CN115105639A

CN115105639A - Composite membrane for periodontal tissue repair and preparation method thereof

Info

Publication number: CN115105639A
Application number: CN202210641470.0A
Authority: CN
Inventors: 林昌健; 李雅娴; 郭文熹; 张艳梅; 董骧; 段红平
Original assignee: Beijing Leiden Biology Material Co ltd; Xiamen University
Current assignee: Beijing Leiden Biology Material Co ltd; Xiamen University
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-09-27
Anticipated expiration: 2042-06-08
Also published as: CN115105639B

Abstract

The invention belongs to the field of biomedical engineering and biomedical materials, and particularly relates to a composite membrane for periodontal tissue repair and a preparation method thereof. The invention provides a composite membrane for periodontal tissue repair, which comprises a porous membrane layer and a magnesium wire network layer, wherein the magnesium wire network layer is positioned between two adjacent porous membrane layers, and the porous membrane layers comprise 20-80% of chitosan, 20-80% of gelatin and 0-20% of nano-hydroxyapatite. The composite membrane has good biodegradability, biocompatibility, bioactivity, mechanical property and antibacterial property, the comprehensive performance can meet the requirements of periodontal tissue repair treatment in various aspects, and the composite membrane is expected to be clinically applied and has good market prospect.

Description

Composite membrane for periodontal tissue repair and preparation method thereof

Technical Field

The invention belongs to the field of biomedical engineering and biomedical materials, and particularly relates to a composite membrane for periodontal tissue repair and a preparation method thereof.

Background

Periodontal tissue is a highly stratified organ, consisting of the soft tissue gingival and periodontal ligament, as well as the hard tissue and alveolar bone, which supports the teeth and plays an important role in transmitting the mechanical forces experienced during chewing. Periodontitis is a chronic inflammatory disease caused by oral bacterial biofilms, resulting in destruction of periodontal soft tissue and possibly permanent loss of teeth. The ultimate goal of periodontitis treatment is the regeneration of periodontal defect tissue, involving the functional reattachment of the periodontal ligament to the newly formed cementum and alveolar bone. This requires a highly coordinated healing response, including reattachment of periodontal ligament fibers to the previously contaminated root surface, cementum formation, and bone formation at the site of the periodontal defect. Modern methods of periodontal treatment are inspired by the guided tissue regeneration, i.e. the use of degradable polymer membranes to obtain a separate, physically protected surgical site, which recruits local stem cells at the site of injury and supports their differentiation into fibroblasts and osteoblasts. But the existing periodontal tissue repair material is expensive, lacks of bacteriostatic ability and has insufficient operability of clinical operation. Therefore, there is a need to further develop multifunctional composite membrane materials that can be used for periodontal tissue repair.

Disclosure of Invention

The chitosan is a biopolymer of natural polysaccharide chitin with partial acetyl removed, has the characteristics of good biocompatibility, biodegradability, antibiosis, blood coagulation and the like, and degradation products of the chitosan are nontoxic, non-antigen, non-immunogenicity and non-carcinogenic. The surface is positively charged and hydrophilic, which is favorable for cell adhesion, growth, proliferation and differentiation. However, chitosan has a need to improve its osteoconductivity.

The invention provides a composite membrane for periodontal tissue repair, which has rich related material sources, good biological activity and antibacterial performance, and particularly has appropriate mechanical property of a repair membrane layer, strong shape plasticity and convenient operation of clinical operation. The prepared composite membrane component can be rapidly degraded and absorbed in a human body or form small molecules harmless to the human body to be discharged out of the human body after the support tissue is regenerated, and the biodegradation performance is excellent.

The composite membrane for periodontal tissue repair comprises porous membrane layers and a magnesium wire network layer, wherein the magnesium wire network layer is positioned between two adjacent porous membrane layers, and the porous membrane layers comprise 20-80% of chitosan, 20-80% of gelatin and 0-20% of nano-hydroxyapatite by mass.

