of BMSCs. (like Figure 24 (As shown)

[0166] The results of the alizarin red semi-quantitative experiment showed the same trend as the staining results of mineralized nodules. Compared with the control group, the osteogenic differentiation capacity of BMSCs increased under the intervention of the compound growth factor, and the difference was statistically significant (p<0.0001). Among them, the compound growth factor group 2 (day 7): BMP-2 500pg / ml, TGF-β1 6 ng / ml, IGF-1 200ng / ml, showed a significant increase in the amount of calcified deposits, indicating that its osteogenic activity was better (as shown in Table 19). Figure 25 (As shown).

[0167] Table 19 Quantitative Analysis of Calcium Salt Deposition after 21Days of Application of Different Combination Growth Factors( ±s,n=3)

[0168]

[0169] Notes:Control VS Compound Growth factor 1 a ;Control VS Compound Growth factor 2 b ;Control VS Compound Growth factor 5 c; Compound Growth factor 1 VSCompound Growth factor 2 d ; Compound Growth factor 1 VS Compound Growth factor 5 e ; Compound Growth factor 2 VS Compound Growth factor 5 f ;

[0170] Multiple growth factors have been detected in the extracellular matrix of bone, which are either locally produced or encapsulated within the bone matrix and play a crucial role in regulating bone growth and remodeling. Existing research shows that hydroxyapatite crystals can inhibit the release of growth factors from dentin materials, leading to reduced osteoinductive properties or delayed expression.

[27] The chemical compositions of bone matrix and dentin matrix are very similar. Obtaining growth factors from them requires demineralization of bone tissue to remove inorganic matrix. Urist et al. initially believed that partially demineralized bone had low osteogenic induction capacity, while nearly or completely demineralized bone exhibited higher osteogenic induction properties. By observing the effects of dentin matrix particle size and demineralization conditions on its properties, they found that increasing the degree of demineralization reduces the inorganic content in dentin matrix particles, but has a relatively small impact on collagen content.

[28] Therefore, this application completely demineralizes the mandibular DBM of SD rats to fully open its growth factor release channels and observes the release of growth factors under complete demineralization conditions.

[0171] BMSCs can differentiate into different cell lineages, including bone, cartilage, tendon, fat, and nerve cells.

[29] Its differentiation capacity depends on its microenvironment; under appropriate conditions, it can be directed to differentiate into cartilage and bone, making it a major source of stem cells in bone tissue engineering.

[30] BMP-2, TGF-β1, and IGF-1 are important growth factors in bone tissue engineering research, promoting cell proliferation, differentiation, and migration, thereby accelerating bone regeneration and osseointegration. However, due to the short biological half-life and rapid local clearance of growth factors, high physiological doses are often required, placing a financial burden on patients and leading to numerous complications. Furthermore, numerous studies have shown that the efficacy of growth factors is closely related to their concentration. Due to safety and cost considerations, further research is needed to optimize the dosage of growth factors and determine their long-term safety.

[0172] As research on bioscaffold materials continues to deepen, studies on how growth factors, supported by bioscaffold materials, promote and induce the proliferation and osteogenic and chondrogenic differentiation of mesenchymal-derived adult stem cells are becoming increasingly in-depth. In particular, their effects are more significant in the early stages of osteogenic formation, but the optimal concentration for their effects varies considerably.

[0173] Diefenderfer DL etc.

[31] Studies have shown that BMP-2 can promote the expression levels of osteogenic-related markers such as RUNX2, ALP, and OPN in stem cells, thereby promoting their differentiation into osteoblasts.

[0174] Studies have measured the effects of different concentrations of BMP-2 (50 ng / ml and 100 ng / ml) on ALP activity in MC3T3-E1 cells, with the 50 ng / ml group showing the most significant effect.

[32] Some researchers have found that 100 μg / L BMP-2 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by upregulating the expression of alkaline phosphatase, Runt-related transcription factor 2, and Osx.

[33] .

[0175] Studies have shown that culturing mouse bone marrow mesenchymal stem cells (BMSCs) with 10 ng / ml TGF-β1 can affect osteoblast differentiation and bone formation, and increase the mRNA levels of osteoblast differentiation markers and ALP activity in mouse BMSCs.

[34] Taong

[35] And Ota, etc.

[36] In vitro and in vivo experiments have demonstrated that the application of 2 ng / ml TGF-β1 can promote bone regeneration and significantly improve the efficiency of new bone formation. Furthermore, they believe that only high doses of TGF-β1 inhibit osteogenesis in the later stages of osteogenic development, and that many factors influence this effect. (Xu Ping et al.)

[37] Studies have shown that certain concentrations of IGF-1 can significantly increase the number of osteoblasts in rats, and the synthesis of ALP and the formation of calcified nodules are positively correlated with IGF-1 concentration in the range of 0.1-100 ng / ml. Furthermore, the positive effects of IGF-1 on bone injury healing have been confirmed in in vivo studies: in a sheep model, the optimal consolidation rate of distraction osteogenesis was achieved by combining local autologous grafts with local IGF-1 application.

[38] Therefore, the local application of IGF-1 to the defect site may be a promising alternative therapy for promoting the healing of bone tissue defects in clinical practice.

