Biomimetic mineralized collagen nanohydroxyapatite membrane

Abstract

The application discloses a kind of bionic mineralized collagen nanometer osteogenic membrane, belong to biological medical bone tissue engineering material technical field.The osteogenic membrane is modified nanometer collagen as organic matrix, and nanometer hydroxyapatite crystal is grown on the surface of collagen fiber by in-situ bionic mineralization technology, and natural biological crosslinking agent is matched with osteogenic active additive, and the nanometer porous organic-inorganic composite membrane material highly consistent with natural bone tissue structure, component is constructed.The application optimizes raw material ratio and preparation process accurately, solves the technical problems of low bionic degree, poor mechanical property, unbalanced degradation rate, weak osteogenic activity and poor biocompatibility of traditional osteogenic membrane.

Description

[0001] This invention belongs to the fields of biomedical materials and bone tissue engineering technology, specifically relating to a biomimetic mineralized collagen nano-osteoblastic membrane for bone defect repair and bone regeneration induction, and its preparation method. The product of this invention is mainly used in various bone defect repair, fracture healing, and bone augmentation surgeries in orthopedics, dentistry, and maxillofacial surgery, and falls under the category of novel functional biomedical implant materials.

[0002] Bone defects, nonunion, and insufficient bone volume are common conditions in clinical orthopedics and dentistry. Trauma, infection, tumor resection, congenital malformations, and other factors can all cause varying degrees of bone tissue loss, severely impacting patients' limb function and quality of life. Currently, clinical bone repair materials mainly include three categories: autologous bone, allogeneic bone, and synthetic bone repair materials. Among them, autologous bone is the clinical gold standard, but it has drawbacks such as limited availability, secondary trauma, and donor site complications; allogeneic bone has problems such as immune rejection, risk of disease transmission, and insufficient bone-inducing activity, limiting its clinical application.

[0003] Among existing artificial bone repair materials, traditional collagen membranes, hydroxyapatite membranes, and polylactic acid polymer membranes are widely used, but all have obvious technical defects: ordinary collagen membranes lack mineralization structure, have poor mechanical properties, and degrade too quickly, making them unable to match the bone tissue regeneration cycle, and their bone induction and osteoconduction capabilities are weak; single hydroxyapatite is brittle and lacks flexibility, has poor interfacial bonding with host bone tissue, and is prone to peeling and detachment; polymer synthetic membranes have limited biocompatibility, their degradation products easily cause local inflammation, lack biomimetic bone tissue structure, and are difficult to effectively induce cell adhesion, proliferation, and osteogenic differentiation.

[0004] Meanwhile, existing osteoblastic membrane materials are mostly micron-sized structures with simple pore structures and small specific surface areas, resulting in low cell adsorption capacity and nutrient permeability. They cannot simulate the nanoscale collagen-inorganic mineral composite microstructure of natural bone tissue, leading to low osteogenic efficiency and poor osteointegration. In addition, the degradation rate of most osteoblastic membranes is uncontrollable. Premature degradation fails to provide stable support for bone regeneration, while excessively slow degradation hinders the remodeling of new bone tissue, making it difficult to meet the personalized repair needs in clinical practice.

[0005] Based on the structural characteristics of natural bone tissue, which is mainly composed of ordered composites of nano-collagen fibers and nano-hydroxyapatite crystals, developing a nano-biomimetic mineralized collagen osteogenic membrane with excellent biocompatibility, biomimetic microstructure, controllable degradation, good mechanical properties and high osteogenic activity is a current research focus and urgent clinical need in the field of bone tissue engineering materials.

[0006] This invention aims to overcome the shortcomings of existing osteoblastic membranes, such as low biomimicry, poor mechanical properties, mismatched degradation rates, weak osteogenic activity, and unsatisfactory osteointegration, by providing a biomimetic mineralized collagen nano-osteoblastic membrane. This invention constructs a nanoscale organic-inorganic composite structure using in-situ biomimetic mineralization technology, precisely mimicking the microstructure and component ratios of natural bone tissue. It possesses excellent flexibility, mechanical support, controllable degradation, and highly efficient osteogenic induction capabilities, effectively promoting bone defect repair and new bone tissue remodeling, making it suitable for various clinical bone repair scenarios.