According to the advantages and technical effects brought by the composite membrane for periodontal tissue repair of the embodiment of the invention, 1, in the embodiment of the invention, gelatin and hydroxyapatite are added into the porous membrane layer, the gelatin is composed of a unique amino acid sequence, is a natural product derived from collagen hydrolysis, has high biological activity and can promote cell adhesion, the hydroxyapatite is one of important components of teeth and can promote bone differentiation and improve bone conductivity, the addition of the gelatin and the nano hydroxyapatite can improve the bone conductivity of chitosan, and the addition of the chitosan can improve the biocompatibility and antibacterial property of the material; 2. in the embodiment of the invention, a magnesium wire network layer is introduced between the porous film layers, magnesium is an absorbable metal biomaterial, has lower elastic modulus and proper strength, and is combined with the porous film layer consisting of chitosan, gelatin and hydroxyapatite to form a composite film, so that the composite film has good degradability, biological activity, antibacterial performance and mechanical performance; 3. in the embodiment of the invention, the magnesium wire network layer is introduced as the bracket of the composite membrane, so that the composite membrane has good plasticity, the problem of difficult operation of an implantation operation can be solved, and the clinical practicability is enhanced; 4. the related materials of the composite membrane disclosed by the embodiment of the invention are rich in sources, and the components of the composite membrane can be rapidly degraded and absorbed in a human body or form small molecules harmless to the human body to be discharged out of the body after the support tissue is regenerated, so that the composite membrane has good biodegradation performance.

In some embodiments, the porous membrane layer further comprises 0.01-1% chitosan oligosaccharide.

In some embodiments, the mesh size in the magnesium wire network layer is 1 × 1-3 × 3mm, the magnesium wire network layer uses a thick magnesium wire as a main skeleton, the main skeleton is fixed by a thin magnesium wire, the diameter of the thick magnesium wire is 300-600 μm, and the diameter of the thin magnesium wire is 100-200 μm.

The embodiment of the invention also provides a preparation method of the composite membrane for periodontal tissue repair, which comprises the following steps:

(1) preparing a porous membrane layer;

(2) weaving a magnesium wire network and carrying out surface modification on the magnesium wire network to obtain a magnesium wire network layer;

(3) and carrying out adhesion assembly on the magnesium wire network layer and the porous film layer.

According to the advantages and technical effects brought by the preparation method of the composite membrane for periodontal tissue repair, 1, the adopted materials are rich in sources, and the components of the composite membrane can be rapidly degraded and absorbed in a human body or form small molecules harmless to the human body to be discharged out of the body after the supporting tissue is regenerated, so that the composite membrane has good biodegradation performance; 2. in the embodiment of the invention, the weaving of the magnesium wires does not involve any chemical reaction, so that the possibility of oxidation of the magnesium wires due to the chemical reaction is reduced; 3. in the embodiment of the invention, the magnesium wire network layer and the porous film layer are assembled in an adhesion manner, and the degradable biological material and the degradable metal material are combined, so that the composite film has good degradability, biological activity, antibacterial performance and mechanical performance; 4. in the embodiment of the invention, the magnesium wire network layer has good plasticity as the support of the composite film layer, can solve the problem of difficult operation in the process of implantation surgery, and enhances the clinical practicability.

In some embodiments, the preparation of the porous membrane layer comprises the steps of:

a. mixing the chitosan solution, the gelatin solution and the nano-hydroxyapatite, uniformly stirring to obtain a mixed solution, and freezing the mixed solution;

b. immersing the frozen material into a precooled NaOH/ethanol mixed solution, washing the material with an ethanol solution, and placing the material into an MES buffer solution;

c. and c, immersing the material obtained by the treatment in the step b into a mixed solution containing EDC & HCl and NHS for crosslinking reaction to obtain hydrogel, and washing and freeze-drying the hydrogel to obtain the porous membrane layer.

In some embodiments, in step a, the concentration of the chitosan solution is 0.1-30mg/mL and the concentration of the gelatin solution is 0.1-20 mg/mL; the freezing treatment is to refrigerate the mixed solution for 1 to 10 hours at 0 to 4 ℃ and then freeze the mixed solution for 1 to 24 hours at a temperature of between 15 ℃ below zero and 25 ℃ below zero.

In some embodiments, in the step b, the concentration of NaOH in the NaOH/ethanol mixed solution is 1-3mol/L, the ethanol is absolute ethanol, and the precooling temperature of the NaOH/ethanol mixed solution is-15 to-25 ℃; the immersion time is 3-12h, the ethanol solution washing is gradient washing by using ethanol solutions with volume fractions of 70%, 80%, 90% and 100%, and the material is placed in MES buffer solution for 10-30 min.

In some embodiments, in step c, the mixture of EDC · HCl and NHS further contains not more than 30mg/mL of chitosan oligosaccharide, the concentration of EDC · HCl in the mixture of EDC · HCl and NHS is 0.01 to 0.05mol/L, the concentration of NHS is 0.005 to 0.01mol/L, and the time of the crosslinking reaction is 1 to 15 hours; in the step d, the freeze drying is carried out at the temperature of-40-50 ℃ for 10-15 h.

