Chitosan / nano-hydroxyapatite guided bone regeneration membrane with dense and sparse composite structure and preparation method thereof
By preparing a chitosan/nanohydroxyapatite guided bone regeneration membrane with a sparse-dense composite structure, the problems of high price and insufficient bone mineralization capacity of existing guided bone regeneration membranes are solved, achieving low-cost and efficient restoration of dental bone defects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG UNIV
- Filing Date
- 2024-09-25
- Publication Date
- 2026-04-17
AI Technical Summary
Existing guided bone regeneration membranes are expensive, lack bone mineralization capacity, have poor treatment effects, and are mostly derived from animal collagen, making them costly and difficult to afford for the general public.
Chitosan derived from shrimp and crab shells was used as an organic matrix, and phosphate and calcium salts were doped into it. A chitosan/nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure was prepared by freeze drying and mechanical extrusion. The functional groups of chitosan were used to induce the crystallization of nanohydroxyapatite in situ, forming a dense and loose bilayer structure.
The preparation process is simple and low-cost, with good nano-dispersion and bone mineralization effects. The dense layer prevents rapid tissue cells from entering the defect area, while the loose layer guides cell growth and provides an optimal tissue regeneration environment.
Smart Images
Figure CN119139565B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical materials technology, specifically relating to a chitosan / nanohydroxyapatite guided bone regeneration membrane with a sparse-dense composite structure and its preparation method. Background Technology
[0002] The function of a guiding bone regeneration membrane is to act as a barrier, preventing invasion from surrounding soft tissues and allowing osteoblasts on the bone surface sufficient time to proliferate, ultimately achieving tissue regeneration. Since its introduction, various types of guiding bone regeneration membranes have emerged, differing in structure (single-layer, double-layer, and multi-layer), materials (absorbable and non-absorbable), and manufacturing processes (e.g., electrospinning). Currently, most existing guiding bone regeneration membranes are derived from animal collagen, resulting in high costs, and imported products dominate the market, making them unaffordable for most people. Therefore, inventing a new type of guiding bone regeneration membrane to benefit a wider range of people is an urgent problem to be solved.
[0003] To address existing problems, this invention starts with raw materials, selecting chitosan derived from waste shrimp and crab shells as the main raw material. This gives the guided bone regeneration membrane good biocompatibility and biodegradability, while reducing costs. The doping with phosphates and calcium salts effectively mimics the structure of natural bone tissue. Leveraging the abundant functional groups (amino groups) of chitosan, the oriented crystallization and uniform dispersion of nano-hydroxyapatite are induced in situ, thereby enhancing bone mineralization capacity through nanomechanical enhancement. A dense-sparse composite bilayer structure is prepared using a mechanical extrusion method. This simple preparation process allows for large-scale production and holds promise as a novel guided bone regeneration membrane that plays a unique role in the repair of dental bone defects. Summary of the Invention
[0004] The purpose of this invention is to solve the problems of high cost, poor bone mineralization capacity, and poor treatment effect of existing guided bone regeneration membranes. Using chitosan derived from shrimp and crab shells as an organic matrix, calcium nitrate as a calcium source, and dipotassium hydrogen phosphate as a phosphate salt, this invention provides a method for preparing a chitosan / nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure by using freeze-drying technology combined with mechanical extrusion. The preparation process is simple, low-cost, and exhibits good nano-dispersion and bone mineralization effect.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] Utilizing material preparation techniques and methods, a biocompatible, biodegradable chitosan / nanohydroxyapatite guided bone regeneration membrane with a porous composite structure was designed for the repair of dental bone defects. Using chitosan as the organic matrix, calcium salts and phosphates were uniformly mineralized and crystallized on the surface of the chitosan polymer chains through freeze-drying to form nanohydroxyapatite, enhancing bone mineralization capacity through nanomechanical enhancement. A simple mechanical extrusion method was used to create an asymmetric double-layer structure. This is because one side of the membrane contacts a smooth mechanical surface, compacting all the original pores into a dense layer, while the other side contacts a gridded polytetrafluoroethylene mold, preserving most of the original pores during extrusion. The dense layer primarily acts as a shield, effectively preventing rapidly growing tissue cells from entering the root (bone) defect area, providing sufficient space for osteogenic formation in the defect area; the porous layer guides the recruitment of periodontal ligament (or bone) cells and provides them with growth space, forming a microenvironment conducive to tissue regeneration. A chitosan / nano-hydroxyapatite guided bone regeneration membrane with a porous-dense composite structure was thus prepared. The specific steps included:
[0007] (1) Prepare an acetic acid solution by dissolving an appropriate amount of chitosan in this weak acid solution and stirring in a water bath to mix it evenly, thus obtaining a chitosan acetic acid solution.
