Preparation method of gradient biomimetic mineralized collagen-based artificial periosteum
By employing a segmented mineralization and porosity gradient construction method, a gradient biomimetic mineralized collagen-based artificial bone membrane was prepared, which solved the problems of insufficient mechanical strength and bioactivity of collagen membranes in existing technologies and achieved the effect of bone defect repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-03-24
AI Technical Summary
The homogeneous mineralized collagen membranes prepared by existing technologies cannot replicate the gradient structure of the natural periosteum, resulting in insufficient mechanical strength and limited bioactivity in the repair of complex bone defects.
A segmented mineralization and pH gradient construction method was adopted. Calcium salt and phosphate solutions were simultaneously added to collagenic acid solution to form a high-medium-low mineralization gradient. A porosity gradient was constructed by adding pore-forming agents in layers and controlling the freezing rate. Finally, a gradient mineralized collagen membrane was formed through cross-linking and molding.
A gradient mineralization and porosity structure similar to the natural periosteum was successfully constructed, which meets the requirements of mechanical support and osteogenic guidance, reduces immunogenicity, and promotes bone tissue regeneration.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical materials, and more particularly to a method for preparing a gradient biomimetic mineralized collagen-based artificial bone membrane. Background Technology
[0002] Bone defect repair is one of the major challenges facing clinical orthopedics. For critical-sized bone defects, their self-healing capacity is limited, often requiring the implantation of bone repair materials to guide and promote bone tissue regeneration. Among various bone repair strategies, guided bone regeneration technology has been widely used. Its core principle is to use a barrier membrane to separate the bone defect area from the surrounding soft tissue, creating a protected space for preferential bone growth. This barrier membrane is commonly referred to as an artificial periosteum.
[0003] An ideal artificial periosteum should have dual functions: first, as a physical barrier, it should prevent soft tissues such as fibroblasts from growing into the bone defect area, thus avoiding interference with the bone repair process; second, as a bioactive scaffold, it should guide and promote the migration, proliferation and differentiation of osteoblasts, and ultimately integrate or degrade them into new bone tissue.
[0004] Collagen is a preferred material for preparing artificial bone membranes due to its excellent biocompatibility, biodegradability, and ability to mimic the natural extracellular matrix. However, pure collagen membranes suffer from insufficient mechanical strength, rapid degradation in bodily fluids, and limited bioactivity, making them unsuitable for repairing complex bone defects. To enhance its osteogenic activity, researchers often introduce hydroxyapatite into the collagen matrix to prepare mineralized collagen composites, mimicking the chemical composition of natural bone.
[0005] Currently, collagen mineralization methods mostly employ blending or in-situ precipitation. However, these traditional methods often result in a uniform distribution of minerals within the collagen matrix. This is inconsistent with the complex structure of the natural periosteum. The natural periosteum itself exhibits a significant structural and functional gradient: the outer layer near the bone cortex is dense and tough, rich in blood vessels and nerves, primarily serving protective, connecting, and nourishing functions; while the inner layer near the bone surface is more porous, containing a large number of osteoblasts, which are crucial for bone growth and repair.
[0006] The homogeneous mineralized collagen membranes prepared by existing technologies cannot reproduce this natural gradient structure. Summary of the Invention
[0007] The main objective of this invention is to solve the technical problem that homogeneous mineralized collagen membranes in the prior art cannot reproduce this natural gradient structure. A method for preparing a gradient biomimetic mineralized collagen-based artificial bone membrane includes the following steps: A method for preparing a gradient biomimetic mineralized collagen-based artificial bone membrane is as follows: (1) Preparation of collagenic acid solution: Collagen powder was dispersed and dissolved in acetic acid solution to obtain a homogeneous collagen acid solution. (2) Segmented mineralization and pH gradient construction: Under low temperature stirring conditions, calcium salt solution and phosphate solution are simultaneously added dropwise to the collagen acid solution at a constant molar ratio, and at least three mineralization stages are carried out in sequence. In each stage, the pH of the solution is adjusted to a different set range so that calcium and phosphate minerals gradually form a high-medium-low mineralization gradient in the collagen fibers, thereby obtaining a mineralized collagen system with a mineralization distribution gradient. (3) Settling, concentration and ion washing: The mineralized collagen system obtained in step (2) was subjected to static aging, fractionation centrifugation, and washing with buffer solution containing trace amounts of calcium and phosphorus to maintain the formed mineralization gradient structure. (4) Pore gradient construction and shaping: The mineralized collagen gel obtained in step (3) is subjected to molding treatment. Different amounts of pore-forming agents are added in layers for molding or frozen by using the temperature difference of the bottom-up program, so as to form a porosity gradient structure corresponding to the mineralization gradient during the molding process. (5) Freeze-drying: The molded sample was pre-frozen and freeze-dried to obtain a gradient mineralized collagen sponge with a stable three-dimensional pore structure. (6) Crosslinking and re-drying: The sponge was washed after contact with the crosslinking agent and then freeze-dried again to obtain a stable morphology for the material. (7) Calendering film formation: The dried material is subjected to temperature-controlled and distance-controlled roller pressing to form a uniform and flexible gradient collagen-based artificial bone membrane.