In some embodiments, in step (2), the surface modification of the magnesium wire network is electrochemical cathodic deposition of the magnesium wire network in an acetic acid solution containing chitosan under an applied voltage of 1-10V for 1-30 min.

In some embodiments, in step (3), the magnesium wire network layer and the porous film layer are adhesively assembled by adhering the magnesium wire network layer between two porous film layers by using a PVA solution or a PCL solution.

Drawings

FIG. 1 is a schematic diagram of a magnesium wire network layer;

FIG. 2 is a degradation curve of the composite films prepared in examples 1-5 and comparative examples 1-2 degraded in artificial saliva for 30 d;

fig. 3 is an absorbance value measured by a microplate reader after 6 hours of co-culture of staphylococcus aureus (s. aureus) and escherichia coli (e.coli) for the composite films prepared in examples 1-5 and comparative examples 1-2;

FIG. 4 is a graph showing the cell viability of the composite membrane extract prepared in examples 1-5 and comparative examples 1-2 after culturing the cells for 24 hours.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The composite membrane for periodontal tissue repair comprises porous membrane layers and a magnesium wire network layer, wherein the magnesium wire network layer is positioned between two adjacent porous membrane layers, and the porous membrane layers comprise 20-80% of chitosan, 20-80% of gelatin and 0-20% of nano-hydroxyapatite.

According to the composite membrane for periodontal tissue repair provided by the embodiment of the invention, gelatin and hydroxyapatite are added in the porous membrane layer, the gelatin is composed of a unique amino acid sequence, is a natural product derived from collagen hydrolysis, has high biological activity and can promote cell adhesion, the hydroxyapatite is one of important components of teeth and can promote bone differentiation and improve bone conductivity, the addition of the gelatin and the nano hydroxyapatite can improve the bone conductivity of chitosan, and the addition of the chitosan can improve the biocompatibility and antibacterial property of the material; in the embodiment of the invention, the magnesium wire network layer is introduced between the porous film layers, the performance of magnesium basically meets the requirements of the porous scaffold for bone tissue engineering, namely, the magnesium has lower elastic modulus and proper strength, good biocompatibility, biodegradability, absorbability and the like, and the magnesium wire network layer is combined with the porous film layer consisting of chitosan, gelatin and hydroxyapatite to form the composite film, so that the composite film has good degradability, biological activity, bacteriostatic performance and mechanical performance; in the embodiment of the invention, the magnesium wire network layer is introduced as the bracket of the composite membrane, so that the composite membrane has good plasticity, the problem of difficult operation of an implantation operation can be solved, and the clinical practicability is enhanced; the composite membrane provided by the embodiment of the invention has rich material sources, and the components of the composite membrane can be rapidly degraded and absorbed in a human body or form small molecules harmless to the human body to be discharged out of the body after the support tissue is regenerated, so that the composite membrane has good biodegradability.

In some embodiments, preferably, the porous membrane layer further comprises 0.01-1% chitosan oligosaccharide, preferably 0.1-0.5%, and more preferably 0.1-0.3%. In the embodiment of the invention, chitosan oligosaccharide is further introduced into the porous film layer, a new idea is provided for tissue engineering by using the chitosan oligosaccharide as a periodontal tissue regeneration material, and the antibacterial property and biocompatibility of the composite film, especially the properties of resisting gram-negative bacteria and positive bacteria, are effectively improved under the combined action of the chitosan oligosaccharide, chitosan, gelatin and nano hydroxyapatite. In the embodiment of the present invention, the content of chitosan oligosaccharide is further preferable, and if the content is too small, the effect of the crosslinking reaction is affected, and if the content is too large, the stability of the composite membrane is lowered.

In some embodiments, preferably, the mesh size in the magnesium wire network layer is 1 × 1-3 × 3mm, the magnesium wire network layer uses thick magnesium wires as a main skeleton, the main skeleton is fixed by thin magnesium wires, the diameter of the thick magnesium wires is 300-600 μm, and the diameter of the thin magnesium wires is 100-200 μm. In the embodiment of the invention, the magnesium wire network is formed by adopting the thick magnesium wires and the thin magnesium wires, and the size of the grid is optimized, so that the shaping and mechanical properties of the magnesium wire network are improved, and the clinical application is facilitated.

