A recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, a preparation method and application thereof

Abstract

The application belongs to the technical field of bone tissue engineering, and particularly relates to a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, a preparation method and application. The bone repair scaffold material is formed into a one-way ordered three-dimensional scaffold by a directional freezing method through recombinant collagen, chitosan and PEG. Nano-hydroxyapatite is uniformly deposited in the inside and surface through a special mineralization process. The prepared material has a very good bionic natural bone structure and composition, a good reticular structure and mechanical strength, good biocompatibility and cell activity, a significant repair effect on critical size bone defects, a good osteogenic induction effect and bone conductivity. The bone repair scaffold material provides a new potential treatment method for severe bone defects, and has a wide application in the fields of artificial bone, artificial cartilage, biological scaffolds and the like.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering technology, specifically relating to a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, its preparation method, and its application. Background Technology

[0002] The causes of skull defects include congenital defects such as skeletal abnormalities and acquired injuries such as tumors and neuromaxillofacial surgeries. They are often accompanied by serious complications, including skull defect syndrome, hydrocephalus, and hemiplegia. Clinically, treatment methods for skull defects mainly include autologous bone grafting, allogeneic / xenogeneic bone grafting, and artificial bone substitutes. Autologous bone grafting, taken from the patient's own bone tissue, can cause secondary trauma and inflammation at the donor site; allogeneic bone grafting carries the potential risks of disease transmission and immune reactions, and its availability is limited. Metal alloy scaffold materials lack bioactivity, are non-degradable, and must be surgically removed; bioceramics suffer from high brittleness and slow degradation. Bone tissue engineering biomimetic scaffold materials have advantages such as good biocompatibility and biodegradability, and therefore have attracted much attention in the treatment of skull defects.

[0003] Skeleton is an organic-inorganic composite with good microstructure and mechanical properties, formed by a mesh-like scaffold of collagen fibers and nano-hydroxyapatite uniformly attached to the scaffold. Therefore, the preparation of composites of three-dimensional scaffold materials and nano-hydroxyapatite has always been one of the important approaches to biomimetic artificial bone. For example, Chinese patent CN111097068A discloses a biomimetic hydroxyapatite powder / gelatin / sodium alginate composite 3D printed scaffold and its preparation method, and Chinese patent CN108478880A discloses a nano-hydroxyapatite / chitosan porous composite scaffold material and its biomimetic dialysis mineralization preparation method and application. Both of these methods have prepared three-dimensional scaffolds. However, the above preparation methods involve printing nano-hydroxyapatite after mixing it with other materials, which has problems such as loose structure, poor mechanical strength, and rapid degradation.

[0004] To address the aforementioned technical problems, the present invention aims to provide a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, its preparation method, and its application. The bone repair scaffold material is prepared from recombinant collagen, chitosan, PEG, and nano-hydroxyapatite, exhibiting a layered structure longitudinally and an interconnected porous structure transversely. The recombinant collagen, chitosan, and PEG are prepared using a directional freezing method to form an ordered recombinant collagen-chitosan-PEG three-dimensional scaffold (CCP). The nano-hydroxyapatite is uniformly deposited inside and on the surface of the CCP scaffold through a special mineralization process. The bone repair scaffold material of the present invention has excellent... The biomimetic structure and composition of natural bone exhibit excellent mesh structure and mechanical strength. The bone repair scaffold material possesses good biocompatibility and cell activity, significantly promoting the proliferation, adhesion, and differentiation of bone mesenchymal stem cells (BMSCs) and the expression of osteogenic genes in BMSCs. This bone repair scaffold material demonstrates significant repair effects on critical-sized bone defects, exhibiting excellent osteogenic induction and osteoconductivity, enabling it to guide bone tissue regeneration even without periosteum and growth factors. The bone repair scaffold material of this invention provides a novel and promising treatment method for severe bone defects and has wide applications in the fields of artificial bone, artificial cartilage, and biological scaffolds. Summary of the Invention

[0005] Specifically, it includes the following:

[0006] The primary objective of this invention is to provide a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, which is prepared by the following method:

[0007] (1) Prepare a mixed solution containing recombinant collagen, chitosan and PEG in proportion, degas under vacuum and let stand at 4 ℃;

[0008] (2) The mixed solution obtained in step (1) is placed in a polytetrafluoroethylene mold, the bottom of the mold is sealed, and it is placed on liquid nitrogen to freeze the mixed solution. After demolding, it is freeze-dried under vacuum.

[0009] (3) The material obtained in step (2) is immersed in an ethanol solution containing EDC-NHS for crosslinking, washed, and freeze-dried to obtain a three-dimensional scaffold CCP;

[0010] (4) The three-dimensional scaffold CCP obtained in step (3) is mineralized for 1-40 days to obtain bone repair scaffold material;

[0011] The mineralization method described in step (4) is as follows: the three-dimensional scaffold CCP obtained in step (3) is placed in a sealed container connected with two silicone tubes, and the mineralization solution flows in and out through the silicone tubes; a pump is used to promote the flow of the mineralization solution.

