CN111714696A

CN111714696A - Composite tendon anti-adhesion membrane and preparation method thereof

Info

Publication number: CN111714696A
Application number: CN202010574129.9A
Authority: CN
Inventors: 李茹冰; 丘海轶; 赵月; 吴云; 李宇璐; 魏照星; 刘泗容; 何楝楝; 邹银萍
Original assignee: Chinese Academy Of Science Shenzhen Hospital, University of
Current assignee: Chinese Academy Of Science Shenzhen Hospital, University of
Priority date: 2020-06-22
Filing date: 2020-06-22
Publication date: 2020-09-29

Abstract

The invention provides a composite tendon anti-adhesion membrane and a preparation method thereof, wherein the composite tendon anti-adhesion membrane is a chitosan-modified nanofiber membrane, and collagen is loaded on the nanofiber membrane; the loading capacity of the collagen is 5-9%. The preparation method of the composite tendon anti-adhesion membrane comprises the preparation of a chitosan-modified nanofiber membrane and the loading of collagen. The composite tendon anti-adhesion membrane prepared by the invention can ensure good biological adhesion to promote endogenous repair more effectively, has excellent flexibility and mechanical property, prolongs the service life of the adhesion membrane and reduces adhesion formed by tendon peripheral tissues.

Description

Composite tendon anti-adhesion membrane and preparation method thereof

Technical Field

The invention belongs to the field of biological materials, and particularly relates to a composite tendon anti-adhesion membrane and a preparation method thereof.

Background

Adhesion after tendon injury is one of the difficulties faced in hand surgery, and is an abnormal structure formed by the connective tissue fiber band combined with adjacent tissues or organs. After surgery, adhesion is easy to occur in the process of tissue healing, and the performance and function recovery of the operation part are affected. In particular, the problem of adhesion is very common in tendon regions, mainly caused by the contact of fibroblasts with the tendon, which is a hyperplastic connective tissue membrane surrounding the tendon. The tendon injury recovery time is relatively long, and the adhesion phenomenon can seriously affect the recovery of the normal function of the tendon.

The development of a product that reduces the adhesion formation of tissue around the tendon (i.e., exogenous healing), promotes the ability of tenocytes to divide and proliferate themselves, and heals without forming an adhesion (i.e., endogenous healing) is crucial to the success of tendon surgery.

Through search, relevant application publications are disclosed in the prior art for research on anti-adhesion biomembranes, for example, the application with Chinese patent application number 201310358048.5 and publication date 2015.10.28 discloses an anti-adhesion biomembrane and a preparation method thereof, wherein SIS is used as a scaffold material, and a dry film formed by compounding hyaluronic acid, functional protein and anti-inflammatory drugs is in a light white to light yellow semitransparent shape and has the thickness of 0.01-0.5 mm. Compared with the prior art, the anti-adhesion biomembrane has excellent biocompatibility, has good physical barrier anti-adhesion effect, has the effects of resisting inflammation, stopping bleeding and promoting fiber degradation, and has the effect obviously superior to that of various existing anti-adhesion biomembrane products. The anti-adhesion biofilm of this application, although having excellent biocompatibility, does not take much into account the flexibility and mechanical strength of the overall material.

Based on the defects of the prior art, a new composite tendon anti-adhesion membrane which can effectively prevent the formation of adhesion without influencing the normal healing of wounds and has certain flexibility and mechanical strength needs to be invented.

Disclosure of Invention

1. Problems to be solved

Aiming at the problem that the anti-adhesion membrane in the prior art cannot simultaneously ensure excellent biocompatibility and mechanical property, the invention provides the chitosan modified nanofiber membrane, wherein collagen is loaded on the nanofiber membrane, and the chitosan modified nanofiber membrane serving as the composite tendon anti-adhesion membrane has excellent biocompatibility, excellent flexibility and mechanical property and is beneficial to tendon injury recovery.

2. Technical scheme

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the invention provides a composite tendon anti-adhesion membrane which is a chitosan-modified nanofiber membrane, wherein collagen is loaded on the nanofiber membrane.

Preferably, the loading of the collagen is 5-9%.

