Method for producing active rhBMP9 bone repair material based on double-transgene bombyx mori silk gland biosynthesis system as well as product and application of active rhBMP9 bone repair material

Abstract

The invention discloses a method for producing an active rhBMP9 bone repair material based on a double-transgene bombyx mori silk gland biosynthesis system as well as a product and application of the active rhBMP9 bone repair material, and realizes cooperative expression of complex precursor protein Pro-mhBMP9 and precursor protein invertase hFurin in bombyx mori posterior silk glands. An hBMP9 / hFurin double-gene biosynthesis system capable of completing precursor protein shearing, maturation and functionalized silk material synthesis in vivo is established, the rhBMP9 / CaP / Sericin composite scaffold is further constructed by extracting silk protein of double-gene transformation bombyx mori, and the rhBMP9 / CaP / Sericin composite scaffold shows excellent bone repair and angiogenesis capacities in both the cell level and the animal level, and can be used for preparing a bone repair material. And a solid experimental and theoretical basis is provided for a functional protein-material-tissue regeneration integrated strategy based on a silk gland biosynthesis system.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and genetic engineering, specifically to a method for producing active rhBMP9 bone repair material based on a double transgenic silkworm silk gland biosynthesis system, and also to the active rhBMP9 bone repair material obtained by the method and its application in the preparation of medical devices or biomaterials for promoting bone tissue regeneration and repairing bone defects. Background Technology

[0002] The silkworm is an important economic insect in my country, and its silk glands, possessing extremely high protein synthesis and secretion capabilities, are known as "natural protein processing plants." With the rapid development of genetic engineering and synthetic biology technologies, the silkworm silk gland is gradually evolving into a highly malleable and promising bioreactor, widely used to synthesize various bioactive recombinant proteins and functional substances. Meanwhile, silk fibroin materials, due to their excellent biocompatibility, mechanical properties, and processability, have gained widespread attention in tissue repair and biomaterials engineering. However, to meet diverse application needs, continuous exploration and optimization are still required in the functionalization of silk fibroin and the performance improvement of silk gland expression systems.

[0003] Not all secreted proteins acquire full biological activity directly under high-level expression conditions. Many growth factors and cytokines are initially synthesized in larger precursor forms, requiring hydrolysis and cleavage by specific proteases to release their active mature domains. This post-translational processing not only determines the protein's spatial conformation but also directly affects its secretion efficiency and functional stability. Among these, human bone morphogenetic proteins (hBMPs) are the most representative family of secreted proteins. Taking hBMP9 as an example, it plays a crucial role in osteogenic differentiation and tissue regeneration, but its precursor form must be cleaved by Furin enzymes to be converted into a biologically active dimer mature protein. Due to the extremely low endogenous expression level of Furin homologs in the posterior silk glands of silkworms, expressing the hBMP9 precursor alone is insufficient to obtain a sufficient amount of active product, thus limiting its biological function.

[0004] Although silkworm silk glands possess a powerful protein synthesis capacity, existing systems generally suffer from problems such as low exogenous gene expression efficiency, limited post-translational processing mechanisms, and difficulty in maintaining stable activity of target functional proteins. These issues severely restrict the in-depth expansion of silk proteins into high-value-added applications. Therefore, establishing a silkworm exogenous protein biosynthesis system with precise post-translational processing capabilities is a key technological breakthrough urgently needed to achieve innovative leaps in the sericulture industry. Summary of the Invention

[0005] In view of this, the present invention aims to provide a method for producing active rhBMP9 bone repair material based on a double transgenic silkworm silk gland biosynthesis system; a second objective of the present invention is to provide the active rhBMP9 bone repair material prepared by the method; and a third objective of the present invention is to provide the application of the active rhBMP9 bone repair material in the preparation of medical devices or biomaterials for promoting bone tissue regeneration and repairing bone defects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. A method for producing active rhBMP9 bone repair material based on a double-transgenic silkworm silk gland biosynthesis system, comprising the following preparation steps: (1) Prepare Pro-mhBMP9 transgenic silkworm line, so that its genome integrates the nucleic acid sequence Pro-mhBMP9 encoding the human BMP9 precursor; (2) Prepare an hFurin transgenic silkworm strain, integrating the nucleic acid sequence hFurin encoding human Furin protease into its genome; the nucleotide sequence of hFurin is shown in SEQ ID NO.2; (3) By hybridizing the Pro-mhBMP9 transgenic silkworm strain and the hFurin transgenic silkworm strain, a double transgenic silkworm strain that can co-express Pro-mhBMP9 and hFurin in the posterior silk gland of the silkworm is obtained, so that Pro-mhBMP9 is cleaved into a biologically active mature rhBMP9 dimer in the silk gland. (4) Extract silk protein containing active rhBMP9 from the cocoons of double transgenic silkworm strains and prepare active rhBMP9 bone repair material.

[0007] In some embodiments of the present invention, the Pro-mhBMP9 is a sequence optimized by the silkworm codon, as shown in SEQ ID NO.1.

[0008] In some embodiments of the present invention, the hFurin sequence is optimized using the silkworm codon, as shown in SEQ ID NO. 2.

[0009] In some embodiments of the present invention, the expression of Pro-mhBMP9 or hFurin is regulated by the hr3 CQ enhancer, the FibH promoter, and the Ser1pA terminator.

[0010] In some embodiments of the present invention, the specific preparation method in step (4) is as follows: First, extract sericin containing active rhBMP9 from the cocoons of double transgenic silkworms using calcium salt solution, and mix it with phosphate after dialysis with deionized water to induce the formation of a mineralized composite material rhBMP9 / CaP / Sericin; then extract fibroin from the remaining silk, and mix it with the composite material rhBMP9 / CaP / Sericin after molding and freeze-drying to obtain an active rhBMP9 bone repair material.