[0008] (2) Add calcium nitrate solution to the above chitosan acetic acid solution and stir continuously until uniform. Then add an appropriate amount of dipotassium hydrogen phosphate solution and stir continuously for a period of time to obtain the precursor solution for guiding bone regeneration membrane.
[0009] (3) The pre-frozen solution for guiding bone regeneration membrane was transferred to a refrigerator for pre-freezing, and then freeze-dried for several hours in a freeze dryer. After that, it was soaked in an ethanol / deionized water solution of sodium hydroxide to achieve in-situ mineralization and crystallization on the surface of chitosan to achieve uniform distribution of nano-hydroxyapatite.
[0010] (4) Rinse repeatedly with deionized water until neutral, transfer to a refrigerator for pre-freezing, and then freeze dry in a freeze dryer for several hours.
[0011] (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a grid and press it for several hours under a certain mechanical strength to obtain a chitosan / nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure.
[0012] Furthermore, in the chitosan acetic acid solution of step (1), the amount of acetic acid used is 0.5 mL, the amount of water used is 49.5 mL, and the mass of chitosan powder is 1 g.
[0013] Furthermore, in step (2), the concentration of the calcium nitrate solution is 2 mol / L, and the volume is 2 mL. The concentration of the dipotassium hydrogen phosphate solution is 1.2 mol / L, and the volume is 2 mL.
[0014] Furthermore, in step (3), the pre-freezing method is to place it at 4°C for 1 hour and at -80°C for 24 hours.
[0015] Further, in step (3), the sodium hydroxide in the ethanol / deionized water solution contains 1 g of sodium hydroxide, 125 mL of ethanol, and 125 mL of deionized water. The soaking time is 10 h.
[0016] Furthermore, in step (4), the pre-freezing method is to directly transfer it to -80°C for placement.
[0017] Furthermore, in step (5), the mechanical extrusion is performed at a strength of 0.2 MPa to 5 MPa.
[0018] Furthermore, in step (5), the mechanical extrusion time is 12h to 96h.
[0019] Furthermore, in step (5), during mechanical extrusion, one side of the membrane contacts a smooth mechanical surface, and the pores are compacted into a dense layer, while the other side of the membrane contacts a polytetrafluoroethylene mold with a grid. During the extrusion process, the original pores are preserved while the surface becomes more porous.
[0020] A chitosan / nanohydroxyapatite guided bone regeneration membrane with a sparse-dense composite structure prepared by the method described above.
[0021] The significant advantages of this invention are:
[0022] (1) This invention utilizes the abundant functional groups (amino groups) of chitosan to induce the orientation crystallization and uniform dispersion of nano-hydroxyapatite in situ, thereby enhancing the bone mineralization capacity through nanomechanical enhancement.
[0023] (2) The bone regeneration membrane prepared by the present invention has a dense-sparse composite double-layer structure. Its dense layer can effectively prevent fast-growing tissue cells from entering the tooth root (bone) defect area, while the loose porous layer can guide the recruitment of periodontal ligament (or bone) cells and provide them with growth space, thus ensuring the best tissue regeneration effect.
[0024] (3) The present invention uses mechanical extrusion, which has low preparation cost, simple method, green and environmentally friendly, and can be mass-produced. Attached Figure Description
[0025] Figure 1 Structural diagram of a PTFE mold with a grid; (a) top view of the mold, (b) side view of the mold;
[0026] Figure 2 These are surface morphology photographs of Examples 1-5, where S-1 to S-5 correspond to Examples 1 to 5, respectively;
[0027] Figure 3The image shows a depth-of-field electron microscope image of the double-sided dense chitosan / nanohydroxyapatite guided bone regeneration membrane prepared in Example 1.
[0028] Figure 4 Scanning electron microscope images of chitosan / nanohydroxyapatite guided bone regeneration membranes with a porous-dense composite structure prepared in Examples 2-5: (a) porous surface, (b) dense surface, (c) cross-section.
