A mineralized collagen-glycosaminoglycan bone repair scaffold material, preparation method and application

Abstract

The application belongs to the technical field of bone tissue engineering, and particularly relates to a mineralized collagen-polysaccharide bone repair scaffold material, a preparation method and application. The bone repair scaffold material is obtained by cross-linking of uniformly mixed mineralized collagen and polysaccharide; the concentration ratio of the mineralized collagen and the polysaccharide is 10-80wt%:1-20wt%, and the balance is water; the mineralized collagen is obtained by reaction of 0.1-10wt% collagen, 0.0334-3.34mol / L calcium salt and 0.02-2mol / L phosphate; the bone repair scaffold material can well imitate the composition and structure of natural bone, and significantly promotes the proliferation, adhesion and differentiation of BMSCs cells, and has good bone induction effect; the mineralized collagen-polysaccharide bone repair scaffold material has high biological activity and bone induction, and has great application potential in the field of bone tissue engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone tissue engineering, and in particular relates to a mineralized collagen-polysaccharide bone repair scaffold material, a preparation method and an application thereof. Background Art

[0002] Bones, the human body's framework, possess a continuous adaptability that allows them to repair small bone defects. However, defects exceeding a critical size threshold (typically >2 cm) are difficult to repair on their own. Severe bone defects caused by traffic accidents, aging, tumor resection, or congenital factors require clinical treatment. Bioscaffold materials, due to their excellent biocompatibility and bioactivity, are widely used to treat bone defects, a procedure clinically known as cranioplasty.

[0003] At present, different types of biological scaffold materials such as bioceramics, metal alloys, high molecular polymers, and hydrogels have been developed, but how to mimic the composition, structure, and function of natural bones remains a huge challenge. Mineralized collagen (MC) is a composite of collagen and nanohydroxyapatite, which is the main structural unit of natural bones. Therefore, the use of MC with good micromorphology and biological activity to prepare bone repair scaffold materials has always attracted much attention. Chinese patent CN104096268A discloses a MC-polylactic acid bone repair scaffold, but the scaffold material lacks a connected mesh structure, which may provide a poor growth environment for cell growth; invention patent CN106421927A discloses a MC-polylactic acid / polycaprolactone bone repair scaffold and its preparation method, but the degradation cycle of the scaffold material is too long, and there is a risk of cytotoxicity caused by residual organic solvents.

[0004] In response to the above technical problems, the object of the present invention is to provide a mineralized collagen-polysaccharide bone repair scaffold material, a preparation method and an application thereof; the bone repair scaffold material is obtained by uniformly mixing mineralized collagen and polysaccharide and then cross-linking them with a cross-linking agent; the mineralized collagen-sodium alginate bone repair scaffold material forms an interconnected mesh structure with a pore size of approximately 135 μm and a porosity of approximately 82%; the bone repair scaffold material has good biocompatibility and bioactivity, and can significantly promote the proliferation, adhesion and differentiation of BMSCs cells; in animal experiments on the bone repair scaffold material for treating critical-sized skull defects, MRI and Micro-CT results showed that the MRCHA scaffold material has good osteoinduction and osteoconductivity; H&E and Masson staining results showed that the MRCHA scaffold material was completely degraded 12 weeks after surgery and can significantly promote bone tissue regeneration; the mineralized collagen-sodium alginate bone repair scaffold material provided by the present invention has excellent bone defect treatment effect and has great application potential in the field of bone tissue engineering. Summary of the Invention

[0005] The primary purpose of the present invention is to provide a mineralized collagen-polysaccharide bone repair scaffold material, which is obtained by uniformly mixing mineralized collagen and polysaccharide and then cross-linking. The concentration ratio of the mineralized collagen and polysaccharide is 10-80 wt%: 1-20 wt%, and the balance is water.

[0006] Preferably, the polysaccharide is one or more of sodium alginate, chondroitin sulfate, and hyaluronic acid.

[0007] Preferably, the polysaccharide is sodium alginate.

[0008] Preferably, the mineralized collagen material is obtained by reacting 0.1-10 wt% collagen, 0.0334-3.34 mol / L calcium salt and 0.02-2 mol / L phosphate.

[0009] Preferably, the mineralized collagen material is obtained by reacting 1 wt% collagen, 0.1336 mol / L calcium salt and 0.08 mol / L phosphate.

[0010] Preferably, the collagen is recombinant collagen and / or animal collagen, and the animal collagen includes one or more of type I, type II and type III.

[0011] Preferably, the collagen is recombinant collagen.

[0012] Preferably, the calcium salt is anhydrous calcium chloride solution, calcium gluconate solution, calcium dihydrogen phosphate solution, calcium nitrate solution or calcium bicarbonate solution; the phosphate is disodium hydrogen phosphate solution, sodium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, ammonium dihydrogen phosphate solution or phosphoric acid solution.