(1) preparing a porous membrane layer;

According to the preparation method of the composite membrane for repairing the periodontal tissue, the source of the used materials is rich, the components of the composite membrane can be rapidly degraded and absorbed in a human body or form small molecules harmless to the human body to be discharged out of the body after the supporting tissue is regenerated, and the composite membrane has good biodegradability; in the embodiment of the invention, the weaving of the magnesium wires does not involve any chemical reaction, so that the possibility of oxidation of the magnesium wires due to the chemical reaction is reduced; in the embodiment of the invention, the magnesium wire network layer and the porous film layer are assembled in an adhesion manner, and the degradable biological material and the degradable metal material are combined, so that the composite film has good degradability, biological activity, antibacterial performance and mechanical performance; in the embodiment of the invention, the magnesium wire network is used as a stent of the composite film layer, has good plasticity and support property, and enhances the operability of a clinical implantation operation.

In some embodiments, the method of preparing the porous membrane layer comprises the steps of:

b. immersing the frozen material into a precooled NaOH/ethanol mixed solution, washing the material by using an ethanol solution, and placing the material into an MES buffer solution;

In the method for preparing the porous membrane layer, chitosan has higher biocompatibility, biodegradability, antibacterial property and blood coagulation property, but the osteoconductivity needs to be improved. The addition of gelatin and nano-hydroxyapatite can improve the osteoconductivity of chitosan, the biological activity of the gelatin is high, the cell adhesion can be promoted, and the nano-hydroxyapatite can promote the bone differentiation and improve the osteoconductivity. The three materials have the characteristics of wide sources, low cost and excellent biological properties. The porous spongy structure is formed by a freeze drying method, which is favorable for guiding the formation of the bone structure and enables the porous film layer to better play a role in periodontal repair.

In some embodiments, in step a, the chitosan solution has a concentration of 0.1-30mg/mL and the gelatin solution has a concentration of 0.1-20mg/mL, and further preferably, the chitosan solution is an aqueous acetic acid solution containing chitosan. Preferably, the volume ratio of the chitosan solution to the gelatin solution is 1: 1. further preferably, in the step a, the freezing is to refrigerate the mixed solution at 0-4 ℃ for 1-10h, and then freeze the mixed solution at-15 to-25 ℃ for 1-24 h. In the embodiment of the present invention, the freezing treatment is preferably performed after the cold storage treatment, so that the mixed solution is already in a gel state during freezing, which is favorable for the directional growth of ice crystals in a low temperature state to perform freezing gelation.

In some embodiments, in the step b, the concentration of NaOH in the NaOH/ethanol mixed solution is 1 to 3mol/L, the ethanol is absolute ethanol, i.e. the concentration is 100 v/v%, and the precooling temperature of the NaOH/ethanol mixed solution is-15 to-25 ℃; the immersion time is 3-12h, the ethanol solution washing is gradient washing with 70%, 80%, 90% and 100% ethanol solution at normal temperature, and the solution is placed in MES (morpholine ethanesulfonic acid) buffer solution for 10-30 min. Preferably, in the gradient washing, the time length of each washing is 10-20 min. Further preferably, the concentration of the MES buffer is 0.05-0.2mol/L, and the pH is 5.5-6.6. In the embodiment of the invention, the frozen material is immersed in preferably NaOH/ethanol mixed solution, the gelatinous porous membrane can be washed from acidity to alkalinity, then gradient washing is carried out by adopting ethanol solution, NaOH is washed off to enable the porous membrane to be neutral and reduce the pore diameter of the porous membrane, and the porous membrane is placed in MES buffer solution after washing, ethanol can be washed off, and carboxyl of a sample can be activated to prepare for a crosslinking reaction.

In some embodiments, the mixture of EDC & HCl (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) in step c further comprises not more than 30mg/mL, preferably 0.1 to 30mg/mL, of chitosan oligosaccharide, EDC & HCl solution at a concentration of 0.01 to 0.05mol/L, NHS solution at a concentration of 0.005 to 0.01mol/L, and the crosslinking reaction time is 1 to 15 hours. In the embodiment of the invention, chitosan oligosaccharide is introduced into the porous membrane, and is added into the mixed solution of EDC, HCl and NHS, so that the chitosan oligosaccharide can be crosslinked on the porous membrane layer in the crosslinking reaction process, the antibacterial property and the biocompatibility of the scaffold are improved together with other components, and particularly, the infection of gram-negative bacteria and gram-positive bacteria can be effectively inhibited.