[0029] Figure 5 The water absorbency of the guiding bone regeneration membranes prepared in Examples 1-5 was determined.
[0030] Figure 6 Biocompatibility test of the guiding bone regeneration membrane prepared in Example 5 (n=4, *p<0.05, **p<0.01, ***p<0.001). Detailed Implementation
[0031] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments. However, the following examples are merely examples of this invention and do not represent the scope of protection of this invention. The scope of protection of this invention is determined by the claims.
[0032] Example 1
[0033] (1) Prepare 50 mL of 1% (V / V) acetic acid solution, accurately weigh 1 g of chitosan and dissolve it in this weak acid solution, and stir it in a water bath at 60°C for 4 h to make it evenly mixed.
[0034] (2) Add 2 mL of 2 mol / L calcium nitrate solution to the above chitosan acetic acid solution and stir continuously for 2 h. Then add 2 mL of 1.2 mol / L dipotassium hydrogen phosphate solution and stir continuously for 4 h to obtain the precursor solution for guiding bone regeneration membrane.
[0035] (3) Pre-freeze in a refrigerator: place at 4℃ for 1 hour, then at -80℃ for 24 hours. Freeze dry at -80℃ for 48 hours using a freeze dryer. Afterward, soak in an ethanol / deionized water solution of sodium hydroxide (sodium hydroxide mass is 1 g, ethanol volume is 125 mL, and deionized water volume is 125 mL) for 10 hours to achieve in-situ mineralization and crystallization on the surface of chitosan, thus achieving uniform distribution of nano-hydroxyapatite.
[0036] (4) Rinse repeatedly with deionized water until neutral. Transfer to a refrigerator and pre-freeze at -80℃ for 24 hours, then freeze dry at -80℃ for 48 hours.
[0037] (5) After freeze-drying, the membrane is extruded for 24 hours under a mechanical strength of 0.66 MPa to obtain a dense chitosan / nanohydroxyapatite guided bone regeneration membrane on both sides.
[0038] Example 2
[0039] (1) Prepare 50 mL of 1% (V / V) acetic acid solution, accurately weigh 1 g of chitosan and dissolve it in this weak acid solution, and stir it in a water bath at 60°C for 4 h to make it evenly mixed.
[0040] (2) Add 2 mL of 2 mol / L calcium nitrate solution to the above chitosan acetic acid solution and stir continuously for 2 h. Then add 2 mL of 1.2 mol / L dipotassium hydrogen phosphate solution and stir continuously for 4 h to obtain the precursor solution for guiding bone regeneration membrane.
[0041] (3) Pre-freeze in a refrigerator: place at 4℃ for 1 hour, then at -80℃ for 24 hours. Freeze dry at -80℃ for 48 hours using a freeze dryer. Afterward, soak in an ethanol / deionized water solution of sodium hydroxide (sodium hydroxide mass is 1 g, ethanol volume is 125 mL, and deionized water volume is 125 mL) for 10 hours to achieve in-situ mineralization and crystallization on the surface of chitosan, thus achieving uniform distribution of nano-hydroxyapatite.
[0042] (4) Rinse repeatedly with deionized water until neutral. Transfer to a refrigerator and pre-freeze at -80℃ for 24 hours, then freeze dry at -80℃ for 48 hours.
[0043] (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a 16-mesh grid and press it for 24 hours under a mechanical strength of 0.66 MPa to obtain the chitosan / nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure.
[0044] Example 3
[0045] (1) Prepare 50 mL of 1% (V / V) acetic acid solution, accurately weigh 1 g of chitosan and dissolve it in this weak acid solution, and stir it in a water bath at 60°C for 4 h to make it evenly mixed.
[0046] (2) Add 2 mL of 2 mol / L calcium nitrate solution to the above chitosan acetic acid solution and stir continuously for 2 h. Then add 2 mL of 1.2 mol / L dipotassium hydrogen phosphate solution and stir continuously for 4 h to obtain the precursor solution for guiding bone regeneration membrane.
