Polyelectrolyte uniformly modified fiber intra-mineralized scaffold material and preparation method and application thereof

Abstract

The present invention discloses a polyelectrolyte uniformly modified intra-fiber mineralized scaffold material, a preparation method and an application thereof, and belongs to the field of biomaterial technology. The preparation method of the present invention comprises: mixing a collagen solution and a polyelectrolyte solution, reacting to precipitate flocs; dripping an acid into the mixed solution to dissolve the flocs; placing the redissolved mixed solution under buffer solution conditions to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation; then freeze-drying, cross-linking, secondary freeze-drying, mineralization, and tertiary freeze-drying to obtain a polyelectrolyte uniformly modified intra-fiber mineralized scaffold material. The present invention discloses an intra-fiber mineralized scaffold material obtained by the above-mentioned preparation method, and the application of the intra-fiber mineralized scaffold material in the preparation of materials for repairing bone defects. The present invention creatively achieves collagen fiber reorganization and polyelectrolyte complexation simultaneously, efficiently and uniformly binds polyelectrolytes to the surface of collagen fibers, and constructs a collagen biomimetic scaffold material with good intra-fiber mineralization characteristics.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and particularly relates to a polyelectrolyte uniformly modified fiber intra-mineralized scaffold material, a preparation method and an application thereof. Background Art

[0002] The repair of bone defects is a difficult problem in clinical treatment. At present, autologous bone transplantation is still the gold standard for bone defect repair, but there are problems such as limited bone volume in the donor area and secondary trauma. As an alternative, allogeneic bone transplantation and xenogeneic bone transplantation have other risks such as immune rejection and disease transmission. Since the 19th century, traditional artificial bone transplantation materials such as alloys, ceramics, and polymers have been used for bone defect repair, but their main mechanism is space occupation and mechanical support, and their bone induction, bone conduction, and bone integration capabilities are insufficient. On this basis, bone tissue engineering has gradually developed into a research hotspot for bone defect repair. It aims to repair and reconstruct bone tissue through the development of biological functional tissues. It is one of the most promising ways to solve the problem of bone defect repair.

[0003] Intrafiber mineralization is the most important characteristic of mineralized collagen in natural bone tissue. Therefore, developing biomimetic scaffolds based on intrafiber-mineralized collagen is a hot research topic in bone tissue engineering. However, intrafiber mineralization of collagen scaffolds is often difficult to achieve and exhibits insufficient mineralization. Polyelectrolytes, with their anionic properties and calcium ion affinity, promote intrafiber mineralization in collagen and hold promise as a solution to these problems. However, efficient and uniform incorporation of polyelectrolytes onto the collagen fiber surface remains a challenge facing existing methods for constructing biomimetic intrafiber-mineralized collagen scaffolds. Polyelectrolyte complexation methods utilize the spontaneous complexation and precipitation of oppositely charged polyelectrolytes upon mixing to prepare scaffolds. These methods offer simple, reproducible, and high polyelectrolyte binding capacity. However, due to the precipitation of collagen from the solution during the complexation process, the preparation of collagen scaffolds with recombinant properties is not feasible. Chemical crosslinking methods also utilize chemical crosslinkers to crosslink polyelectrolytes onto the collagen surface, offering broad applicability and structural stability. However, these methods suffer from low and uneven polyelectrolyte binding capacity and are susceptible to steric hindrance within the three-dimensional collagen scaffold.

[0004] Therefore, providing an intrafiber mineralized scaffold material that can efficiently and evenly bind polyelectrolytes to the surface of collagen fibers has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a method for preparing a polyelectrolyte uniformly modified intrafibrous mineralized scaffold material. The method is simple and easy to operate. The intrafibrous mineralized scaffold material prepared by this method is closer to the hierarchical structure characteristics of natural bone tissue than traditional co-deposited mineralized collagen scaffold materials and has better bone defect repair effect.

[0006] The second object of the present invention is to provide an intra-fiber mineralized scaffold material obtained by the preparation method.

[0007] The third object of the present invention is to provide an application of an intra-fiber mineralized scaffold material in the preparation of a material for repairing bone defects.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The present invention discloses a method for preparing a polyelectrolyte uniformly modified fiber intra-mineralized scaffold material, comprising the following steps:

[0010] S1. Reaction: Mixing the collagen solution and the polyelectrolyte solution to react and precipitate floccules;

[0011] S2. Redissolve: Then add acid dropwise to the mixed solution to dissolve the flocculent to obtain a mixed solution after re-dissolution;

[0012] S3. Reconstitution and doping: The reconstituted mixed solution is placed in a buffer solution to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation;

[0013] S4 freeze-drying: The recombinant and doped solution in step S3 is freeze-dried to obtain a polyelectrolyte uniformly modified recombinant collagen scaffold material;

[0014] S5. Cross-linking: placing the polyelectrolyte-modified recombinant collagen scaffold material in a cross-linking solution for cross-linking reaction;

[0015] S6. Secondary freeze-drying: freeze-drying the scaffold material after the cross-linking reaction;

[0016] S7. Mineralization: Placing the secondary freeze-dried scaffold material in a mineralization solution for mineralization reaction;

[0017] S8. three freeze-drying steps: freeze-drying the scaffold material after the mineralization reaction to obtain a mineralized scaffold material uniformly modified with polyelectrolyte in the fiber.

[0018] In some embodiments of the present invention, the collagen comprises type I collagen, and the polyelectrolyte comprises chondroitin sulfate A;

[0019] Preferably, the collagen solution and the polyelectrolyte solution are prepared separately using acetic acid solution;

[0020] The concentration of the acetic acid solution is preferably 0.1 to 1 M, more preferably 0.5 M;

[0021] The concentration of the collagen solution is preferably 1-20 mg / mL, more preferably 10 mg / mL;

[0022] The concentration of the polyelectrolyte solution is preferably 0.01-0.5 mg / mL, more preferably 0.1 mg / mL;

[0023] Preferably, the mass of collagen and polyelectrolyte is 1 to 10% of the mass of collagen;

[0024] Preferably, in step S1, the reaction is carried out at room temperature;

[0025] Preferably, the reaction time is 5 to 30 minutes.

[0026] In some embodiments of the present invention, the acid in step S2 comprises hydrochloric acid.

[0027] In some embodiments of the present invention, in step S3, the reconstituted solution is placed in a dialysis bag, which is then immersed in a buffer solution to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation;

[0028] Preferably, the molecular weight cut-off of the dialysis bag is greater than or equal to 1000;

[0029] Preferably, the dialysis bag is immersed in a buffer solution and placed at room temperature for 2-48 hours, preferably 24 hours, to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation;

[0030] Preferably, an alkaline substance is added to the buffer solution, and the amount of the alkaline substance is such that it can neutralize the acid added dropwise in step S2, so that the collagen can be reconstituted under neutral to weakly alkaline conditions;

[0031] Preferably, the base includes sodium hydroxide or potassium oxide.

[0032] In some embodiments of the present invention, in step S4, the recombined and doped solution is placed in a mold of a certain shape and freeze-dried to obtain a scaffold material.

[0033] In some embodiments of the present invention, the cross-linking solution comprises an EDC / NHS cross-linking solution;

[0034] The concentration of EDC in the cross-linking solution is preferably 0.01-0.04 mM, more preferably 0.02 mM;

[0035] The concentration of NHS in the cross-linking solution is preferably 0.006-0.024 mM, more preferably 0.012 mM.

[0036] In some embodiments of the present invention, in step S5, the cross-linking reaction is carried out at room temperature;

[0037] Preferably, the cross-linking solution is used in an amount that at least completely immerses the scaffold material.