[0013] Preferably, the preparation method of the mineralized collagen is as follows:

[0014] (1) preparing an aqueous solution of a calcium salt solution and a collagen solution in proportion, mixing and reacting for 10-120 minutes, and then adding a phosphate solution dropwise in proportion to obtain a mixed solution;

[0015] (2) The pH of the mixed solution in step (1) is adjusted, the mixture is stirred for reaction, the mixture is allowed to stand in a water bath, the mixture is centrifuged, and the mixture is washed to obtain mineralized collagen as a white colloid.

[0016] Preferably, the mixing reaction time in step (1) is 30-120 min.

[0017] Preferably, the pH in step (2) is 7.4, the reaction is stirred for 12-36 hours, and then allowed to stand at a constant temperature of 37° C. for 12-96 hours.

[0018] Preferably, the stirring reaction in step (2) is carried out for 24 hours and then allowed to stand for 72 hours;

[0019] A second object of the present invention is to provide a method for preparing the bone repair scaffold material, comprising the following steps:

[0020] (1) Mixing mineralized collagen and polysaccharide solution in proportion, homogenizing, injection molding, vacuum degassing, and freeze drying;

[0021] (2) Immersing the material prepared in step (1) in an ethanol solution containing a cross-linking agent for cross-linking, washing, and freeze-drying to obtain a bone repair scaffold material.

[0022] Preferably, the washing in step (2) is performed with deionized water, and the cross-linking agent is EDC-NHS.

[0023] 1. The third object of the present invention is to provide the application of the bone repair scaffold material in the preparation of artificial bone repair, biomaterials, and medical devices.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention provides a mineralized collagen-polysaccharide bone repair scaffold material that mimics the composition, structure, and function of natural bones and forms an interconnected mesh structure with a pore size of approximately 135 μm and a porosity of approximately 82%;

[0026] (2) The mineralized collagen-polysaccharide bone repair scaffold material of the present invention has good biocompatibility and bioactivity, and can significantly promote the proliferation and adhesion of BMSCs cells; the expression level of osteogenic differentiation marker genes in the bone repair scaffold material is significantly increased, indicating that the bone repair scaffold material has good osteoinductivity in vitro;

[0027] (3) The mineralized collagen-polysaccharide bone repair scaffold material of the present invention was transplanted into a SD rat skull defect model. MRI and Micro-CT results showed that the bone repair scaffold material had good osteoinduction and osteoconductivity. H&E and Masson staining results showed that the bone repair scaffold material was completely degraded 12 weeks after surgery and could significantly promote bone tissue regeneration.

[0028] (4) The mineralized collagen-sodium alginate bone repair scaffold material provided by the present invention has excellent bone defect treatment effect and has great application potential in the field of bone tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Characterization of mineralized recombinant collagen

[0030] (a) XRD pattern of mineralized recombinant collagen; (b) TEM image of mineralized recombinant collagen (inset: selective area electron diffraction pattern (SAED)); (c) EDX spectrum; (d) HAADF-STEM image; (e) EDS mapping of C, O, P and Ca in mineralized recombinant collagen.

[0031] Figure 2 Characterization of mineralized recombinant collagen-sodium alginate scaffold for bone repair

[0032] (ad) SEM images and element distribution diagrams of recombinant collagen-sodium alginate bone repair scaffold material; (e) TGA image of recombinant collagen-sodium alginate bone repair scaffold material; (f) DTA image of recombinant collagen-sodium alginate bone repair scaffold material.

[0033] Figure 3 Micromorphology and in vitro degradation properties of scaffold materials

[0034] (a1-a2) RCHA; (b1-b2) MRCHPCL; (c1-c2) MCHA; (d1-d2) MRCHA. (a1-c1) Photographs of the scaffolds; (a2-c2) FESEM images of the scaffolds (inset: pore size distribution of the scaffolds); (e) Scaffold mass after in vitro degradation in PBS solution for different time periods; (f) pH of the degradation medium after in vitro degradation in PBS solution for different time periods.

[0035] Figure 4 Cell activity of scaffold materials

[0036] (a) Cytotoxicity of scaffold extracts; (b) CCK-8 assay for BMSC proliferation; (c) Live and dead cell staining of BMSCs after 1 and 4 days of culture on the scaffold; (d) CLSM images of BMSCs after 4 days of culture on the scaffold. (☆ indicates significant difference, p < 0.05).

[0037] Figure 5 In vitro osteogenic properties of scaffold materials

[0038] (a) ALP activity was detected using the pNPP assay when BMSC cells were cultured on the scaffold material for 1, 4, 7, and 11 days; (b) RT-qPCR was used to detect the expression of osteogenic markers by BMSC cells after 7 and 14 days of culture on the scaffold material; (f) ARS quantification; (g) ARS method was used to evaluate the osteogenic capacity of BSC cells; (☆ indicates significant difference, p < 0.05).

[0039] Figure 6 MRI examination of scaffold materials repairing critical-size skull defects in SD rats

[0040] Figure 7 Micro-CT examination of scaffold materials repairing critical-size skull defects in SD rats

[0041] (a) Coronal and sagittal scan images; (b) bone mineral density (BMD); (c) bone volume fraction; (d) trabecular number (☆ indicates significant difference, p < 0.05).