In some embodiments, the freeze-drying in step d is drying at-40 to-50 ℃ for 10-15 h. In the embodiment of the invention, the freeze drying treatment is adopted, so that the moisture in the sample is directly sublimated from ice to achieve the drying purpose, the freeze drying process is free from the action of surface tension, the sample is not deformed, loose and porous, and the original chemical composition can be kept.

In some embodiments, in step (2), the surface modification of the magnesium wire network is electrochemical cathodic deposition of the magnesium wire network in an acetic acid solution containing chitosan under an applied voltage of 1-10V for 1-30 min. Preferably, the content of the chitosan is 0.1-0.8 mg/mL. Preferably, in the step (3), the assembling of the surface-modified magnesium wire network on the porous membrane layers is to adhere the magnesium wire network between two porous membrane layers by using a PVA (polyvinyl alcohol) solution or a PCL (polycaprolactone) solution to form a sandwich structure. In the embodiment of the invention, in the preparation process of the porous membrane layer, the molecular weight of the chitosan is reduced after the chitosan is dissolved, the chitosan is straight-chain polysaccharide without side chains, and the amino in the chitosan can adsorb hydrogen ions, so that the chitosan is positively charged. During the electrochemical cathode deposition process, the positively charged chitosan will undergo a reduction reaction at the cathode and deposit on the surface of the magnesium wire network. The chitosan deposited on the surface of the magnesium wire network can regulate the degradation rate of the magnesium wire in the body, improve the biocompatibility of the composite membrane and make the composite membrane more suitable for clinical use. In the embodiment of the invention, the PVA solution or the PCL solution is adopted to adhere the magnesium wire network layer between the porous membranes, and the PVA or the PCL is a biodegradable polymer, has good biocompatibility and high solution viscosity, and can play a role of a binder.

In some embodiments, in the step (2), the magnesium wire network layer is woven by using a thick magnesium wire as a main skeleton, and the main skeleton is fixed by using a thin magnesium wire, wherein the diameter of the thick magnesium wire is 300-. Preferably, the size of the mesh in the magnesium wire network is 1 x 1-3 x 3 mm. Referring to fig. 1, it is further preferred that the magnesium wire network is woven by first criss-crossing two magnesium wires with a diameter of 500 μm and fixing two turns of the magnesium wire with a diameter of 100 μm around the "8" in a counterclockwise direction from top left to bottom right. A500-micron magnesium wire is longitudinally added, and the same magnesium wire with the diameter of 100 microns is fixed by winding 8 in the same way. And a magnesium wire is transversely added, and the same magnesium wire with the diameter of 100 mu m is fixed for one circle around the '8' in the counterclockwise direction from the left lower part to the right upper part. The weaving is continued until a 3 x 3mm magnesium wire network is knitted. In the embodiment of the invention, the used magnesium wire is a medical implantable magnesium wire, and the weaving of the magnesium wire network does not involve any chemical reaction, so that the possibility of oxidation of the magnesium wire caused by the chemical reaction is reduced. In the embodiment of the invention, the chitosan oligosaccharide and the magnesium wires are added to inhibit the infection of gram-negative bacteria and gram-positive bacteria, and the magnesium wire network interlayer can obviously improve the mechanical plasticity of the composite film layer, so that the operability of clinical operation is greatly enhanced.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1

The preparation method of the composite membrane for periodontal tissue repair provided by the embodiment comprises the following steps:

(1) preparing a porous membrane layer: 200 μ L of acetic acid was pipetted into 20mL of deionized water to give a 1% (v/v) acetic acid solution. The acetic acid solution was heated to 40 ℃ and 0.4g of chitosan powder was added thereto, and the chitosan was dissolved by stirring to obtain a chitosan solution of 2 mg/mL. Another 20mL of deionized water was added to 0.2g of gelatin, heated to 40 ℃ and stirred to dissolve it, thus obtaining a 1mg/mL gelatin solution. Mixing a 2mg/mL chitosan solution and a 1mg/mL gelatin solution according to a volume ratio of 1: 1, adding 100mg of nano-hydroxyapatite into 40ml of the obtained mixture, and stirring vigorously for 48 hours to ensure that the hydroxyapatite is uniformly distributed in the mixed solution without precipitation. The mixture was poured into a 60mm petri dish and first refrigerated at 4 ℃ for 3h and then frozen at-20 ℃ for 24 h. Mixing a 2.5mol/L NaOH solution and an absolute ethyl alcohol solution according to a volume ratio of 1: 1, mixing uniformly, filling into a container, and precooling to-20 ℃. The porous membrane after being frozen for 24h is immersed in the NaOH/ethanol solution for 12h, taken out and washed by 70 percent, 80 percent, 90 percent and 100 percent ethanol solution in sequence at normal temperature in a gradient way, the washing time is 15min for each time, and then the material is placed in MES buffer solution with 0.1mol/L, pH being 6 for 15 min. 0.9585g of EDC & HCl and 0.1151g of NHS were weighed out and dissolved in 100mL of deionized water to obtain a mixed solution of 0.05mol/L EDC & HCl and 0.01mol/L NHS, and the material was immersed therein for a crosslinking reaction for 12 hours. And then washing the bracket with deionized water, and finally freeze-drying at-40 ℃ for 12h to obtain the porous membrane layer, wherein the mass content of chitosan in the prepared porous membrane layer is 57.14%, the mass content of gelatin is 28.57%, and the mass content of hydroxyapatite is 14.29%.

