CN116077743A

CN116077743A - Degradation-controllable absorbable medical anti-adhesion film and preparation method thereof

Info

Publication number: CN116077743A
Application number: CN202211729810.1A
Authority: CN
Inventors: 王永会; 谢艺; 李冲
Original assignee: Zhejiang Zhuji Juyuan Biotechnology Co ltd
Current assignee: Zhejiang Zhuji Juyuan Biotechnology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2023-05-09

Abstract

The invention discloses a degradation-controllable absorbable medical anti-adhesion membrane and a preparation method thereof. The method comprises the steps of mixing recombinant humanized collagen with a cross-linking agent for in-situ cross-linking, adding glycerol, pouring into a mold to form a prefabricated glue, and finally preparing the absorbable medical anti-adhesion film through secondary drying and primary rinsing. The absorbable medical anti-adhesion film prepared by the invention has controllable degradation period, good flexibility and deformation resistance, strong water absorption capacity and good biocompatibility, and the complete degradation product is amino acid, can be completely absorbed by human body, has high safety, and has the advantage of once use without secondary operation.

Description

Degradation-controllable absorbable medical anti-adhesion film and preparation method thereof

Technical Field

The invention belongs to the technical field of medical anti-adhesion materials, and relates to a degradable and controllable absorbable medical anti-adhesion film and a preparation method thereof.

Background

Adhesions are abnormal structures formed by the binding of connective tissue fibrous tapes to adjacent tissues or organs, which are both common clinical phenomena after surgery and the process that a patient must undergo during the healing process. If the adhesion phenomenon occurs in the operations of abdominal cavity, pelvic bones and the like, serious complications such as adhesive intestinal obstruction and female infertility caused by pelvic tissue adhesion are caused; adhesion after thyroid surgery causes recurrent laryngeal nerve injury.

The anti-adhesion membrane is a membrane material which has moderate softness and can physically isolate an affected part from surrounding tissues, plays a role in preventing tissue adhesion in the tissue healing process, can be automatically degraded and absorbed in vivo after operation, and has good tissue compatibility. Medical absorbable anti-blocking film products on the market are largely divided into two categories: the natural polymer material is mainly hyaluronic acid, cellulose derivatives and their complexes, chitosan and modified products thereof; one is derived from artificially synthesized polymer materials, mainly polylactic acid, lactic acid-glycolic acid copolymer (PLGA), polyethylene glycol (PEG) and their complexes. Chinese patent application 201310232435.4 discloses a film for preventing postoperative tissue adhesion, which comprises a mixed solution of poly-L-glutamic acid/chitosan/polyethylene glycol as a main component, wherein the main component chitosan has certain elasticity, but the main component chitosan is easy to cause the occurrence of conjunctivitis, with or without adverse phenomena such as fever, blood leukocyte increase and the like of a patient after gynecological operation. Chinese patent application 201310380614.2 discloses an anti-adhesion hemostatic membrane which is prepared by adopting an electrostatic spinning technology after crosslinking, has a complex production process, does not remove cross-linking agent residues, and has undefined biocompatibility.

The recombinant humanized collagen is used as a novel absorbable and degradable biological material, and is increasingly widely applied in the biomedical field by virtue of the unique biosafety and the excellent tissue regeneration promoting capability. The recombinant humanized collagen is obtained by fermentation by utilizing a biological genetic engineering technology based on the characteristics of main functional sequences of human-like collagen. Compared with the traditional natural collagen, the collagen has the following advantages: (1) strong workability: the recombinant humanized collagen can form a triple-double spiral structure, and has better processability; (1) controllable quality: the genetic engineering technology can express human-like collagen fragments with specific molecular weight, and the extracted collagen is a product with widely distributed molecular weight, which is not beneficial to quality control; (3) Water solubility: through construction and structural transformation, the recombinant humanized collagen can be dissolved in water, and has higher development value; (4) lower rejection: human-like collagen is constructed based on human collagen genes, and has less immune rejection than animal-derived collagen. However, the degradation of single recombinant humanized collagen in vivo is rapid and not consistent with the time of adhesion formation. In addition, because of its small molecular weight, it is impossible to form a flexible biofilm by itself. Therefore, the molecular weight of the anti-adhesion membrane is increased by adopting a chemical crosslinking method, the degradation time is prolonged, and the anti-adhesion membrane with certain toughness and controllable degradation is prepared.

Disclosure of Invention

The invention aims to provide a degradation-controllable absorbable medical anti-adhesion membrane and a preparation method thereof. The method takes recombinant humanized collagen as a raw material, takes water-soluble carbodiimides as a cross-linking agent, adopts an in-situ cross-linking mode to carry out cross-linking, and prepares the degradable controllable absorbable medical anti-adhesion membrane with excellent biocompatibility and good mechanical property.