[0011] In some embodiments of the present invention, the calcium salt is calcium chloride, and the phosphate source is disodium hydrogen phosphate.

[0012] 2. Active rhBMP9 bone repair material prepared by the method described above.

[0013] 3. The application of the active rhBMP9 bone repair material in the preparation of medical devices or biomaterials for promoting bone tissue regeneration and repairing bone defects.

[0014] The beneficial effects of this invention are as follows: For the first time, the co-expression of the complex precursor protein Pro-mhBMP9 and the precursor protein converting enzyme hFurin in the posterior silk gland of silkworms was achieved, establishing an hBMP9 / hFurin dual-gene biosynthesis system capable of in vivo synthesis of precursor protein cleavage, maturation, and functionalized silk materials. This system, through gene hybridization, yielded double-positive silkworms carrying both red and green fluorescent labels, realizing the co-expression and enzyme-mediated processing of Pro-mhBMP9 and hFurin in posterior silk gland cells. Molecular and protein-level detection showed that hFurin can specifically recognize and cleave the RRKR site of Pro-mhBMP9, promoting its correct folding and dimerization to form a biologically active mature rhBMP9 protein, thus verifying the feasibility and effectiveness of the silk gland system in the post-translational processing of complex eukaryotic proteins.

[0015] Functional validation results showed that rhBMP9 cocoons obtained from the Pro-mhBMP9×hFurin dual-gene hybrid line significantly induced upregulation of alkaline phosphatase (ALP) activity and calcium nodule formation in rBMSCs cells, exhibiting osteogenic induction effects comparable to commercially available exogenous recombinant rhBMP9 protein. Further in vitro experiments demonstrated that rhBMP9 cocoon extract could activate the expression of osteogenic-related transcription factors (Runx2, COL1A1, OCN, etc.), significantly promoting early osteogenic differentiation and late mineralization, indicating that rhBMP9 maintains a stable structure and biological activity in the silken environment.

[0016] Building upon this foundation, this chapter further constructs an rhBMP9 / CaP / Sericin composite material and systematically characterizes its structure, composition, and biological properties. Scanning electron microscopy and energy dispersive spectroscopy (EDS) analysis revealed that a uniform calcium-phosphorus deposition layer forms on the surface of the rhBMP9 composite material, exhibiting good mineralization characteristics and blood compatibility. Cell experiments demonstrated that this composite material not only promotes early ALP activity and late mineralization formation in rBMSCs but also significantly upregulates the expression of osteogenic (ALP, Osx, COL1, OPN, OCN) and angiogenesis (VEGFA, ANGPT2, PDGFA) related genes and proteins, achieving a triple synergistic regulation of osteogenic-angiogenic-matrix processes. Transcriptome analysis further revealed that the rhBMP9 / CaP / Sericin composite material can activate key signaling pathways such as BMP, IGF, PDGF, and VEGF, promoting synergistic bone-vascular remodeling and extracellular matrix dynamic balance, providing a favorable microenvironment for bone regeneration.

[0017] In animal experiments, the rhBMP9 / CaP / Sericin composite scaffold demonstrated significant bone repair capacity in a rat model of a 5 mm skull defect. Micro-CT and histological analysis showed that the rhBMP9 group had significantly higher new bone formation, bone density, and angiogenesis than the control group, and the defect area was almost completely filled with new bone at 8 weeks. Immunohistochemical results further confirmed that COL1, Runx2, OCN, and VEGFA proteins were significantly upregulated in the rhBMP9 group, indicating that this material simultaneously promotes osteogenic and angiogenesis in vivo, achieving synergistic reconstruction of bone tissue structure and function.

[0018] In summary, this invention successfully verified the feasibility of the silkworm silk gland biosynthesis system in the post-translational processing and functional expression of complex secretory proteins, and constructed rhBMP9 functionalized silk materials with bioactivity and medical potential. The rhBMP9 / CaP / Sericin composite material exhibited excellent bone repair and angiogenesis capabilities at both cellular and animal levels, providing a solid experimental and theoretical foundation for an integrated "functional protein-material-tissue regeneration" strategy based on the silk gland biosynthesis system. Attached Figure Description