(2) Weaving a magnesium wire network: magnesium wires with the diameter of 500 mu m are used as a main framework, and the distance between every two magnesium wires is 1 mm. Two magnesium wires with the diameter of 500 mu m are crossed, and the magnesium wires with the diameter of 100 mu m are wound around the 8-shaped wire in the counterclockwise direction from the upper left to the lower right for two circles. Adding a magnesium wire with the diameter of 500 mu m in the longitudinal direction, winding the same magnesium wire with the diameter of 100 mu m for one circle around the 8 in the same direction, adding a magnesium wire in the transverse direction, and winding the same magnesium wire with the diameter of 100 mu m for one circle around the 8 in the counterclockwise direction from the left to the right. The braiding was continued until a 3 x 3cm network of magnesium filaments was formed.

(3) Surface modification of the magnesium wire network: placing the magnesium wire mesh in acetic acid solution containing 0.2mg/mL chitosan under 2V of applied voltage for electrochemical cathode deposition for 10 min.

(4) Adhering and assembling the surface-modified magnesium wire network and the porous film layer: 2g of PVA powder was added to 20mL of deionized water to give a 10mg/mL PVA solution. Coating PVA solution on the surface of one side of the porous film layer, attaching a magnesium wire network, covering and adhering another porous film layer, and assembling the composite film of the porous film layer/the magnesium wire network/the porous film layer.

Example 2

The same procedure as in example 1, except that: when crosslinking, the sample was immersed in a mixed solution of 0.05mol/L EDC. HCl and 0.01mol/L NHS containing 10mg/mL chitosan oligosaccharide.

In the porous membrane layer prepared in the embodiment, the mass content of chitosan is 57.06%, the mass content of gelatin is 28.53%, the mass content of hydroxyapatite is 14.27%, and the mass content of chitosan oligosaccharide is 0.14%.

Example 3

The same procedure as in example 2 was repeated, except that a porous film containing 49.94% of chitosan, 29.92% of gelatin, 20.00% of hydroxyapatite and 0.14% of chitosan oligosaccharide was obtained.

Example 4

The same procedure as in example 2 was repeated, except that the porous film was prepared to have 20.4% chitosan, 71.1% gelatin, 8.2% hydroxyapatite, and 0.3% chitosan oligosaccharide.

Example 5

The same procedure as in example 2 was repeated, except that the porous film was prepared to contain 78.16% of chitosan, 21.83% of gelatin, 0% of hydroxyapatite and 0.01% of chitosan oligosaccharide.

Comparative example 1

The same method as in example 1 except that steps (2), (3) and (4) were omitted, and the porous film produced in step (1) was the repair film produced in comparative example 1.

Comparative example 2

The same as in example 2 except that steps (2), (3) and (4) were eliminated, and the porous film produced in step (1) was the repair film produced in comparative example 1.

Comparative example 3

The same procedure as in example 1, except that: to the acetic acid solution were added 0.4g of chitosan and 0.4g of chitosan oligosaccharide.

In comparative example 3, chitosan oligosaccharide was directly added, and was not crosslinked, and only chitosan oligosaccharide connected by hydrogen bond was dissolved in NaOH/ethanol solution, and the sample could not form hydrogel state, and could not be made into a qualified sample.

The composite membranes prepared in examples 1 to 5 and the repair membranes prepared in comparative examples 1 to 2 were subjected to degradation, bacteriostasis and in vitro cytotoxicity tests, and the test results are shown in fig. 2 to 4.