Preferably, the preparation method of the composite tendon anti-adhesion membrane comprises the following steps:

1) preparing a chitosan modified nanofiber membrane: dissolving chitosan and cellulose derivatives in a mixed solvent, stirring uniformly, standing for defoaming to obtain an electrostatic spinning solution, and then performing electrostatic spinning to obtain a chitosan-modified nanofiber membrane;

2) loading of collagen: and (3) carrying out vacuum drying treatment on the chitosan-modified nanofiber membrane to remove residual solvent in the fiber membrane, then placing the fiber membrane in a collagen hydrolysate solution for soaking treatment, and then carrying out freeze drying to obtain the composite tendon anti-adhesion membrane.

Preferably, the concentration of the electrostatic spinning solution in the step 1) is 220-275 g/L.

Preferably, the molecular weight of the chitosan is 2-50 kDa, and the cellulose derivative is cellulose acetate.

Preferably, the mass ratio of the chitosan to the cellulose derivative in the step 1) is 1: 2.

preferably, the electrostatic spinning process conditions are as follows: the voltage is 18-24kV, the distance from the needle point to the roller is 10-15cm, and the flow rate of the solution is 0.5-1.0 mL/h.

Preferably, the mixed solvent is a mixture of any two or more of trifluoroacetic acid, formic acid, acetic acid, acetone, N-dimethylformamide, dimethyl sulfoxide, hexafluoroisopropanol, trifluoroethanol and trichloromethane.

Preferably, the collagen hydrolysate solution is obtained by dissolving hydrolyzed collagen in water, and the mass concentration of the collagen hydrolysate solution is 5-15%. The hydrolyzed collagen was purchased from Shijiazhuang Xuermei Biotech limited.

Preferably, the soaking time in the step 2) is 18-25 h.

3. Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) the composite tendon anti-adhesion membrane provided by the invention is prepared by dissolving chitosan and cellulose derivatives in a mixed solvent to prepare an electrostatic spinning solution, then performing electrostatic spinning to obtain a chitosan-modified nanofiber membrane, and loading collagen by soaking the chitosan-modified nanofiber membrane in a collagen hydrolysate solution.

(2) According to the composite tendon anti-adhesion membrane provided by the invention, the collagen is loaded on the nanofiber membrane layer, so that the good fitting property and endogenous healing promotion function of the collagen can be effectively utilized, the chitosan modified nanofiber membrane layer is adopted, the degradation time of the whole material can be effectively prolonged, an effective isolation effect is achieved, the chitosan serving as an effective component simultaneously has a good hemostatic and antibacterial function, and the whole composite tendon anti-adhesion membrane has good flexibility after being wetted, and on the other hand, the polylactic acid-based chitosan nanofiber membrane has the characteristics of high porosity and large specific surface area, so that the composite tendon anti-adhesion membrane not only has better flexibility and mechanical property, but also is beneficial to transmitting nutrient substances and promoting the transportation of the nutrient substances. The synergistic effect of the components in the composite tendon anti-adhesion membrane provided by the invention can better accord with the mechanism of promoting endogenous healing, and is close to the ideal effect of a tendon anti-adhesion product.

Drawings

FIG. 1 is a scanning electron micrograph of the complex tendon anti-adhesion membrane prepared in example 1 (protein loading is 5%);

FIG. 2 is the observation result of HE pathological section of the compound tendon anti-adhesion membrane animal experiment prepared in example 1;

fig. 3 is a scanning electron micrograph of the complex tendon anti-adhesion membrane provided in example 2 (protein loading 6%).

Detailed Description

It should be noted that the terms "upper", "lower", "left", "right" and "middle" used in the present specification are for the sake of clarity, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; as used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, measure or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art.

As used herein, at least one of the terms "is intended to be synonymous with one or more of. For example, "at least one of A, B and C" explicitly includes a only, B only, C only, and combinations thereof, respectively.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limit values of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all of the aforementioned values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or feature being described.

Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims.

The invention is further described with reference to specific examples.

Example 1

The invention provides a composite tendon anti-adhesion membrane which is a chitosan-modified nanofiber membrane, wherein collagen is loaded on the nanofiber membrane, and the loading amount of the collagen is 5%.