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 A schematic diagram illustrating the creation of the Pro-mhBMP9×hFurin double-gene hybrid silkworm strain; Figure 2Screening of Pro-mhBMP9 and hFurin transgenic silkworms and detection of rhBMP9 expression (A: Fluorescence screening of Pro-mhBMP9 and hFurin individuals; B: Detection of Pro-mhBMP9 mRNA in the posterior silk gland of positive individuals; C: Detection of hFurin mRNA in the posterior silk gland of positive individuals; D: Detection of hFurin protein in the middle and posterior silk glands; E: Detection of rhBMP9 protein in the cocoons of hybrid silkworms). Figure 3 Qualitative analysis of ALP staining in C3H10T1 / 2 cells treated with different silkworm cocoons; Figure 4 ALP staining and quantitative analysis of C3H10T1 / 2 cells induced by silkworm cocoon extract rhBMP9 (A: ALP staining of C3H10T1 / 2 cells in different treatment groups; B: Statistical analysis of ALP activity quantification). Figure 5 Analysis of mineralization formation and osteogenic differentiation-related protein expression in C3H10T1 / 2 cells induced by rhBMP9 silkworm cocoon extract (A: staining of mineralized nodules in C3H10T1 / 2 cells from different treatment groups; B: Western blot detection of osteogenic differentiation-related proteins). Figure 6 Characterization and biocompatibility analysis of rhBMP9 / CaP / Sericin composite material (A: Appearance and SEM observation, scale bar represents 50µm; B: Zeta potential analysis; C: Particle size distribution; D: Energy dispersive spectroscopy, scale bar represents 10µm; E: Hemolysis test). Figure 7 ALP and Alizarin Red S staining results after treating rBMSCs cells with non-traditional silkworm silk composite materials; Figure 8 Multidimensional validation of the effect of rhBMP9 / CaP / Sericin silk composite on osteogenic differentiation of rBMSCs (A: ALP gene transcription in different treatment groups; B: Osx gene transcription in different treatment groups; C: VEGFA in different treatment groups) Gene transcription status; D: OPN gene transcription status in different treatment groups; EF: ALP staining and quantification in different treatment groups; GH: Alizarin Red S staining and quantification in different treatment groups; IJ: Detection and quantification of bone differentiation-related proteins in different treatment groups. Figure 9Evaluation of the repair effect of rhBMP9 / CaP / Sericin composite scaffold in a rat skull defect model (A: rhBMP9 / CaP / Sericin composite scaffold bone repair evaluation design; B: Microcomputed tomography evaluation; C: Changes in bone volume fraction; D: Changes in bone mineral density; E: Changes in trabecular separation). Figure 10 Histological and immunofluorescence analysis of the rhBMP9 / CaP / Sericin composite scaffold in a skull defect model (A: H&E staining; B: Masson staining; C: CD31 immunofluorescence staining; D: quantitative analysis of regenerated angiogenesis). Figure 11 Analysis of osteogenic and vascular-related protein expression in rhBMP9 / CaP / Sericin composite scaffold during cranial defect repair (AB: immunohistochemical staining of COL1, VEGFA, Runx2 and OCN; quantitative analysis of CF-positive cells). Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Example 1. Construction of Pro-mhBMP9 and hFurin expression vectors and creation of transgenic silkworms The recombinant Pro-mhBMP9 protein sequence (423 amino acids) in this invention consists of Lys22-Arg297, Ser320-Arg429 and an 8×His-FLAG tag from the full-length sequence of human bone morphogenetic protein 9 (hBMP9), wherein the tag sequence is located in the middle of the two BMP9 fragment sequences. In addition, the recombinant hFurin protein sequence consists of Gln27-Leu794 and a C-terminal fusion of 6×His from the full-length sequence of human furin (hFurin). These two sequences were optimized according to the codon preference of silkworm and were obtained by the company through synthesis (SEQ ID NO.1-4). The synthesized recombinant Pro-mhBMP9 and hFurin genes were inserted into the p-HFRS vector (for details of the vector, see application number 202110312874.0, "Establishment and Application of an Efficient Silk Fiber Heavy Chain Promoter Secretory Expression System"). Subsequently, the Pro-mhBMP9 expression cassette was inserted into the pBac[3×P3-DsRedaf] vector, and the hFurin expression cassette was inserted into the pBac[3×P3-EGFPaf] (Horn and Wimmer 2000, Thomas et al. 2002) vector, finally generating the transgenic expression vectors pBac-HF-Pro-mhBMP9S and pBac-HF-hFurinS.

[0022] The construction process of the Pro-mhBMP9×hFurin hybrid line based on the silk gland of silkworm is as follows: Figure 1 As shown, the pBac-HF-Pro-mhBMP9S and pBac-HF-hFurinS vectors were transformed into non-diapause Dazao silkworms to obtain two transgenic silkworm lines expressing Pro-mhBMP9 and hFurin, respectively. Individuals with high expression levels in the Pro-mhBMP9 and hFurin lines were selected for preservation, and their offspring were hybridized. After reaching the cocooning stage, fluorescence selection was performed. Individuals with eyes exhibiting both red and green fluorescence were selected under a fluorescence microscope. Then, male and female individuals with red and green fluorescence in their eyes (Pro-mhBMP9×hFurin) were mated. After more than three generations of mating, when all offspring individuals showed red and green fluorescence in their eyes, this line was named the Pro-mhBMP9×hFurin hybrid line, realizing the co-expression and in vivo processing of hBMP9 precursor protein and hFurin enzyme in the posterior silk gland. Figure 1The diagram illustrates the mechanism of hFurin-mediated cleavage of the hBMP9 precursor protein: Pro-mhBMP9 is cleaved intracellularly via a specific RRKR recognition site to generate a biologically active mature dimer (mhBMP9). Mature mhBMP9 is secreted into the silk gland lumen and further integrated into the silk, ultimately forming rhBMP9-functionalized silk material with osteogenic activity.

[0023] To verify the construction results of transgenic silkworms and the expression of target proteins in the silk glands, two transgenic silkworm lines, Pro-mhBMP9 and hFurin, underwent multi-stage screening and molecular-level detection. Positive transgenic silkworm cocoons were shredded and added to 8 M urea solution to a final concentration of 20 mg / mL. After shaking at room temperature for 30 min, the extract was centrifuged at 12000g for 5 min, and the supernatant was collected. Subsequently, protein samples were separated and analyzed by SDS-PAGE and Western blot. Rabbit anti-hBMP9 and hFurin primary antibodies were diluted 1:3000 and incubated overnight at 4℃. Goat anti-rabbit secondary antibody was diluted 1:8000 and incubated at room temperature for 90 min. The results are as follows: Figure 2 As shown, both strains exhibited obvious red or green fluorescent signals during the egg, larval, and pupal stages. Figure 2 A). qPCR analysis showed that Pro-mhBMP9 and hFurin were significantly higher in transgenic silkworms than in wild-type (WT), indicating that the target gene was efficiently transcribed in the posterior silk gland cells. Figure 2 ,BC), and hFurin protein is highly expressed in posterior filament cells ( Figure 2 D).

