CN1319604C - Double layer composite collagen base guide tissue regeneration film and its preparing method - Google Patents

Abstract

The invention relates to a double-layer composite collagen-based guided tissue regeneration material and a preparation method thereof. It uses collagen and hyaluronic acid or its sodium salt as main raw materials to prepare a double-layer composite material with a dense layer and a loose layer structure. The mass composition For: the dense layer is 0.6-0.8% malonate collagen swelling solution. The loose layer is composed of 0.2-0.4% malonate collagen swelling solution and 5-20% hyaluronic acid or sodium salt of collagen dry weight. The invention has excellent biocompatibility, one side has a loose structure, and the other side has a dense structure, so that the product has the ability to prevent fibroblasts from growing into the defect, and at the same time promote the regeneration and repair of the defect tissue, and has corresponding biological properties. Functional, degradable and absorbable, no need for secondary surgery to take out the remarkable features of implanted materials. It is suitable for tissue defects caused by trauma, tumor surgery, etc., and is used for guided regeneration and repair of defective tissues to achieve regenerative healing of defective tissues.

Description

Double-layer composite collagen matrix guided tissue regeneration membrane and preparation method thereof

technical field

The invention belongs to the field of biomedical materials, in particular to a double-layer composite collagen-based guided tissue regeneration material and a preparation method thereof. The invention is applicable to tissue defects caused by trauma, tumor surgery, etc., and is used for guided regeneration and repair of defective tissues, so as to realize regenerative healing of defective tissues. As a functional biomaterial, the material prepared by the invention can also be used as a tissue engineering scaffold for the research and development of tissue engineering artificial organs.

technical background

Medical guided tissue regeneration materials are an important part of medical biomaterials and a new type of biomaterials developed in recent years. The concept of guided tissue regeneration (guided tissue regeneration, GTR) was first proposed by Nyman et al. in the 1980s. It refers to relying on mechanical barriers and other functions to selectively guide cells to attach and proliferate to damaged parts to achieve the purpose of tissue repair. The key to realizing tissue disease (defect) repair and tissue-guided regeneration is the development and application of functional repair materials.

As a new treatment method to promote tissue regenerative healing, guided tissue regeneration cleverly uses the protection and barrier effect of membrane materials to achieve morphological and functional tissue regeneration or reconstruction. The rise of this technology has broken the limitations of traditional surgical therapy. The key to this technology is the application of guided tissue regeneration membranes. Most of the existing guided recycled materials are prepared from a single material, which can be divided into two categories: degradable and non-degradable. From the late 1970s to the early 1980s, Nyman, Gottlow, Becker and others successively studied non-degradable materials such as polyacetal, polytetrafluoroethylene (PTFE), silicone membrane, etc. Good biocompatibility, but because it cannot be absorbed by the tissue, a second operation is required to remove the material, which increases the chance of trauma.

The traditional guided tissue regeneration mainly utilizes the membrane effect formed by certain biological materials, so that the damaged tissue is partially protected from the surrounding fibrous connective tissue, etc., which is conducive to tissue regeneration and repair, but it is not very good for the tissue that needs to be repaired and regenerated. promotion. In order to be more conducive to tissue repair and regeneration, it is necessary to develop new materials. The new GTR material developed should be able to prevent fibroblasts from growing into the defect, and should also promote the regeneration and repair of the defect tissue, which has biological functions. This goal can be achieved by preparing guided tissue regeneration materials with a multilayer structure, and some research work has made progress. In these research works, due to the limitation of the main material (such as the selection of type II collagen to make it more suitable for cartilage tissue) or the instability of the preparation process (such as the preparation of multilayer film materials by mechanical pressing, there may be split Separation of the layer structure) affects the practicality of these developed products. The widely used type I collagen is selected as the main material, and at the same time, the biological function of promoting cell adhesion, proliferation and differentiation of hyaluronic acid or its sodium salt is used, and a more stable and simple preparation process can be used to better realize the guidance. Clinical needs for tissue regeneration and repair.

Contents of the invention

The purpose of the present invention is to provide a double-layer composite collagen matrix guided tissue regeneration material and its preparation method, which is a medical guided tissue regeneration material prepared from collagen extracted from bovine tendon as the main raw material. The novel medical guided tissue regeneration membrane produced by the present invention has the characteristics of collagen material and hyaluronic acid or its sodium salt material, and has excellent biocompatibility. The prepared material has different structures on both sides, one side is a loose structure, and the other One side is a dense structure, so that the product can prevent fibroblasts from growing into the defect, and at the same time promote the regeneration and repair of the defect tissue. It has corresponding biological functions, can be degraded and absorbed, and does not need a second operation to remove the implant material. Notable features.