[0038] In some embodiments of the present invention, the mineralization solution is prepared by mixing calcium mother liquor, PAA mother liquor, and phosphorus mother liquor in a volume ratio of 1:0.5-2:0.5-2; preferably 1:1:2;

[0039] The calcium mother solution is a calcium salt solution, preferably a calcium chloride solution, with a concentration of 3-12 mM, preferably 6-7 mM;

[0040] The PAA mother solution is obtained by dissolving PAA powder in deionized water; the concentration of PAA in the mineralization solution is 12.5-200 μg / mL, more preferably 100 μg / mL;

[0041] The phosphorus mother solution is prepared by dissolving NaH2PO4 in PBS buffer; the concentration of Na2HPO4 in the mineralization solution is 0.5-2mM, preferably 1mM; the concentration of PBS in the mineralization solution is 0.5-1×PBS, preferably 0.85×PBS.

[0042] In some embodiments of the present invention, in step S7, the mineralization reaction is carried out at room temperature;

[0043] The mineralization time is preferably 1-10 days, more preferably 3-7 days, and even more preferably 7 days;

[0044] The amount of mineralizing liquid used is at least enough to completely immerse the scaffold material.

[0045] In some embodiments of the present invention, in step S4, the reconstituted and doped solution is placed in a mold of a certain shape, first frozen at -20°C overnight, then frozen at -80°C for 12-48 hours, preferably 24 hours, and finally freeze-dried;

[0046] In step S6, the scaffold material after the cross-linking reaction is first washed with deionized water to remove the residual cross-linking agent, and then freeze-dried;

[0047] In step S7, the scaffold material after the mineralization reaction is first washed with deionized water to remove the residual mineralization liquid, and then freeze-dried.

[0048] Preferably, the scaffold material after washing away the residual cross-linking agent or mineralization solution is immersed in deionized water, first frozen at -20°C overnight, then frozen at -80°C for 12-48 hours, preferably 24 hours, and finally freeze-dried.

[0049] The invention discloses an intra-fiber mineralized scaffold material prepared by the preparation method.

[0050] The invention discloses an application of the intra-fiber mineralized scaffold material in preparing a material for repairing bone defects.

[0051] The Chinese names corresponding to the English and Chinese versions of this invention are:

[0052] COL1: type I collagen; CS-A: chondroitin sulfate A; rCOL1: recombinant type I collagen fibrils; FTIR: Fourier transform infrared spectroscopy; SEM: scanning electron microscopy; TEM: transmission electron microscopy; XRD: X-ray diffraction; TGA: thermogravimetric analysis; rBMSCs: rat bone marrow mesenchymal stem cells; HE: hematoxylin-eosin staining; PAA: polyacrylic acid; ACP: amorphous calcium phosphate; BMSCs: bone marrow mesenchymal stem cells; HAP: hydroxyapatite; Gly: glycine; CS: chondroitin sulfate; DMMB: dimethylmethylene blue; MES: 4-morpholinoethanesulfonic acid; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide; β-GB: sodium β-glycerophosphate; DM: dexamethasone; Vc: ascorbic acid.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] The present invention is scientifically designed and ingeniously conceived. It creatively and simultaneously realizes collagen fiber reorganization and polyelectrolyte complexation, efficiently and evenly binds polyelectrolytes to the surface of collagen fibers, and constructs a collagen biomimetic scaffold material with good intrafiber mineralization characteristics.

[0055] The method described in this invention, used to construct recombinant collagen scaffolds, boasts comparable polyelectrolyte utilization to polyelectrolyte complexation methods. Furthermore, the polyelectrolyte is evenly distributed along the collagen fibers, enabling the incorporation of polyelectrolytes into the scaffold at a predetermined concentration. This uniform incorporation of polyelectrolytes provides additional anionic properties and calcium ion affinity to the collagen fiber surface, promoting high intrafiber mineralization of the collagen scaffold.

[0056] The polyelectrolyte-modified intrafibrous mineralized scaffold prepared by this method also exhibits significant advantages over traditional co-deposited mineralized collagen scaffolds, more closely resembling the hierarchical structure of natural bone tissue and demonstrating improved bone defect repair efficacy. Animal studies have demonstrated that the intrafibrous mineralized scaffold prepared by this method exhibits excellent safety and is effective in repairing bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This diagram illustrates the polyelectrolyte complexation process and the reconstitution phenomenon caused by pH adjustment. The leftmost vial contains a collagen solution; the middle vial contains a mixed solution of collagen and polyelectrolyte solutions, with a white complex precipitated; and the rightmost vial contains the mixed solution after acid addition and reconstitution.

[0058] Figure 2 The turbidity change curve of different concentrations of PAA within 7 days.

[0059] Figure 3TEM observation images of turbidity measurement of PAA at different concentrations, where b, c, and d are the mineral morphologies in the solution after 5 min, 24 h, and 7 d of reaction with the mineralization solution without PAA, respectively; e, f, and g are the mineral morphologies in the solution after 5 min, 24 h, and 7 d of reaction with the mineralization solution containing 100 μg / mL PAA, respectively (the white target indicates the diffraction position).

[0060] Figure 4 The following are transmission electron micrographs and elemental scanning images of recombinant collagen scaffolds uniformly modified with different concentrations of polyelectrolytes. The pure group is a recombinant collagen scaffold without CS-A, while the 1%, 5%, and 10% groups are recombinant collagen scaffolds containing corresponding concentrations of CS-A. Figure 4 In the figure, each row from left to right is a low-magnification image of recombinant collagen fibers, a high-magnification image of recombinant collagen fibers, a bright-field image of recombinant collagen fibers, N element distribution, O element distribution, and S element distribution.

[0061] Figure 5 Appearance images of CS-A / COL1 blended mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 intrafiber mineralized scaffolds; (a) Cs-A / COL1 blended mineralized scaffold; (b) 1% CS-A / rCOL1 intrafiber mineralized scaffold; (c) 5% CS-A / rCOL1 intrafiber mineralized scaffold; (d) 10% CS-A / rCOL1 intrafiber mineralized scaffold.

[0062] Figure 6 SEM morphologies of CS-A / COL1 blended mineralized scaffolds and 1% CS-A / rCOL1 intra-fiber mineralized scaffolds at 3d and 7d of mineralization, at low magnification (×200) and high magnification (×200000);

[0063] Figure 7 The TEM morphology and elemental scans of the mineralized scaffolds within the 1%, 5% and 10% CS-A / rCOL1 fibers after 7 days of mineralization are shown. The first row of each group, from left to right, shows the mineralized collagen fibers within the fibers, selected area electron diffraction positions, bright field imaging, overall elemental distribution map, selected area electron diffraction pattern, and elemental distribution maps of N, O, Ca, P, and S.

[0064] The second row of each group, from left to right, is the elemental surface scans of N (dark blue), O (yellow), Ca (green), P (red), and S (light blue); the white circles indicate the diffraction positions, low magnification (×8000) and high magnification (×40000).

[0065] Figure 8 FTIR images of CS-A / COL1 blended mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 intra-fiber mineralized scaffolds after 7 days of mineralization.

[0066] Figure 9 XRD patterns of CS-A / COL1 blended mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 fiber mineralized scaffolds after 7 days of mineralization.

[0067] Figure 10 Figure 3 shows the TGA results of CS-A / COL1 co-mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 intrafiber mineralized scaffolds after 7 days of mineralization. (a) CS-A / COL1 co-mineralized scaffold; (b) 1% CS-A / rCOL1 intrafiber mineralized scaffold; (c) 5% CS-A / rCOL1 intrafiber mineralized scaffold; and (d) 10% CS-A / rCOL1 intrafiber mineralized scaffold.

[0068] Figure 11 This is the Alizarin Red staining image of Experiment 3, where the lower image is a partial enlarged image of the upper image.