[0042] Figure 8 Histological staining of scaffold materials repairing critical-sized skull defects in SD rats

[0043] (a) H&E staining; (b) Masson staining. Specific implementation plan

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings. Obviously, the described embodiments are only part of the present invention, not the entire invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0045] The recombinant collagen described in the following embodiments is collagen obtained by using transgenic technology and gene recombination technology in an animal, plant or microbial expression system.

[0046] The recombinant collagen described in the following examples is obtained by fermentation with Escherichia coli, but is not limited to the above method and may also be recombinant collagen prepared by other methods, including recombinant collagen fermented with Pichia pastoris, recombinant source collagen, etc.

[0047] In the following embodiments, chemical cross-linking refers to the process in which macromolecular chains are linked together by chemical bonds under the action of light, heat, high-energy radiation, mechanical force, ultrasound, and cross-linking agents to form a network or body-shaped structure of the macromolecule.

[0048] Example 1. Preparation and characterization of mineralized recombinant collagen

[0049] 1. Preparation of Mineralized Recombinant Collagen

[0050] Prepare 1wt% recombinant collagen, 0.1336mol / L Ca 2+ and 0.08mol / LPO4 3- The mixed solution was adjusted to pH 7.4 with sodium hydroxide, the reaction system was stirred for 24 hours, and the mixture was allowed to stand in a 37°C water bath for 72 hours. The white colloid was collected by centrifugation, and the precipitate was washed three times with ultrapure water to remove soluble salts to obtain mineralized recombinant collagen (MRC).

[0051] 2. Preparation of Mineralized Animal Collagen

[0052] Prepare 1wt% yak collagen, 0.1336mol / L Ca 2+ and 0.08mol / LPO4 3- The mixed solution was adjusted to pH 7.4 with sodium hydroxide, the reaction system was stirred for 24 hours, and the mixture was allowed to stand in a 37°C water bath for 72 hours. The white colloid was collected by centrifugation, and the precipitate was washed three times with ultrapure water to remove soluble salts to obtain mineralized yak collagen (MC).

[0053] 3. Characterization

[0054] The mineralized recombinant collagen was characterized by powder X-ray polycrystalline diffraction (XRD), transmission electron microscopy (TEM), electron diffraction and energy dispersive X-ray.

[0055] The results are as follows Figure 1 As shown in Figure 2, a is the XRD spectrum of mineralized recombinant collagen, and the diffraction peaks at 2θ=25.52° and 31.95° correspond to the (002) and (211) crystal planes of hydroxyapatite (HA), indicating that nano-hydroxyapatite was successfully prepared using recombinant collagen as a biological template; b is a TEM image, and the mineralized collagen presents a good nanofiber structure; the related selective area electron diffraction (SAED) image shows the (211), (112) and (310) crystal planes of hydroxyapatite crystals, indicating that the nano-hydroxyapatite The arrangement of hydroxyapatite is very orderly. (c) Elemental mapping results from energy-dispersive X-ray spectroscopy (EDS) show the presence of C, O, P, and Ca, with C originating from recombinant collagen and O, P, and Ca from hydroxyapatite. (d) High-angle annular dark-field scanning TEM (HAADF-STEM) images show that the synthesized mineralized collagen has a well-defined fibrous structure. (e) Electron diffraction (EDX) images further demonstrate that C, O, P, and Ca are uniformly distributed within the mineralized collagen. These results demonstrate the successful preparation of a recombinant collagen-nanohydroxyapatite composite with well-defined fibrous morphology, i.e., mineralized collagen.

[0056] Example 2: Characterization of bone repair scaffold material MRCHA

[0057] 1. Preparation of MRCHA Bone Repair Scaffold

[0058] MRCHA: A 2% sodium alginate solution was mixed with 40% mineralized recombinant collagen. The mixture was homogenized using a laboratory high-shear emulsifier to form a uniform, viscous slurry. The slurry was then poured into a polytetrafluoroethylene mold, degassed under vacuum, pre-cooled at -20°C overnight, and freeze-dried. The freeze-dried sample was then cross-linked by immersing it in a 95% ethanol solution containing EDC-NHS. The scaffold was then washed with deionized water to remove excess cross-linker and cross-linking byproducts, and then freeze-dried.

[0059] 2. Internal structure of bone repair scaffold material MRCHA

[0060] The bone repair scaffold material MRCHA was characterized by scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS).

[0061] The results are as follows Figure 2 As shown in ad, a is the FESEM image of the spatial structure of the cross-section of the bone repair scaffold material, indicating that the interior of the bone repair scaffold material is a porous structure that is interconnected; c and d are EDS images of the bone repair scaffold material, indicating that the Ca / P ratio in the bone repair scaffold material is 1.67, which is the same as the Ca / P in hydroxyapatite in bones.

[0062] 3. Thermogravimetric Analysis of Bone Repair Scaffold Material MRCHA

[0063] The bone repair scaffold material MRCHA was characterized by thermogravimetric analysis.