The invention comprises the collagen protein extracted from the connective tissue of animals by protease digestion method, treated with 0.3% malonic acid solution to swell the collagen protein, and then adjusting the solid content with 0.3% malonic acid solution after mixing. That is, a collagen solution is obtained. Collagen solutions with different content ratios were prepared respectively, wherein hyaluronic acid or its sodium salt was mixed in the collagen solution with a low content ratio, and the dosage of hyaluronic acid or its sodium salt was the dry weight of collagen in the collagen solution with a corresponding low content ratio The 5-20% of the film is formed by the usual method of freeze-drying and pressure molding, and the collagen and hyaluronic acid or its sodium salt after film formation are cross-linked by conventional cross-linking methods, and finally dried again or freeze-dried. Cross-linked composite collagen membrane material.

In order to achieve the purpose of the present invention, firstly, connective tissues of mammals such as bovine tendon, cowhide, pig tendon, pig skin, etc. are used for enzyme treatment to prepare a collagen solution, see Chinese patent ZL94118836.1. The specific content includes extracting type I collagen from bovine tendon tissue by enzymatic digestion, and removing the antigenic telopeptide from the collagen fiber, so that it has good biocompatibility. Medical grade hyaluronic acid or its sodium salt is selected and treated with oxidation to make it have good composite performance. Through blending, multiple times, multi-stage freeze-drying techniques and chemical cross-linking, a composite material with a double-layer structure is prepared. One side of the material is a loose layer containing collagen and a moderate proportion of hyaluronic acid or its sodium salt, and the other side is a loose layer. It is a dense layer containing a high concentration ratio of collagenous material. Specifically, take an appropriate amount of collagen swelling solution in proportion, pre-freeze after appropriate dilution, freeze-dry under suitable conditions, and process the freeze-dried material into a dense structure; weigh collagen swelling solution and hyaluronic acid or its Dilute and dissolve the sodium salt separately, slowly add hyaluronic acid or its sodium salt solution to the evenly dispersed collagen diluent, stir at a low speed while adding to mix the two, and pre-freeze for an appropriate time to form a viscous ice-water structure. Finally, the prepared collagen dense layer is tightly covered on it, and then frozen and freeze-dried to form a relatively loose/dense structure; the freeze-dried composite material is chemically cross-linked, washed and freeze-dried to prepare a finished product. The cross-linking agent used for chemical cross-linking can be selected from commonly used cross-linking agents such as aldehydes and diimines. The obtained products are cut into desired shapes, packed with aluminum foil or other flexible packaging materials, and then sterilized by gamma-ray irradiation. During the preparation process, the thickness of the dense layer and the loose layer can be adjusted to adjust the thickness of the final product as required.

The specific embodiment of the present invention is: the double-layer composite collagen-based guided tissue regeneration material is a double-layer composite material with dense layer and loose layer structure prepared from collagen and hyaluronic acid or its sodium salt as the main raw materials, and the mass composition is:

The dense layer is 0.6-0.8% malonate collagen swelling solution.

The loose layer is hyaluronic acid or its sodium salt with 0.2-0.4% malonate collagen swelling solution and 5-20% collagen dry weight content.

The preparation method of the described double-layer composite collagen-based guided tissue regeneration material comprises the following steps:

(1) Extract and prepare collagen swelling liquid according to the method described in patent 94118836.1, and prepare 0.6-0.8% malonate collagen swelling liquid with 0.3% malonic acid according to the metering;

(2) Pour the malonic acid collagen swelling solution into a suitable container and place it in a refrigerator at -35°C for 2 hours, with a thickness of 1-3cm, and then freeze-dry it in a vacuum freeze dryer;

(3) place the collagen film between two flat plates of appropriate size, and press it mechanically to form a relatively dense layer;

(4) At 4°C, oxidize the medical grade hyaluronic acid or its sodium salt with an oxidant sodium periodate solution for 18 hours, and then reduce the unreacted residual oxidant with sodium bisulfite until it is colorless;

(5) At 4°C, a large amount of water was dialyzed for 72 hours, then pre-heated at -30°C, and freeze-dried in a vacuum freeze dryer for use.

(6) Take 0.4-0.8% of evenly dispersed malonic acid collagen swelling solution and dissolve the above-mentioned treated hyaluronic acid or its sodium salt with 0.3% malonic acid or water to form a solution, hyaluronic acid or its sodium salt The dosage is 5-20% of the dry weight of collagen. Add hyaluronic acid or its sodium salt solution to the collagen swelling solution to make the final concentration of the collagen swelling solution 0.2-0.4%. Stir and mix well, transfer to a suitable container with a thickness of 1- 3cm, pre-freeze at -30°C for 30 minutes; then cover the dense layer of collagen that has been prepared above tightly, continue to freeze at -30°C and perform lyophilization;

(7) cross-linking the above-mentioned formed composite material with a common cross-linking solution, after cross-linking, fully wash with distilled water and soak overnight until neutral; then place it in a suitable container, and freeze-dry by freeze-drying method;

(8) After taking it out from the lyophilizer, place it on two flat plates of suitable size, press it flat, and become the final product;

(9) Cutting into different specifications, packaging, and then sterilizing with gamma-ray irradiation to obtain a composite collagen-based guided tissue regeneration material.