[0007] A biomimetic mineralized collagen nano-osteoblastic membrane is disclosed. The osteoblastic membrane is a nano-scale collagen-hydroxyapatite organic-inorganic composite membrane material, which is composed of a modified nano-collagen matrix and in-situ grown nano-hydroxyapatite crystals. The overall structure is a porous network nanostructure with a thickness of 0.1–0.5 mm, a porosity of 60%–85%, and a nanopore size distribution of 50–500 nm.

[0008] The raw material components of the biomimetic mineralized collagen nano osteogenic membrane, by mass percentage, include: 40%–65% modified nano collagen, 30%–55% nano hydroxyapatite, 1%–3% biocompatible crosslinking agent, and 0.5%–2% osteogenic active agent, with the total mass percentage of each component being 100%.

[0009] The modified nano-collagen is a nanoscale collagen fiber obtained by enzymatic modification of fish-derived type I collagen. The fiber diameter is 20–100 nm. Compared with ordinary collagen, it has higher purity, better dispersibility and cell affinity, and can stably support the growth of inorganic mineral crystals.

[0010] The nano-hydroxyapatite is a needle-shaped nanocrystal generated by in-situ biomimetic mineralization. The crystals are 50–200 nm in length and 10–30 nm in width, and are orderly deposited on the surface and inside the pores of nano-collagen fibers. The composition is highly consistent with that of natural bone minerals.

[0011] The biocompatible crosslinking agent is genipin, which is low in toxicity and highly efficient. It can achieve gentle crosslinking of collagen molecules, improve the overall mechanical stability and degradation resistance of the membrane material, and avoid cytotoxicity and inflammatory reactions caused by chemical crosslinking agents.

[0012] The osteogenic active agent is a compound system of sodium β-glycerophosphate and vitamin C, which can synergistically regulate the adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, and further improve osteogenic efficiency.

[0013] This invention also provides a method for preparing the biomimetic mineralized collagen nano-osteoblastic membrane, comprising the following steps: Step 1: Preparation of modified nano-collagen: Take purified fish-derived type I collagen and use trypsin for low-temperature and mild enzymatic hydrolysis at a temperature of 4℃–8℃ for 12–24h. After hydrolysis, dialysis purification and freeze-drying are performed to obtain modified nano-collagen fibers with a diameter of 20–100nm for later use.

[0014] Step 2: Preparation of biomimetic mineralization precursor solution: Disperse the modified nano-collagen in deionized water to prepare a collagen dispersion with a mass concentration of 0.5%–2%. After stirring evenly, add sodium β-glycerophosphate and vitamin C in sequence, and continue stirring for 30–60 minutes to obtain a uniformly mixed precursor solution.

[0015] Step 3: In-situ biomimetic mineralization reaction: Slowly add calcium chloride solution to the precursor solution, adjust the pH of the system to 7.2–7.6 (physiological neutral environment), and stir at 37℃ for 4–8 hours. Through in-situ mineralization reaction, needle-like nano-hydroxyapatite crystals are generated on the surface of nano-collagen fibers, and mineralized collagen composite solution is obtained.

[0016] Step 4, cross-linking and molding: Add genipin cross-linking agent to the mineralized collagen composite liquid, and let it stand at room temperature for 2–4 hours to allow the composite system to fully cross-link and solidify, forming a stable nanocomposite system.

[0017] Step 5, Post-film treatment: The cross-linked composite liquid is spread into a film, freeze-dried under vacuum, and after sterile cleaning and low-temperature sterilization, a biomimetic mineralized collagen nano-osteoblastic membrane is obtained.