[0008] The present invention has the following beneficial effects: This invention successfully constructs a dual structure of gradient mineralization and gradient porosity similar to the natural periosteum. The continuous gradient change from the side closer to the bone tissue to the side farther away from the bone tissue can simultaneously meet the biological requirements of mechanical support, osteogenic guidance, and vascular infiltration.
[0009] This invention benefits from the refining process of the source collagen raw material (DNA residue as low as 19.47 ng / mg, no C- or N-terminal peptides detected) and the mild low-concentration ethanol cross-linking system, resulting in a final product with extremely low immunogenicity. Detailed Implementation
[0010] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification and claims of this invention are used to distinguish similar objects and are not necessarily used to describe a particular order or sequence. It should be understood that such data used can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0011] Preparation method of collagen powder: 1) Take fresh pig skin (from genetically modified pigs), remove the grease and hair, wash, and cut into small pieces of 1cm×1cm. Add 0.01mol / L sodium hydroxide solution at a solid-liquid ratio of 1:30, soak at 6℃ for 2 hours, filter, and wash with water until neutral. 2) After the above treatment, the pig skin was added to 600 mg / L papain at 37°C, and the pH was adjusted to 6.5 with 0.1 mol / L hydrochloric acid for 2 hours. Then it was immersed in a mixture of 0.6 mol / L acetic acid (pH 2.0) and 600 mg / L pepsin and stirred continuously for 20 hours. The pig skin after the above treatment was soaked in acetic acid at 25°C for 1 hour at pH 3.0. 3) Then add 0.5 mol / L sodium hydroxide solution to adjust to neutral; filter the neutralized liquid with gauze, take the filtrate, centrifuge at 4℃, 4000 rpm for 20 minutes, and take the supernatant; select a 30 kDa regenerated cellulose ultrafiltration membrane, rinse 3 times with deionized water, separate the membrane at 25 degrees Celsius, and retain the retentate; 4) The above liquid was freeze-dried at -80 degrees Celsius.
[0012] Circular dichroism spectroscopy analysis revealed a negative absorption peak around 194 nm and a positive absorption peak around 220 nm, indicating that the raw material possesses a complete natural triple helix structure. SDS-PAGE electrophoresis showed the α chain (around 100 kDa), β chain (around 200 kDa), and γ chain (around 300 kDa) of collagen, with a collagen purity of 99%. The collagen retains a complete natural triple helix structure, making it an ideal material for bioactive collagen. The residual DNA content of the collagen raw material was 19.47 ng / mg. The concentrations of C- and N-terminal peptides were below the detection limit of the kit (Monkey Type I Collagen N-terminal Peptide (NTX) Kit), and no C- and N-terminal peptides were detected in porcine collagen, indicating a high antigen clearance rate and low immunogenicity.
[0013] Calcium salt solution: Calcium chloride solution, concentration 50mM, prepared with deionized water.
[0014] Phosphate solution: Disodium hydrogen phosphate solution, concentration 30mM, prepared with deionized water.
[0015] pH adjustment solution: sodium hydroxide solution, concentrations of 0.1M and 1.0M; Hydrochloric acid (HCl) solution, concentration 0.1M.
[0016] For ease of understanding, the specific process of the embodiments of the present invention is described below. The first embodiment of the gradient biomimetic mineralization collagen-based artificial bone membrane method of the present invention includes: 1. Preparation of collagenic acid solution Pre-cool the 0.5M acetic acid solution to 4 degrees Celsius. Under ice bath conditions and magnetic stirring at approximately 300 rpm, slowly disperse the collagen powder in the pre-cooled acetic acid solution to a final concentration of 5 mg / mL.
[0017] Stir continuously at 4 degrees Celsius for at least 24 hours until the collagen is completely dissolved, forming a homogeneous, clear, and viscous collagenic acid solution. Let it stand in a 4-degree Celsius refrigerator to defoam before use.
[0018] 2. Segmented dripping mineralization and pH gradient construction The 5 mg / mL collagenic acid solution prepared in step 1 was placed in an ice bath and magnetically stirred at a low speed of 200 rpm. The initial pH value was approximately 2.5-3.0.
[0019] First stage of mineralization (bottom layer, high mineralization): Two peristaltic pumps were started to simultaneously add calcium salt solution and phosphate solution to collagenic acid solution at a rate of 0.5 mL / min, with the Ca / P molar ratio always maintained at 1.67.