[0069] Figure 12 Figure 3. Evaluation of the osteogenic differentiation ability of rBMSCs at the late stage (14 days) by Alizarin red staining. At the same time point, there is no statistical difference between the same letters, but there is a statistical difference between different letters (α=0.05).

[0070] Figure 13 This is the HE staining image of the heart, liver, spleen, lung, and kidney of a rat 3 months after surgery. The English letters in the image correspond to the Chinese characters: heart: heart; liver: liver; spleen: spleen; lung: lung; kidney: kidney.

[0071] Figure 14 This is a Micro-CT three-dimensional reconstruction image 3 months after surgery; 3M in the figure means 3 months; full view represents the panoramic view of the sample, control represents the blank control bone defect without implantation of material, and material represents the bone defect implanted with the corresponding scaffold material.

[0072] Figure 15 Statistical analysis at 3 months after surgery. **** indicates P < 0.0001. Symbols with the same letter indicate no statistical difference; symbols with different letters indicate statistical difference (α = 0.05). Control represents a blank bone defect without implantation of a material; Material represents a bone defect with implantation of the corresponding scaffold material.

[0073] Figure 16Results of CS-A utilization measurements for three scaffold construction methods. (a) CS-A utilization in the polyelectrolyte complexation method; (b) CS-A utilization in the chemical cross-linking method; (c) CS-A utilization in the modified polyelectrolyte complexation method; (d) CS-A mass per unit mass of COL1 in the polyelectrolyte complexation method; (e) CS-A mass per unit mass of COL1 in the chemical cross-linking method; (f) CS-A mass per unit mass of COL1 in the modified polyelectrolyte complexation method.

[0074] In the figure, blend refers to CS-A / COL1 blended mineralized scaffold, 1%, 5%, and 10% refer to 1% CS-A / rCOL1 intrafiber mineralized scaffold, 5% CS-A / rCOL1 intrafiber mineralized scaffold, and 10% CS-A / rCOL1 intrafiber mineralized scaffold, respectively. DETAILED DESCRIPTION

[0075] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0076] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0077] Example 1

[0078] This embodiment discloses a method for preparing a polyelectrolyte-uniformly modified fiber intra-mineralized scaffold material of the present invention, specifically:

[0079] 1. Prepare collagen solution and polyelectrolyte solution: Use 0.5 M acetic acid to prepare 10 mg / mL collagen (COL1) solution and 0.1 mg / mL chondroitin sulfate A (CS-A) solution, respectively.

[0080] 2. Reaction and redissolution: Mix 20 mL of COL1 solution and 20 mL of CS-A solution in a 50 mL centrifuge tube and let it stand. A white flocculent complex will precipitate in the tube. Then slowly add HCl dropwise to the tube until the complex is completely dissolved. Transfer the liquid to a dialysis bag (molecular weight cut-off 1000). The polyelectrolyte complexation process and the redissolution phenomenon caused by pH adjustment in this step are shown in the attached figure. Figure 1 shown.

[0081] 3. Recombination and doping: The dialysis bag is immersed in 2L of 2×PBS buffer solution and placed at room temperature for 24 hours to simultaneously achieve COL1 recombinant (rCOL) and CS-A doping; sodium hydroxide is added to the 2×PBS buffer solution to neutralize the hydrochloric acid added dropwise in step 2. In the present invention, collagen recombinant needs to be in a neutral or weakly alkaline environment. Hydrochloric acid is used in the process of adjusting the pH to redissolve the complex, so it is necessary to add the corresponding sodium hydroxide to the buffer system. Since sodium hydroxide is added to the buffer system and not directly added to the inside of the dialysis bag, no precipitation will be produced. As the dialysis process proceeds, the pH in the dialysis bag gradually becomes neutral, and the collagen gradually achieves self-assembly.

[0082] 4. Lyophilization: Use a sterile plastic pipette to blow the contents of the dialysis bag evenly and transfer them to a 24-well plate with 2 mL per well. Then, freeze the plate in a -20°C refrigerator overnight, then place it in a -80°C refrigerator for 24 hours, and finally lyophilize to obtain a 1% CS-A / rCOL1 scaffold.

[0083] 5. Preparation of EDC / NHS chemical crosslinking solution: First, use 80% ethanol to prepare a 0.02 mM EDC solution, then add NHS powder to the solution until the NHS concentration in the solution is 0.012 mM after dissolution, and let it stand to obtain the EDC / NHS chemical crosslinking solution.

[0084] 6. Cross-linking: Add 12 mL of EDC / NHS chemical cross-linking solution to a 6-well plate in advance, then transfer the lyophilized 1% CS-A / rCOL1 scaffolds from step 4 to the 6-well plate, 3 per well, and let stand at room temperature for 24 hours to complete cross-linking.

[0085] 7. Secondary Lyophilization: Rinse the cross-linked scaffold repeatedly with deionized water to remove residual crosslinker. Place the rinsed scaffold back into a new 24-well plate. Add deionized water to each well to submerge the scaffold. Freeze the plate overnight at -20°C, then at -80°C for 24 hours. Finally, freeze the plate to obtain a 1% CS-A / rCOL1 scaffold.

[0086] 8. Prepare the mineralization solution: First, mix the calcium and PAA mother solutions, then add the phosphorus mother solution and mix thoroughly to obtain the mineralization solution. The volume ratio of calcium, PAA, and phosphorus mother solutions is 1:1:2. The mineralization solution contains 1.67 mM CaCl2, 1 mM Na2HPO4, 0.85× PBS (8.5 mM phosphate, 131.7 mM NaCl), and 100 μg / mL PAA.

[0087] The phosphorus mother solution was prepared as follows: first, 10× PBS buffer was prepared and its pH was adjusted to 7.7. 85 mL of the pH-adjusted PBS buffer was diluted to 500 mL with deionized water, and then 0.14196 g of anhydrous Na₂PO₄ powder was added and stirred to dissolve, thereby preparing the phosphorus mother solution.

[0088] The calcium mother solution was prepared as follows: 370.6732 mg of anhydrous CaCl2 was weighed and dissolved in 500 mL of deionized water to obtain a 6.68 mM CaCl2 solution;

[0089] The preparation method of PAA mother solutions of different concentrations is as follows: PAA powder of corresponding content is dissolved in deionized water to obtain a 400 μg / mL PAA solution.

[0090] 9. Mineralization: Use a 6-well plate as a mineralization container. Add 10 mL of the mineralization solution prepared in step 8 to each well. Immerse the 1% CS-A / rCOL1 scaffolds constructed in step 7 in the mineralization solution. Place one cylindrical scaffold in each well. Mineralize the plates in a 37°C incubator for 7 days, replacing the mineralization solution daily.

[0091] 10. Triple freeze-drying: After mineralization, rinse the scaffold repeatedly with deionized water. Place the cleaned cylindrical scaffold material in a 24-well plate. Add deionized water to each well to submerge the scaffold material. Freeze the 24-well plate in a -20°C refrigerator overnight, then place it in a -80°C refrigerator for 24 hours, and finally freeze-dry to obtain a 1% CS-A / rCOL1 intrafiber mineralized scaffold.

[0092] Example 2

[0093] Compared with Example 1, the concentration of the chondroitin sulfate A (CS-A) solution in step 1 was 0.5 mg / mL, and the other conditions were the same. In this example, a 5% CS-A / rCOL1 intrafibrous mineralized scaffold was prepared.

[0094] Example 3

[0095] Compared with Example 1, the concentration of the chondroitin sulfate A (CS-A) solution in step 1 was 1 mg / mL, and the other conditions were the same. In this example, a 10% CS-A / rCOL1 intrafibrous mineralized scaffold was prepared.