(1) The method for testing the degradation performance comprises the following steps: the sample was cut (n ═ 3) and weighed (W) ₁ ) And degraded in artificial saliva at 37 ℃. Changing degradation liquid every 2 days, collecting sample every 5 days, washing with deionized water, freeze drying, and weighing (W) ₀ )。

Residual mass (%) ═ W ₁ /W ₀ ×100

FIG. 2 is a degradation curve of the composite films prepared in examples 1 to 5 and the repair films prepared in comparative examples 1 to 2, respectively, degraded in artificial saliva for 30 d. As can be seen from FIG. 2, in 5d, the chitosan and gelatin which were not crosslinked were rapidly degraded, resulting in rapid degradation in the early stage but smooth degradation in the later stage in each of the examples and comparative examples. The degradation rate of the composite membrane can be increased by adding the grafted chitosan oligosaccharide and the magnesium wire network, so that the degradation rate of the composite membrane is the fastest in example 2, and the degradation rate of the composite membrane is the slowest in comparative example 1. Since the periodontal tissue regeneration membrane must satisfy a certain degradation rate, which may not be too slow or too fast, the degradation experiment of the composite membrane 30d of examples 1 to 5 shows that it satisfies the condition that the periodontal tissue regeneration membrane can support and guide tissue growth in a certain time and can be completely degraded when the tissue growth is mature.

(2) The testing method of the antibacterial performance comprises the following steps: recovering the strain at room temperature, sucking 50 μ l of the strain liquid into a conical flask containing 50ml of LB liquid culture medium, shaking the conical flask at 37 ℃ for 12h at 180 rpm. And (3) sucking 100 mu l of bacteria turbid liquid, testing the absorbance value at 600nm by using an enzyme-labeling instrument, carrying out three-time parallel test on each bacteria to obtain an average value, and then obtaining the bacteria concentration according to a bacteria standard curve. Bacteria were diluted to 1X 10 using LB medium ⁷ cfu/ml ³ 1ml of the diluted bacterial suspension is absorbed by a pipette into a 24-well plate filled with the sample, and then the plate is placed into a shaker, and the absorbance value is measured after shaking for 6 hours at 37 ℃ and 150 rpm.

FIG. 3 shows the absorbance measured by an microplate reader after 6 hours of co-culture of Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) in the composite membranes prepared in examples 1 to 5 and the repair membranes prepared in comparative examples 1 to 2, respectivelyThe bacterial planting density is 1 x 10 ⁷ and/mL. The greater the absorbance value, the poorer the bacteriostatic ability of the sample. As can be seen from FIG. 3, the absorbance values of Staphylococcus aureus were not significantly different in the co-cultivation for 6h in the different examples and the comparative example due to the excellent anti-gram-positive properties of chitosan itself. However, the difference between each example of cocultivation with E.coli and the comparative example is significant, wherein the antibacterial activity of the comparative example 1 is the worst, because the antibacterial ability of chitosan is poor in a neutral environment. In examples 1 to 5, however, the magnesium is rapidly degraded in the bacterial culture medium due to the presence of the magnesium wire network, and a slightly alkaline environment is formed, so that the bacterial growth can be inhibited by changing the pH value of the bacterial growth environment. Examples 2 to 5 contain chitosan oligosaccharide and a magnesium filament network, and chitosan oligosaccharide has both gram-negative bacteria and gram-positive bacteria resistance, so the antibacterial properties of examples 2 to 5 have excellent gram-negative bacteria and gram-positive bacteria resistance. Comparative example 2 also has a remarkable antibacterial activity against E.coli, and since chitosan oligosaccharide has a weaker antibacterial activity than magnesium, comparative example 2 has a weaker antibacterial ability than examples 1 to 5. The antibacterial experiment shows that the composite membrane added with the magnesium wire network or chitosan oligosaccharide crosslinking has a certain promotion effect on bacteriostasis.

(3) In vitro cytotoxicity assay

According to the ISO 10992-12 standard, except the culture medium absorbed by the material swelling, the sample is leached according to the proportion of 0.1g/mL of leaching liquor at 37 ℃ for 24h to prepare a sample leaching liquor. Selecting 100% leaching liquor as leaching stock solution, diluting the leaching stock solution to 75%, 50% and 25%, and adopting leaching medium as blank control group; MC3T3-E1 cells were cultured in the leach solution according to the method of ISO 10992-5 and tested for viability using CCK-8 reagent, and if the viability of the cells is higher than 70% of that of the blank control group, the material is not considered to have obvious potential toxicity.