[0018] Compared with the prior art, the present invention has the following significant advantages: 1. Highly biomimetic structure with excellent osteogenic activity: This invention uses in-situ biomimetic mineralization technology to construct a nanoscale collagen-hydroxyapatite composite structure, which accurately simulates the composition, microstructure and pore structure of natural bone tissue. The high specific surface area of ​​the nanoporous structure can effectively adsorb cells and enrich growth factors, significantly promoting osteogenic differentiation of stem cells. Its osteoconduction and osteoinduction properties are far superior to traditional osteoblastic membrane materials.

[0019] 2. Strong performance adaptability: Through nanostructure regulation and mild cross-linking modification, this invention takes into account both the flexibility and mechanical support of the membrane material, which can be arbitrarily applied to the bone defect wound surface. At the same time, it has good tensile and tear resistance properties and can stably maintain the space for bone defect repair. The degradation cycle is 3-6 months, which perfectly matches the growth and remodeling cycle of new bone tissue, with no residue and no stress obstruction.

[0020] 3. High biocompatibility: All raw materials are biomedical grade, using genipin natural cross-linking agent instead of traditional chemical cross-linking agent. It has no cytotoxicity, no immune rejection, no inflammatory reaction, and excellent biocompatibility. After implantation, it can be gradually degraded and absorbed by the body, and the degradation products can participate in bone tissue metabolism.

[0021] 4. Simple and controllable process: The preparation process of this invention is mild and the conditions are easy to control. It does not require high temperature, high pressure and strong chemical reaction. The mineralization, porosity and membrane thickness can be precisely controlled. It can be mass-produced and adapted to the clinical repair needs of bone defects of different parts and sizes. It has a wide range of applications.

[0022] The present invention will be further described in detail and completely below with reference to specific embodiments. The following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Example

[0023] A biomimetic mineralized collagen nano-osteoblastic membrane, the raw material components by mass percentage are: modified nano-collagen 55%, nano-hydroxyapatite 42%, genipin 2%, and osteogenic active agent 1%.

[0024] Preparation method: 1. Preparation of modified nano-collagen: Purified fish-derived type I collagen was enzymatically hydrolyzed with trypsin at 4℃ for 18h, purified by dialysis, and then freeze-dried to obtain modified nano-collagen fibers with an average diameter of 50nm.

[0025] 2. Preparation of precursor solution: Disperse the modified nano-collagen in deionized water to prepare a collagen dispersion with a mass concentration of 1%. After stirring for 40 min, add 0.6% sodium β-glycerophosphate and 0.4% vitamin C, and mix well.

[0026] 3. In-situ mineralization: 0.1 mol / L calcium chloride solution was slowly added dropwise to adjust the pH of the system to 7.4. The reaction was carried out at a constant temperature of 37℃ for 6 hours to complete the in-situ growth of nano-hydroxyapatite.

[0027] 4. Cross-linking molding: Add 2% genipin cross-linking agent, let stand at room temperature for 3 hours for cross-linking, freeze dry under vacuum to form the final product, and sterilize to obtain the osteoblastocyst.

[0028] The osteogenic membrane prepared in this embodiment has a thickness of 0.3 mm, a porosity of 75%, a nanopore size of 80–300 nm, excellent flexibility, and moderate mechanical strength. In vitro cell experiments show that the cell adhesion rate and proliferation rate are significantly better than those of ordinary collagen membranes, and the osteogenic differentiation ability is outstanding. Example

[0029] A biomimetic mineralized collagen nano-osteoblastic membrane, the raw material components by mass percentage are: modified nano-collagen 45%, nano-hydroxyapatite 52%, genipin 2.5%, and osteogenic active agent 0.5%.

[0030] The preparation method is the same as in Example 1, with the mineralization reaction time controlled at 7 hours and the crosslinking time at 3.5 hours. The finished osteoblastic membrane has a thickness of 0.4 mm, a porosity of 70%, a higher mineral content, and better mechanical support properties, making it suitable for repairing bone defects in weight-bearing areas. Example

[0031] A biomimetic mineralized collagen nano-osteoblastic membrane, the raw material components by mass percentage are: modified nano-collagen 62%, nano-hydroxyapatite 35%, genipin 1.5%, and osteogenic active agent 1.5%.