[0096] Example 4

[0097] This example discloses a test to investigate the concentration of PAA in the mineralization solution of the present invention.

[0098] 1. Prepare mineralization solution:

[0099] 1.1 Preparation of phosphorus mother solution and calcium mother solution: prepare phosphorus mother solution and calcium mother solution respectively according to the method of Example 1;

[0100] 1.2 Preparation of PAA mother solutions of different concentrations: PAA powder of corresponding contents was dissolved in deionized water to obtain PAA solutions of 50, 100, 200, 400, and 800 μg / mL, respectively.

[0101] 2. Turbidity measurement experiment

[0102] A 96-well plate was used as the reaction vessel, with three replicate wells per concentration. Using a dispenser, 50 μL of calcium stock solution was first added to each well. Then, 50 μL of PAA stock solution of varying concentrations was added to each well according to grouping (50 μL of deionized water was added to the blank control). After 5 minutes of stagnation, 100 μL of phosphorus stock solution was added to each well to obtain mineralization solutions containing different PAA concentrations (0, 12.5, 25, 50, 100, and 200 μg / mL). Subsequently, 50 μL of sterile mineral oil was added to all wells to prevent water evaporation during incubation. The 96-well plate was then quickly transferred to a multi-functional microplate reader. The absorbance of each well at 550 nm was measured after a 5-minute incubation at 37°C. The instrument was then set to automatically shake and measure absorbance every 10 minutes for a total of 9 hours. The plate was then transferred to a 37°C incubator, and absorbance was measured daily until day 7. This experiment was repeated three times.

[0103] The turbidity test results are shown in the attached Figure 2 As shown, the blank group mineralization solution without PAA ( Figure 2 The turbidity change trend of the control group in the mineralization solution is as follows: after the calcium and phosphate solutions are mixed, the turbidity suddenly rises to a peak value, and then the turbidity suddenly drops, and then the turbidity slowly increases and finally remains relatively constant. The whole process can be divided into three stages: the first stage (the initial turbidity sudden rise stage) is the stage of cluster and ACP formation and accumulation before nucleation; the second stage (the turbidity sudden drop stage) is the stage when the ACP concentration accumulates to a certain level, and then it quickly fuses and crystallizes to form HAP and precipitates; the third stage (the final turbidity slowly rises and finally remains relatively constant) is the stage when HAP continues to grow and mature. With the gradual increase of the PAA concentration in the mineralization solution, the peak values ​​of the turbidity of the first and third stages of the mineralization solution gradually decrease, and the turbidity sudden drop characteristics of the second stage gradually weaken until they disappear, indicating that with the increase of PAA concentration, the stabilizing effect on the ACP formed in the first stage solution gradually increases. When the stabilizing effect reaches a certain threshold, the ACP in the solution no longer converts into HAP but remains in the ACP state for a long time. From Figure 2 It can be seen from the graph that when the PAA concentration reaches 100 μg / mL, the peak value of the turbidity of the mineralized solution no longer decreases with the increase of the PAA concentration, indicating that for the calcium and phosphorus concentrations of the mineralized solution in the experiment, 100 μg / mL of PAA can completely stabilize ACP within the observation time.

[0104] 3.TEM and selected area electron diffraction

[0105] A mineralization solution containing 100 μg / mL PAA was used as the experimental group, and a mineralization solution without PAA was used as the control group. After incubation in the mineralization solution for 5 minutes, 24 hours, and 7 days, a small amount of liquid was drawn from the mineralization solution and dropped onto a copper grid. After natural drying, TEM observation was performed, and an appropriate area was selected for electron diffraction detection.

[0106] The results of TEM and selected area electron diffraction are consistent with the turbidity determination experiment, e.g. Figure 3 As shown in the figure, at 5 minutes, the mineral morphology in the blank group was ACP particles, and the corresponding diffraction pattern was amorphous. These ACP particles crystallized into irregular flake crystals after 24 hours, and diffraction rings of 002 and 211 crystal planes were visible in the corresponding diffraction pattern. After 7 days, they grew into larger flake crystals, and diffraction rings of 002, 211 and 004 crystal planes appeared in the diffraction. When the mineralization solution containing 100 μg / mL PAA reacted for 5 minutes, the mineral morphology in the solution was smaller pre-nucleation clusters and ACP particles, and the corresponding diffraction pattern was amorphous. After 24 hours and 7 days of reaction, the electron density of these particles gradually increased and the particles gradually aggregated, but the diffraction pattern still showed an amorphous state, proving that 100 μg / mL PAA can stabilize ACP within 7 days.

[0107] Test Example 1

[0108] The 1% CS-A / rCOL1 scaffold prepared in step 7 of Example 1, the 5% CS-A / rCOL1 scaffold prepared in step 7 of Example 2, and the 10% CS-A / rCOL1 scaffold prepared in step 7 of Example 3 were respectively taken for TEM observation, and O, N, and S elemental surface scanning was performed.

[0109] A CS-A-free rCOL1 scaffold was prepared by preparing a 10 mg / mL collagen (COL1) solution in 0.5 M acetic acid. 20 mL of the COL1 solution was placed in a dialysis bag (molecular weight cutoff 1000). The dialysis bag was immersed in 2 L of 2× PBS buffer at room temperature for 24 hours to achieve COL1 reconstitution (rCOL). The remaining steps were the same as steps 4-7 of Example 1. The CS-A-free rCOL1 scaffold was subjected to TEM observation and O, N, and S elemental scanning.

[0110] The results are as attached Figure 4 The results showed that polyelectrolytes with different concentration gradients were introduced into the collagen scaffold.

[0111] Test Example 2

[0112] In this experimental example, the CS-A / rCOL1 intrafibrous mineralized scaffold was characterized and a CS-A / COL1 blended mineralized scaffold was prepared as a control group.

[0113] 1. Preparation of CS-A / rCOL1 Intrafibrous Mineralized Scaffolds

[0114] According to the methods of Examples 1-3, 1% CS-A / rCOL1 intrafiber mineralized scaffolds with mineralization of 7 days were prepared respectively; and then according to the methods of Examples 1-3, the mineralization time was reduced to prepare 1% CS-A / rCOL1 intrafiber mineralized scaffolds with mineralization of 3 days.

[0115] 2. Preparation of CS-A / COL1 Blended Mineralized Scaffolds

[0116] A 10 mg / mL collagen (COL1) solution was prepared using 0.5 M acetic acid, and CaCl2 powder was added to a CaCl2 concentration of 3.34 mM. A phosphorus mother solution was prepared according to the method in Example 1, and a 1 mg / mL CS-A solution was prepared using the phosphorus mother solution. Equal volumes of the COL1 solution containing 3.34 mM CaCl2 and the CS-A solution were mixed and placed in a 37°C incubator for mineralization and sedimentation for 24 hours. The contents of the dialysis bag were then blown evenly using a sterile plastic pipette and transferred to a 24-well plate, with 2 mL per well. The plate was first frozen at -20°C overnight, then at -80°C for 24 hours, and finally lyophilized to obtain a CS-A / COL1 blended scaffold. Following the methods of Steps 5-7 in Example 1, an EDC / NHS chemical crosslinking solution was prepared, and the CS-A / COL1 blended scaffold was crosslinked and lyophilized to obtain a CS-A / COL1 blended mineralized scaffold.

[0117] The appearance of CS-A / COL1 blended mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 fiber mineralized scaffolds after 7 days of mineralization are shown in the attached figure. Figure 5 shown.

[0118] 3. Each scaffold was subjected to SEM observation, TEM observation and element surface scanning, FTIR test, XRD test, TGA test, porosity, water absorption rate and degradation rate determination.