[0012] Preferably, the mineralization solution in step (4) is an SBF solution or a Ca solution. 2+ Mixed with PBS, or Ca 2+ and PO4 3- Mixed solution.

[0013] Preferably, the mineralization time in step (4) is 14-40 days.

[0014] Preferably, the bone repair scaffold material has a layered structure in the longitudinal direction and an interconnected porous structure in the transverse direction.

[0015] Preferably, the mixed solution in step (1) is obtained by reacting 0.1-5 wt% recombinant collagen, 0.1-5 wt% chitosan and 0.1-5 wt% polyethylene glycol.

[0016] Preferably, in step (2), a metal copper plate is used to seal the mold and the solution is frozen in one direction.

[0017] Preferably, the ethanol solution containing EDC-NHS mentioned in step (3) is a 50-100% ethanol solution containing 0.01-10 mol / L EDC and 0.0025-2.5 mol / L NHS.

[0018] A second objective of this invention is to provide the application of the aforementioned bone repair scaffold material in bone tissue regeneration.

[0019] A third objective of this invention is to provide the application of the aforementioned bone repair scaffold material in the preparation of artificial bone repair materials.

[0020] A fourth objective of this invention is to provide the bone repair scaffold material for use as or in the preparation of one or more bone defect filling materials, including: bone defects in maxillofacial surgery, bone defects in dental surgery, skull defects, bone defects from various closed fractures of the limbs, and vertebral bone defects.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) This invention provides a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, which is prepared from recombinant collagen, chitosan, PEG and nano-hydroxyapatite. It has a layered structure in the longitudinal direction and an interconnected porous structure in the transverse direction. The nano-hydroxyapatite is uniformly deposited in the interior and surface of the CCP scaffold through a special in-situ mineralization process. The bone repair scaffold material has a good biomimetic structure and composition of natural bone, and has a good network structure and mechanical strength.

[0023] (2) The bone repair scaffold material has good biocompatibility and cell activity, and can significantly promote the proliferation and adhesion of BMSCs cells; RT-qPCR results show that osteogenic differentiation marker genes are highly expressed in the bone repair scaffold material, and the bone repair scaffold material has excellent osteoinduction effect in vitro.

[0024] (3) The bone repair scaffold material has a significant repair effect on critical-sized bone defects. MRI, Micro-CT and tissue staining results show that the bone repair scaffold material has good osteogenic induction and osteoconductivity. Tissue staining results show that the bone repair scaffold material can be biodegraded, can induce the formation of new bone and promote bone tissue regeneration.

[0025] (4) This invention provides a bone repair scaffold material, which offers a new and promising treatment method for severe bone defects and has wide applications in the fields of artificial bone, artificial cartilage, and biological scaffolds. Attached Figure Description

[0026] Figure 1 Characterization of recombinant collagen-chitosan-PEG three-dimensional scaffolds and recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold materials

[0027] (a) Photograph of the recombinant collagen-chitosan-PEG three-dimensional scaffold CCP; (b, c) FESEM images of the recombinant collagen-chitosan-PEG three-dimensional scaffold CCP; (d) EDX image of the recombinant collagen-chitosan-PEG three-dimensional scaffold CCP; (e) Photograph of the recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold CMCCP; (f, g) FESEM images of the recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold CMCC; (h) EDX image of the recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold CMCC; (i) XRD pattern; (j) FTIR pattern; (k) TGA pattern.

[0028] Figure 2 Effect of mineralization time on the preparation of nano-hydroxyapatite

[0029] (aj) FESEM plots for different mineralization times, (a, f) 2 days; (b, g) 7 days; (c, h) 14 days; (d, i) 26 days; (e, j) 30 days; (k) TGA plots for different mineralization times; (l) Content of organic matrix and inorganic minerals in the scaffold material at different mineralization time periods.

[0030] Figure 3 Mechanical properties of bone repair scaffold materials

[0031] (a) Stress-strain curves of the CCP stent in different directions; (b) Stress-strain curves of the SMCCP stent material in different directions; (c) Stress-strain curves of the CMCCP stent material in different directions; (d) Compressive strength of the material; (e) Compressive modulus of the material.

[0032] Figure 4 Cell activity and biocompatibility of bone repair scaffold materials

[0033] (a) Cytotoxicity of scaffold material extract; (b) Cell proliferation assay using CCK-8 assay; (c) Staining of live and dead cells of BMSCs after 1 and 4 days of culture on scaffold material; (d) CLSM plot of BMSCs after 4 days of culture on scaffold material; (★ indicates significant difference, p < 0.05).