[0024] Silkworm cocoons from the Pro-mhBMP9×hFurin hybrid line were minced for protein extraction. Reducing and non-reducing SDS-PAGE electrophoresis were performed, combined with Western blot analysis to determine the polymeric form of recombinant rhBMP9. Results showed that specific bands were detected in the hybridized silk samples. Under non-reducing conditions (NR), rhBMP9 existed in a dimer form, and only under reducing conditions (R) was the monomeric form detected. Figure 2 (E). These results demonstrate that, after co-processing with hFurin, the BMP9 precursor protein is correctly folded and dimerized within the silk gland of silkworms to form the biologically active mature rhBMP9 protein. Compared to the Pro-mhBMP9 silkworm line expressing only the precursor form, the dual-expression line achieved in vivo transformation from the precursor to the mature active structure, fully validating the effectiveness of the Furin-mediated post-translational processing mechanism in the silkworm silk gland system.

[0025] Example 2. Activity detection of silkworm cocoons from Pro-mhBMP9×hFurin hybrid strains To evaluate the osteogenic activity of rhBMP9 in the cocoons of Pro-mhBMP9×hFurin hybrid silkworms, we co-cultured mouse embryonic fibroblast cell line C3H10T1 / 2 cells with cocoons from different strains of silkworms or with commercially available exogenous recombinant hBMP9 protein (Recombinant Human BMP-9 Protein CF, manufacturer: R&D Systems, concentration: 10 ng / mL) for 7 days. Alkaline phosphatase (ALP) staining was then performed to assess the osteogenic activity of rhBMP9 in the cocoons of the Pro-mhBMP9×hFurin hybrid. The cocoon treatment process was as follows: cocoons from the three transgenic silkworm strains in Example 1 were sterilized with UV light, punched into 5 mm diameter discs, and two discs were placed in each well of a 24-well plate. 0.5 mL of MEM complete medium was added to each well, and the medium was replenished every two days in 0.5 mL increments.

[0026] The results are as follows Figure 3 As shown, the hybrid silkworm cocoon group exhibited a significant ALP staining reaction, forming a large amount of deep purple-black precipitate, and its positive cell density was significantly higher than other silkworm cocoon groups. However, compared with the commercially available exogenous recombinant hBMP9 protein treatment group, the staining intensity of the hybrid silkworm cocoon group was slightly lower. In contrast, the Pro-mhBMP9 silkworm cocoon group showed only mild staining, while the WT and hFurin groups showed low staining intensity, almost identical to the untreated group (CK). This indicates that although rhBMP9 silk can effectively induce early osteogenic differentiation of C3H10T1 / 2 cells, verifying its significant in vitro osteogenic activity, its effect has not yet reached the optimal level of commercial products. We speculate that this may be because the concentration of rhBMP9 released from the silk in the co-culture system is temporarily lower than the concentration used in the commercial hBMP9 protein group.

[0027] Example 3. Pro-mhBMP9×hFurin hybrid silkworm cocoon extract rhBMP9 induces osteogenic differentiation of C3H10T1 / 2 cells. To further verify the promoting effect of rhBMP9, a silkworm cocoon extract from the Pro-mhBMP9×hFurin hybrid strain, on osteogenic differentiation, C3H10T1 / 2 cells were co-cultured with the silkworm cocoon extract (extracted with a 2.5 mol / L urea solution containing 0.3% papain at pH 2.5, with 20 mg / mL of hybrid silk added, extracted at 50℃ for 2 h, centrifuged at 12000 rpm for 5 min, the supernatant was collected, the papain was removed by passing the supernatant through a nickel column, and the urea was removed by dialysis with deionized water or by ultrafiltration tube replacement. The cell experiment added rhBMP9 at a concentration of 10 ng / mL, and the commercial exogenous recombinant hBMP9 protein group was also added at 10 ng / mL) for 7 days, followed by ALP staining and quantitative analysis of ALP enzyme activity. The staining procedure was as follows: After washing cells with PBS, they were fixed with 4% paraformaldehyde for 15 min, and then reacted with BCIP / NBT chromogenic solution for 20 min. The blue-purple deposits were observed under a microscope. The ALP enzyme activity assay was as follows: The pNPP substrate method was used. The reaction was terminated after incubating the cell lysis buffer with the substrate for 30 min, and the absorbance was detected at a wavelength of 405 nm.

[0028] The results are as follows Figure 4 As shown, the control group only showed a light-colored background, while the silkworm cocoon extract of the Pro-mhBMP9×hFurin hybrid strain showed a large amount of purplish-black precipitate in the cells due to the presence of active rhBMP9, indicating that ALP activity was significantly enhanced. Figure 4 Quantitative results showed that the ALP enzyme activity in this group was significantly higher than that in the control group, and comparable to that in the commercially available exogenous recombinant rhBMP9 protein group. Figure 4 B).

[0029] To evaluate the effect of silkworm cocoon extract rhBMP9 on late osteogenic differentiation of C3H10T1 / 2 cells, Alizarin Red S staining and osteogenic marker protein detection were performed. Alizarin Red S (ARS) detection was performed on days 14 and 21 of treatment. Staining procedure: After cell fixation, 2% ARS solution (pH 4.2) was added, and staining was performed at room temperature for 30 min. Cells were washed three times with PBS to remove free dye, and red calcium salt deposition was observed under a microscope. Quantitative analysis: 10% CPC (cetylpyridinium chloride) solution was added to elute the bound dye, and the supernatant was collected after shaking for 30 min. The absorbance was measured at 560 nm.

