CN105688288A - Collagen based composite dental restoration membrane material and preparation method thereof - Google Patents

Abstract

The invention discloses a collagen based composite dental restoration membrane material and a preparation method thereof. The method comprises steps as follows: type I collagen is subjected to crosslinking modification by oxidized carboxymethyl chitosan firstly, meanwhile, self-assembly of the collagen is promoted with a thermal driving method, a collagen based material is subjected to further crosslinking and pre-calcification sequentially, finally, biomimetic mineralization is performed in 1-10 times of an SBF (simulated body fluid) solution with trace PAA (polyacrylic acid) solution added, a product is subjected to freeze-drying and sterilization, and the collagen based composite dental restoration membrane material is obtained. The self-assembly of the collagen and the biomimetic mineralization are adopted, a forming process of natural teeth of a human body is simulated in the aspect of the preparation method, so that the membrane material has good biocompatibility, meanwhile, a membrane has good mechanical property and antibacterial property and an appropriate and controllable degradation rate, the raw materials are cheap and available, and the collagen based composite dental restoration membrane material can be widely applied to the field of dental restoration.

Description

Collagen-based composite oral repair membrane material and preparation method thereof

technical field

The invention relates to a collagen-based composite oral repair membrane material and a preparation method thereof, belonging to the field of oral medical materials.

Background technique

The oral repair membrane creates a good regenerative repair space by establishing a biological barrier between the oral soft tissue and the bone defect. Oral wound healing. Obviously, a good oral repair membrane should have mechanical barrier effect, vascularization effect, participation in metabolic process and induction effect, etc.

Collagen is a structural protein with biological functions, accounting for 1/3 of the total protein in the human body, and is the main component of connective tissues or organs such as skin, cartilage, ligaments, and tendons. Natural type I collagen has good mechanical properties and important biological properties, biodegradability and good biocompatibility, and is also very beneficial to cell adhesion, proliferation and differentiation in tissue culture. Biomedical field has a wide range of application value. Although type I collagen has so many advantages, it still has poor mechanical strength and antigenicity of pure collagen gel, which limits its application. Therefore, researchers are looking for various methods to improve its performance to make up for its shortcomings. Carboxymethyl chitosan is a water-soluble chitosan derivative, and the presence of carboxymethyl groups is beneficial to the deposition of Ca ²⁺ ions in the later stage. Carboxymethyl chitosan also inherits many advantages of chitosan, such as antibacterial and antibacterial properties, anti-tumor properties, and promotion of wound healing. The oxidized carboxymethyl chitosan introduces aldehyde groups, which can undergo Schiff base reaction with amino groups on collagen, in situ cross-linking, and has high reactivity, which can improve the mechanical properties of collagen-based composite membrane materials, and can Degradation absorbency etc.

Self-assembly is an important property of collagen. The basic organic framework of human natural teeth is composed of type I collagen molecules, and its formation process mainly includes: synthesis of collagen matrix, extracellular assembly and biomineralization. During the formation process, the type Ⅰ collagen molecule itself undergoes a series of multi-level and orderly self-assembly, and its hierarchical structure is: three-dimensional network structure of procollagen molecule→collagen microfibril→collagen fiber→mineralized matrix. Collagen fibers provide the substrate for the deposition of hydroxyapatite in the later stage, and the non-collagen components in the pore area of the collagen fibers provide sites for the nucleation of hydroxyapatite and arrange the formation orientation, which can act as a bridge between hydroxyapatite and collagen effect.

At present, oral restoration membrane materials are mainly simple composites of some materials. For example, the patent CN104922732A discloses a preparation method of oral biofilm. Collagen and chondroitin sulfate are configured into a slurry, vacuum freeze-dried and pressed into a collagen composite film, and then the collagen-chondroitin sulfate slurry is sprayed into the two-layer collagen composite film. The collagen-chondroitin sulfate composite oral biofilm is obtained after freeze-drying, high-temperature vacuum cross-linking and sterilization. Patent CN101081314B discloses a preparation method of a collagen/chitosan scaffold composited with hydroxyapatite, which first prepares a cross-linked collagen/chitosan scaffold, and then uses chemical deposition to compound hydroxyapatite to the scaffold Finally, the active composite bone scaffold was obtained by mineralization.

In the above method, there are the following deficiencies: (1) Most of the collagen raw materials are collagen molecules or hydrolyzed products of collagen, which are different from the collagen fiber components in human natural teeth or have lost the biologically active triple helical structure of collagen itself; (2) The composite material and collagen cannot form a large amount of effective cross-linking, which affects the mechanical properties and degradable absorption of the repair membrane material; (3) The composite material itself does not have excellent performance, and cannot endow the repair membrane material with new and more excellent (4) The biomimetic mineralization process is simple, and no other elements are added to regulate the mineralization.