[0034] Figure 5 In vitro osteogenic properties of bone repair scaffold materials

[0035] (a) ALP activity was detected using the pNPP assay after BMSC cells were cultured on scaffold material for 1, 4, 7, and 11 days. (b) RT-qPCR was used to detect osteogenic marker genes expressed by BMSC cells after 7 and 14 days of culture on scaffold material: (b) Runx-2; (c) ALP; (d) Collagen I; (e) OCN. (f) ARS quantification. (g) ARS assay was used to assess the osteogenic capacity of BMSC cells. (★ indicates significant difference, p < 0.05)

[0036] Figure 6 MRI results of bone repair scaffold material used to treat skull defects in SD rats

[0037] Figure 7 Micro-CT results of bone repair scaffold material used to treat skull defects in SD rats

[0038] (a) Coronal and sagittal scans; (b) Bone volume; (c) Bone mineral density (BMD); (d) Number of trabeculae (☆ indicates significant difference, p < 0.05).

[0039] Figure 8 Tissue staining results of bone repair scaffold material for treating cranial defects in SD rats

[0040] (a) H&E staining; (b) Masson staining Detailed Implementation Plan

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the present invention, and not the entirety of the invention. 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.

[0042] The recombinant collagen described in the following embodiments is collagen obtained in animal, plant or microbial expression systems using transgenic technology and gene recombination technology.

[0043] The recombinant collagen described in the following examples is recombinant collagen obtained by Escherichia coli fermentation, but it is not limited to the above method. It can also be recombinant collagen prepared by other methods, including recombinant collagen fermented by Pichia pastoris, recombinant human collagen, etc.

[0044] Example 1: Preparation of Materials

[0045] 1. Preparation of recombinant collagen-chitosan-PEG three-dimensional scaffold (CCP)

[0046] A solution of 1.5 wt% recombinant collagen, 1 wt% chitosan, and 1.5 wt% PEG was thoroughly mixed, degassed under vacuum, and incubated overnight at 4 °C. The mixture was then injected into a polytetrafluoroethylene (PTFE) mold, the bottom of which was sealed with a copper plate. A foam box containing liquid nitrogen was placed at the bottom of the mold; the evaporation of the liquid nitrogen uniformly froze the solution. The completely frozen sample was removed from the mold and freeze-dried using a vacuum freeze dryer. The dried recombinant collagen-chitosan-PEG composite matrix was then crosslinked by immersing it in a 95% ethanol solution containing EDC-NHS. The matrix was then washed with deionized water to remove excess crosslinking agent and crosslinking byproducts. A stable and water-insoluble recombinant collagen-chitosan-PEG three-dimensional scaffold (referred to as CCP in the following examples) was prepared by freeze-drying.

[0047] 2. Preparation of CCP-nanohydroxyapatite composite bone repair scaffold material

[0048] The CCP scaffold was placed in a polytetrafluoroethylene mold with two silicone tubes for the inflow and outflow of the mineralization solution. The mold was then sealed, and a peristaltic pump was used to drive the SBF solution through the matrix. Mineralization lasted for 26 days, with the mineralization solution being replaced after 2, 7, and 14 days. After mineralization, the material was immersed in deionized water to remove soluble salts. The resulting bone repair scaffold material is referred to as CMCCP in the following examples.

[0049] The CCP scaffold was immersed in SBF mineralization solution for 26 days, with the mineralization solution being replaced after 2, 7, and 14 days of mineralization. After mineralization, the material was immersed in deionized water to remove soluble salts. This yielded a mineralized recombinant collagen-chitosan composite matrix, abbreviated as SMCCP in the following examples.

[0050] Example 2: Characterization of bone repair scaffold materials

[0051] 1. Microstructure of the material

[0052] Materials were prepared according to the method in Example 1. The morphology of the samples was characterized using a Hitachi S-4800 field emission scanning electron microscope. The pore size of each group was calculated from multiple FESEM images using pixel measurement software (E-ruler), and 50 data points were randomly selected from each group to describe the range and distribution of the pore size.

[0053] Figure 1 Characterization of recombinant collagen-chitosan-PEG three-dimensional scaffolds and recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold materials. Figure 1 a is a photograph of the CCP, showing the successful fabrication of a three-dimensional scaffold with a complete morphology; Figure 1 b is the FESEM image of CCP in the L direction, showing that CCP forms a well-structured layered structure with a spacing of about 40 μm between each layer; Figure 1 c is the FESEM image of CCP in the R direction, showing that CCP forms a uniform, interconnected porous structure with a pore size distribution of approximately 110-180 μm; Figure 1 d is the EDS plot of CCP, indicating the presence of C and O elements in the three-dimensional scaffold. These results demonstrate that a recombinant collagen-chitosan-PEG three-dimensional scaffold with good microstructure and ordered orientation was successfully prepared by directional freezing.

[0054] Figure 1 e is a photograph of the CMCCP scaffold material, showing the formation of a scaffold material with a complete morphology; Figure 1 f and g are FESEM images of the CMCCP scaffold material, showing that the nano-hydroxyapatite is uniformly distributed; Figure 1 h is an EDS image showing C, O, P, and Ca elements. C and O elements are derived from the CCP three-dimensional scaffold, while P and Ca elements are derived from nano-hydroxyapatite. These results demonstrate that nano-hydroxyapatite uniformly distributed on the CCP scaffold surface was successfully prepared using CCP as a biological template via a special in-situ mineralization method.