[0119] 3.1SEM

[0120] Each bracket was fixed on the sample stage using conductive tape, sprayed with gold for 30 seconds, and observed by SEM at an acceleration voltage of 10 keV. Images were taken at low magnification (200 times) and high magnification (200,000 times), respectively.

[0121] The results are as attached Figure 6As shown, at low magnification (×200), all four groups of mineralized scaffolds exhibited a loose, porous, sponge-like structure. At high magnification (×200,000), the CS-A / COL1 blended mineralized scaffolds displayed a homogeneous organic-inorganic co-deposited membrane structure with no apparent fibrous structure. However, mineralized collagen fibers were evident in the 1%, 5%, and 10% CS-A / rCOL1 intrafibrous mineralized scaffolds at 3 and 7 days of mineralization. Within the same group of scaffolds, rCOL1 at 7 days of mineralization exhibited a denser mineral distribution and larger diameter than that at 3 days. At the same mineralization time, increasing CS-A concentrations led to a gradual increase in the density and diameter of the mineralized rCOL1.

[0122] 3.2TEM

[0123] During the mineralization process of the scaffold, a small amount of collagen fibers was torn off from the scaffold after 7 days of mineralization using tweezers and placed on a copper mesh. After drying, TEM observation was performed and a suitable area was selected for surface scanning of O, N, Ca, P, and S elements.

[0124] The results are as attached Figure 7 As shown, after 7 days of mineralization, high electron density of mineralized rCOL1 was observed in all three scaffolds under TEM observation. The corresponding electron diffraction patterns showed diffraction rings of the 002, 004, and 211 crystal planes, with the 002 and 004 crystal planes oriented parallel to the long axis of rCOL1, indicating that rCOL1 in all three scaffolds underwent significant intrafiber mineralization. Major element surface scanning results showed that the distribution of N, O, Ca, P, and S was consistent with the orientation of the fibers, confirming that the high-density fibers in the field of view were indeed rCOL1. Furthermore, Ca, P, and S were evenly distributed throughout the rCOL1, indicating relatively uniform mineralization within the rCOL1 fibers and a uniform distribution of CS-A within the rCOL1 fibers. Furthermore, the signal intensities of Ca and P in the 5% and 10% CS-A concentration groups were significantly stronger than those in the 1% concentration group, indicating that the degree of mineralization within the rCOL1 fibers increased with increasing CS-A concentration at the same mineralization time.

[0125] 3.3 FTIR

[0126] The freeze-dried scaffolds (CS-A / COL1 blended mineralized scaffolds and 1%, 5%, and 10% CS-A / rCOL1 intrafiber mineralized scaffolds after 7 days of mineralization) were mixed with KBr powder, ground, and pressed into tablets for FTIR characterization. The test wavelength was 400-4000 cm-1 with an accuracy of 4 cm -1 .

[0127] The results are as attached Figure 8 As shown, 555, 600 and 1021 cm -1 The peaks at 555 and 600 cm are the characteristic peaks of phosphate. -1The two characteristic peaks merge into a single peak. In the FTIR spectra of the four mineralized scaffolds, these two peaks do not merge, indicating a high degree of HAP crystallization in all four scaffolds. Furthermore, a higher intensity of the phosphate characteristic peak indicates a higher degree of scaffold mineralization, with the 10% concentration group achieving the highest concentration.

[0128] 3.4XRD

[0129] Each freeze-dried scaffold was evenly flattened on a glass slide. The parameters were set as follows: voltage 40 kV, current 40 mA, and rotation angle 10-60°. XRD detection was performed and the experimental results were analyzed using JADE 5.0 software.

[0130] The results are as attached Figure 9 As shown, the XRD patterns of the four mineralized scaffolds (CS-A / COL1 blended mineralized scaffolds and 7-day-old 1%, 5%, and 10% CS-A / rCOL1 intrafiber mineralized scaffolds) all conform to the characteristic spectra of HAP, indicating that the minerals in all four scaffolds are HAP. Furthermore, the spectra of all four scaffolds show strong peaks characteristic of the 002, 211, and 004 crystal planes, indicating a relatively high degree of HAP crystallinity. The 10% scaffold exhibits the highest intensity and exhibits more characteristic peaks of crystal planes, such as 112, 300, 202, 310, 222, and 213, indicating the highest degree of crystallinity.

[0131] 3.5TGA

[0132] Approximately 3.5 mg of each freeze-dried scaffold (CS-A / COL1 blended mineralized scaffold and 1%, 5%, and 10% CS-A / rCOL1 intrafiber mineralized scaffolds mineralized for 7 days) was weighed for TGA. In a nitrogen atmosphere (80 mL / min), the sample was heated to 800°C at a rate of 5°C / min to detect the change in sample mass.

[0133] like Figure 10As shown, the DTG curves of the four groups of scaffolds all show three extrapolated peaks, corresponding to three stages of rapid mass loss in the TGA curves, i.e., the thermal degradation of the three different components in the scaffolds. The first thermal degradation temperatures in the curves are 77.16-80.98°C (blend), 66.18-67.38°C (1%), 68.46-70.49°C (5%), and 69.72-77.18°C (10%), respectively. The mass loss in this stage is caused by the loss of water molecules in the scaffolds. The second stage of thermal degradation occurs at temperatures of 328.36-329.30°C (blend), 330.40-331.93°C (1%), 333.98-334.78°C (5%), and 335.23-335. 98℃ (10%). The mass loss at this stage is caused by the degradation of COL1 in the scaffold. The degradation temperature of this stage is ranked as 10%>5%>1%>blend. It is speculated that the COL1 in the blended mineralized scaffold group has not been reorganized, and the molecular thermal stability is poor. In addition, the minerals in the scaffold are all extra-fiber minerals, which have poor protection for COL1 molecules, so the thermal degradation temperature is the lowest. The COL1 in the 1%, 5% and 10% scaffolds have all been reorganized, and the thermal stability is relatively high. In addition, intra-fiber mineralization has occurred in all of them. Studies have shown that intra-fiber mineralization can improve the thermal stability of the scaffold. The stability of rCOL1 is high, so the thermal degradation temperatures of these three groups of scaffolds are relatively high. Among them, the thermal degradation temperature of the 10% scaffold group is the highest, indicating that its degree of mineralization in the fiber is the highest; the thermal degradation temperatures of the third stage are 498.13-499.25℃ (blend), 500.44-502.48℃ (1%), 500.62-501.18℃ (5%) and 502.02-502.93℃ (10%), respectively. The mass loss in this stage is caused by the degradation of CS-A in the scaffold, and the degradation temperature of this stage is ranked 10%. The reason for this is that the degradation temperature of COL1 molecules in the blended mineralized scaffolds is lower. This degradation of COL1 molecules reduces the stability of the covalently cross-linked CS-A molecules, resulting in a lower thermal degradation temperature. In the other three scaffolds, COL1 undergoes reorganization, resulting in higher thermal degradation temperatures, and thus, higher thermal degradation temperatures for the CS-A molecules. Furthermore, the intensities of the extrapolated peaks are ranked as blend > 10% > 5% > 1%, indicating that the CS-A content in the four scaffolds follows the order of blend > 10% > 5% > 1%, consistent with previous studies. Finally, the final residual masses of the four scaffolds at 800°C were 49.77% (blend), 49.50% (1%), 61.70% (5%), and 62.48% (10%), respectively, ranking 10% > 5% > blend > 1%, indicating that the mineral content in the scaffolds follows the order of 10% > 5% > blend > 1%.

[0134] 3.6 Porosity

[0135] The porosity of each scaffold was determined by the liquid (ethanol) displacement method.

[0136] 3.7. Water absorption

[0137] The water absorption of each scaffold was measured by gravimetric method.