[0047] (3) Pre-freeze in a refrigerator: place at 4℃ for 1 hour, then at -80℃ for 24 hours. Freeze dry at -80℃ for 48 hours using a freeze dryer. Afterward, soak in an ethanol / deionized water solution of sodium hydroxide (sodium hydroxide mass is 1 g, ethanol volume is 125 mL, and deionized water volume is 125 mL) for 10 hours to achieve in-situ mineralization and crystallization on the surface of chitosan, thus achieving uniform distribution of nano-hydroxyapatite.
[0048] (4) Rinse repeatedly with deionized water until neutral. Transfer to a refrigerator and pre-freeze at -80℃ for 24 hours, then freeze dry at -80℃ for 48 hours.
[0049] (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a 16-mesh grid and press it under a mechanical strength of 0.66 MPa for 48 hours to obtain the chitosan / nanohydroxyapatite guided bone regeneration membrane with a sparse-dense composite structure.
[0050] Example 4
[0051] (1) Prepare 50 mL of 1% (V / V) acetic acid solution, accurately weigh 1 g of chitosan and dissolve it in this weak acid solution, and stir it in a water bath at 60°C for 4 h to make it evenly mixed.
[0052] (2) Add 2 mL of 2 mol / L calcium nitrate solution to the above chitosan acetic acid solution and stir continuously for 2 h. Then add 2 mL of 1.2 mol / L dipotassium hydrogen phosphate solution and stir continuously for 4 h to obtain the precursor solution for guiding bone regeneration membrane.
[0053] (3) Pre-freeze in a refrigerator: place at 4℃ for 1 hour, then at -80℃ for 24 hours. Freeze dry at -80℃ for 48 hours using a freeze dryer. Afterward, soak in an ethanol / deionized water solution of sodium hydroxide (sodium hydroxide mass is 1 g, ethanol volume is 125 mL, and deionized water volume is 125 mL) for 10 hours to achieve in-situ mineralization and crystallization on the surface of chitosan, thus achieving uniform distribution of nano-hydroxyapatite.
[0054] (4) Rinse repeatedly with deionized water until neutral. Transfer to a refrigerator and pre-freeze at -80℃ for 24 hours, then freeze dry at -80℃ for 48 hours.
[0055] (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a 16-mesh grid and press it for 24 hours under a mechanical strength of 0.88 MPa to obtain the chitosan / nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure.
[0056] Example 5
[0057] (1) Prepare 50 mL of 1% (V / V) acetic acid solution, accurately weigh 1 g of chitosan and dissolve it in this weak acid solution, and stir it in a water bath at 60°C for 4 h to make it evenly mixed.
[0058] (2) Add 2 mL of 2 mol / L calcium nitrate solution to the above chitosan acetic acid solution and stir continuously for 2 h. Then add 2 mL of 1.2 mol / L dipotassium hydrogen phosphate solution and stir continuously for 4 h to obtain the precursor solution for guiding bone regeneration membrane.
[0059] (3) Pre-freeze in a refrigerator: place at 4℃ for 1 hour, then at -80℃ for 24 hours. Freeze dry at -80℃ for 48 hours using a freeze dryer. Afterward, soak in an ethanol / deionized water solution of sodium hydroxide (sodium hydroxide mass is 1 g, ethanol volume is 125 mL, and deionized water volume is 125 mL) for 10 hours to achieve in-situ mineralization and crystallization on the surface of chitosan, thus achieving uniform distribution of nano-hydroxyapatite.
[0060] (4) Rinse repeatedly with deionized water until neutral. Transfer to a refrigerator and pre-freeze at -80℃ for 24 hours, then freeze dry at -80℃ for 48 hours.
[0061] (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a 16-mesh grid and press it for 48 hours under a mechanical strength of 0.88 MPa to obtain the chitosan / nanohydroxyapatite guided bone regeneration membrane with a dense-sparse composite structure.
[0062] The polytetrafluoroethylene mold with 16-mesh raised grid used in Examples 2-5 is shown below. Figure 1 (a) is a top view of the mold, and (b) is a side view of the mold. The cuboid protrusions (1mm*1mm*0.3mm) on the top of the mold form the grid structure of the mold. The gaps between the protrusions can effectively preserve most of the original loose structure of the guide bone regeneration membrane.
[0063] The morphological images of the guided bone regeneration membrane prepared in Examples 1-5 are shown below. Figure 2 .
[0064] The depth-of-field electron microscope image of the preparation of the guided bone regeneration membrane in Example 1 is shown below. Figure 3 .