The technical scheme for realizing the purpose of the invention is as follows:

the preparation method of the degradation-controllable absorbable medical anti-adhesion film comprises the following specific steps:

(1) Adding a cross-linking agent water-soluble carbodiimide solution into the recombinant humanized collagen solution under continuous stirring, and continuously stirring to obtain a mixed solution with the cross-linking degree of 3% -6%;

(2) Adding glycerol into the mixed solution in the step (1), and stirring until the glycerol and the mixed solution are uniformly mixed to obtain a mixed solution with the volume concentration of the glycerol of 1-5% and the mass concentration of the recombinant humanized collagen of 9-13%;

(3) Transferring the mixed solution obtained in the step (2) into a mold, standing at room temperature, and gelatinizing to form a prefabricated glue;

(4) Performing primary drying on the prefabricated glue to obtain a primary dried film;

(5) Placing the membrane after primary drying in PBS buffer solution for rinsing to obtain transparent gel;

(6) Performing secondary drying on the transparent gel to obtain a secondary dried film;

(7) Cutting, packaging and sterilizing the film after secondary drying to obtain the absorbable medical anti-adhesion film.

The crosslinking degree in the invention refers to the mass ratio of the crosslinking agent to the recombinant humanized collagen.

Preferably, in the step (1), the mass concentration of the recombinant humanized collagen solution is 10% -20%, and the mass concentration of the water-soluble carbodiimide solution is 5% -15%.

Preferably, in step (1) or (2), the stirring speed is 100 to 500rpm, and the stirring time is 1 to 5 minutes.

Preferably, in step (1), the molecular weight of the recombinant humanized collagen is 56-112 Kda.

Preferably, in step (1), the water-soluble carbodiimides include, but are not limited to dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the like.

Preferably, in step (3), the height of the mixed solution transferred into the mold is 1.5mm to 4mm, more preferably 2mm to 3mm.

Preferably, in the step (3), the mold is made of stainless steel or polytetrafluoroethylene or glass.

Preferably, the first drying in step (4) and the second drying in step (6) are performed in the same manner, and are vacuum drying or air drying. When vacuum drying is adopted, the first drying parameters are as follows: the temperature is 45-65 ℃, the vacuum degree is-0.4X100 KPa to-0.7X100 KPa, and the time is 18-28 h; the second drying parameters were: vacuum drying parameters: the temperature is 45-55 ℃, the vacuum degree is-0.1X100 KPa to-0.4X100 KPa, and the time is 3-7 h. When adopting the forced air drying, the first drying parameter is: the temperature is 40-65 ℃ and the time is 1.5-3 h; the second drying parameters were: the temperature is 35-55 ℃ and the time is 1-2.5 h.

Preferably, in the step (5), the PBS buffer solution is prepared from 0.05M-1M sodium dihydrogen phosphate solution and 0.05M-1M disodium hydrogen phosphate solution according to the volume ratio of 1-7: 3-9, and the pH value is 6.5-7.9, more preferably, the pH value is prepared from 0.1M sodium dihydrogen phosphate solution and 0.1M disodium hydrogen phosphate solution according to the volume ratio of 3:7, PBS buffer solution prepared by the method.

Preferably, in the step (5), each rinsing time is 1-5 min, and the rinsing times are 3-5 times.

Preferably, in step (7), the sterilization mode is radiation sterilization, and the dosage is 10-20 KGy, more preferably 15KGy.

Compared with the prior art and the method, the invention has the following advantages:

(1) The invention takes the recombinant humanized collagen as the raw material, has high biological safety, can be completely degraded in vivo, and the degradation products are amino acid and water, and can be completely absorbed by human body as nutritional ingredients; the water-soluble carbodiimide is used as a cross-linking agent, and is cross-linked in an in-situ cross-linking mode, so that the water-soluble carbodiimide cannot be retained in collagen molecules, but is converted into a water-soluble urea derivative with extremely low cytotoxicity, and the whole cross-linking process has good cross-linking effect without introducing toxic substances.

(2) The preparation process of twice drying and once rinsing is adopted, so that the residues of the cross-linking agent can be effectively removed, the pH value can be controlled, the process requirement difficulty is low, and the stability is good; PBS buffer solution is used as rinsing liquid to ensure that the pH value of the medical anti-adhesion film after rinsing is maintained to be neutral, and the medical anti-adhesion film is suitable for human bodies.

(3) The absorbable medical anti-adhesion membrane prepared by the invention controls the degradation starting time to be 4-6 days and the complete degradation time to be 8-14 days by regulating and controlling the concentration and crosslinking degree of the recombinant humanized collagen, meets the requirements of different tissue parts for preventing adhesion, and is matched with the in-vivo adhesion occurrence period to achieve the effect of preventing adhesion.

(4) The absorbable medical anti-adhesion film prepared by the invention has strong water absorption capacity, and the water absorption rate can reach 1300% -2100%; has good flexibility and deformation resistance, tensile strength of 1-20 MPa and elastic modulus of 7-800 kgf/cm ² Provides a proper microenvironment for cell growth, can inhibit inflammatory reaction, and has excellent effects in preventing tissue adhesion and promoting damaged tissue repair.

Drawings

FIG. 1 is a graph showing the tensile strength of the anti-blocking films prepared in examples 1 to 6.

FIG. 2 is a graph showing the water absorption of the anti-blocking films prepared in examples 1 to 6.