Contents of the invention

The purpose of the present invention is to provide a collagen-based composite material with good biocompatibility for the field of oral medical materials. Bacteriostasis and suitable and controllable degradation and absorption, raw materials are cheap and easy to obtain, and have good application prospects.

The purpose of the present invention is achieved by the following technical measures:

(1) Preparation of oxidized carboxymethyl chitosan: Add 10-100 parts by weight of deionized water into a stainless steel reactor, and then add carboxymethyl chitosan and oxidant at a mass ratio of 1:0.1-2.5 , and dissolve it completely. Next, stir and react in the dark at 0~20°C for 10~50h, then add 5~50 parts by weight of sodium chloride and 50~1000 parts by volume of absolute ethanol, let it stand, and mix the bottom liquid at 10000~15000r/min Centrifuge for 10-30 minutes, finally dialyze in deionized water for 3-5 days, and freeze-dry to obtain oxidized carboxymethyl chitosan;

(2) Heat-driven type I collagen self-assembly: Dissolve type I collagen in buffer solution so that the concentration of type I collagen is 1-5 mg/ml. After incubating the well-stirred type I collagen solution at 37°C for 10-24 hours, add oxidized carboxymethyl chitosan and stir for 3-6 hours, then continue to incubate at 37°C for 10-24 hours, and finally freeze-dry the product for later use;

(3) Pretreatment of collagen-based materials: immerse the collagen-based materials obtained in step (2) in a cross-linking agent solution for cross-linking for 0.5-12 hours, and then immerse the materials in 0.1-0.5 mol/L Ca(NO ₃ ) In the ₂ solution for 4~24h, replace the Ca(NO ₃ ) ₂ solution every 12h, take it out and rinse it gently with deionized water 3 times;

(4) Biomimetic mineralization of collagen-based materials: the collagen-based materials obtained in step (3) were immersed in 1-10 times the SBF solution at 37 °C, and a small amount of polyacrylic acid (PAA) solution was added to make the concentration 20 ~60μg/ml, biomimetic mineralization for 12~336h, replace 1~10 times of SBF solution every 12h, take it out and rinse gently with deionized water for 3 times, freeze-dry and sterilize to get collagen-based composite oral restoration membrane material.

The collagen-based composite oral repair membrane material and its preparation method are characterized in that the oxidizing agent is one or both of sodium periodate and sodium perchlorate.

The collagen-based composite oral repair membrane material and its preparation method are characterized in that the buffer is a phosphate buffer with a pH value of 7-7.5.

The collagen-based composite oral repair membrane material and its preparation method are characterized in that the mass ratio of oxidized carboxymethyl chitosan to type I collagen is 1-3:10.

The collagen-based composite oral repair film material and its preparation method are characterized in that the crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N- Either or both of hydroxysuccinimide, genipin, proanthocyanidins, and glutaraldehyde.

The collagen-based composite oral repair membrane material and its preparation method are characterized in that the mass ratio of the collagen-based material to 1-10 times the SBF solution is 1:25-300.

The key performance indicators of the collagen-based composite dental repair membrane material prepared by the above method should meet the following requirements:

Appearance: white or yellowish film, soft, without visible impurities;

Heavy metal content: ≤10μg/ml;

Cytotoxicity: the cytotoxic reaction is not greater than grade 1;

Sterility test: sterile;

Sensitization test: no delayed hypersensitivity reaction;

Intradermal test: primary irritation index PII<0.4.

Compared with prior art, the present invention has following advantage:

(1) The present invention uses thermal drive to promote collagen self-assembly, and simulates the formation process of human natural teeth in the preparation method, so that the collagen-based composite material has good biocompatibility;

(2) The present invention uses oxidized carboxymethyl chitosan to compound and modify type I collagen, and oxidized carboxymethyl chitosan and type I collagen produce in-situ cross-linking, which can significantly improve the stability of type I collagen and can Degradation of absorbency, while the presence of carboxymethyl groups is conducive to the deposition of Ca ²⁺ ions in the later stage. According to the theory of synergy, the collagen-based composite membrane material of the present invention also has the advantages of antibacterial and antibacterial properties, anti-oxidation properties, and promotion of wound healing;

(3) The present invention adopts the method of combining pretreatment with biomimetic mineralization of human physiological body fluids, and introduces biologically active hydroxyapatite into the collagen-based material, which is similar to the composition and structure of human natural teeth, which is beneficial to oral restoration ;

(4) The present invention adds a small amount of polyacrylic acid (PAA) solution in the biomimetic mineralization process, which is beneficial to construct a multi-level ordered structure from nanometer to micrometer on the collagen-based material, and can further improve the biocompatibility of the membrane material.

detailed description

The present invention will be specifically described below by implementing it. It is necessary to point out that this embodiment is only used to further illustrate the present invention, and can not be interpreted as limiting the protection scope of the present invention. Those skilled in the art can according to the above-mentioned The content of the invention makes some non-essential improvements and adjustments.