[0064] The results are as follows Figure 2 As shown in ef, Figure 2 e is the TGA diagram of MRCHA, a bone repair scaffold material. Figure 2 f is the DTA graph of the scaffold material. Three temperature transition stages are observed from the DTA graph. The first stage is when the temperature is below 100°C, which is mainly due to the weight loss caused by the evaporation of H2O. Figure 2 Figure f shows that the weight loss of the bone repair scaffold material in this stage is 2-4%; the second weight loss stage occurs at 250-300 ° C. The weight loss in this stage is mainly caused by the thermal decomposition of the organic components (collagen and sodium alginate) in the bone repair scaffold material. Figure 2 f shows that its weight loss is 15-20%; the third temperature transition stage is 530℃, which is mainly caused by the loss of bound water in hydroxyapatite. Figure 2 Figure f shows that the content of hydroxyapatite is about 60-70%. The above results show that the content of organic components and inorganic components in the prepared bone repair scaffold material MRCHA is about 15-20% and 60-70%, respectively, which is a good biomimetic of the composition of natural bones.

[0065] Example 3: Synthesis of scaffold materials and characterization of micromorphology

[0066] 1. Preparation of Scaffold Materials

[0067] RCHA: A 2% sodium alginate solution was mixed with a 40% mixture of medical-grade hydroxyapatite and recombinant collagen. The mixture was homogenized using a laboratory high-shear homogenizer to form a uniform, viscous slurry. The slurry was poured into a polytetrafluoroethylene mold, degassed under vacuum, pre-cooled at -20°C overnight, and freeze-dried. The freeze-dried sample was then immersed in a 95% ethanol solution containing EDC-NHS for cross-linking. The scaffold was then washed with deionized water to remove excess cross-linker and cross-linking byproducts, and then freeze-dried.

[0068] MRCHPCL: A 2% polycaprolactone (PCL) solution was mixed with 40% mineralized recombinant collagen. The mixture was homogenized using a laboratory high-shear homogenizer to form a uniform, viscous slurry. The slurry was poured into a polytetrafluoroethylene mold, degassed under vacuum, pre-cooled at -20°C overnight, and freeze-dried. The freeze-dried sample was then cross-linked by immersing it in a 95% ethanol solution containing EDC-NHS. The scaffold was then washed with deionized water to remove excess cross-linker and cross-linking byproducts, and then freeze-dried.

[0069] MCHA: A 2% sodium alginate solution was mixed with 40% mineralized yak collagen. The mixture was homogenized using a laboratory high-shear homogenizer to form a uniform, viscous slurry. The slurry was poured into a polytetrafluoroethylene mold, degassed under vacuum, pre-cooled at -20°C overnight, and freeze-dried. The freeze-dried sample was cross-linked by immersing it in a 95% ethanol solution containing EDC-NHS. The scaffold was washed with deionized water to remove excess cross-linker and cross-linking byproducts, and then freeze-dried.

[0070] MRCHA: A 2% sodium alginate solution was mixed with 40% mineralized recombinant collagen. The mixture was homogenized using a laboratory high-shear emulsifier to form a uniform, viscous slurry. The slurry was then poured into a polytetrafluoroethylene mold, degassed under vacuum, pre-cooled at -20°C overnight, and freeze-dried. The freeze-dried sample was then cross-linked by immersing it in a 95% ethanol solution containing EDC-NHS. The scaffold was then washed with deionized water to remove excess cross-linker and cross-linking byproducts, and then freeze-dried.

[0071] 2. Micromorphology of scaffold materials

[0072] The uniformly mixed slurry was poured into a polytetrafluoroethylene circular mold and freeze-dried to prepare a cylinder with a diameter of 1.5 cm and a thickness of 1.2 cm.

[0073] Figure 3a1-d1 are photos of four stent materials: RCHA, MRCHPCL, MCHA, and MRCHA. The RCHA stent has visible hydroxyapatite particles on its surface that easily fall off, and the cylindrical structure exhibits some deformation. The MRCHPCL, MCHA, and MRCHA stents have smooth surfaces and well-maintained cylindrical structures.

[0074] 3. Micromorphology and pore size distribution of scaffold materials

[0075] The micromorphology of the scaffold material prepared above 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 in each group were randomly selected to describe the range and distribution of the pore size. The liquid replacement method was used to quantitatively measure the porosity of different scaffold materials. The mass and volume of the freeze-dried scaffold material were marked as m1 and V1, respectively. The scaffold material was infiltrated with ultrapure water under vacuum, and the wet weight of the material was marked as m2. The weight of water in the material was calculated and marked as m3 (m3=m2-m1). The pore volume of the scaffold The porosity of the scaffold is Each sample was assayed in triplicate, and the data were expressed as mean ± standard deviation (SD).

[0076] Figure 3 a2-d2 are FESEM images of the micromorphology of RCHA, MRCHPCL, MCHA and MRCHA scaffold materials.

