阅读说明：本技术 一种高强度细胞外基质海绵及其制备方法 (High-strength extracellular matrix sponge and preparation method thereof ) 是由 高秀岩 郑红霞 姜红 任孝敏 王京燕 董平格 高英娇 曲梁静 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种高强度细胞外基质海绵及其制备方法,这种高强度细胞外基质海绵呈白色蜂窝状结构,其制备方法包括以下步骤：取哺乳动物腹膜组织,机械方式联合碳酸氢钠溶液浸泡去除脂肪,EDTA-NaOH溶液去除细胞核等免疫原性物质,采用不同浓度梯度的NaOH溶液洗脱,强酸处理,纯化水洗涤至pH值4-7,冻干,辐照灭菌形成一种高强度细胞外基质海绵。(The invention discloses a high-strength extracellular matrix sponge and a preparation method thereof, wherein the high-strength extracellular matrix sponge is of a white honeycomb structure, and the preparation method comprises the following steps: taking peritoneal tissues of mammals, soaking the tissues in a mechanical way in combination with a sodium bicarbonate solution to remove fat, removing immunogenic substances such as cell nucleuses and the like by an EDTA-NaOH solution, eluting the tissues by NaOH solutions with different concentration gradients, treating the tissues by strong acid, washing the tissues by purified water to a pH value of 4-7, freeze-drying the tissues, and performing irradiation sterilization to form the high-strength extracellular matrix sponge.)

1. A preparation method of a high-strength extracellular matrix sponge is characterized by comprising the following steps:

1) taking fresh peritoneal tissue of mammal, mechanically removing large fat on the surface, soaking for 6-12h with 1-3% NaHCO3, washing for 24h with running water, and removing residual fat;

2) transferring the tissue obtained in the step 1) into 0.5mol/L EDTA-2Na-2.5% NaOH solution, shaking for 15h at room temperature, then shaking for 2h at room temperature by using 2% NaOH solution, and repeating the operation for 1 time;

3) transferring the tissue in the step 2) into 0.5 percent NaOH solution, shaking for 30min at room temperature, and repeating for 3 times;

4) transferring the tissue in the step 3) into hydrochloric acid solution with the pH value less than 1;

5) washing the tissue in the step 4) in purified water until the pH value is 4-7;

6) and (3) low-temperature freeze drying: freezing at-80 deg.C for 24-48 h, and vacuum-drying;

7) and (5) sterilizing.

2. The method of claim 1, wherein the mammals in step 1) include pigs, cows, dogs, sheep, rabbits and mice, preferably calves.

3. The preparation method according to claim 1, wherein in the step 4), the concentration of the hydrochloric acid solution is 0.1mol/L to 0.5mol/L, and the treatment time is 2 to 3 hours, preferably 1 to 5 hours.

4. A high-strength extracellular matrix sponge obtained by the production method according to any one of claims 1 to 3.

5. The high strength extracellular matrix sponge according to claim 4, wherein the high strength extracellular matrix sponge has a white honeycomb structure and a thickness of 1mm to 3 mm.

Technical Field

The invention relates to preparation of medical materials, in particular to a high-strength extracellular matrix sponge and a novel preparation method thereof, belonging to the field of medical biomaterials.

Background

Extracellular matrix is a macromolecular substance synthesized by cells and secreted into extracellular matrix, and mainly consists of three types of components: 1. structural proteins: such as collagen, elastin, and basement membrane fibronectin, etc.; 2. polysaccharide: such as glycosaminoglycan, proteoglycan, hyaluronic acid, chondroitin sulfate, etc.; 3. adhesion protein: such as fibronectin, laminin, and the like, and in addition, some lipids, growth factors, and the like. The extracellular matrix can be obtained after the treatment such as decellularization and the like of the same or different tissues, and the extracellular matrix is applied to tissue repair, provides a regeneration environment for tissue cells, and can effectively promote the tissue repair.

Collagen, which is a main component of extracellular matrix, has low antigenicity, degradability, good biocompatibility, and the effect of promoting cell proliferation, and is commonly processed into collagen sponge, however, the use thereof is limited to some extent due to the insufficient mechanical strength.