FIG. 4 is a graph showing the survival rate of 24h cells cultured from the composite membranes prepared in examples 1 to 5 and the reparative membrane extract prepared in comparative examples 1 to 2. As can be seen from FIG. 4, the cell viability of each example and comparative example increased as the concentration of the leaching solution decreased. The survival rate of cells can be improved by grafting COS (chitosan oligosaccharide), but the cell survival rate is reduced by increasing the pH of the leaching solution and reducing the cell survival rate by adding the magnesium wire network, so the cell survival rate of the leaching solution of example 1 in different concentrations is lower. However, example 1 still had a cell viability of greater than 70% in the leach liquor, indicating that the composite membrane is not potentially cytotoxic. Although example 5 does not contain hydroxyapatite, the addition of chitosan oligosaccharide improves the cell survival rate, and the cell survival rate in the leaching stock solution is still higher than 70%. According to the embodiment of the invention, the magnesium wire network is modified by the chitosan film on the surface, so that the degradation speed of the magnesium wire network can be regulated and controlled, and the biocompatibility of the composite film is further improved.

In the present disclosure, the terms "one embodiment," "some embodiments," "an example," "a specific example," or "some examples" and the like mean that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although the above embodiments have been shown and described, it should be understood that they are exemplary and should not be construed as limiting the present invention, and that many changes, modifications, substitutions and alterations to the above embodiments may be made by those of ordinary skill in the art without departing from the scope of the present invention.

Claims

1. A composite membrane for periodontal tissue repair comprising a porous membrane layer and a magnesium wire network layer, the magnesium wire network layer being located between two adjacent porous membrane layers, wherein the porous membrane layers comprise 20-80% by mass of chitosan, 20-80% by mass of gelatin and 0-20% by mass of nano-hydroxyapatite.

2. A composite membrane for periodontal tissue repair according to claim 1, wherein said porous membrane layer further comprises 0.01-1% of chitosan oligosaccharide.

3. The composite membrane for periodontal tissue repair according to claim 1, wherein the mesh size in the magnesium wire network layer is 1 x 1-3 x 3mm, the magnesium wire network layer uses thick magnesium wires as a main framework, the main framework is fixed by thin magnesium wires, the diameter of the thick magnesium wires is 300-600 μm, and the diameter of the thin magnesium wires is 100-200 μm.

4. A method of preparing a composite membrane for periodontal tissue repair according to any of claims 1 to 3, comprising the steps of:

(1) preparing a porous membrane layer;

5. The method of preparing a composite membrane for periodontal tissue repair according to claim 4, wherein the preparation of the porous membrane layer comprises the steps of:

6. The method of preparing a composite membrane for periodontal tissue repair according to claim 5, wherein in step a, the concentration of the chitosan solution is 0.1-30mg/mL, and the concentration of the gelatin solution is 0.1-20 mg/mL; the freezing treatment is to refrigerate the mixed solution for 1 to 10 hours at 0 to 4 ℃ and then freeze the mixed solution for 12 to 48 hours at a temperature of between 15 ℃ below zero and 25 ℃ below zero.

7. The method for preparing a composite membrane for periodontal tissue repair according to claim 5, wherein in step b, the concentration of NaOH in the NaOH/ethanol mixed solution is 1-3mol/L, ethanol is absolute ethanol, and the precooling temperature of the NaOH/ethanol mixed solution is-15 to-25 ℃; the immersion time is 3-12h, the ethanol solution washing is gradient washing by using ethanol solutions with volume fractions of 70%, 80%, 90% and 100%, and the material is placed in MES buffer solution for 10-30 min.

8. The method of claim 5, wherein in the step c, the mixture of EDC-HCl and NHS further comprises chitosan oligosaccharide of not more than 30mg/mL, the mixture of EDC-HCl and NHS has EDC-HCl concentration of 0.01-0.05mol/L and NHS concentration of 0.005-0.01mol/L, and the time of the cross-linking reaction is 1-15 h; in the step d, the freeze drying is carried out for 10-15h at the temperature of-40 to-50 ℃.

9. The method of preparing a composite membrane for periodontal tissue repair according to claim 4, wherein in the step (2), the surface modification of the magnesium filament network is electrochemical cathodic deposition of the magnesium filament network in an acetic acid solution containing chitosan under an applied voltage of 1 to 10V for 1 to 30 min.

10. The method of preparing a composite membrane for periodontal repair according to claim 1, wherein in step (3), the magnesium wire network layer is adhesively assembled with the porous membrane layers by adhering the magnesium wire network layer between the two porous membrane layers using a PVA solution or a PCL solution.