[0055] 2. XRD testing of materials

[0056] Materials were prepared according to the method in Example 1. The CCP scaffold and CMCCP scaffold materials were tested using a concave method on a Rigaku D / max-2400 X-ray diffractometer with Cu Ka radiation (40 kV, 40 mA) at a scan rate of 0.02 ° / s and a scan range of 20–80 °. The results are as follows. Figure 1 As shown in Figure i, the XRD pattern of CCP shows a broad peak, indicating that CCP has an amorphous structure, consistent with its inherent amorphous fibrous properties. The XRD pattern of CCPCCP shows obvious hydroxyapatite (HA) crystallization peaks; the characteristic diffraction peaks at 25.52 ° and 31.95 ° correspond to the (002) and (211) crystal planes of hydroxyapatite, indicating successful preparation of hydroxyapatite. The relatively broad diffraction peaks in the figure indicate that the synthesized hydroxyapatite has a low crystallinity, similar to that of hydroxyapatite in natural bone.

[0057] 3. Infrared characterization of materials

[0058] The material was prepared according to the method in Example 1, and Fourier transform infrared spectroscopy characterization was performed. The results are as follows: Figure 1 As shown in j. The FT-IR image of the CCP three-dimensional scaffold shows that at 3400 cm... -1 The vibrational peak observed at 2926 cm⁻¹ can be attributed to the NH bond. -1 and 1650 cm -1 The vibrational peaks at 1032 cm⁻¹ are the vibrational absorption peaks of the CH and C=O bonds, respectively. In the FT-IR image of the CMCCP scaffold material, the peak at 1032 cm⁻¹ is... -1 The strong infrared peak at 566 cm⁻¹ corresponds to the characteristic absorption peaks of vibration and stretching of PO. -1 and 603 cm -1 The vibrational peak can be attributed to the bending vibration of O=PO-bridged phosphorus, indicating the presence of phosphate in the CMCCP scaffold material. Compared to CCP, the CH4 concentration in the CMCCP scaffold material increases from 2926 cm⁻¹. -1 Changed to 2870 cm -1 The C=O bond position is 1650 cm. -1 Changed to 1630 cm -1 The presence of nHAp indicates a redshift. FT-IR results demonstrate the successful preparation of CCP-nanohydroxyapatite composite scaffold material.

[0059] 4. Thermogravimetric analysis (TGA) of materials

[0060] The material was prepared according to the method in Example 1, and thermogravimetric analysis was performed to characterize it. The results are as follows: Figure 1As shown in Figure k, three weight loss stages were observed in the TGA images. The first stage occurred below 200 °C, primarily due to the evaporation of moisture in the material. The results indicated that the water content in the CCP and CMCCP scaffold materials was approximately 16% and 9%, respectively. A second weight loss stage occurred between 280 and 350 °C, mainly caused by the thermal decomposition of organic components. The weight losses in the CCP composite matrix and CMCCP scaffold material were 68% and 20%, respectively. The third weight loss stage occurred at 600 °C, with the remaining mass of the CCP composite matrix at 0, and the mass of the residue in the CMCCP scaffold material approximately 65%, i.e., the nHAp content was approximately 65%. The TGA results indicate that the content of each component in the CMCCP bone repair scaffold material is similar to that of natural bone.

[0061] Example 3: Effect of mineralization time on the amount of inorganic minerals deposited

[0062] Materials were prepared according to the method in Example 1, and the morphology of the samples was characterized using a Hitachi S-4800 field emission scanning electron microscope. Thermogravimetric analysis (TGA) was performed on the samples in a nitrogen atmosphere (40 mL / min) using a TGA / NETZSCH STA449 F3 instrument at a heating rate of 10 °C / min, with an analysis temperature range of 25 °C–800 °C. The content of nano-hydroxyapatite in the CCP three-dimensional scaffold was compared after 2, 7, 14, 26, and 30 days of mineralization.

[0063] The results are as follows Figure 2 As shown. Figure 2 a and e are FESEM images taken 2 days after mineralization, showing a small number of nano-hydroxyapatite particles attached to the surface of the CCP 3D scaffold. Figure 2 b and g are FESEM images of mineralization after 7 days; Figure 2 c and h are FESEM images of mineralization after 14 days; Figure 2 d and i are FESEM images taken after 26 days of mineralization; Figure 2 e and j are FESEM images after 30 days of mineralization. The FESEM images show that the amount of hydroxyapatite deposited in the CCP 3D scaffold gradually increases with increasing mineralization time. Figure 2 i is the thermogravimetric curve of the mineralized material. The weight loss below 200 °C can be attributed to the evaporation of moisture in the material, while the weight loss between 200-500 °C can be attributed to the decomposition of organic components in the material. The weight above 500 °C represents the hydroxyapatite in the material. Figure 2k is a graph showing the changes in organic and inorganic components in the material over mineralization time. Calculations show that the organic component content at 2, 7, 14, 26, and 30 days is 66.98%, 49.39%, 33.95%, 22.94%, and 21.46%, respectively; while the inorganic component content is 20.61%, 38.69%, 56.33%, 69.71%, and 69.92%, respectively. The results indicate that in-situ mineralization was completed at 26 days, with hydroxyapatite fully deposited in the CCP three-dimensional scaffold. The composition of each component in the CMCCP bone repair scaffold material is similar to that of natural bone, effectively mimicking its composition.