Patent application No. 201110022008.4 describes a collagen sponge and a method for preparing the same, which is prepared by radiation crosslinking of an aqueous solution of collagen, followed by freeze-drying, and sterilization is performed while crosslinking, thereby avoiding biosafety problems caused by the preparation process. Patent application No. 200910059363.1 discloses a method for preparing a cartilage-derived collagen sponge scaffold and a collagen sponge scaffold obtained by the method. The invention method comprises the following steps: the obtained collagen sponge scaffold has good biocompatibility and extremely low antigenicity, can be prepared from cartilages derived from a plurality of individuals of the same animal, has controllable pore diameter and porosity, can provide a proper growth and proliferation environment for chondrocytes and mesenchymal stem cells, particularly has a good repairing effect on cartilage defect parts, and is more beneficial to standardized production and clinical use. Patent application No. 200710040444.8 discloses a method for preparing porous collagen sponge, which comprises dissolving collagen in acetic acid solution, dissolving chitosan in acetic acid solution, mixing the two solutions, and freeze drying to obtain sponge-like material. According to the invention, the porous collagen sponge with uniform pore diameter is obtained by accurately controlling the parameters of the freeze drying process in the preparation process of the porous collagen sponge. The existing preparation technology of the collagen sponge still does not solve the problem of poor mechanical strength of the sponge.

Disclosure of Invention

The invention provides a high-strength extracellular matrix sponge and a preparation method thereof, aiming at the problem of poor mechanical strength of the existing collagen sponge. The method comprises the steps of taking peritoneal tissues of mammals, removing impurities and immunogen substances, swelling with strong acid, and freeze-drying to obtain the high-strength extracellular matrix sponge. The sponge prepared in this way retains various components of the extracellular matrix which are beneficial for tissue regeneration, and the fiber structure of the matrix is less damaged so that the strength thereof is maintained. The fluffy three-dimensional structure provides a good bracket structure for the regeneration of damaged tissue cells. In particular, the sponge does not decrease in strength but increases in strength after absorbing liquid, and is suitable for suturing when used.

The strategy for preparing the biomembrane capable of inducing bone regeneration comprises the following steps: taking peritoneal tissues of mammals, soaking the tissues in a mechanical way in combination with a sodium bicarbonate solution to remove fat, removing immunogenic substances such as cell nucleuses and the like by an EDTA-NaOH solution, eluting the tissues by NaOH solutions with different concentration gradients, treating the tissues by strong acid, washing the tissues by purified water to a pH value of 4-7, freeze-drying the tissues, and performing irradiation sterilization to form the high-strength extracellular matrix sponge.

Further, the high-strength extracellular matrix sponge prepared by the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for preparing a high-strength extracellular matrix sponge, comprising:

1) taking fresh peritoneal tissue of mammal, mechanically removing large fat on the surface, soaking for 6-12h with 1-3% NaHCO3, washing for 24h with running water, and removing residual fat;

2) transferring the tissue obtained in the step 1) into 0.5mol/LEDTA-2Na-2.5% NaOH solution, shaking for 15h at room temperature, then shaking for 2h at room temperature by using 2% NaOH solution, and repeating the operation for 1 time;

3) transferring the tissue in the step 2) into 0.5 percent NaOH solution, shaking for 30min at room temperature, and repeating for 3 times;

4) transferring the tissue in the step 3) into hydrochloric acid solution with the pH value less than 1;

5) washing the tissue in the step 4) in purified water until the pH value is 4-7;

6) and (3) low-temperature freeze drying: freezing for 24-48 h at the temperature of minus 80 ℃, and carrying out vacuum freeze-drying;

7) and (5) sterilizing.

Optionally, the mammals in step 1) include pigs, cows, dogs, sheep, rabbits and mice, preferably calves.

Optionally, in the step 4), the concentration of the hydrochloric acid solution is 0.1mol/L to 0.5mol/L, and the treatment time is 2 to 3 hours, preferably 1 to 5 hours.

In another aspect, the invention provides a high-strength extracellular matrix sponge prepared by the preparation method.

Optionally, the high-strength extracellular matrix sponge is white honeycomb-shaped and suture-resistant.

The invention has the following beneficial effects:

1. the high-strength extracellular matrix sponge obtained by the preparation method provided by the invention has high strength, can tolerate the traction of clinical suture, and has the suture strength of 10N in a wet state;

2. the high-strength extracellular matrix sponge provided by the invention effectively removes the immunogenicity of the material, retains the fibrous structure of the original tissue, and ensures the stability of the mechanical property of the sponge;

3. the preparation method of the high-strength extracellular matrix sponge provided by the invention is simple and effective, the used reagent has small risk of environmental influence, and the reagent is easy to remove and is suitable for preparing medical materials.

Drawings

FIG. 1 is a photograph of a sample of high-strength extracellular matrix sponge.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the following description is only exemplary of the present invention, and is not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.