[0065] SEM images of the guided bone regeneration membrane prepared in Examples 2-5 are shown below. Figure 4 .
[0066] The water absorption properties of the guided bone regeneration membrane prepared in Examples 1-5 are shown in the figure. Figure 5 .
[0067] The biocompatibility test of the guided bone regeneration membrane prepared in Example 5 is shown in [reference needed]. Figure 6 .
[0068] Topography of the bone regeneration membrane (guided bone regeneration membrane) Figure 2 It can be seen that the guide bone regeneration membrane prepared in the experiment is a disc-shaped membrane with a diameter of about 1.2 cm and a thickness of about 0.5~1.2 mm, which is loose on one side and dense on the other. The guide bone regeneration membrane after freeze-drying is white in color. As the pressing time and pressing pressure increase, the thickness of the membrane gradually decreases. It was found that there was no significant difference in the surface of the five groups of bone membranes.
[0069] Through depth-of-field electron microscopy images ( Figure 3 The guiding bone regeneration membrane prepared in Example 1 exhibits a structure with compacted pores.
[0070] SEM revealed that all four groups of guiding bone regeneration membranes exhibited significantly different densities. Figure 4 The loose surface of the membrane exhibits a porous structure (a), while the dense surface exhibits a structure where the pores are compacted (b). Furthermore, with increasing pressing time and pressure, the porous characteristics of the loose surface become less pronounced, while the dense surface becomes even denser. This change is consistent with the characteristics of the mechanical extrusion method of this invention.
[0071] With appropriate water absorption capacity, it helps the periosteum maintain a moist environment at the site of tissue loss, providing a favorable external environment for cell growth and reproduction. Figure 5 The results of the water absorption analysis of the periosteum show that all five groups of periosteum materials exhibit high water absorption, with absorption rates exceeding 300% (S-1: 361%, S-2: 758%, S-3: 485%, S-4: 719%, S-5: 456%). Such high absorption rates are sufficient to maintain the dynamic balance of nutrients and active factors in the microenvironment of the defect site, ensuring the transport and metabolism of substances in newly formed tissue. Among them, S-2, S-3, S-4, and S-5 have higher water absorption than S-1, mainly due to the porous structure of their loose surfaces. However, with the extension of pressing time and the increase of pressing pressure, the water absorption of the membrane gradually decreases, primarily because the porosity of the loose surface of the membrane becomes less pronounced. This change is consistent with the characteristics of the mechanical extrusion method of this invention.
[0072] The CCK-8 reagent can rapidly test the effect of periosteum on the proliferation of MC3T3-E1 cells. By measuring the OD value of the culture plate at 450 nm using a microplate reader and then calculating using a formula, the cell number and viability can be visually displayed. The cell viability after co-culturing MC3T3-E1 cells with periosteum extract is shown below. Figure 6 As shown. From Figure 6It was observed that the number of cells increased with prolonged culture time. At day 1 of co-culture, the cell proliferation in the experimental group was slightly lower than that in the control group, but significant cell proliferation was observed on days 3 and 5. Compared to the control group, the experimental group showed higher cell viability, and the experimental results were significantly different. This is attributed to the chitosan / nanohydroxyapatite system's ability to promote cell proliferation. This indicates that chitosan / nanohydroxyapatite has good biocompatibility and no toxic effects on cells. Therefore, the experimental results demonstrate that the chitosan / nanohydroxyapatite-guided bone regeneration membrane has excellent biocompatibility and helps promote the adhesion and proliferation of MC3T3-E1 cells.
[0073] Water absorption test:
[0074] The dried guiding bone regeneration membranes from different groups were weighed and recorded as W1. They were then placed in 50 mL centrifuge tubes containing 30 mL of distilled water, labeled, and placed in a constant-temperature shaker at 37°C and 150 r / min for 24 hours. Excess surface moisture was absorbed with filter paper and the membranes were weighed and recorded as W2. Each sample was repeated three times. The water absorbency (A) of the prepared guiding bone regeneration membranes was calculated as follows.
[0075] A(%) = (W2 - W1) / W1 * 100%
[0076] In the formula, W2 represents the weight of the support after water absorption, and W1 represents the mass of the support before water absorption.