[0077] in Figure 3 a2 is the FESEM image of the scaffold material RCHA, which shows that the scaffold material has no porous structure, the RCH particles are unevenly dispersed in the sodium alginate, and there is obvious agglomeration of RCH particles; the RCHA scaffold material is relatively loose and easily collapses when cut; the FESEM morphology of the RMCHPCL scaffold material is as follows Figure 3 As shown in b2, it presents an interconnected pore structure with a pore size distribution between 16-50 μm and a porosity of approximately 29.2±3.3%; Figure 3 c2-d2 are FESEM images of MCHA and MRCHA scaffold materials, showing interconnected porous structures with pore sizes ranging from 50-165 μm and 50-160 μm, respectively (see inset), and porosities of 82.3±3.4% and 82.6±2.2%, respectively; at the same time, MCH and MRCH nanoparticles are uniformly dispersed in sodium alginate.

[0078] 4. In vitro degradation of scaffold materials

[0079] The scaffold material was cut into a cylinder with a diameter of 12 mm and a height of 10 mm. After weighing (W0), it was immersed in 20 mL of 0.01 M PBS buffer solution with pH = 7.4 and placed in a 37°C shaker with a rotation speed of 60 r / min. During the entire 12W in vitro degradation experiment, the PBS buffer solution was replaced every week, and each degradation period was 2W. At the end of each degradation period, three samples were taken, washed thoroughly with distilled water, and vacuum freeze-dried (W t ) The degradation rate was expressed as the mass loss of the scaffold material loss = (W0-W t ) / W0x 100%. The pH value during the degradation process was also measured at the set time points.

[0080] The results are shown in Figure 3 e. RCHA, MRCHPCL, MCHA, and MRCHA scaffold materials can all be degraded, but the degradation rate of MCHA and MRCHA scaffold materials is significantly higher than that of RCHA and MRCHPCL scaffold materials; the remaining mass of RCHA, MRCHPCL, MCHA, and MRCHA after 12 weeks is 76.58 ± 3.09%, 85.27 ± 3.13%, 52.03 ± 4.67%, and 51.99 ± 3.23%, respectively. The results show that MRCHA and MCHA scaffold materials have an ideal degradation rate. The pH value of the degradation products during the degradation of the scaffold material was detected, and the results are shown in Figure 3 f. With the extension of the degradation time, the pH values of RCHA, MCHA, and MRCHA scaffold materials are always around 7.4, maintaining a physiological environment conducive to cell growth; while the pH value of MRCHPCL scaffold material is significantly reduced from 7.4 to 6.7, which is not conducive to cell growth.

[0081] These results show that MRCHA and MCHA scaffold materials have ideal micro-morphology, porous structure, and degradation performance.

[0082] Example Four, Cell Activity of Scaffold Material

[0083] 1. Cytotoxicity of Scaffold Material

[0084] The porous scaffold material was soaked in α-MEM medium for 1 and 4 days, and the leaching liquor was collected. The in vitro cytotoxicity of the scaffold material leaching liquor was evaluated by CCK-8 method. 100 μL of BMSCs cell suspension was added to each well at a concentration of 5x10 5Cells were plated at a density of 100 μL in a 96-well cell culture plate and cultured for 24 hours to allow attachment. Then, 100 μL of the extract of each scaffold material was added. An equal volume of α-MEM medium was added to the other wells as a control group. After 24 hours of culture, 10 μL of CCK-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazole monosodium salt) was added to each well, and the 96-well plate was incubated in a cell culture incubator for 2 hours. Absorbance at 450 nm was measured using a Tecan Infinite F200 / M200 multi-function microplate reader (Tecan, Mannedorf, Switzerland). Cell viability was calculated by dividing the mean absorbance of three measurements for each condition by the mean absorbance of the control group.

[0085] The results are as follows Figure 4 As shown in a, the extracts of RCHA, MRCHPCL and MRCHA scaffold materials immersed in α-MEM culture medium for 1 day and 4 days showed no cytotoxicity, indicating that the synthesized scaffold materials have high biocompatibility.

[0086] 2. Cell Proliferation on Scaffold Materials

[0087] The proliferation of BMSCs cells in the scaffold material was detected by CCK-8 method. BMSCs cells were plated at 5×10 5 Cells were seeded at a density of 100 μL in 96-well plates. Cell proliferation was observed on days 1, 4, 7, and 11. The medium was changed every two days. On the day of experiment, the medium was replaced with basal medium containing 10% (v / v) CCK-8 reagent and cultured for an additional 6 hours. The absorbance of 100 μL of supernatant was measured at 450 nm using a Tecan Infinite F200 / M200 multi-function microplate reader (Tecan, Mannedorf, Switzerland).

[0088] The results are as follows Figure 4 As shown in b, the CCK-8 assay results of the MRCHA scaffold material group showed that the absorbance value at 450 nm increased from 0.38 to 3.03; the results of the MRCHPCL scaffold material group showed that it increased from 0.38 to 2.62, and its cell proliferation effect was significantly higher than that of the RCHA (0.38 to 2.42) and blank groups (0.36 to 1.54).

[0089] Figure 4c is the live-dead cell staining experiment of the scaffold material. On the first day, there is no significant difference in the number of live and dead cells on the RCHA, MRCHPCL and MRCHA scaffold materials. At this time, the cells just adhere to the wall and are in the adaptation period. Four days after implanting the cells, there is a significant difference in the number of cells on the scaffold materials. The number of cells on the MRCHPCL and MRCHA scaffold materials is significantly more than that on the RCHA, indicating that the MRCHPCL and MRCHA scaffold materials can promote cell proliferation.