The loose layer is composed of 0.2-0.4% collagen swelling solution and 5-20% hyaluronic acid or sodium salt of collagen dry weight.

In the preparation step of the loose layer, hyaluronic acid or its sodium salt solution is added to the collagen swelling solution, the pH value can also be adjusted to 7-9 with 0.1M Tris base or 0.1M sodium bicarbonate solution, and the temperature is kept at 20°C Stir in an air bath at a low speed, <60 rpm, for 24 hours to cause a cross-linking reaction between the two. After the reaction, carefully discard the excess liquid, and pre-freeze the remaining solid suspension at -30°C until it forms Loose layer.

The novel medical guided tissue regeneration membrane produced by the present invention has the characteristics of collagen material and hyaluronic acid or its sodium salt material, and has excellent biocompatibility. The prepared material has different structures on both sides, one side is a loose structure, and the other One side is a dense structure, so that the product can prevent fibroblasts from growing into the defect, and at the same time promote the regeneration and repair of the defect tissue. It has corresponding biological functions, can be degraded and absorbed, and does not need a second operation to remove the implant material. Notable features. The invention is applicable to tissue defects caused by trauma, tumor surgery, etc., and is used for guided regeneration and repair of defective tissues, so as to realize regenerative healing of defective tissues. As a biological material, the material prepared in the present invention can also be used as a tissue engineering scaffold for the research and development of tissue engineering artificial organs.

Description of drawings

Fig. 1 SEM image of the dense side surface of the bilayer film (×50).

Fig. 2 SEM image of the dense side surface of the bilayer film (×50).

Detailed ways

Example 1

Adopt the method described in Chinese patent ZL94118836.1 (CN 1086145C) to extract and prepare the collagen swelling solution, the swelling solution is diluted to 0.6% with 0.3% malonic acid, and 100 grams of the swelling solution diluted with collagen protein is poured into a diameter of 100 mm and a height of 100 mm. 2.5cm glass dish. The petri dish was first placed in a refrigerator at -35°C for 2 hours. After taking it out, the frozen petri dish was quickly placed on the shelf of a vacuum freeze dryer and freeze-dried for 50 hours under a vacuum of 10-100u. Take out the lyophilized collagen material from the lyophilizer and place it between two polytetrafluoroethylene plates, and flatten it with a smooth object such as a roller to form a relatively dense layer.

Dissolve 1 gram of medical-grade sodium hyaluronate in 50ml of water, oxidize with 50ml of 20mM sodium periodate solution, 5ml of 0.2M sodium perchlorate, 18 hours at 4°C, and slowly add 0.04M hydrogen sulfite dropwise after the reaction is completed Sodium reduces the unreacted residual oxidant. Determine the amount to be added according to the color change until the color is colorless. Dialyze a large amount of water at 4°C for 72 hours. Change the dialysis water 4 times a day. Transfer to a suitable container for pre-freezing at -30°C. Freeze-dry in a vacuum freeze dryer for later use.

Take collagen swelling solution and treated sodium hyaluronate respectively by appropriate ratio, dilute and dissolve respectively, make collagen dilution swelling solution concentration be 0.6% 50ml, sodium hyaluronate consumption is 15% (mass) relative to collagen dry weight content ratio), use 0.3% malonic acid or water to dissolve in 50ml solution, slowly add sodium hyaluronate solution to the uniformly dispersed collagen diluent, stir at low speed while adding to mix the two, and pre-freeze at -30°C for an appropriate time (about 30 minutes), a viscous ice-water structure is formed, and the prepared collagen dense layer is tightly covered on it, and then frozen and freeze-dried to become a composite material with different densities on both sides.

The molded composite material prepared by the above method is crosslinked for 120 minutes with a formaldehyde solution with a pH of 8.4, and after crosslinking, it is fully washed with distilled water for 10-15 times and soaked overnight until neutral. Then it is placed in a glass plate, pre-frozen and then freeze-dried. After removal from the lyophilizer, the membrane was again placed between two Teflon plates and flattened to become the final product.