[0138] 3.8 Degradation rate

[0139] The degradation rate of each stent was determined by the weight loss method. A cylindrical stent was randomly selected, weighed with a weighing balance and recorded as M1, immersed in 20 mL of sterile PBS solution, incubated at 37°C, and the PBS solution was replaced once a week. After 4 weeks, the stent was removed and washed with deionized water 3 times. Most of the surface moisture was gently absorbed with filter paper, and then placed in an oven at 37°C for drying. The weight was weighed again and recorded as M2. Five cylindrical stents were measured for each stent and the average degradation rate was calculated. The calculation formula for the degradation rate (L) is as follows:

[0140] L=(M1-M2) / M1×100%

[0141] As shown in Table 3-1, the order of porosity is blend>10%>1%>5%; the order of water absorption is>5%>1%>blend; and the order of degradation rate is blend>1%>5%>10%.

[0142] Table 1 General properties of the four groups of mineralized scaffolds (mean ± SD, n = 5)

[0143] Grouping Porosity (P) Water absorption (W) Degradation rate (L) blend 92.61±4.31％ 1349.84±33.75％ 12.52±0.34％ 1％ 73.43±6.08％ 1433.16±42.55％ 2.15±0.53％ 5％ 72.02±3.71％ 1440.25±38.25％ 2.01±0.42％ 10％ 74.61±2.11％ 1461.55±35.65％ 1.85±0.36％

[0144] Experimental Example 3: Alizarin Red Staining to Evaluate the Late Osteogenic Differentiation Ability of Bone Marrow Mesenchymal Stem Cells Induced by Different Scaffold Materials

[0145] Experimental groups: NC group (10% α-MEM culture medium); OM group (osteoblastic differentiation induction culture medium group); blend group (CS-A / COL1 blended mineralized scaffold group); 1% group (1% CS-A / rCOL1 intrafibrous mineralized scaffold group mineralized for 7 days); 5% group (5% CS-A / rCOL1 intrafibrous mineralized scaffold group mineralized for 7 days); 10% group (10% CS-A / rCOL1 intrafibrous mineralized scaffold group mineralized for 7 days).

[0146] The CS-A / COL1 blended mineralized scaffold, 1% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days, 5% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days, and 10% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days were all prepared according to the method of Experimental Example 2.

[0147] Experimental methods:

[0148] (1) Liquid preparation: 10 mg of scaffold material was immersed in 10 mL of α-MEM medium and incubated at 37°C for 24 h. The extract was filtered using a syringe and a 0.22 μm sterile filter (in a clean bench) to obtain sterile extracts of different scaffold materials. The NC group used 10% α-MEM medium as the extract.

[0149] Fetal bovine serum, double antibody, β-GB, DM, and vitamin C were added to the above extracts to prepare an extract osteogenic differentiation induction medium containing 10% fetal bovine serum, 1% double antibody, 10 mM β-GB, 0.1 μM DM, and 50 μg / mL vitamin C. For the OM group, an osteogenic differentiation induction medium containing 10% fetal bovine serum, 1% double antibody, 10 mM β-GB, 0.1 μM DM, and 50 μg / mL vitamin C was prepared.

[0150] (2) resuscitation of rBMSCs;

[0151] (3) When the cells grew to 80% confluence, they were digested, centrifuged, resuspended, and the cells were counted. 4 Cells were seeded at a density of 1 mL / well in a 12-well plate, with 1 mL per well and 3 replicates per group, and cultured at 37°C and 5% CO2; the 1% group, 5% group, 10% group, and NC group were cultured with the corresponding extract prepared in step (1) in the osteogenic differentiation induction medium, and the OM group was cultured with the osteogenic differentiation induction medium.

[0152] (4) Change the medium after 24 hours, and then change the medium every 2 days;

[0153] (5) After 14 days, the cells were gently washed with sterile PBS, fixed with 4% paraformaldehyde for 30 min, and then gently washed again with PBS;

[0154] (6) Staining: Add 200 μl of Alizarin Red staining solution to each well and incubate at room temperature for 30 min. Aspirate and discard the staining solution, gently wash with sterile PBS solution, and observe and photograph under a microscope.

[0155] (7) ImageJ software was used to calculate the average gray value of Alizarin Red staining and perform statistical analysis.

[0156] The results are as attached Figure 11 and 12As shown in the figure, after 14 days of culture, the calcium nodules were stained red with alizarin red. The average gray value was calculated using ImageJ software and statistical analysis was performed. The results showed that the average gray value of the four groups of extracts and the OM group was higher than that of the NC group, with statistical differences (P<0.05). Among them, the 10% group, the blend group, and the 5% group were all significantly higher than the OM group, with statistical differences (P<0.05). This shows that the scaffold of the present invention can promote the formation of calcium nodules in the late stage of rBMSCs osteogenic, and the 10% group has the best effect.

[0157] Experimental Example 5: Investigation of the repair effect of CS-A / rCOL1 intrafibrous mineralized scaffold on bone defects in vivo

[0158] 1. Construction of a Bilateral Self-Controlled Critical Bone Defect Model in the Rat Skull

[0159] Experimental groups: blend group (CS-A / COL1 blended mineralized scaffold group); 1% group (1% CS-A / rCOL1 intrafiber mineralized scaffold group); 5% group (5% CS-A / rCOL1 intrafiber mineralized scaffold group); 10% group (10% CS-A / rCOL1 collagen intrafiber mineralized scaffold group).

[0160] The CS-A / COL1 blended mineralized scaffold, 1% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days, 5% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days, and 10% CS-A / rCOL1 intrafiber mineralized scaffold mineralized for 7 days were all prepared according to the method of Experimental Example 2.

[0161] Sixty-four SD rats were randomly divided into 4 groups, 16 rats in each group. Each group was further divided into two subgroups, 2 months and 3 months old, 8 rats in each group.

[0162] The rats were fasted but not watered for 12 hours before surgery. The rats were weighed, anesthetized, and fixed on the operating table in a prone position. The skin was prepared in the center of the rat's skull using a razor and disinfected after preparation.

[0163] Use a straight blade to cut open the skin, fascia, and periosteum of the rat's skull to expose the bone surface. Locate the skull, expose the bone surface, find the anterior and posterior edges of the "H"-shaped suture on the skull, retract the tissue with a tissue retractor, and carefully drill holes between the anterior and posterior bregma on both sides of the sagittal suture using a 5 mm diameter trephine drill, avoiding any suture damage and avoiding damage to intracranial tissue.

[0164] The round skull piece drilled out was removed. No material was implanted in the left defect, while the corresponding scaffold material was implanted in the right defect. The fascia was sutured with purple absorbable sutures and the skin was sutured with black non-absorbable sutures. The surgical area was disinfected with iodine and cleaned.

[0165] After surgery, rats were given an intramuscular injection of 200,000 U of penicillin G to prevent wound infection.

[0166] The rats were killed 2 months and 3 months after surgery, and skull samples and major organ samples were collected and fixed with 4% paraformaldehyde for later use.

[0167] 2. Routine blood test

[0168] Two and three months after surgery, all blood routine indicators in the four groups of rats were normal. Counts of major blood cells, such as red blood cells, white blood cells, and platelets, were within normal ranges, and there were no significant abnormalities in hemoglobin, monocytes, and granulocytes. This indicates that none of the rats in each group developed significant anemia, bacterial or viral infection, poisoning, or inflammation. This suggests that none of the four groups of scaffolds had significant systemic toxicity within two and three months.

[0169] 3. Serum Biochemical Analysis

[0170] Two and three months after surgery, liver and kidney function biochemical results in all four groups of rats were normal. Key indicators such as aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, and creatinine showed no significant abnormalities, indicating that the scaffold materials in the four groups had no significant liver and kidney toxicity within two and three months.