[0090] 3. Immunofluorescence experiment of the scaffold material

[0091] Place the thin slice of the scaffold material on the glass slide, and place the BMSCs cells on the scaffold material at a density of 400 cells / mm 2 Implant into the scaffold material, and incubate at 37°C for 24 hrs. Wash with PBS for three times to remove the unadhered cells. Then, fix the adhered cells with 4% paraformaldehyde for 10 min, and permeabilize with 0.1% Triton X-100 for 5 min, and then block with 1% bovine serum albumin (BSA) for 30 min. Incubate the cells with the phalloidin-tetramethylrhodamine isothiocyanate at 37°C for 1 hrs, and then add DAPI (Sigma-Aldrich) to incubate at 37°C for 10 min for cytoskeleton and nucleus staining, and obtain the images on the fluorescence microscope.

[0092] Figure 4 d is the CLSM image of the BMSCs cells co-cultured with the scaffold material for 4 days. The results show that on the RCHA scaffold material, the cells are spindle-shaped and polygonal, the diffusion area is small, and the number is small; on the MRCHPCL scaffold material, the number of cells is large, and the diffusion area is large; compared with the RCHA and MRCHPCL scaffold materials, the number of cells on the MRCHA scaffold material is the largest, and the diffusion area is the largest. These results show that the scaffold material can significantly promote the adhesion and diffusion of the BMSCs cells. The results of cell toxicity, proliferation and adhesion show that the MRCHA scaffold material has excellent biocompatibility and biological activity.

[0093] Example Five, in vitro osteogenic performance of the scaffold material

[0094] 1. ALP activity detection of the scaffold material

[0095] Implant the BMSCs cells into the scaffold material at a density of 1 x 10 5The density of the cells was seeded on the scaffold materials and cultured for 1 day, 4 days, 7 days and 11 days. The total protein and ALP activity of the cells were determined using BCA kit and ALP kit. The activity of ALP was normalized to the level of total protein according to the kit instruction. At each time point, the scaffold material with seeded cells was digested with trypsin for 3-4 min, and fetal bovine serum was added to stop the digestion. The cells adhered to the scaffold material were completely detached by slow blowing with a pipette, and the resulting liquid was transferred to an EP tube and centrifuged at 600 r / min for 8 min. The cells were collected. The cells were washed with PBS to remove trypsin. The cells were resuspended with PBS and lysed with cell lysis solution. The supernatant was centrifuged at 3000 rpm / min for 10 min to remove all insoluble debris. 50 μL of the sample was added to a 96-well plate, 50 μL of the matrix solution was added, and the plate was incubated at 37°C for 10 min. Then 100 μL of the termination solution was added, and the OD value of each well was determined at 405 nm.

[0096] The results are shown in Figure 2. Figure 5 As shown in Figure 2, the quantitative analysis results show that the ALP activity of the cells on the MRCHA scaffold material increased from 0.46 to 2.37, the ALP activity of the cells on the MRCHPCL scaffold material increased from 0.51 to 2.17, and the ALP activity of the cells on the RCHA scaffold material increased from 0.36 to 1.37. The results show that the ALP activity on the MRCHA scaffold material is significantly higher than that on the RCHA and MRCHPCL scaffold materials.

[0097] 2. Scaffold material specific osteogenic gene expression

[0098] BMSCs cells were seeded on the scaffold materials at a density of 1 x 10 5Cells were seeded at a density of 100 μg / mL in 24-well plates immobilized with scaffold material. After 7 and 14 days of culture, the relative expression of osteoblast-specific genes (Runx-2, ALP, OCN, Collagen I, and β-Actin) was 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 repeatedly pipetted to detach the cells. The supernatant was transferred to an EP tube and allowed to stand at room temperature for 5 minutes. 200 μL of chloroform was added and vortexed, and the tube was allowed to stand at room temperature for 5 minutes. The tube was centrifuged at 12,000 g and 4°C for 15 minutes. The supernatant was carefully transferred to a new tube, 500 μL of isopropanol was added, and the tube was mixed thoroughly. The tube was allowed to stand at room temperature for 10 minutes. The tube was centrifuged at 12,000 g and 4°C for 10 minutes. The supernatant was discarded, the pellet was collected, dried, and dissolved in DEPC-treated water for RNA concentration measurement. cDNA was obtained by reverse transcription, and real-time PCR reactions were performed using standard reaction conditions of the Thermal Cycler Dice Real Time System. After the reaction is completed, confirm the amplification curve and melting curve of Real Time PCR, and prepare a standard curve for PCR quantification. -ΔΔCt Calculate the expression level of each target gene relative to the previous time point. Verify primer amplification efficiency and quantitatively compare gene expression.