[0030] Western blot analysis was used to detect the expression levels of osteogenic factors ALP (alkaline phosphatase), Runx2 (Runt-related transcription factor 2), OCN (osteocalcin), and OPN (osteopontin). The internal control protein was GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Results were analyzed using 2... -ΔΔCt Methods analysis was performed, with the NC group serving as a control. After treatment, cells were collected and lysed using RIPA lysis buffer (containing a PMSF inhibitor). Cells were incubated on ice for 30 min, centrifuged at 12000 g for 15 min, and the supernatant was used to determine protein concentration using the BCA method. 30 μg of protein was added to each well, separated by SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 1 h, and then incubated overnight at 4°C with primary antibodies (ALP, Runx2, OCN, OPN, 1:1000-1:3000; GAPDH, 1:10000). The next day, HRP-labeled secondary antibody (1:10000) was added, and the membrane was incubated at room temperature for 1 h. Exposure was performed using a high-sensitivity chemiluminescent substrate. Finally, grayscale analysis was performed using ImageJ software.

[0031] result Figure 5 As shown, the control group showed almost no calcium salt deposition, while the rhBMP9 cocoon group prepared in this invention and the commercially available exogenous recombinant rhBMP9 protein group showed a large number of red calcium nodules, indicating a significant increase in cell mineralization level. Figure 5 Further Western blot analysis showed that the expression of ALP, Runx2, OCN, and OPN proteins was significantly upregulated after treatment with the rhBMP9 silkworm cocoon extract prepared in this invention, indicating that this material can activate osteogenic differentiation-related signaling pathways. Figure 5 (B). The combined results indicate that furin-processed rhBMP9 maintains stable biological activity in the silk glands of silkworms and can effectively promote osteogenic differentiation and mineralization of mesenchymal stromal cells.

[0032] Example 4. Preparation and characterization of recombinant rhBMP9 / CaP / Sericin composite material First, the cocoons of wild-type, large-scale silkworms producing red fluorescent protein silk (LG-HFRS-F2, whose construction process is supported by Chinese patent CN202110426146.2), Pro-mhBMP9, hFurin, and Pro-mhBMP9×hFurin hybrids were cut into small pieces and added to 3 M CaCl2 solution at a ratio of 25 mg / mL. Sericin was extracted by shaking at room temperature for 30 min. Then, the mixture was centrifuged at 12000 g for 10 min at room temperature. The remaining silk was reserved for later use (Example 7). The supernatant was placed in a dialysis bag (molecular weight cutoff 3500 Da) for dialysis using 20 mM CaCl2 solution. The dialysis solution was changed every 6 h, for a total of 4 changes. After dialysis, the complex from the dialysis bag was collected, and 12 mM Na2HPO4 solution was added in equal volumes and mixed thoroughly. Sonic induction was then performed to obtain CaP / Sericin, RFP / CaP / Sericin, Pro-mhBMP9 / CaP / Sericin, hFurin / CaP / Sericin, and rhBMP9 / CaP / Sericin composite materials, respectively. CaP material was obtained by mixing equal volumes of 20 mM CaCl2 solution and 12 mM Na2HPO4 solution, followed by ultrasonic induction. All materials were sterilized by gamma irradiation before use.

[0033] To verify the structural characteristics and composition of the rhBMP9 / Sericin / CaP composite material, scanning electron microscopy and energy dispersive spectroscopy (EDS) analyses were performed. The results are as follows: Figure 6 As shown, the surface of rhBMP9 silk exhibits a rough and granular structure, which contrasts sharply with the smooth morphology of WT silk, suggesting significant mineralization deposition on the material surface. Figure 6 EDS energy dispersive spectroscopy and elemental distribution analysis further showed that the Ca and P elemental signals in the rhBMP9 group samples were significantly enhanced and uniformly distributed, confirming that the expression of BMP9 protein can promote calcium and phosphorus deposition and biomineralization formation in the silk fibroin matrix. Figure 6 (D). Furthermore, hemolysis experiments showed that the rhBMP9 / Sericin / CaP composite material did not cause significant damage to erythrocytes, with a hemolysis rate of less than 5%, meeting the safety standards for biomedical materials. This indicates that the material has good blood compatibility and biosafety. Figure 6 E).

[0034] Example 5. rhBMP9 / CaP / Sericin composite material induces osteogenic differentiation of rBMSCs cells Rat bone marrow mesenchymal stem cells (rBMSCs) used in the experiments were purchased from Cyagen Biosciences. Cell identification reports showed that CD29, CD44, and CD90 were positive (>70%), while CD34, CD45, and CD11b were negative. Under a microscope, the cells exhibited typical spindle-shaped or polygonal morphology, adherent growth, and arranged in a whorled or radial pattern. To ensure the differentiation potential of the cells, all experiments strictly used cells passaged for 3-5 generations. rBMSCs were cultured in OriCell® rat bone marrow mesenchymal stem cell complete medium. The culture environment and passage method were similar to those for C3H10T1 / 2 cells. After cells adhered to the culture vessel 12 h after inoculation, they were cultured for 7 days with silk composites from four silkworm strains at a final concentration of less than 10 ng / mL. The grouping information was as follows: CK (Negative Control) group: untreated control, conventional culture only; CaP group: CaP material prepared in Example 4 added to the culture medium; RFP group: RFP / CaP / Sericin composite material prepared in Example 4 added to the culture medium; hFurin group: hFurin / CaP / Sericin composite material prepared in Example 4 added to the culture medium; Pro-mhBMP9 group: Pro-mhBMP9 / CaP / Sericin composite material prepared in Example 4 added to the culture medium; rhBMP9 group: rhBMP9 / CaP / Sericin composite material prepared in Example 4 added to the culture medium. All experiments were conducted under the same conditions to ensure the rationality of the controls. Samples from different groups were collected for molecular biological detection and osteogenic function testing.