[0176] This application uses CCK-8, ALP, and ARS experiments to observe BMSC cell proliferation, ALP activity expression, and mineralized nodule formation, thereby evaluating the effects of combined application of BMP-2, TGF-β1, and IGF-1 on BMSC cell proliferation and osteogenic differentiation. CCK-8 results showed that the combined application of these three growth factors promoted BMSC proliferation. Based on the previous study, the compound growth factor group showed significantly higher proliferation than the single growth factor group and the control group. However, in this CCK-8 experiment, the control group showed faster proliferation than the compound growth factor group on day 1. This may be due to errors caused by manipulation or degradation of the growth factors due to long storage time; further verification will be conducted. ALP activity, ARS staining, and semi-quantitative results revealed that the compound growth factor group had a large number of sand-like calcium nodules, significantly higher than the control group. Among different combinations of compound growth factors, the compound growth factor group constructed from naturally occurring bone matrix contents of 500 pg / ml BMP-2, 6 ng / ml TGF-β1, and 200 ng / ml IGF-1 at day 7 showed significantly increased calcium nodule formation compared to other compound groups, and was superior to the compound growth factor group constructed from the optimal concentration of a single growth factor. Compared to the compound growth factor group constructed from naturally secreted growth factors in the bone matrix at day 7, the compound group with the same BMP-2 and TGF-β1 concentrations differed in IGF-1 concentration. When the BMP-2 and TGF-β1 concentrations were the same, the compound group with an IGF-1 concentration of 200 ng / ml exhibited better bone performance than that with 300 ng / ml, suggesting that the combined application of growth factors is not a simple addition of doses, and that the dosage affects osteogenic potential. Different growth factors exert synergistic effects when combined in certain ratios; sometimes, even small doses of combined growth factors can achieve good results, indicating that the effects of compound growth factors are complex and multidimensional, requiring comprehensive consideration of the interactions and influences between different factors. The semi-quantitative results of alizarin red were consistent with the staining results, which intuitively reflected that the combined use of growth factors could promote the osteogenic differentiation of BMSCs. The best osteogenic effect was obtained when the dosage of BMP-2 was 500 pg / ml, TGF-β1 was 6 ng / ml, and IGF-1 was 200 ng / ml.

[0177] This application reveals that using the highest individual dose of each growth factor in combination does not necessarily yield optimal osteogenic results. This may be due to interactions between different growth factors or regulation by intracellular signaling pathways. Therefore, in bone tissue engineering, it is necessary to comprehensively consider the interactions between different growth factors and the balance between dosages to achieve the best therapeutic effect.

[0178] In summary, the combination of BMP-2, TGF-β1, and IGF-1 can not only promote the proliferation of BMSCs but also significantly enhance their ability to differentiate into osteoblasts, providing a new approach and method for the combined application of these three growth factors. In the future, our research group will continue to conduct further studies at the protein level and in vivo animal experiments, hoping to make breakthroughs in key issues related to the pathway mechanisms of the combined application of growth factors.

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[0218] The above-described embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application, and should all be included within the protection scope of this invention.

Claims

1. A complex growth factor composition that can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, The composition comprises the following components: Bone morphogenetic protein-2: concentration 400–600 pg / ml; Transforming growth factor-β1: concentration 5–7 ng / ml; Insulin-like growth factor-1: concentration 150–250 ng / ml; The composition was obtained by simulating the natural secretion pattern of bone matrix on day 7, and its osteogenic induction effect was significantly better than that of single factors and high-dose combinations.

2. The composite growth factor composition according to claim 1, characterized in that, In the composition: The concentration of BMP-2 was 500 pg / ml; the concentration of TGF-β1 was 6 ng / ml; and the concentration of IGF-1 was 200 ng / ml.

3. The composite growth factor composition according to claim 1 or 2, characterized in that, The composition is derived from decalcified bone matrix extract or artificially formulated using recombinant proteins.

4. The composite growth factor composition according to any one of claims 1 to 3, characterized in that, The composition is used to induce the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro.

5. A method for constructing the composite growth factor composition as described in claim 1, characterized in that, Includes the following steps: Preparation of decalcified bone matrix and establishment of growth factor time-release curves; The natural secretion concentration on day 7, a critical time window for bone healing, was selected as the baseline for the combination. BMP-2, TGF-β1, and IGF-1 were prepared at a ratio of 400–600 pg / ml: 5–7 ng / ml: 150–250 ng / ml.

6. The construction method according to claim 5, characterized in that, The concentrations of each factor in the composition are: BMP-2500 pg / ml, TGF-β1 6 ng / ml, and IGF-1 200 ng / ml.

7. The application of the composite growth factor composition according to any one of claims 1 to 4 in the preparation of bone tissue engineering materials, and in the repair of bone tissue and bone defects, characterized in that, The uses include: Used to reduce BMP-2 dosage by ≥90%; Simultaneously, it increases ALP activity by ≥50% and calcium nodule formation by ≥25%; Significantly reduces the risk of heterotopic ossification.

8. The use according to claim 7, characterized in that, The composition was used to induce mineralization of BMSCs in vitro, and the amount of calcium nodule formation was increased by ≥50% compared with the control group.

9. The composite growth factor composition according to claim 1, characterized in that, The concentration deviation of each component is less than ±10%, the protein purity is ≥95%, and it meets the pharmaceutical grade quality standard.

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