[0020] When the pH of the system naturally rises to approximately 4.5, the dropwise addition is paused. At this point, the pH is finely adjusted using 0.1M NaOH solution to stabilize it at 5.0 ± 0.1, and the calcium phosphate solution is continued to be added dropwise at this pH for approximately 30 minutes. This stage aims to induce the extensive nucleation and growth of minerals within the collagen fibers.
[0021] Second stage of mineralization (middle layer, medium mineralization): Continue adding calcium salt solution and phosphate solution dropwise, while gradually increasing the pH of the system to 6.0±0.1 with 0.1M NaOH, and maintaining this pH for about 20 minutes.
[0022] Third stage mineralization (top layer, low mineralization): Continue adding calcium phosphate solution dropwise, while rapidly raising the pH of the system to 7.4 ± 0.1 with 0.1 M NaOH, and continue adding at this final pH for about 10 minutes to ensure complete mineralization reaction.
[0023] The entire dropwise addition process must be carried out in an ice bath at 4 degrees Celsius, with a total dropwise addition time of approximately 2-3 hours. The final result is a milky white, opaque mineralized collagen mixture.
[0024] 3. Settling, Concentration, and Washing The above-mentioned mineralized collagen mixture was transferred to centrifuge tubes and aged at 4 degrees Celsius for 12 hours.
[0025] Perform fractional centrifugation: First stage: low speed centrifugation (1000g, 10 minutes, 4 degrees Celsius), carefully remove the supernatant.
[0026] Second stage: Centrifuge at medium speed (3000g, 15 minutes, 4 degrees Celsius) to remove most of the supernatant and concentrate the system into a viscous gel.
[0027] The gel was resuspended in a pre-cooled wash buffer (pH 7.4) containing 0.1 mM calcium chloride and 0.06 mM disodium hydrogen phosphate solution, and centrifuged (5000 g, 20 min, 4°C). This washing step was repeated 2-3 times to thoroughly remove free ions and acetate ions, while avoiding disruption of the established mineral gradient due to solvent displacement. The final product was a structurally stable mineralized collagen gel.
[0028] 4. Layered casting and porosity gradient construction Example 1: Divide the mineralized collagen gel obtained in step 3 into three equal portions.
[0029] Add different masses of sucrose as pore-forming agents and gently stir until homogeneous: Bottom layer (high mineralization side): Add 10% (w / w) sucrose, corresponding to low porosity.
[0030] Middle layer: Add 30% (w / w) sucrose, corresponding to mesoporous structure.
[0031] Top layer (low mineralization side): Add 50% (w / w) sucrose, corresponding to high porosity.
[0032] In the order of bottom layer → middle layer → top layer, slowly inject the different formulas of gel into the mold to avoid mixing between layers.
[0033] Example 2: The complete gel obtained in step 3 is injected into a tall mold.
[0034] The mold is placed in a programmed cooling freezer and frozen at a specific cooling rate, for example, rapidly cooling from the highly mineralized side at the bottom of the mold (-5 degrees Celsius / min) while maintaining a slow cooling rate (-1 degrees Celsius / min) on the low-mineralized side at the top. Ice crystals grow to different sizes and shapes at different cooling rates, thus forming a gradient structure with gradually increasing porosity from bottom to top.
[0035] 5. Initial freeze-drying After molding, the sample should be quickly transferred to an ultra-low temperature freezer at -80 degrees Celsius for pre-freezing for more than 4 hours to ensure complete freezing.
[0036] The sample was then placed in a freeze dryer with a cold trap temperature below -50 degrees Celsius and a vacuum degree below 10 Pa.
[0037] Main drying stage: -20 degrees Celsius, 24 hours.
[0038] Analysis and drying stage: gradually increase the temperature to 25 degrees Celsius and maintain it for 24-48 hours.
[0039] The final product is a mineralized collagen sponge with a gradient pore structure.
[0040] 6. Crosslinking and washing Prepare a 0.05 wt% glutaraldehyde ethanol solution as a crosslinking agent.
[0041] The freeze-dried sponge was completely immersed in the crosslinking agent solution to ensure thorough saturation, and crosslinked at room temperature for 12 hours.
[0042] After cross-linking is complete, the sponge is removed and placed in a flowing pure water device for continuous washing at a flow rate of 100 mL / min for 48 hours to thoroughly remove residual byproducts.
[0043] 7. Final shaping The washed, wet cross-linked sponge was freeze-dried again (under the same conditions as in step 5).
[0044] The dried gradient sponge is passed through a pair of precisely temperature- and spacing-controlled roller presses, where slight pressure is applied and the sponge is rolled 1-2 times to densify it and form a final film product with uniform thickness and flexibility.
[0045] Experiment 1: Verifying the effect of gradient structures on bone regeneration in vivo (animal experiment) Animal model: A New Zealand white rabbit (n=6 / group) model of critical size skull defect (8mm in diameter) was used.