[0171] 4. HE staining results of the main organs of rats were normal 2 months and 3 months after surgery

[0172] like Figure 13 As shown, 2 months and 3 months after surgery, the myocardial cells of the rats in each group were regularly arranged, with clear boundaries and normal morphology, and no obvious necrotic areas, indicating that the stent had no obvious cardiotoxicity; the liver tissue structure of the rats in each group was normal, the hepatic cords were radial and neatly arranged, the cytoplasm was evenly stained, and the cell nuclei were clear, indicating that the stent had no obvious hepatotoxicity; the spleen cortical-medullary junction of the rats in each group was clear, and lymphocytes were densely distributed in the white pulp and red pulp, indicating that the stent had no obvious spleen toxicity; the alveolar wall epithelial cells of the rats in each group were neatly arranged, with clear boundaries, and the alveoli were evenly distributed, indicating that the stent had no obvious lung toxicity; the kidneys of the rats in each group had no obvious morphological changes, the glomeruli and renal tubules had clear structures, and the renal tubular epithelial cells were neatly arranged, indicating that the stent had no obvious renal toxicity.

[0173] 5. Micro-CT 3D Reconstruction and Analysis of Skull Samples

[0174] The collected skull samples were fixed in 4% paraformaldehyde for 24 hours and wet-scanned using Micro-CT at 70 kVp, 200 μA, and a resolution of 10 μm. The scan files were reconstructed and selected regions were analyzed for bone volume fraction (BV / TV).

[0175] Two months after surgery, no significant new bone formation was observed in the blank control bone defects (left side) of the skull specimens in each group, indicating that the critical bone defect model was successfully constructed. Significant new bone formation was observed in the 10% experimental group bone defect (right side), almost completing the repair of the entire bone defect. However, only a small amount of new bone formation was observed in the experimental group bone defects (right side) of the other three groups. Statistical analysis of the bone volume fraction of the newly formed bone in each group showed that the blend group was 5.541±3.5%, the 1% group was 8.900±3.9%, the 5% group was 12.541±4.1%, and the 10% group was 52.900±4.5%. The results of statistical analysis showed that there was no statistical difference in the new bone volume fraction between the experimental group and the blank control group in the blend group, indicating that the blend group scaffold had no significant repair effect at 2 months, while the new bone volume fraction of the bone defects in the 1%, 5% and 10% intrafibrous mineralized scaffold experimental groups was significantly higher than that in the blank control bone defects (P < 0.05), indicating that the 1%, 5% and 10% groups showed certain bone defect repair effects, and the 10% group showed the best bone defect repair effect among the 4 groups.

[0176] like Figure 14 and Figure 15 As shown, three months after surgery, no significant new bone formation was observed in the blank control bone defects (left side) of the skull specimens in each group, further demonstrating the successful establishment of the critical bone defect model. Different amounts of new bone formation were observed in the experimental bone defects (right side) of the four groups. Statistical analysis of the bone volume fraction of the newly formed bone in each group revealed that the blend group had a value of 45.400±2.4%, the 1% group had a value of 35.090±4.9%, the 5% group had a value of 42.127±7.1%, and the 10% group had a value of 81.042±7.8%. Statistical analysis showed that the newly formed bone volume fraction of the bone defects in the four scaffold experimental groups was significantly higher than that in the blank control bone defects (P < 0.05), indicating that all four scaffold groups demonstrated good bone defect repair efficacy at three months, with the 10% group showing the most significant effect and exceeding the other three groups (P < 0.05).

[0177] Test Example 6

[0178] This test example investigated the utilization of CS-A by the traditional polyelectrolyte complexation method, the chemical crosslinking method, and the improved polyelectrolyte complexation method of the present invention. This test example used the DMMB method to determine the concentration of CS-A.

[0179] (1) Preparation of DMMB test solution and drawing of standard curve: 1.520 g Gly and 1.185 g NaCl were added to 500 mL deionized water in sequence and stirred until completely dissolved. 8 mg DMMB powder was added to the solution and the pH of the solution was adjusted to 3.0 with NaOH and HCl to obtain DMMB test solution. The solution was stored in the dark. 5, 10, 20, 30, 40, 50 and 60 μg / mL CS-A solutions were prepared using deionized water. 40 μL of CS-A solutions of different concentrations were taken and 1 mL DMMB test solution was added. The solution was mixed and allowed to stand for 5 min. The absorbance of the solution was measured at a wavelength of 525 nm and recorded. After all concentrations were measured, a standard curve was drawn with the concentration as the horizontal axis and the absorbance as the vertical axis.

[0180] (2) Investigating the utilization rate of CS-A by polyelectrolyte complexation

[0181] 10 mg / mL COL1 solution and 0.2, 0.5, 1, 1.5, 2, 3, 5, 10, 15, and 20 mg / mL CS-A solutions were prepared using 0.5 M acetic acid.

[0182] 10 mg / mL COL1 solution and 0.2, 0.5, 1, 1.5, 2, 3, 5, 10, 15, and 20 mg / mL CS-A solutions were prepared using 0.5 M acetic acid.

[0183] Mix 0.5 mL of COL1 solution and 0.5 mL of CS-A solution with varying concentrations, let stand for 5 minutes, and centrifuge at 15,000 g for 5 minutes at 4°C. Aspirate 40 μL of the supernatant, add 1 mL of DMMB detection solution, mix thoroughly, let stand for 5 minutes, and measure the absorbance of the solution at 525 nm. (The supernatant of the high-concentration group was diluted to within the range of the standard curve before measurement.) Calculate the CS-A utilization rate and the mass of CS-A bound per mg of COL1 using the CS-A standard curve.

[0184] (3) Investigating the utilization rate of CS-A by chemical cross-linking

[0185] Prepare the EDC / NHS chemical crosslinking solution: First, prepare 0.1M MES buffer and adjust the pH to 5.5 using NaOH and HCl. Use the above MES buffer to prepare the EDC / NHS chemical crosslinking solution: First, add EDC powder to the MES buffer to an EDC concentration of 0.02mM. Stir until the EDC is completely dissolved. Then, add NHS powder to the solution to an NHS concentration of 0.012mM. Stir until the NHS is completely dissolved. Let it stand for 15 minutes and adjust the pH to 7.2-7.5 using NaOH and HCl.

[0186] The EDC / NHS crosslinking solution was then used to prepare CS-A solutions at concentrations of 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2.5, 5, 7.5, and 10 mg / mL. For the experiment, 5 mg of COL1 sponge was added to 1 mL of the CS-A solution at each concentration gradient. The mixture was incubated at 37°C for 2 hours, centrifuged at 15,000 g for 5 minutes at 4°C, and 40 μL of the supernatant was aspirated and added with 1 mL of DMMB detection solution. The mixture was mixed and allowed to stand for 5 minutes. The absorbance at 525 nm was measured and recorded (the supernatant of the high-concentration group was diluted to the range of the standard curve). The CS-A standard curve was used to calculate the CS-A utilization rate and the mass of CS-A bound per mg of COL1.

[0187] (4) Investigating the utilization rate of CS-A by the improved polyelectrolyte complexation method

[0188] Preparation of EDC / NHS chemical crosslinking solution: Prepare EDC / NHS chemical crosslinking solution according to the method in (2) Investigating the utilization rate of CS-A by chemical crosslinking method.

[0189] 10 mg / mL COL1 solution and 0.2, 0.5, 1, 1.5, 2, 3, 5, 10, 15, and 20 mg / mL CS-A solutions were prepared using 0.5 M acetic acid.

[0190] Mix 5 mL of COL1 solution and 5 mL of gradient concentration CS-A solution in a 15 mL centrifuge tube and let it stand for 5 minutes. A white flocculent complex will precipitate in the tube. Then, slowly add HCl to the tube until the complex is completely dissolved. Transfer the liquid to a dialysis bag (molecular weight cutoff 1000).