The preparation method of the composite tendon anti-adhesion membrane comprises the following steps:

1) preparation of chitosan modified nanofiber membrane

Dissolving chitosan and cellulose acetate (the mass ratio of the chitosan to the cellulose acetate is 1: 2) in a mixed solvent, uniformly stirring, standing and defoaming to obtain an electrostatic spinning solution with the solution concentration of 220-275g/L, and then performing electrostatic spinning; obtaining a chitosan-modified nanofiber membrane; the molecular weight of the chitosan is 2-50 kDa;

the electrostatic spinning process conditions are as follows: the voltage is 18-24kV, the distance from the needle point to the roller is 10-15cm, and the flow rate of the solution is 0.5-1.0 mL/h;

the mixed solvent is a mixture of any two or more of trifluoroacetic acid, formic acid, acetic acid, acetone, N-dimethylformamide, dimethyl sulfoxide, hexafluoroisopropanol, trifluoroethanol and trichloromethane.

2) Collagen loading

And (2) carrying out vacuum drying treatment on the chitosan-modified nanofiber membrane at 37 ℃ to remove residual solvent in the fiber membrane, then placing the fiber membrane in collagen hydrolysate solution for soaking treatment for 18-25h, and then carrying out freeze drying to obtain the chitosan/cellulose-collagen composite tendon anti-adhesion membrane.

The collagen hydrolysate solution is obtained by dissolving hydrolyzed collagen in water, and the mass concentration of the hydrolyzed collagen is 5-15%; the hydrolyzed collagen was purchased from Shijiazhuang Xuermei Biotech limited.

The mechanical property test data of the composite tendon anti-adhesion membrane (chitosan/cellulose-collagen nanofiber membrane) prepared in this example are shown in table 1. The scanning electron micrograph of the composite tendon anti-adhesion membrane is shown in FIG. 1.

TABLE 1 mechanical Properties test data of Chitosan/cellulose-collagen nanofiber membranes

As can be seen from Table 1, the composite tendon anti-adhesion membrane prepared by the invention has excellent flexibility and mechanical properties. As can be seen from figure 1, the collagen-loaded composite tendon anti-adhesion membrane prepared by the method has a developed pore structure, has the characteristics of high porosity and large specific surface area, and therefore has better flexibility and mechanical properties, and is beneficial to transmitting nutrients, and the transportation of the nutrients is promoted in the actual use process.

In order to verify the practical application effect of the composite tendon anti-adhesion membrane prepared in this example, animal experiments were performed, and the experimental procedures were as follows:

experimental animals: adult SPF SD rats, 40, half male and female, with weight of 180-200 g, were purchased from Guangdong province medical laboratory animal center and were subjected to adaptive feeding for 3-7 days.

Materials: surgical instruments, anesthetics (Uratin), gauze, surgical cotton swab, iodophor, alcohol, surgical drape, penicillin, suture needle with thread (Ningbo medical suture needle Co., Ltd., nylon 3/040 cm double needle, 1/2 arc 4X 12), absorbable electrospun polymer membrane

The instrument and equipment method comprises the following steps: 1) preparation of tendon adhesion animal model: after anesthetizing the animal, the animal's hind limbs were shaved, draped for routine sterilization, and the animal was mounted on an operating table. Disinfecting feet with iodophor and deiodinating with alcohol, exposing the 3 rd-toe deep flexor tendon on both sides of the experimental animal by adopting a side incision and performing an operation to transect the middle part of the tendon by 50%, then performing an operation to suture the tendon broken ends by using a suture needle with threads and a splayed suture method, preparing a tendon adhesion model, and suturing the skin; different groups were set, and the experimental group used the composite tendon anti-adhesion membrane prepared in example 1, and the control group was a control without any adhesion membrane and was fixed with plaster or externally fixed with a brace. And (5) returning the experimental animal after the operation to a cage for single cage feeding. At the end of the 1 week period, 12 each of the groups were subjected to gross anatomical and histological observations.

2) And E, observation of HE pathological section: taking tendon sheaths and tendon tissues within the range of 0.5cm on two sides of a tendon suture opening, embedding the tendon sheaths and the tendon tissues by conventional paraffin, slicing tendon cross sections, and observing tissue adhesion, tendon repair and inflammatory cell infiltration conditions at each time point by HE staining.