[0046] Experimental Groups: Experimental group: Product of this invention (possessing both mineralization gradient and porosity gradient) Comparative Example 1: Materials without mineralization gradient (only porosity gradient) Comparative Example 2: Materials without porosity gradient (only mineralization gradient) Comparative Example 3: Materials without gradient (uniform mineralization and porosity) Blank control group: No materials were implanted in the defect area. 3. Experimental methods: Implantation surgery: Four symmetrical defects are created in the skull, and different materials are randomly implanted. Postoperative care follows standard procedures.
[0047] Observation time points: week 4 and week 8 post-implantation.
[0048] Detection indicators and methods: Micro-CT Scanning and Analysis: Instrument: High-resolution Micro-CT scanner Analysis parameters: bone volume / tissue volume (BV / TV, %), i.e. new bone formation rate; bone mineral density (BMD); three-dimensional reconstruction to observe the morphology and distribution of new bone.
[0049] Histological analysis: Sample processing: Skull specimens were taken, decalcified, paraffin-embedded, and sectioned.
[0050] dyeing: Hematoxylin-eosin (H&E) staining: to observe the overall tissue morphology and cell distribution.
[0051] Masson trichrome staining: distinguishes between collagen (blue) and mineralized bone (red), visually indicating the maturity of new bone.
[0052] Image analysis: The proportion of new bone area was quantified using image analysis software. The expected results and data analysis at 4.8 weeks are shown in Table 1.
[0053] Table 1 The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the embodiments of the present invention.
Claims
1. A method for preparing a gradient biomimetic mineralized collagen-based artificial bone membrane, characterized in that, The method includes: (1) Preparation of collagenic acid solution: Collagen powder was dispersed and dissolved in acetic acid solution to obtain a homogeneous collagen acid solution. (2) Segmented mineralization and pH gradient construction: Under low temperature stirring conditions, calcium salt solution and phosphate solution are simultaneously added dropwise to the collagen acid solution at a constant molar ratio, and at least three mineralization stages are carried out in sequence. In each stage, the pH of the solution is adjusted to a different set range so that calcium and phosphate minerals gradually form a high-medium-low mineralization gradient in the collagen fibers, thereby obtaining a mineralized collagen system with a mineralization distribution gradient. (3) Settling, concentration and ion washing: The mineralized collagen system obtained in step (2) was subjected to static aging, fractionation centrifugation, and washing with buffer solution containing trace amounts of calcium and phosphorus to maintain the formed mineralization gradient structure. (4) Pore gradient construction and shaping: The mineralized collagen gel obtained in step (3) is subjected to molding treatment. Different amounts of pore-forming agents are added in layers for molding or frozen by using the temperature difference of the bottom-up program, so as to form a porosity gradient structure corresponding to the mineralization gradient during the molding process. (5) Freeze-drying: The molded sample was pre-frozen and freeze-dried to obtain a gradient mineralized collagen sponge with a stable three-dimensional pore structure. (6) Crosslinking and re-drying: The sponge was washed after contact with the crosslinking agent and then freeze-dried again to obtain a stable morphology for the material. (7) Calendering to form a film: The dried material is subjected to temperature-controlled and distance-controlled roller pressing to form a uniform and flexible gradient collagen-based artificial bone membrane.
2. The method according to claim 1, wherein, The concentration of the acetic acid solution in step (1) is 0.1M to 0.5M, and the final concentration of collagen is 3–10 mg / mL.
3. The method according to claim 1, wherein, In step (2), the calcium salt solution added simultaneously is a calcium chloride solution, and the phosphate solution is a disodium hydrogen phosphate solution.
4. The method according to claim 1, wherein, In step (2), the molar ratio of calcium salt solution to phosphate solution is maintained at Ca / P = 1.
67.
5. The method according to claim 1, wherein, In step (2), the three mineralization stages control the solution pH in the ranges of 4.5–5.0, 5.5–6.0, and 7.2–7.4, respectively.
6. The method according to claim 1, wherein, The washing solution in step (3) contains 0.05–0.2 mM calcium chloride and 0.03–0.1 mM disodium hydrogen phosphate.
7. The method according to claim 1, wherein, In step (4)(a), the pore-forming agent is sucrose, and its addition amount is 5% to 60% of the gel mass.
8. The method according to claim 1 or 7, wherein, In step (4)(a), the amount of pore-forming agent added increases layer by layer along the mineralization gradient direction to form a porosity gradient in the opposite direction to the mineralization gradient.
9. The method according to claim 1, wherein, In step (4)(b), the cooling process is as follows: the cooling rate on the high mineralization side is 3–8°C / min, and the cooling rate on the low mineralization side is 0.5–2°C / min.
10. A collagen-based artificial bone membrane prepared by the method of any one of claims 1-9.