Example 1

(1) Preparation of oxidized carboxymethyl chitosan: Add 60 parts by weight of deionized water into a stainless steel reactor, then add carboxymethyl chitosan and oxidant at a mass ratio of 1:1, and make it Dissolve completely. Next, stir and react in the dark at 20°C for 48 hours, then add 5 parts by weight of sodium chloride and 600 parts by volume of absolute ethanol, let it stand still, centrifuge the bottom liquid at 15,000 r/min for 20 minutes, and finally dialyze it in deionized water for 5 days , freeze-drying to obtain oxidized carboxymethyl chitosan;

(2) Heat-driven self-assembly of type I collagen: Dissolve type I collagen in the buffer so that the concentration of type I collagen is 3 mg/ml. After incubating the well-stirred type I collagen solution at 37°C for 20 hours, add oxidized carboxymethyl chitosan and stir for 5 hours, then continue to incubate at 37°C for 20 hours, and finally freeze-dry the product for later use;

(3) Pretreatment of collagen-based materials: immerse the collagen-based materials obtained in step (2) in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide Crosslink in the solution for 4 hours, then immerse the material in 0.2mol/L Ca(NO ₃ ) ₂ solution at 37°C for 6 hours, take it out and rinse it gently with deionized water for 3 times;

(4) Biomimetic mineralization of collagen-based materials: The collagen-based materials obtained in step (3) were immersed in 5 times the SBF solution at 37°C, and a small amount of polyacrylic acid (PAA) solution was added to make the concentration 30 μg/ml , Biomimetic mineralization for 168 hours, replace the 5 times SBF solution every 12 hours, take it out and rinse it gently with deionized water for 3 times, freeze-dry and sterilize to obtain the collagen-based composite oral repair membrane material.

Example 2

(1) Preparation of oxidized carboxymethyl chitosan: Add 80 parts by weight of deionized water into a stainless steel reactor, then add carboxymethyl chitosan and oxidant at a mass ratio of 1:0.5, and make it Dissolve completely. Next, stir and react in the dark at 10°C for 24 hours, then add 10 parts by weight of sodium chloride and 500 parts by volume of absolute ethanol, let it stand still, centrifuge the bottom liquid at 10,000 r/min for 20 minutes, and finally dialyze it in deionized water for 5 days , freeze-drying to obtain oxidized carboxymethyl chitosan;

(2) Heat-driven self-assembly of type I collagen: Dissolve type I collagen in buffer so that the concentration of type I collagen is 5 mg/ml. After incubating the well-stirred type I collagen solution at 37°C for 20 hours, add oxidized carboxymethyl chitosan and stir for 6 hours, then continue to incubate at 37°C for 20 hours, and finally freeze-dry the product for later use;

(3) Pretreatment of collagen-based materials: immerse the collagen-based materials obtained in step (2) in glutaraldehyde solution for cross-linking for 4 hours, and then immerse the materials in 0.1mol/L Ca(NO ₃ ) ₂ solution at 37°C For 16 hours, replace the Ca(NO ₃ ) ₂ solution every 12 hours, take it out and rinse it gently with deionized water for 3 times;

(4) Biomimetic mineralization of collagen-based materials: the collagen-based materials obtained in step (3) were immersed in 1.5 times the SBF solution at 37°C, and a small amount of polyacrylic acid (PAA) solution was added to make the concentration 50 μg/ml , Biomimetic mineralization for 240 hours, replace 1.5 times of SBF solution every 12 hours, take it out and rinse it gently with deionized water for 3 times, freeze-dry and sterilize to obtain the collagen-based composite oral repair membrane material.

Example 3

(1) Preparation of oxidized carboxymethyl chitosan: Add 50 parts by weight of deionized water into a stainless steel reactor, then add carboxymethyl chitosan and oxidant at a mass ratio of 1:1.5, and make it Dissolve completely. Next, stir and react at 4°C for 12 hours in the dark, then add 15 parts by weight of sodium chloride and 700 parts by volume of absolute ethanol, let it stand, centrifuge the bottom liquid at 15,000 r/min for 15 minutes, and finally dialyze it in deionized water for 5 days , freeze-drying to obtain oxidized carboxymethyl chitosan;