[0064] Example 4 Mechanical properties of the support material

[0065] The compressive strength and elastic modulus of the material were tested using a universal testing machine. The sample used for the compressive strength test was a cylinder with a diameter of 15 mm and a height of 12 mm. The compressive strength test was conducted using the universal testing machine at a speed of 1 mm / min. The elastic modulus was calculated based on the slope of the linear range of the load-displacement curve.

[0066] The results are as follows Figure 3 As shown. Figure 3 a is the stress-strain curve of the CCP three-dimensional scaffold. Figure 3 b is the stress-strain curve of the SMCCP material. Figure 3 c is the stress-strain curve of the CMCCP material. Figure 3 d is the compressive strength of the material. Figure 3 e represents the compressive modulus of the material. The compressive strength diagram shows that the compressive strengths of the CCP scaffold, SMCCP scaffold, and CMCCP scaffold materials in the L direction are 110.22 kPa, 377.07 kPa, and 601.51 kPa, respectively, and in the R direction are 51.12 kPa, 202.18 kPa, and 408.87 kPa, respectively, indicating a significant increase in the mechanical properties of the scaffold materials after mineralization. The higher compressive strength in the L direction compared to the R direction is mainly due to the ordered arrangement of the composite matrix in the L direction, while it exhibits a porous structure in the R direction, demonstrating that an ordered structure can significantly improve the mechanical properties of the material.

[0067] Example 5: In vitro biocompatibility and activity of the material

[0068] 1. Cytotoxicity

[0069] The in vitro cytotoxicity of the scaffold material extract was evaluated using the CCK-8 assay. 100 μL of BMSCs cell suspension was added at 5 × 10⁻⁶ cells per well. 3Cells were placed at a density of 100 μL in 96-well cell culture plates and incubated for 24 hours to allow them to adhere. Then, 100 μL of extraction buffer for each scaffold material was added. An equal volume of α-MEM culture medium was added to the remaining wells as a control group. After 24 hours of incubation, 10 μL of CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) was added to each well, and the 96-well plate was incubated for another 2 hours. The absorbance at 450 nm was measured using a Tecan Infinite F200 / M200 multi-mode microplate reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated by dividing the average absorbance of three measurements under each condition by the average absorbance of the control group.

[0070] The results are as follows Figure 4 As shown in Figure a, the extracts of CCP scaffold, SMCCP scaffold material and CMCCP scaffold material all exhibit similar high cell viability, indicating that the synthesized scaffold material has high biocompatibility.

[0071] 2. Cell proliferation

[0072] The proliferation of BMSCs in the scaffold material was detected using the CCK-8 assay. BMSCs were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates. Cell proliferation was observed on days 1, 4, 7, and 11, with the medium changed every two days. At the end of the experimental period, the medium was replaced with basal medium containing 10% (v / v) CCK-8 reagent, and cultured for another 6 hours. The absorbance of 100 μL of supernatant was measured at 450 nm using a Tecan Infinite F200 / M200 multi-plate reader (Tecan, Mannedorf, Switzerland).

[0073] The results are as follows Figure 4 As shown in b, cell proliferation was good on all samples with prolonged culture time. On the first day after cell implantation, there was no significant difference in cell number among the groups. After the fourth day, the cell proliferation rates of CMCCP and SMCCP were significantly higher than those of CCP and the control group. Figure 4 c is a live-cell-dead-cell staining pattern of the materials, showing that there was no significant difference in the number of live and dead cells on the three materials on the first day of cell implantation, indicating that the scaffold materials have no toxic side effects on the cells. Four days after cell implantation, differences in the number of cells on the three groups of materials emerged, with CMCCP and SMCCP showing significantly higher numbers than CCP, indicating that CMCCP and SMCCP scaffold materials can significantly promote the proliferation of BMSC cells.

[0074] 3. Immunofluorescence

[0075] A thin sheet of scaffold material was placed on a glass slide, and BMSCs cells were arranged at a density of 400 cells / mm². 2 Cells were implanted into scaffold material and cultured at 37 °C for 24 hours. They were washed three times with PBS to remove non-adhering cells. Adhering cells were then fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% bovine serum albumin (BSA) for 30 minutes. Cells were incubated with phalloidin-tetramethylrhodamine isothiocyanate at 37 °C for 1 hour, followed by incubation with DAPI (Sigma-Aldrich) at 37 °C for 10 minutes for staining the cell actin cytoskeleton and nuclei. Images were acquired using a fluorescence microscope.