[0035] rBMSCs were treated with different materials and then stained with ALP. The results are as follows: Figure 7 As shown, the rhBMP9 group exhibited the deepest blue-purple reaction, with significantly higher ALP activity than other groups, indicating that the composite material effectively promotes early osteogenic differentiation of cells. Further Alizarin Red S staining after induction culture revealed that the rhBMP9 group formed numerous red calcified nodules, with significantly increased nodule density and area, demonstrating stronger mineralization capacity. In contrast, the hFurin and CaP groups showed lower mineralization levels, while the Pro-mhBMP9 group, although promoting ALP expression, did not show the same level of mineralization as the mature rhBMP9 group. These results demonstrate that the rhBMP9 / CaP / Sericin composite material significantly enhances the osteogenic differentiation and mineralization capacity of bone marrow mesenchymal stem cells.

[0036] To further evaluate the induction of osteogenic differentiation of bone marrow mesenchymal stem cells (rBMSCs) by the rhBMP9 / CaP / Sericin composite material (rhBMP9 group), multi-layered validation of early and late osteogenic markers was performed. rBMSCs treated with different materials were collected, and total RNA was extracted using the Total RNA Kit II according to the manufacturer's instructions. cDNA was synthesized using a reverse transcription kit as a template. The total volume of the qPCR reaction system was 20 μL, including: 10 μL of 2×NovoStart®SYBR qPCR Super Mix Plus, 1 μL each of forward and reverse primers (10 μM), 2 μL of template cDNA (200 ng / mL), and the remaining volume was made up with RNase-free water. The reaction was performed on a real-time quantitative PCR instrument with the following program settings: 95℃ pre-denaturation for 30 s; followed by 40 cycles: 95℃ for 3 s, 60℃ for 30 s. Relative gene expression levels were measured using 2... -ΔΔCt The formula was calculated, and GAPDH was used as an internal reference gene in the experiment. The primer sequences used are shown in Table 1. The grouping information is as follows: NC (Negative Control) group: untreated control, only routine culture; WT group: the culture medium was supplemented with the CaP / Sericin composite material prepared in Example 4; rhBMP9 group: the culture medium was supplemented with the rhBMP9 / CaP / Sericin composite material prepared in Example 4; PC (Positive Control) group: the culture medium was supplemented with commercial rhBMP9 protein (Recombinant Human BMP-9 Protein CF, manufacturer R&D Systems).

[0037] Table 1 Primers used for qPCR As shown in Figure 8, the transcriptional levels of osteogenic marker genes ALP, Osx (Osterix), and OPN were significantly upregulated in the rhBMP9 group, all higher than in the WT group, with the transcriptional level of OPN slightly higher than in the PC group. This indicates that the composite material can effectively activate osteogenic differentiation-related pathways. Figure 8 (A, B, and D). Notably, the transcription of the VEGFA gene (vascular endothelial growth factor A) was also significantly increased, indicating that rhBMP9 not only promotes the upregulation of osteogenic genes but also induces the activation of angiogenesis signals, suggesting that it plays a dual regulatory role in promoting bone-angiogenic synergistic regeneration. Figure 8 C).

[0038] ALP staining results showed that the NC and WT groups had the weakest reactions, while the rhBMP9 group showed a strong deep blue-purple reaction. Quantitative results further confirmed that the rhBMP9 group had the highest ALP activity, and there was no significant difference compared with the PC group, indicating that this material can significantly promote early osteogenic differentiation. Figure 8 ,EF).

[0039] Alizarin Red S staining showed that the rhBMP9 group formed the densest red mineralized nodules with a wide coverage. The WT group showed almost no mineralization trend and its intensity was significantly lower than that of the rhBMP9 group, suggesting that the rhBMP9 composite material also has an advantage in promoting the late mineralization stage, and there was no significant difference compared with the PC group. Figure 8 ,GH).

[0040] Protein detection results showed that the expression levels of COL1 (type I collagen), Runx2, OPN, and OCN proteins were significantly upregulated in the rhBMP9 group. Quantitative analysis showed that the protein levels of COL1A1, COL1A2, and Runx2 in the rhBMP9 group were approximately 1.3 times higher than those in the WT group, while the protein levels of OCN and OPN were approximately 3.7 times and 2.5 times higher, respectively, further validating the promoting effect of the material on osteogenic differentiation. Figure 8 (IJ).

[0041] In summary, the rhBMP9 / CaP / Sericin composite material not only effectively induces early osteogenic differentiation of rBMSCs but also continuously promotes late-stage mineralization and matrix maturation. Notably, the composite material prepared in this invention exhibits comparable biological effects in osteogenic and angiogenesis-related responses to the group treated with commercially available rhBMP9 alone, with no significant difference between the two. This result demonstrates that the rhBMP9 / Sericin / CaP composite material can efficiently deliver and stably maintain the bioactivity of rhBMP9, achieving a combination of sustained release and localized continuous action while maintaining its osteogenic induction function, providing a new and feasible strategy for the materialized delivery of bioactive proteins.

[0042] Example 6. Preparation of rhBMP9 / CaP / Sericin composite scaffold and its effect on promoting the repair of skull defects in rats. Preparation of CaP / Sericin composite scaffolds and recombinant rhBMP9 / CaP / Sericin composite scaffolds: The remaining silk fibers after sericin extraction from wild-type Dazao silkworms and Pro-mhBMP9×hFurin strains (Example 4) were washed with deionized water and air-dried at room temperature. After drying, they were dissolved in 9.3 M LiBr solution at a ratio of 50 mg / mL. After complete dissolution, the solutions were centrifuged at 12000 g for 10 min. The supernatant was placed in a dialysis bag and dialyzed with deionized water. The dialysate was changed every 6 h, for a total of 4 times. After dialysis, the liquid was collected and centrifuged at 12000 g for 10 min. The supernatant was the silk fibroin solution, and its concentration was measured. After determining the concentration, 1% silk fibroin was added to 48-well plates and freeze-dried. After freezing, the plates were annealed in water for 12-24 h to obtain the silk fibroin scaffolds. The specific steps of water annealing are as follows: At room temperature, place sterile water in a sealed container, then place the material on top of the sterile water without direct contact. Vacuum the container using a vacuum pump and let it stand for 12-24 hours. Finally, place the gamma-ray sterilized scaffold into the CaP / Sericin and rhBMP9 / CaP / Sericin composite materials prepared in Example 4, respectively, and incubate overnight at 4°C to allow for full absorption, obtaining the CaP / Sericin composite scaffold and the recombinant rhBMP9 / CaP / Sericin composite scaffold.