(2) Heat-driven type I collagen self-assembly: Dissolve type I collagen in the buffer so that the type I collagen concentration is 1 mg/ml. After incubating the well-stirred type I collagen solution at 37°C for 20 hours, add oxidized carboxymethyl chitosan and stir for 5 hours, then continue to incubate at 37°C for 20 hours, and finally freeze-dry the product for later use;

(3) Pretreatment of collagen-based materials: immerse the collagen-based materials obtained in step (2) in genipin solution for cross-linking for 6 hours, and then immerse the materials in 0.1mol/L Ca(NO ₃ ) ₂ solution at 37°C 10h, after taking it out, rinse it gently with deionized water for 3 times;

(4) Biomimetic mineralization of collagen-based materials: immerse the collagen-based materials obtained in step (3) in 2 times the SBF solution at 37 °C, and add a small amount of polyacrylic acid (PAA) solution to make the concentration 40 μg/ml , Biomimetic mineralization for 288 hours, replace 2 times the SBF solution every 12 hours, take it out and rinse it gently with deionized water for 3 times, freeze-dry and sterilize to obtain the collagen-based composite oral repair membrane material.

Claims (7)

1. collagen-based compound Oral Repair membrane material and preparation method thereof, its Key Performance Indicator is as follows:

Outward appearance: white or slightly yellow membranoid substance, soft, without naked eyes visible foreign；

Content of beary metal :≤10 μ g/ml;

Cytotoxicity: cell-cytotoxic reaction is not more than 1 grade；

Sterility test: aseptic；

Sensitization test (STT): without tardus allergy；

Intradermoreaction is tested: constitutional stimulation index PII < 0.4。

2. the collagen-based compound Oral Repair membrane material described in claim 1, it is characterised in that preparation method comprises the following steps:

(1) preparation of oxidated carboxymethyl chitosan: the deionized water of 10 ~ 100 weight portions is joined in stainless steel cauldron, then carboxymethyl chitosan and oxidant are added with the mass ratio of 1:0.1 ~ 2.5, and so as to dissolve completely, then, lucifuge stirring reaction 10 ~ 50h at 0 ~ 20 DEG C, add the sodium chloride of 5 ~ 50 weight portions and the dehydrated alcohol of 50 ~ 1000 parts by volume, stand, by bottom liquid with the centrifugal 10 ~ 30min of 10000 ~ 15000r/min, last 3 ~ the 5d that dialyses in deionized water, namely lyophilization obtains oxidated carboxymethyl chitosan；

(2) thermal drivers NTx self assembly: NTx is dissolved in buffer, making NTx concentration is 1 ~ 5mg/ml, after the NTx solution stirred is hatched 10 ~ 24h at 37 DEG C, add oxidated carboxymethyl chitosan stirring 3 ~ 6h, hatching 10 ~ 24h is continued again, finally by standby for product lyophilization at 37 DEG C；

(3) pretreatment of collagen-based material: step (2) gained collagen-based material is immersed crosslinking 0.5 ~ 12h in cross-linking agent solution, then material is immersed at 37 DEG C the Ca (NO of 0.1 ~ 0.5mol/L₃)₂4 ~ 24h in solution, every 12h change a Ca (NO₃)₂Solution, rinses with deionized water 3 times after taking-up gently；

(4) biomimetic mineralization of collagen-based material: step (3) gained collagen-based material is immersed in the SBF solution of 1 ~ 10 times at 37 DEG C, and add trace polyacrylic acid (PAA) solution, making its concentration is 20 ~ 60 μ g/ml, biomimetic mineralization 12 ~ 336h, every 12h changes the SBF solution of 1 ~ 10 times, taking-up deionized water rinses 3 times gently, freeze-dried, sterilization treatment, obtains collagen-based compound Oral Repair membrane material。

3. collagen-based compound Oral Repair membrane material described in claim 2 and preparation method thereof, it is characterised in that described oxidant is one or both in sodium metaperiodate or sodium perchlorate。

4. collagen-based compound Oral Repair membrane material described in claim 2 and preparation method thereof, it is characterised in that described buffer is pH value is the phosphate buffer of 7 ~ 7.5。

5. collagen-based compound Oral Repair membrane material described in claim 2 and preparation method thereof, it is characterised in that the mass ratio of described oxidated carboxymethyl chitosan and NTx is 1 ~ 3:10。

6. collagen-based compound Oral Repair membrane material described in claim 2 and preparation method thereof, it is characterized in that, described cross-linking agent be 1-ethyl-3-(3-DimethylAminopropyl)-carbodiimides, N-hydroxy-succinamide, genipin, procyanidin, glutaraldehyde any one or two kinds of。

7. collagen-based compound Oral Repair membrane material described in claim 2 and preparation method thereof, it is characterised in that the mass ratio of described collagen-based material and 1 ~ 10 times of SBF solution is 1:25 ~ 300。