Cut into different specifications, pack with aluminum foil or other soft packaging materials, and then sterilize with gamma-ray irradiation at a dose of 25kGy to obtain a composite collagen matrix-guided tissue regeneration material. Fig. 1 SEM image of the dense side surface of the bilayer film (×50). Fig. 2 SEM image of the dense side surface of the bilayer film (×50).

Example 2

The dense collagen layer and the treatment with sodium hyaluronate are the same as in Example 1.

The preparation process of the loose layer is as follows: Weigh an appropriate amount of collagen swelling solution and treated sodium hyaluronate, dilute and dissolve respectively, the concentration and ratio are the same as in Example 1, add the sodium hyaluronate solution into the collagen swelling solution, and use 0.1M Adjust the pH value to 7-9 with Tris base or 0.1M sodium bicarbonate solution, and shake it in a constant temperature air bath at 20°C for 24 hours at a low speed (<60 rpm) to make the oxidized sodium hyaluronate molecules and collagen A crosslinking reaction occurs. After the reaction, carefully discard the excess liquid, and prefreeze the remaining solid suspension according to the conditions in Example 1 until a certain viscous ice-water structure is formed to tightly cover the prepared dense layer, and continue to prefreeze until it is completely formed. Ice-like structure, freeze-dried. The freeze-dried sample was pressed into a mechanical device, sliced, packaged, and sterilized by gamma-ray irradiation at a dose of 25 kGy to obtain a composite collagen-based guided tissue regeneration material.

Claims (3)

1, a kind of double layer composite collagen base guide tissue regeneration material, it is made up of the collagen layer and the weaker zone of compact texture, and the collagen layer that it is characterized in that described compact texture is to be formed by 0.6-0.8% malonic acid collagen swelling solution lyophilizing compacting; Described weaker zone is that the malonic acid or the aqueous solution lyophilizing of malonic acid collagen swelling solution and hyaluronic acid or its sodium salt forms, hyaluronic acid or its sodium salt consumption 5-20% that is the collagen dry weight wherein, the concentration of collagen in final mixed solution is 0.2-0.4%; The collagen compacted zone closely is overlying on repressed again molding after the continuation lyophilization on the weaker zone.

2, the said double layer composite collagen base guide tissue regeneration preparation methods of claim 1 is characterized in that it comprises the steps:

(1) method described in the Chinese patent ZL94118836.1 of pressing is extracted preparation collagen swelling solution, is mixed with malonic acid collagen swelling solution 0.6-0.8% by the malonic acid that measures with 0.3%;

(2) malonic acid collagen swelling solution is poured in the container into the refrigerator 2 hours that places-35 ℃, then in vacuum freeze drier, lyophilization;

(3) collagem membrane is placed two be fit to flatten between the big or small flat board, make into relative compacted zone;

Under (4) 4 ℃, hyaluronic acid and sodium salt thereof are carried out oxidation processes 18 hours with the oxidant sodium periodate solution, use the remaining oxidant of bisulfite sodium reduction unreacted then, till colourless;

Under (5) 4 ℃, in a large number to water dialysis 72 hours, ℃ pre-freeze then-30, lyophilizing is stand-by in the vacuum freeze drier;

(6) get homodisperse malonic acid collagen swelling solution by metering and through hyaluronic acid or its sodium salt of above-mentioned processing, this kind hyaluronic acid or its sodium salt are become solution with 0.3% malonic acid or water dissolution, add hyaluronic acid or its sodium salt solution in the collagen swelling solution, stirring and evenly mixing,-30 ℃ of pre-freezes 30 minutes, then the above-mentioned collagen compacted zone that has prepared is closely covered thereon ,-30 ℃ are continued freezing and carry out lyophilizing and handle;

(8) composite of above-mentioned molding is crosslinked with cross-linking agent solution commonly used, crosslinked back uses distilled water thorough washing 10-15 time and soaked overnight to neutral; And then be placed in the container freeze-drying method lyophilizing;

(9) from freeze dryer, take out after, film is placed between two flat boards, machinery adds and flattens, and becomes end article;

(10) cut into different specifications, packing is shone sterilization with gamma-radiation then, promptly obtains the composite collagen base guide tissue regeneration material.

3, double layer composite collagen base guide tissue regeneration preparation methods according to claim 2, it is characterized in that in the described weaker zone preparation process that hyaluronic acid or its sodium salt solution add in the collagen swelling solution, be to regulate pH value to 7-9 with the Tris alkali of 0.1M or the sodium bicarbonate solution of 0.1M, put stirring at low speed in 20 ℃ of Tempeerature-constant air bath cabinets,＜60 commentaries on classics/per minutes, 24 hours, make the generation cross-linking reaction, after reaction finishes, discard too much liquid, remaining solid suspension-30 ℃ pre-freeze is to forming weaker zone.

Images