FIG. 2 is the observation result of HE pathological section of the compound tendon anti-adhesion membrane animal experiment prepared in example 1; where panel a is normal group: shows that the tendon has normal shape and structure and does not have abnormal conditions such as inflammatory cell infiltration; panel b is a pathological section of the control group three days after surgery, and adhesion can be observed to begin to appear; the graph c is a pathological section graph of the control group at 1-2 weeks after the operation, and a large amount of inflammatory cell infiltration can be observed, and the graph d and the graph e are pathological section graphs of the experimental group at 1-2 weeks after the operation, and a small amount of inflammatory cell infiltration can be observed.

Example 2

The invention provides a composite tendon anti-adhesion membrane which is a chitosan-modified nanofiber membrane, wherein collagen is loaded on the nanofiber membrane, and the loading amount of the collagen is 6%. FIG. 2 is the electron microscope image of the composite tendon anti-adhesion membrane prepared in this example.

1) preparation of chitosan modified nanofiber membrane

Dissolving chitosan (molecular weight is 2-50 kDa) and cellulose acetate (mass ratio of the chitosan to the cellulose acetate is 1: 2) in a mixed solvent, uniformly stirring, standing and defoaming to obtain an electrostatic spinning solution with solution concentration of 220-275g/L, and then performing electrostatic spinning; obtaining a chitosan-modified nanofiber membrane;

2) Collagen loading

Example 3

The invention provides a composite tendon anti-adhesion membrane which is a chitosan-modified nanofiber membrane, wherein collagen is loaded on the nanofiber membrane, and the loading amount of the collagen is 9%.

1) preparation of chitosan modified nanofiber membrane

Dissolving chitosan (with the molecular weight of 2-50 kDa) and cellulose acetate (with the mass ratio of 1: 2) in a mixed solvent, uniformly stirring, standing and defoaming to obtain 275g/L electrostatic spinning solution with the solution concentration of 220-; obtaining a chitosan-modified nanofiber membrane;

2) Collagen loading

Claims

1. The utility model provides a compound anti-adhesion membrane of tendon which characterized in that: the composite tendon anti-adhesion membrane is a chitosan-modified nanofiber membrane, and collagen is loaded on the nanofiber membrane.

2. The composite tendon anti-adhesion membrane of claim 1, wherein: the loading capacity of the collagen is 5-9%.

3. The method for preparing the composite tendon anti-adhesion membrane of any one of claims 1 or 2, wherein: the method comprises the following steps:

4. The method for preparing the composite tendon anti-adhesion membrane according to claim 3, wherein: the concentration of the electrostatic spinning solution in the step 1) is 220-275 g/L.

5. The method for preparing the composite tendon anti-adhesion membrane according to claim 4, wherein: the molecular weight of the chitosan is 2-50 kDa, and the cellulose derivative is cellulose acetate.

6. The method for preparing the composite tendon anti-adhesion membrane according to claim 3, wherein: the mass ratio of the chitosan to the cellulose derivative in the step 1) is 1: 2.

7. the method for preparing the composite tendon anti-adhesion membrane according to claim 6, wherein: the electrostatic spinning process conditions are as follows: the voltage is 18-24kV, the distance from the needle point to the roller is 10-15cm, and the flow rate of the solution is 0.5-1.0 mL/h.

8. The method for preparing the composite tendon anti-adhesion membrane according to claim 7, wherein: the mixed solvent is a mixture of any two or more of trifluoroacetic acid, formic acid, acetic acid, acetone, N-dimethylformamide, dimethyl sulfoxide, hexafluoroisopropanol, trifluoroethanol and trichloromethane.

9. The method for preparing the composite tendon anti-adhesion membrane according to claim 6 or 7, wherein: the collagen hydrolysate solution is obtained by dissolving hydrolyzed collagen in water, and the mass concentration of the collagen hydrolysate solution is 5-15%.

10. The method for preparing the composite tendon anti-adhesion membrane according to claim 9, wherein: the soaking time is 18-25 h.