[0076] The results are as follows Figure 4 As shown in Figure d, the CLSM diagram revealed a lower cell count on the CCP scaffold, while the SMCCP scaffold exhibited a higher cell count and a larger diffusion area. Compared to both the CCP and SMCCP scaffolds, the CMCCP scaffold showed the highest cell count and the largest diffusion area. These results indicate that the CMCCP bone repair scaffold provides a favorable environment for the growth of BMSCs.

[0077] Example 6: In vitro osteogenic properties of the scaffold material

[0078] 1. ALP activity detection

[0079] BMSCs cells were loaded at a rate of 1×10 5 Cells were seeded onto scaffold material at a specific density. After incubation for 1, 4, 7, and 11 days, total cellular protein and ALP activity were measured using the BCA and ALP kits. ALP activity was normalized to the total protein level according to the kit instructions. At each time point, the scaffold material seeded with cells was digested with trypsin for 3–4 minutes. Fetal bovine serum was added to stop the digestion. Cells were gently pipetted to completely detach from the scaffold material. The resulting liquid was transferred to EP tubes and centrifuged at 600 rpm for 8 minutes to collect the cells. Cells were washed with PBS to remove trypsin. Cells were resuspended in PBS, and cell lysis buffer was added to induce complete lysis. The supernatant was centrifuged at 3000 rpm for 10 minutes to remove all insoluble debris. 50 μL of the sample was added to a 96-well plate, followed by 50 μL of matrix buffer. The plate was incubated at 37 °C for 10 minutes, and then 100 μL of stop buffer was added. The OD value of each well was measured at 405 nm.

[0080] The results are as follows Figure 5As shown in Figure a, quantitative analysis results indicate that the ALP activity of BMSCs cultured on each material group increased over time. On the first day, there was no significant difference in ALP activity among the different material groups. After the seventh day, the ALP activity on CMCCP and SMCCP scaffold materials was significantly higher than that on the CCP scaffold.

[0081] 2. Osteoblastic gene expression

[0082] BMSCs cells were loaded at a rate of 1×10 5 Cells were seeded at a density in 24-well plates with a fixed scaffold material. After 7 and 14 days of culture, the relative expression levels of specific genes (Runx-2, ALP, OCN, Collagen I, β-Actin) in osteoblasts were assessed using real-time quantitative PCR. The cell-scaffold complex was washed with PBS, and 1 ml of RNAex was added. The culture plate was gently shaken, and cells were repeatedly pipetted to detach them. The supernatant was transferred to EP tubes and incubated at room temperature for 5 minutes. 200 μL of chloroform was added, and the mixture was vortexed and incubated at room temperature for 5 minutes. The cells were then centrifuged at 12000 g at 4 °C for 15 minutes. The supernatant was carefully transferred to a new centrifuge tube, and 500 μL of isopropanol was added. The mixture was thoroughly mixed and incubated at room temperature for 10 minutes. The cells were then centrifuged at 12000 g at 4 °C for 10 minutes. The supernatant was discarded, the precipitate was collected, dried, and dissolved in DEPC-treated water. The RNA concentration was measured. cDNA was obtained by reverse transcription and Real-Time PCR was performed under standard reaction conditions using the Thermal Cycler Dice Real-Time System. After the reaction, confirm the amplification and melting curves of Real-Time PCR. Prepare a standard curve for PCR quantification using 2... -△△Ct Calculate the expression level of each target gene relative to the previous time point. Verify the amplification efficiency of the primers and quantitatively compare gene expression.

[0083] The results are as follows Figure 5 As shown in the results, RT-qPCR at 7 and 14 days of culture revealed that the expression levels of four osteogenic genes—Col-I, ALP, Runx-2, and OCN—were significantly higher in the CMCCP scaffold material than in the CCP and SMCCPL scaffold materials. At 14 days, the expression levels of the four genes in the CMCCP scaffold material were 3.4, 5.6, 4.3, and 5.4 times that of the CCP scaffold material, respectively; and 1.65, 2.1, 2.5, and 1.3 times that of the SMCCPL scaffold material, respectively.

[0084] 3. Cellular mineralization

[0085] BMSCs cells were loaded at a rate of 1×10 5The culture medium was seeded at a density in 24-well plates using a fixed scaffold material and incubated at 37°C with 5% CO2. After 7 and 14 days, the culture medium was aspirated from the plates, and the plates were washed three times with PBS buffer. The plates were then fixed with 4% paraformaldehyde for 30 minutes and washed three times with PBS buffer. Alizarin Red S staining solution was added, and the plates were stained at room temperature for 30 minutes. The plates were then thoroughly washed with deionized water and observed under an inverted microscope.

[0086] The results are as follows Figure 5 As shown in Figure g, from day 7 to 14, only a small number of red mineralized nodules were observed in the CCP scaffold group; red, irregularly shaped, and unevenly colored mineralized nodules were observed in the SMCCP scaffold material group; and in the CMCCP scaffold material group, red nodules gradually enlarged to form obvious nodules. Quantitative analysis results showed that the mineralization activity of the CMCCP scaffold material group was significantly higher than that of the CCP and SMCCP groups. In vitro cell experiments showed that the synthesized CMCCP scaffold material has excellent biocompatibility and mineralization induction ability, and can promote osteogenic differentiation.