[0191] Each dialysis bag was immersed in 2L of 2×PBS buffer and placed at room temperature for 24 hours to simultaneously achieve COL1 recombining and CS-A doping. The dialysis bags were then taken out and immersed in 500mL of EDC / NHS chemical crosslinking solution respectively, and reacted at 37°C for 2 hours.

[0192] Transfer the liquid from the dialysis bag to a 50mL centrifuge tube and centrifuge at 5000 rpm for 30 minutes at 4°C. Aspirate 40μL of the supernatant and add 1mL of DMMB detection solution. Mix thoroughly and let stand for 5 minutes. Measure the absorbance of the solution at 525nm and record the absorbance (the supernatant of the high-concentration group should be diluted to the standard curve range before measurement). Calculate the CS-A utilization rate and the mass of CS-A bound per mg of COL1 based on the CS-A standard curve.

[0193] The results showed that in the range of 0 to 60 μg / mL, there was a good linear relationship between the concentration of CS-A and the absorbance at 525 nm. 2 =0.9944.

[0194] The results of the CS-A utilization survey are shown in the attached Figure 16 As shown in the figure, in the polyelectrolyte complexation method, the utilization rate of CS-A gradually increased with increasing CS-A concentration, reaching a peak utilization rate of nearly 100% at 0.75 mg / mL. It then gradually decreased with increasing CS-A concentration. When the CS-A concentration was below 2.5 mg / mL, the utilization rate of CS-A in the polyelectrolyte complexation method remained above 40%. In the chemical crosslinking method, the utilization rate of CS-A gradually decreased with increasing CS-A concentration. When the CS-A concentration was between 0.25 and 1.5 mg / mL, the utilization rate slowly decreased and remained relatively stable. It then rapidly decreased with increasing CS-A concentration. The maximum utilization rate of CS-A in the chemical crosslinking method was approximately 40%, and when the CS-A concentration was below 1.5 mg / mL, the utilization rate remained around 20%. The utilization trend of CS-A in the improved polyelectrolyte complex method is similar to that in the polyelectrolyte complex method. The utilization reaches a peak of approximately 95.39% at 0.75 mg / mL, and then gradually decreases with the increase of CS-A concentration. When the CS-A concentration is lower than 2.5 mg / mL, the utilization of CS-A in the improved polyelectrolyte complex method can be maintained above 50%.

[0195] The binding amount of CS-A is as shown in the attached Figure 16 As shown in the figure, in the polyelectrolyte complexation method, the mass of CS-A that can be bound per milligram of COL1 gradually increased with increasing CS-A concentration, reaching a peak of approximately 0.71481 mg at 5 mg / mL, and then gradually decreased with increasing CS-A concentration. In the chemical cross-linking method, the mass of CS-A that can be bound per milligram of COL1 gradually increased with increasing CS-A concentration, reaching a peak of approximately 0.21898 mg at 1.5 mg / mL, then slightly decreased and remained relatively constant. In the modified polyelectrolyte complexation method, the mass of CS-A that can be bound per milligram of COL1 gradually increased with increasing CS-A concentration, reaching a peak of approximately 1.45278 mg at 7.5 mg / mL, and then remained relatively constant.

[0196] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material, characterized in that: The steps include: S1. Reaction: Mixing a collagen solution and a polyelectrolyte solution to precipitate flocs; preparing a 1-20 mg / mL collagen solution and a 0.01-0.5 mg / mL polyelectrolyte solution using 0.1-1 M acetic acid solution; the polyelectrolyte being chondroitin sulfate A; S2 re-dissolve: hydrochloric acid was added dropwise to the mixed solution to dissolve the flocculent to obtain a re-dissolved mixed solution; S3. Reconstitution and doping: The reconstituted mixed solution is placed in a dialysis bag, which is then immersed in a buffer solution to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation; the dialysis bag has a molecular weight cutoff of greater than or equal to 1000; The buffer solution is added with an alkaline substance in an amount sufficient to neutralize the hydrochloric acid added dropwise in step S2, so that the collagen is reconstituted under neutral to weakly alkaline conditions; S4 lyophilization: The recombinant and doped solution in step S3 was lyophilized to obtain a polyelectrolyte uniformly modified recombinant collagen scaffold material; S5. Crosslinking: The scaffold material after freeze-drying in step S4 is placed in an EDC / NHS crosslinking solution for crosslinking reaction; the concentration of EDC in the crosslinking solution is 0.01-0.04 mM, and the concentration of NHS is 0.006-0.024 mM; S6. Secondary freeze-drying: freeze-drying the cross-linked scaffold material; S7. Mineralization: The secondary freeze-dried scaffold material is placed in a mineralization solution for mineralization reaction; the mineralization solution is composed of a mixture of calcium mother liquor, PAA mother liquor, and phosphorus mother liquor in a volume ratio of 1:0.5-2:0.5-2; the calcium mother liquor is a 3-12 mM calcium salt solution; the PAA mother liquor is prepared by dissolving PAA powder in deionized water; the PAA concentration in the mineralization solution is 12.5-200 μg / mL; the phosphorus mother liquor is prepared by dissolving NaH2PO4 in PBS buffer; the Na2HPO4 concentration in the mineralization solution is 0.5-2 mM; and the PBS concentration in the mineralization solution is 0.5-1×PBS. S8. Three freeze-drying steps: freeze-drying the mineralized scaffold to obtain a mineralized scaffold material uniformly modified with polyelectrolyte in the fiber.

2. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The collagen includes type I collagen.

3. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The concentration of acetic acid solution is 0.5 M.

4. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The concentration of collagen solution was 10 mg / mL.

5. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The concentration of the polyelectrolyte solution was 0.1 mg / mL.

6. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S1, the reaction is carried out at room temperature.

7. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S1, the reaction time is 5 to 30 minutes.

8. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S3, the dialysis bag is immersed in a buffer solution and placed at room temperature for 2-48 hours to simultaneously achieve collagen fiber reorganization and polyelectrolyte complexation.

9. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S3, the dialysis bag is immersed in the buffer solution and left at room temperature for 24 hours.

10. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The alkaline substance includes sodium hydroxide.

11. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S4, the recombined and doped solution is placed in a mold of a certain shape and freeze-dried to obtain a polyelectrolyte uniformly modified recombinant collagen scaffold material.

12. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S5, the concentration of EDC in the cross-linking solution is 0.02 mM; the concentration of NHS is 0.012 mM.

13. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In the step S5, the cross-linking reaction is carried out at room temperature.

14. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S5, the amount of the cross-linking solution is at least enough to completely immerse the scaffold material.

15. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The volume ratio of calcium mother liquor, PAA mother liquor and phosphorus mother liquor is 1:1:

2.

16. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: The calcium mother solution is a calcium chloride solution with a concentration of 6-7 mM; The concentration of PAA in the mineralization solution was 100 μg / mL; The concentration of Na2HPO4 in the mineralization solution was 1 mM; the concentration of PBS in the mineralization solution was 0.85×PBS.

17. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S7, a mineralization reaction is performed at room temperature; the amount of the mineralization liquid is at least enough to completely immerse the scaffold material.

18. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S7, the mineralization time is 1-10 days.

19. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S7, the mineralization time is 3 to 7 days.

20. The method for preparing a polyelectrolyte uniformly modified fiber mineralized scaffold material according to claim 1, characterized in that: In step S7, the mineralization time is 7 days.

21. An intra-fiber mineralized scaffold material prepared according to the preparation method according to any one of claims 1 to 20.

22. Use of the intra-fiber mineralized scaffold material according to claim 21 in preparing a material for repairing bone defects.

Images