[0099] The results are as follows Figure 5 As shown in Figure 3, the expression levels of four osteogenesis-related genes, Runx-2, ALP, Col-I, and OCN, were significantly higher in MRCHA scaffolds than in RCHA and MRCHPCL scaffolds at 7 and 14 days. At 14 days, Runx-2 expression in MRCHA scaffolds reached 1.5-fold and 1.3-fold compared to RCHA and MRCHPCL scaffolds, respectively; ALP expression reached 1.8-fold and 1.6-fold; Col-I expression reached 2.3-fold and 2.1-fold; and OCN expression reached 1.7-fold and 1.5-fold compared to RCHA and MRCHPCL scaffolds, respectively.

[0100] 3. Cellular Mineralization of Scaffold Materials

[0101] BMSCs were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μg / ml in 24-well plates fixed with scaffold material and cultured in a 37°C, 5% CO2 incubator. 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 fixed with 4% paraformaldehyde for 30 minutes and washed three times with PBS buffer. Alizarin Red S staining solution was added and stained for 30 minutes at room temperature. The plates were then washed thoroughly with deionized water and observed under an inverted microscope.

[0102] The results are as follows Figure 5As shown in g, at 7 and 14 days, the number of red mineralized nodules increased in the RCHA scaffold group; the number of nodules in the MRCHPCL scaffold group was higher than that in the RCHA scaffold group, but all appeared as small mineralized spots; while in the MRCHA scaffold group, red, irregularly shaped mineralized nodules were observed, gradually increasing in size to form reddish-brown nodules. The mineralization capacity of BMSCs in the scaffold was quantitatively analyzed by measuring the absorption intensity at 560 nm. Figure 5 f. At 14 days, the MRCHA scaffold group had an absorption intensity of 0.825 at 560 nm, significantly higher than the RCHA (0.274) and MRCHPCL (0.496) scaffold groups. Higher absorption intensities indicate greater calcium matrix release, indicating higher mineralization capacity. These results demonstrate that the MRCHA scaffold has excellent mineralization-inducing ability and can significantly promote osteogenic differentiation.

[0103] Example 6: In vivo osteogenic properties of porous scaffold materials

[0104] 1. MRI Detection of Scaffold Materials in Repairing Critical-Size Skull Defects in SD Rats

[0105] Four, eight, and 12 weeks after surgery, rats were anesthetized with an intraperitoneal injection of 10% (w / v) sodium pentobarbital (0.3 mL / 100 g) before scanning to reduce respiratory movements. After anesthesia, the rats were placed in a supine position, with their heads and abdomens secured directly to the coil using belts. A Siemens MAGNETOM Skyro 3.0T coil with a 70 mm aperture and 8 channels was used for rats (Shanghai Chenguang Medical Technology, China).

[0106] MRI results such as Figure 6 As shown, 4-12 weeks after surgery, the tissue imaging of the defect area in the blank group showed almost no change, and no obvious high-density imaging appeared in the bone wound area, indicating that large-sized skull defects cannot heal themselves; during the repair period of 4-12 weeks after implantation of RCHA, MRCHPCL and MRCHA scaffold materials, the defect area had increased density imaging, indicating the formation of new bone tissue; in the MRCHA scaffold material group, the tissue imaging of the bone defect area was consistent with the surrounding bone, and the regenerated area was almost completely fused with the peripheral bone boundary, indicating that the amount of mature regenerated bone increased significantly.

[0107] 2. Micro-CT Detection of Scaffold Materials in Repairing Critical-Sized Skull Defects in SD Rats

[0108] At 4, 8, and 12 weeks after surgery, the animals were euthanized under general anesthesia according to the previous grouping, and the skulls were collected and fixed in 4% paraformaldehyde solution. The morphology of the reconstructed skull was evaluated using an animal micro-CT scanner (Bruker Skyscan 1176, USA) in high-resolution scanning mode.

[0109] Micro-CT results Figure 7 As shown in the figure, the blank group had a small amount of bone formation in the critical size defect area at 8 and 12 weeks after surgery. Similar results were observed in the RCHA material group, that is, a small amount of new bone formation was observed at the defect edge. At 12 weeks, the MRCHA scaffold material group showed more newly formed bone tissue than the other three groups. Gaps of different sizes were observed from the sagittal scan images, among which the gaps in the MRCHA scaffold material group were the smallest. Quantitative morphological analysis was performed using a Micro-CT analysis system, and the results are shown in the figure. Figure 7 As shown in Figures bd. Bone mineral density (BMD) increased over time in all groups. At 12 weeks postoperatively, BMD in the MRCHA group (0.1585±0.0195 g / cc) was significantly higher than in the RCHA group (0.0839±0.0107 g / cc), the MRCHPCL group (0.11069±0.02813 g / cc), and the control group (0.0125±0.00762 g / cc) (P<0.05). Furthermore, bone volume fraction (BV / TV) in the MRCHA group (89.01±10.67%) was significantly higher than in the RCHA group (52.71±8.28%), the MRCHPCL group (66.07±11.25%), and the control group (27.46±5.33%) (P<0.05). Trabecular bone number (TbN) showed the same trend as BV / TV. These results indicate that MRCHA scaffolds can significantly enhance bone tissue regeneration.