[0087] Example 7: In vivo osteogenic properties of the scaffold material

[0088] 1. MRI detection of osteogenic properties of scaffold materials in vivo

[0089] At 5, 10, and 15 weeks post-surgery, rats were anesthetized by intraperitoneal injection of 10% (w / v) sodium pentobarbital (0.3 mL / 100 g) before scanning, according to the previous grouping, to reduce respiratory movement. After anesthesia, the rats were kept in a supine position, with their heads and abdomens directly fixed to the coil with a belt. A Siemens MAGNETOM Skyro 3.0T laser was used, and the coil was a 70 mm aperture 8-channel rat-specific coil (Shanghai Chenguang Medical Technology, China).

[0090] The results are as follows Figure 6 As shown, in the control group, no obvious soft tissue swelling was observed above the skull defect in rats 5 weeks post-surgery. At 10 and 15 weeks, the tissue imaging layers in the defect area became clearer, and no obvious high-density images were observed within the bone wound. At 15 weeks post-implantation, the tissue imaging in the bone defect area of ​​the CCP scaffold group was slightly clearer than that of the control group. In the SMCCP scaffold material group, there was a clear boundary between the soft tissue and the defect area, and the density of the skull defect area was slightly higher than the surrounding area. In the CMCCP scaffold material group, there was a significant high-density image in the skull defect area. These results indicate that the CMCCP bone repair scaffold material can significantly promote bone tissue regeneration, forming a morphology similar to normal bone.

[0091] 2. Micro-CT Detection of Osteogenic Properties of Scaffold Materials in Vivo

[0092] Five, ten, and fifteen weeks post-surgery, the experimental animals were euthanized under general anesthesia according to their previous groupings. Skull specimens were collected and fixed in 4% paraformaldehyde solution. The morphology of the reconstructed skull was evaluated using an animal micro-computed tomography (Micro-CT) scanner (Bruker Skyscan 1176, USA) in high-resolution scanning mode. Following the Micro-CT scan, the bones were visualized using three-dimensional isosurface rendering.

[0093] The results are as follows Figure 7 As shown in the figure. Micro-CT images of the control group at 5, 10, and 15 weeks postoperatively showed minimal bone formation within the critical-sized defect area, with volumes of 0.53 ± 0.12 mm. 3 1.05 ± 0.17 mm 3 and 1.58 ± 0.72 mm 3 Similar results were observed in the CCP scaffold group and the control group, namely, only a small amount of bone tissue was observed, with a volume of 1.01 ± 0.13 mm. 3 2.48 ± 0.43 mm 3 and 3.54 ± 0.58 mm 3 Conversely, Micro-CT analysis results showed that the CMCCP scaffold material at week 5 (1.79 ± 0.09 mm) 3 Week 10 (6.15 ± 0.26 mm) 3 ) and week 15 (12.21 ± 0.61 mm) 3 Significant bone regeneration was triggered. In the CMCCP scaffold material, BMD increased significantly from 0.086 g / cc (5 weeks) to 0.21 g / cc (15 weeks). In the control group, CCP scaffold, and SMCCP scaffold material groups, BMD was below 0.1 g / cc at all time points, indicating incomplete bone regeneration. Tb.N showed the same trend as BMD (…). Figure 7 (ad). Compared with the SMCCP, CCP, and control groups, the CMCCP scaffold material showed significantly higher BV and BDM at 5, 10, and 15 weeks. These results indicate that the CMCCP scaffold material has good osteoinductive properties and can guide osteogenic differentiation to form new bone.

[0094] 4. Tissue staining of the osteogenic properties of scaffold materials in vivo

[0095] At 5, 10, and 15 weeks post-surgery, rats were euthanized under anesthesia according to the previously grouped procedures. Skull specimens from the euthanized rats were used for histopathological analysis. The rat skull and maxilla were harvested, and the scalp and brain tissue were removed. Bone defects and soft tissue were cut using a scalpel. Tissue samples were fixed in 4% paraformaldehyde solution for 24 hours. The samples were then rinsed with PBS and immersed in decalcification solution for decalcification. Decalcification was checked periodically; complete decalcification was indicated when a needle could penetrate the bone tissue without resistance. Completely decalcified samples underwent gradient dehydration, being dehydrated twice each time with 50%, 75%, 95%, and 100% ethanol for 10 minutes each time. The dehydrated tissue was embedded in paraffin and then sectioned into 5 μm sections using a paraffin sectioning machine. The paraffin sections were stained with hematoxylin and eosin and Masson's stain, respectively, and observed under a microscope.