[0032] The preparation method is the same as in Example 1, with the mineralization reaction time adjusted to 5 hours and the cross-linking time to 2.5 hours. The resulting osteoblastic membrane has a thickness of 0.2 mm and a porosity of 82%, with a more porous structure, resulting in higher efficiency in cell penetration and nutrient transport. It is suitable for superficial bone defects and fine repair of maxillofacial bones.

[0033] The osteoblasts prepared in Examples 1–3 of this invention were subjected to performance testing. The results showed that the osteoblasts of this invention had a cell compatibility grade of I and no cytotoxicity. After implantation into animals, there was no inflammation or rejection reaction. Most of the degradation was completed in 3 months and complete degradation was completed in 6 months, which is highly consistent with the bone regeneration cycle. The osteogenic induction capacity was improved by more than 40% compared with traditional commercial collagen osteoblasts. The newly formed bone tissue was dense, well integrated, and without fibrous encapsulation.

Claims

1. A biomimetic mineralized collagen nano-osteoblastic membrane, characterized in that, The osteogenic membrane is a nano-scale collagen-hydroxyapatite organic-inorganic composite membrane material, composed of a modified nano-collagen matrix and in-situ grown nano-hydroxyapatite crystals. The overall structure is a porous network nanostructure with a thickness of 0.1–0.5 mm, a porosity of 60%–85%, and a nanopore size distribution of 50–500 nm. The raw material components of the osteogenic membrane include, by mass percentage: 40%–65% modified nano-collagen, 30%–55% nano-hydroxyapatite, 1%–3% biocompatible crosslinking agent, and 0.5%–2% osteogenic active agent, with the total mass percentage of each component being 100%.

2. The biomimetic mineralized collagen nano-osteoblastic membrane according to claim 1, characterized in that, The modified nano-collagen is a nano-collagen fiber obtained by low-temperature enzymatic hydrolysis modification of fish-derived type I collagen, with a fiber diameter of 20–100 nm.

3. The biomimetic mineralized collagen nano-osteoblastic membrane according to claim 1, characterized in that, The nano-hydroxyapatite is a needle-shaped nanocrystal with a length of 50–200 nm and a width of 10–30 nm, which is deposited in an orderly manner on the surface and inside the pores of the modified nano-collagen fiber.

4. The biomimetic mineralized collagen nano-osteoblastic membrane according to claim 1, characterized in that, The biocompatible cross-linking agent is genipin, and the osteogenic active agent is a compound system of sodium β-glycerophosphate and vitamin C.

5. A method for preparing the biomimetic mineralized collagen nano-osteoblastic membrane according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Preparation of modified nano-collagen: Take purified fish-derived type I collagen, hydrolyze it with trypsin at 4℃–8℃ for 12–24h, dialyze to purify, and freeze-dry to obtain modified nano-collagen fibers; Step 2, Preparation of biomimetic mineralization precursor solution: Disperse modified nano-collagen in deionized water to prepare a collagen dispersion with a mass concentration of 0.5%–2%, add osteogenic active adjuvant, and stir evenly to obtain the precursor solution; Step 3: In-situ biomimetic mineralization reaction: Add calcium chloride solution dropwise to the precursor solution, adjust the pH of the system to 7.2–7.6, and stir at 37℃ for 4–8 hours to obtain mineralized collagen composite solution; Step 4, Crosslinking and Molding: Add genipin crosslinking agent and allow to crosslink at room temperature for 2–4 hours; Step 5, Post-film formation treatment: The film is laid flat, freeze-dried under vacuum, and sterilized to obtain the finished biomimetic mineralized collagen nano-osteoblastic membrane.

6. The preparation method according to claim 5, characterized in that, The concentration of the calcium chloride solution mentioned in step three is 0.05–0.2 mol / L.

7. The biomimetic mineralized collagen nano-osteoblastic membrane according to claim 1, characterized in that, The osteoblastic membrane has a degradation cycle of 3–6 months, which is compatible with the human bone tissue regeneration and remodeling cycle, and can be used for bone defect repair in orthopedics, dentistry, and maxillofacial surgery.