[0043] Establishment of a rat model of skull defect: The experimental animals were SPF-grade male Sprague-Dawley (SD) rats purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., weighing 280-320 g and 8 weeks old at purchase. Animals were acclimatized for one week before the experiment to maintain good health. The rearing conditions were constant temperature and humidity (22±2℃, 55±5%), 12-hour light / dark cycle, and free access to food and water. This experimental protocol was approved by the university's Experimental Animal Ethics Committee and strictly followed the "Regulations on the Management of Experimental Animals" and the "Guidelines for the Management and Use of Experimental Animals." Minimize animal suffering during surgery. Rats were anesthetized by isoflurane inhalation: induction concentration 3%, maintenance concentration 1.5-2%; after induction in an anesthesia box for 2-3 minutes, the animals were transferred to the operating table and continuously inhaled through a nasal mask; respiratory rate and reflexes were monitored to ensure appropriate depth of anesthesia. Hair in the surgical area was shaved before surgery, and the area was disinfected three times alternately with 70% alcohol and iodine. Animals were placed on a heating pad to maintain body temperature. A 1-2 cm incision was made in the midline of the rat's skull, and the subcutaneous tissue and periosteum were bluntly dissected to expose the parietal bone surface. A standardized 5 mm full-thickness bone defect was prepared using a 5 mm diameter circular burr under continuous irrigation with pre-cooled saline. Care was taken to avoid damaging the dura mater and brain tissue during drilling. Immediately after the operation, the wound was irrigated with saline to remove any remaining bone fragments and blood.

[0044] Implantation of composite scaffolds, such as Figure 9 As shown in Figure A: Animals were randomly divided into three groups (n=6): Blank group: the defect area remained blank, without any filling material; WT group: a CaP / Sericin composite scaffold was implanted; rhBMP9 group: a recombinant rhBMP9 / CaP / Sericin composite scaffold was implanted. All materials were appropriately trimmed under sterile conditions before implantation to ensure full adhesion to the defect edges. After implantation, it was carefully confirmed that the material covered the entire defect area and did not protrude from the skull surface. After implantation, the periosteum and skin were sutured layer by layer. Penicillin was immediately injected subcutaneously for 3 consecutive days postoperatively to prevent infection. Animal weight, activity level, and wound healing were observed daily postoperatively. Any abnormalities were treated promptly. Each group was sacrificed at 4 and 8 weeks postoperatively to obtain skull samples for subsequent testing.

[0045] Micro-CT Assessment: At 4 and 8 weeks, skull specimens were collected after animal euthanasia and fixed in 4% paraformaldehyde fixative at 4°C for 48 hours. Subsequent scanning was performed using a NEMO Micro-CT device with tube voltage of 80 kV, tube current of 0.06 mA, and resolution of 35 μm. The scan results were used for 3D reconstruction using Recon software. The following parameters were analyzed: BV / TV (bone volume fraction), BMD (bone mineral density), and Tb.Sp (trabecular bone separation).

[0046] Micro-CT scans showed that the Blank group exhibited significant bone defects at both time points, with only a small amount of marginal new bone forming; the WT group showed localized bone deposition, but the defect center was not completely closed; while the rhBMP9 group showed continuous bone structure at 4 weeks, and by 8 weeks, the newly formed bone tissue almost completely filled the defect area. Figure 9 B).

[0047] Further micro-CT analysis showed that the rhBMP9 group exhibited significantly better osteogenic formation than the WT and Blank groups. At 4 weeks, the bone volume fraction (BV / TV) in the rhBMP9 group was approximately 4.0 times and 8.1 times that of the WT and Blank groups, respectively; by 8 weeks, it remained 2.2 times and 6.2 times higher, respectively, indicating that this group possessed stronger osteogenic capacity and sustained bone formation activity. Figure 9 C).

[0048] Meanwhile, bone mineral density (BMD) analysis showed that the mineralization level of the rhBMP9 group was significantly higher than that of the control group at all time points. At 4 weeks, its BMD value was 3.1 times and 12.5 times that of the WT group and Blank group, respectively; at 8 weeks, it was still 2.0 times and 7.2 times that of the control group, respectively, indicating that the mineralization degree and maturity of the newly formed bone tissue were significantly improved. Figure 9 D).

[0049] Furthermore, trabecular separation (Tb.Sp) was significantly reduced in the rhBMP9 group, reaching only 0.75 times and 0.79 times that of the WT and Blank groups at 4 weeks, and further decreasing to 0.59 times and 0.65 times at 8 weeks, indicating that the trabecular arrangement was more compact and the continuity was better. Figure 9 In summary, the rhBMP9 composite scaffold can promote rapid new bone formation and mature mineralization in bone defect areas within a short period of time, significantly improving the microstructure and overall mechanical integrity of bone tissue.