[0077] Biocompatibility testing:
[0078] The biocompatibility of the guided bone regeneration membrane was assessed using a CCK-8 assay kit. MC3T3-E1 cells in good growth condition were resuspended and counted to calculate cell concentration. Cells were then seeded at a density of 5000 cells per well in sterile 96-well plates. A suitable amount of complete culture medium was added to ensure a volume of 100 μL per well. Five parallel wells were set up for each group, and the complete culture medium was changed every two days. Cells were stained using a CCK-8 assay kit on days 1, 3, and 5, following the instructions of the CCK-8 assay kit (Shanghai Sangon Biotech). The absorbance at 450 nm was measured using a microplate reader. Note that wells containing only the CCK-8 assay solution but no cells were used as blank wells to subtract background values. DMEM / F-12 complete cell culture medium served as a blank control group. The guided bone regeneration membrane extract group from Example 5 was used as the experimental group. Cell viability (S) was calculated using the following formula:
[0079] S(%) = (Os - Ob / Oc - Ob) * 100%
[0080] In the formula, Os represents the absorbance value of the experimental group, Oc represents the absorbance value of the blank control group, and Ob represents the absorbance value of the blank well containing only CCK-8 detection solution.
[0081] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A method for preparing a chitosan / nanohydroxyapatite guided bone regeneration membrane with a porous-dense composite structure for the repair of dental bone defects, characterized in that: Using chitosan as an organic matrix, calcium salts and phosphates are uniformly mineralized and crystallized on the surface of chitosan polymer chains in situ via freeze-drying to form nano-hydroxyapatite. This is then combined with mechanical extrusion to prepare a chitosan / nano-hydroxyapatite guided bone regeneration membrane with a porous-dense composite structure. The process includes the following steps: (1) Prepare an acetic acid solution by dissolving an appropriate amount of chitosan in the acetic acid solution and stirring in a water bath to obtain a chitosan acetic acid solution; (2) Add calcium nitrate solution to chitosan acetic acid solution and stir continuously until uniform. Then add an appropriate amount of dipotassium hydrogen phosphate solution and stir continuously for a period of time to obtain the precursor solution for guiding bone regeneration membrane. (3) The pre-frozen solution for guiding bone regeneration membrane was transferred to a refrigerator for pre-freezing, and then freeze-dried for several hours in a freeze dryer. After that, it was soaked in an ethanol / deionized water solution of sodium hydroxide to achieve in-situ mineralization and crystallization on the surface of chitosan to achieve uniform distribution of nano-hydroxyapatite. (4) Rinse repeatedly with deionized water until neutral, transfer to a refrigerator for pre-freezing, and then freeze dry in a freeze dryer for several hours; (5) After freeze-drying, place it on the grid surface of a polytetrafluoroethylene mold with a grid and press it for several hours under a certain mechanical strength to obtain a chitosan / nanohydroxyapatite guided bone regeneration membrane with a sparse-dense composite structure. In step (1), the amount of acetic acid used in the chitosan acetic acid solution is 0.5 mL, the amount of water is 49.5 mL, and the mass of chitosan powder is 1 g. In step (2), the concentration of calcium nitrate solution is 2 mol / L and the volume is 2 mL; the concentration of dipotassium hydrogen phosphate solution is 1.2 mol / L and the volume is 2 mL. In step (5), the mechanical extrusion strength is 0.2 MPa to 5 MPa; the mechanical extrusion time is 12 h to 96 h; during mechanical extrusion, one side of the membrane contacts a smooth mechanical surface, and the pores are compacted into a dense layer, while the other side of the membrane contacts a polytetrafluoroethylene mold with a grid. During the extrusion process, the original pores are preserved while the surface becomes more porous.
2. The preparation method according to claim 1, characterized in that: In step (3), the pre-freezing method is to place it at 4℃ for 1 hour and at -80℃ for 24 hours.
3. The preparation method according to claim 1, characterized in that: In step (3), the sodium hydroxide is in an ethanol / deionized water solution with a mass of 1 g, an ethanol volume of 125 mL, and a deionized water volume of 125 mL; the soaking time is 10 h.
4. The preparation method according to claim 1, characterized in that: In step (4), the pre-freezing method is to directly transfer it to -80℃ for placement.
5. A chitosan / nanohydroxyapatite guided bone regeneration membrane with a porous-dense composite structure for the repair of dental bone defects, prepared by the preparation method according to any one of claims 1-4.
Citation Information
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