[0096] Figure 8 A shows the results of hematoxylin and eosin staining. Five weeks after repair, no material was observed in the H&E staining images of the CCP scaffold and SMCCP scaffold material groups, indicating complete degradation of the material after 5 weeks. In the CMCCP scaffold material group, new bone formation at 5 weeks did not begin from the fracture ends of the original bone, but from the defect center, demonstrating the scaffold material's ability to induce new bone formation. At 10 weeks, compared to the CCP and SMCCP material groups, the CMCCP scaffold material group showed a large aggregation of osteocytes and increased new bone tissue. At 15 weeks post-operation, the CMCCP scaffold material group showed complete degradation of the scaffold material and replacement by regenerated tissue, forming mature bone tissue with the same morphology as the original natural bone. H&E staining showed that new bone formation in the CMCCP scaffold material was significantly higher than that in the CCP scaffold, SMCCP scaffold material, and control group at any time point.

[0097] Figure 8 b shows the results of Masson's trichrome staining of the skull cross-section. In the tissue staining image, immature primary bone appears blue, while mature primary bone appears red. Bone tissue increased gradually in a time-dependent manner in all groups. During the 5-15 week repair period, significantly more new bone tissue was observed at the defect site in the CMCCP scaffold material group compared to other groups. In conclusion, H&E staining and Masson's staining results indicate that the CMCCP scaffold material possesses good osteogenic activity and can promote bone regeneration.

[0098] In summary, this invention provides a recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, prepared from recombinant collagen, chitosan, PEG, and nano-hydroxyapatite. It exhibits a layered structure longitudinally and an interconnected porous structure transversely. The nano-hydroxyapatite is uniformly deposited inside and on the surface of the CCP scaffold through a special in-situ mineralization process. This bone repair scaffold material effectively mimics the structure and composition of natural bone, possessing a good network structure and mechanical strength. Furthermore, it demonstrates good biocompatibility and cell activity, significantly promoting the proliferation and adhesion of BMSCs cells. RT-qPCR results are presented in the table. This invention demonstrates that osteogenic differentiation marker genes are highly expressed in bone repair scaffold materials, which exhibit excellent osteoinduction activity in vitro. The scaffold materials show significant repair effects on critical-sized bone defects. MRI, Micro-CT, and tissue staining results indicate that the scaffold materials possess good osteogenic induction and osteoconductivity. Tissue staining results also show that the scaffold materials are biodegradable, capable of inducing new bone formation, and promoting bone tissue regeneration. This invention provides a bone repair scaffold material, offering a novel and promising treatment method for severe bone defects, with wide applications in the fields of artificial bone, artificial cartilage, and biological scaffolds.

Claims

1. A recombinant collagen-chitosan-PEG-hydroxyapatite bone repair scaffold material, characterized in that, The bone repair scaffold material is prepared by the following method: (1) proportionally configuring a mixed solution containing recombinant collagen, chitosan and PEG, vacuum degassing, and standing at 4°C; (2) placing the mixed solution obtained in step (1) in a polytetrafluoroethylene mold, sealing the lower end of the mold, placing it on liquid nitrogen, freezing the mixed solution, demolding, and vacuum freeze-drying; (3) immersing the material obtained in step (2) in an EDC-NHS-containing ethanol solution for crosslinking, washing, and freeze-drying to obtain a recombinant collagen-chitosan-PEG three-dimensional scaffold; (4) mineralizing the three-dimensional scaffold obtained in step (3) for 1-40 days to obtain a bone repair scaffold material; The mineralization method described in step (4) is as follows: the three-dimensional scaffold CCP obtained in step (3) is placed in a closed container with two silica gel tubes, and the mineralization solution flows in and out through the silica gel tubes; a pump is used to promote the flow of the mineralization solution, and the mineralization solution is SBF solution, or a mixed solution of Ca 2+ and PBS, or a mixed solution of Ca 2+ and PO4 3- The mineralization time is 14-26 days. The mixed solution in step (1) is obtained by reacting 1.5 wt% recombinant collagen, 1 wt% chitosan and 1.5 wt% PEG.

2. The bone repair scaffold material of claim 1, wherein, The bone repair scaffold material has a layered structure in the longitudinal direction and a mutually interconnected pore structure in the transverse direction.

3. The bone repair scaffold material of claim 1, wherein In step (2), a copper plate is used to seal the mold and freeze the solution in one direction.

4. The bone repair scaffold material of claim 1, wherein In step (3), the EDC-NHS-containing ethanol solution is a 50-100% ethanol solution containing 0.01-10 mol / L EDC and 0.0025-2.5 mol / L NHS.

5. Use of the bone repair scaffold material according to any one of claims 1-4 in the preparation of a bone tissue regeneration material.

6. Use of the bone repair scaffold material according to any one of claims 1-4 in the preparation of an artificial bone repair material.

7. Use of the bone repair scaffold material according to any one of claims 1-4 as or in the preparation of one or more of the following bone defect filling materials: maxillofacial surgery bone defects, dental surgery bone defects, skull defects, various types of closed fracture bone defects of limbs, and spinal bone defects.

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