[0050] Histological staining results further validated the osteogenic promoting effect of the rhBMP9 / CaP / Sericin composite scaffold in vivo. Figure 10 H&E staining showed that the defect area in the Blank group was mainly filled with fibrous connective tissue, with very limited bone formation; some osteoid deposition was observed in the WT group, but no continuous bone tissue was formed; while a large amount of new bone was observed in the defect area of ​​the rhBMP9 group, showing obvious bone regeneration. Figure 10 Masson staining results further showed that collagen deposition in the rhBMP9 group was dense and uniformly distributed, and the proportion of mineralized matrix was significantly increased, suggesting that this system promoted the formation and maturation of bone matrix. Figure 10 (B). CD31 immunofluorescence staining (Figure 10, C) showed that a large number of new capillaries appeared in the defect area of ​​the rhBMP9 group, with clear vascular lumen structure and CD31 positive signal distribution; angiogenesis was weak in the WT group, and only a small number of positive signals were observed in the Blank group. Statistical results showed that 4 weeks after implantation, the number of new blood vessels in the rhBMP9 group was approximately 5.8 times higher than that in the Blank group and approximately 1.6 times higher than that in the WT group; at 8 weeks, it was approximately 1.8 times higher than that in the Blank group and 1.5 times higher than that in the WT group. Figure 10 Furthermore, at 8 weeks, there was no significant difference in the number of new blood vessels between the Blank and WT groups, suggesting that the rhBMP9 / CaP / Sericin composite scaffold not only promotes osteoogenesis but also significantly induces angiogenesis, providing sufficient nutrition and oxygen supply support for bone regeneration.

[0051] To further verify the role of the rhBMP9 / Sericin / CaP composite scaffold in promoting osteogenic and angiogenesis in vivo, immunohistochemical staining and statistical analysis were performed on the regenerated tissue in the defect repair area. As shown in Figures 11A and 11B, osteogenic differentiation-related markers showed significantly high expression levels in the rhBMP9 group, with distinct spatial distribution characteristics. Type I collagen (COL1), as a major component of the bone matrix, showed significantly increased expression levels in the rhBMP9 group and was densely distributed around the new bone matrix, indicating that the composite scaffold effectively promoted the synthesis and deposition of collagen matrix, providing a solid structural basis for bone regeneration. The key osteogenic differentiation transcription factor Runx2 showed significantly enhanced positive signal in the new bone formation area, confirming that rhBMP9 successfully drove the directed differentiation of osteogenic progenitor cells into osteoblasts. The late osteogenic differentiation marker OCN showed strong positive expression in the new bone site, suggesting that the new bone tissue has entered the mineralization maturation stage, and the bone repair process is transitioning from early matrix accumulation to functional bone tissue.

[0052] Meanwhile, the expression of the angiogenesis marker VEGFA in the rhBMP9 group also showed a significant upregulation trend, increasing by approximately 2.6 times and 49.7 times compared to the WT and Blank groups, respectively, at 8 weeks. Figure 11 (D), confirming the material's excellent angiogenesis-promoting ability.

[0053] Statistical analysis showed that the expression levels of COL1, Runx2, and OCN proteins in the rhBMP9 group were significantly higher than those in the WT and Blank groups. At week 8, the expression levels of these three proteins were approximately 1.5-fold, 12.3-fold, and 83.3-fold higher than those in the Blank group, respectively. Figure 11 (C, E, F). These results indicate that the rhBMP9 / CaP / Sericin composite scaffold can synergistically promote new bone formation and angiogenesis in vivo, providing good support and microenvironment for bone defect repair, thereby achieving efficient bone tissue regeneration.

[0054] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for producing active rhBMP9 bone repair material based on a double-transgenic silkworm silk gland biosynthesis system, characterized in that, The preparation steps include the following: (1) Prepare Pro-mhBMP9 transgenic silkworm line, so that its genome integrates the nucleic acid sequence Pro-mhBMP9 encoding the human BMP9 precursor; (2) Prepare an hFurin transgenic silkworm strain, integrating the nucleic acid sequence hFurin encoding human Furin protease into its genome; the nucleotide sequence of hFurin is shown in SEQ ID NO.2; (3) By hybridizing the Pro-mhBMP9 transgenic silkworm strain and the hFurin transgenic silkworm strain, a double transgenic silkworm strain that can co-express Pro-mhBMP9 and hFurin in the posterior silk gland of the silkworm is obtained, so that Pro-mhBMP9 is cleaved into a biologically active mature rhBMP9 dimer in the silk gland. (4) Extract silk protein containing active rhBMP9 from the cocoons of double transgenic silkworm strains and prepare active rhBMP9 bone repair material.

2. The method according to claim 1, characterized in that, The Pro-mhBMP9 is a sequence optimized using the silkworm codon, as shown in SEQ ID NO.

1.

3. The method according to claim 1, characterized in that, The hFurin sequence optimized using the silkworm codon is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that, The expression of Pro-mhBMP9 or hFurin is regulated by the hr3 CQ enhancer, the FibH promoter, and the Ser1pA terminator.

5. The method according to claim 1, characterized in that, In step (4), the specific preparation method is as follows: First, extract sericin containing active rhBMP9 from the cocoons of double transgenic silkworms using calcium salt solution. After dialysis with deionized water, mix with phosphate to induce the formation of mineralized composite material rhBMP9 / CaP / Sericin. Then, extract fibroin from the remaining silk, shape and freeze-dry it, and mix it with the composite material rhBMP9 / CaP / Sericin to obtain active rhBMP9 bone repair material.

6. The method according to claim 5, characterized in that, The calcium salt is calcium chloride, and the phosphate source is disodium hydrogen phosphate.

7. Active rhBMP9 bone repair material prepared by the method according to any one of claims 1-6.

8. The use of the active rhBMP9 bone repair material of claim 7 in the preparation of medical devices or biomaterials for promoting bone tissue regeneration and repairing bone defects.

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