[0110] 3. Histological Staining of Scaffold Materials for Repair of Critical-Sized Calvarial Defects in SD Rats

[0111] Four, eight, and 12 weeks after surgery, rats were anesthetized and sacrificed according to the pre-defined groupings. Skull specimens were collected for histopathological analysis. The skulls and maxillae were removed, and the skin and brain tissue were removed. Bone defects and soft tissue were excised using a razor blade. Tissue specimens were fixed in 4% paraformaldehyde for 24 hours. The specimens were rinsed with PBS and decalcified in a decalcifying solution. Decalcification was regularly checked, and complete decalcification was confirmed when the needle could penetrate the bone without resistance. Completely decalcified specimens were dehydrated in a gradient of 50%, 75%, 95%, and 100% ethanol, twice for 10 minutes each. The dehydrated tissues were embedded in paraffin and sliced ​​into 5-μm sections using a paraffin sectioner. Paraffin sections were stained with hematoxylin & eosin and Masson staining, respectively, and examined under a microscope.

[0112] H&E tissue staining results Figure 8As shown in Fig. 4a, a large amount of fibrous tissue was formed in the blank group, and only a small amount of pink-stained structure was observed 4-12 weeks after the operation. The bone tissue in the RCHA scaffold material group was slightly more than that in the blank group. In the MRCHPCL material group, obvious undegraded material was observed, which hindered the formation of new bone tissue. In the MRCHA scaffold material group, bone-like tissue was observed in the dura mater adjacent area and the center of the scaffold, and obvious bone trabeculae were observed. The MRCHA scaffold material was almost completely degraded and replaced by regenerated mature bone tissue, which had similar morphology to natural bone tissue.

[0113] The results of the cross-sectional Masson trichrome staining images of the skull are shown in Fig. 4b. Figure 8 As shown in Fig. 4b, the immature primary bone is blue, and the mature primary bone is red. The newly formed bone tissue in all groups gradually increased over time, and the newly formed bone tissue observed in the MRCHA scaffold material group was significantly more than that in the other groups. The H&E staining and Masson staining results show that the MRCHA scaffold material has excellent osteogenic activity and can significantly promote bone tissue regeneration.

[0114] In summary, the present application provides a mineralized collagen-polysaccharide bone repair scaffold material that well mimics the composition, structure and function of natural bone and forms a network structure that is interconnected; the mineralized collagen-polysaccharide bone repair scaffold material has good biocompatibility and bioactivity, can significantly promote the proliferation, adhesion and differentiation of BMSCs cells, and has good bone inductivity in vitro; after the mineralized collagen-polysaccharide bone repair scaffold material is transplanted into the SD rat skull defect model, the MRI and Micro-CT results show that it has good osteogenic induction and bone conduction; the H&E and Masson staining results show that it is completely degraded 12 weeks after the operation and can significantly promote bone tissue regeneration; the mineralized collagen-sodium alginate bone repair scaffold material provided by the present application has excellent bone defect treatment effect and has great application potential in the field of bone tissue engineering.

Claims

1. A mineralized collagen-polysaccharide bone repair scaffold material, characterized in that: The bone repair scaffold material is obtained by uniformly mixing mineralized collagen and polysaccharide and then chemically cross-linking. The concentration ratio of the mineralized collagen and polysaccharide is 40wt%:2wt%, and the balance is water. The polysaccharide is sodium alginate. The mineralized collagen is obtained by reacting 1wt% collagen, 0.1336mol / L calcium salt and 0.08mol / L phosphate. The collagen is recombinant collagen and / or animal collagen. The animal collagen includes one or more of type I, type II and type III. The calcium salt is anhydrous calcium chloride solution, calcium gluconate solution, calcium dihydrogen phosphate solution, calcium nitrate solution or calcium bicarbonate solution; the phosphate is disodium hydrogen phosphate solution, sodium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution or ammonium dihydrogen phosphate solution. The preparation method of the mineralized collagen is as follows: (1) preparing an aqueous solution of a calcium salt solution and a collagen solution in proportion, mixing and reacting for 10-120 minutes, and then adding a phosphate solution dropwise in proportion to obtain a mixed solution; (2) adjusting the pH of the mixed solution in step (1), stirring the mixture, allowing it to stand in a water bath, centrifuging it, and washing it to obtain mineralized collagen as a white colloid; In step (2), the pH is 7.4, the reaction is stirred for 12-36 hours, and the mixture is kept at a constant temperature of 37° C. for 12-96 hours.

2. The method for preparing the bone repair scaffold material according to claim 1, wherein: The following steps are involved: (1) Mixing mineralized collagen and polysaccharide solution in proportion, homogenizing, injection molding, vacuum degassing, precooling, and freeze drying; (2) Immersing the material prepared in step (1) in an ethanol solution containing a cross-linking agent for cross-linking, washing, and freeze-drying to obtain a bone repair scaffold material.

3. The preparation method according to claim 2, wherein The precooling in step (1) is precooling at -20°C overnight; the washing in step (2) uses deionized water, and the crosslinking agent is EDC-NHS.

4. Use of the bone repair scaffold material according to claim 1 in the preparation of biomaterials and medical devices.

Images