FIG. 3 is a graph showing the tensile strength of the anti-blocking films prepared in comparative example 5 and example 5.

Fig. 4 is an external view of the anti-blocking films prepared in comparative example 6 (right) and example 2 (left) after drying.

Detailed Description

The invention will be described in further detail with reference to specific embodiments and drawings.

In the examples described below, materials, reagents and the like used, unless otherwise specified, were commercially available. Recombinant humanized collagen was purchased from Jiangsu Jiangshan poly-source biotechnology limited. The PBS buffer solution is prepared from a 0.1M sodium dihydrogen phosphate solution and a 0.1M disodium hydrogen phosphate solution according to the volume ratio of 3: 7.

Comparative example 1

(1) Preparing a raw material solution: preparing 20% recombinant humanized collagen solution and 10% edc solution respectively using purified water;

(2) Weighing 4ml of 20% recombinant humanized collagen solution, adding 5.38ml of purified water, and stirring; adding 0.32ml of 10% EDC solution, and stirring for 2min to obtain a mixed solution with a crosslinking degree of 4%;

(3) Adding 0.3ml of medical grade glycerol into the mixed solution, continuously stirring for 2min, and stopping to obtain a mixed solution with glycerol concentration of 3% and recombinant humanized collagen concentration of 8%;

(4) Preparing a prefabricated glue: transferring the mixed solution into a pre-sterilized polytetrafluoroethylene mould, wherein the height of the solution is 3mm; standing at room temperature to obtain the prefabricated glue;

(5) Placing the prefabricated glue into a vacuum drying box for primary vacuum drying at the temperature of 60 ℃ and the vacuum degree of-0.5X100 KPa for 26 hours to obtain a primary dried film;

(6) The once dried membrane was rinsed in PBS buffer.

Because the concentration of the recombinant humanized collagen is low, the membrane is quickly dissolved after being put into the rinsing liquid and cannot be taken out.

Comparative example 2

(2) Weighing 7ml of 20% recombinant humanized collagen solution, adding 2.28ml of purified water, and stirring; adding 0.42ml of 10% EDC solution, and stirring for 2min to obtain a mixed solution with a crosslinking degree of 3%;

(3) Adding 0.3ml of medical grade glycerol into the mixed solution, continuously stirring for 2min, and stopping to obtain a mixed solution with glycerol concentration of 3% and recombinant humanized collagen concentration of 14%;

(5) The preformed adhesive is placed into a vacuum drying box for primary vacuum drying, the temperature is 60 ℃, the vacuum degree is minus 0.5X100 KPa, and the time is 26 hours.

Because the concentration of the recombinant humanized collagen is higher, the membrane is shrunk after primary vacuum drying, and the next process cannot be continued.

Comparative example 3

(2) Weighing 5.5ml of 20% recombinant humanized collagen solution, adding 4.06ml of purified water, and stirring; adding 0.44ml of 10% EDC solution, and stirring for 2min to obtain a mixed solution with a crosslinking degree of 4%;

(3) Adding no glycerol, continuing stirring for 2min, and stopping to obtain a mixed solution with 11% of recombinant humanized collagen concentration;

(4) Preparing a prefabricated glue: transferring the mixed solution into a pre-sterilized polytetrafluoroethylene mould, wherein the height of the solution is 2mm; standing at room temperature to obtain the prefabricated glue;

(6) Rinsing the membrane in PBS buffer solution for 4 times and 1 min/time after primary drying, absorbing water by the membrane to form transparent gel, and placing the transparent gel in a drying plate;

(7) And carrying out secondary vacuum drying on the transparent gel, wherein the temperature is 50 ℃, the vacuum degree is-0.2 multiplied by 100KPa, and the time is 4 hours, so as to obtain the secondary dried film to be cut.

As no glycerol is added, the film is hard after secondary vacuum drying, has over-strong rigidity, has no flexibility and does not meet the requirements.

Comparative example 4

(2) Weighing 5.5ml of 20% recombinant humanized collagen solution, adding 3.76ml of purified water, and stirring; 0.44ml of 10% EDC solution is added and stirred for 3min to obtain a mixed solution with the crosslinking degree of 4%;

(3) Adding 0.3ml of medical grade glycerol into the mixed solution, continuously stirring for 2min, and stopping to obtain a mixed solution with glycerol concentration of 3%;

(5) Placing the prefabricated glue into a vacuum drying oven for primary vacuum drying at 60 ℃ and vacuum degree of-0.6X100 KPa for 23 hours; or performing primary air-blast drying in an air-blast drying box at 55 ℃ for 2 hours to obtain a film after primary drying;

(6) Placing the film after primary drying on a stainless steel table top, and cutting by using a utility knife or scissors to obtain a semi-finished absorbable medical anti-adhesion film;

(7) Packaging and sterilizing to obtain the finished product of the absorbable medical anti-adhesion film with 11% of the final concentration of the recombinant humanized collagen.

In comparison with example 2, comparative example 4 was a film prepared by vacuum drying only once, and the inside thereof contained many small bubbles to affect the appearance; in comparison with example 5, the film prepared in comparative example 4 was dried only once by blowing, and was poor in softness and liable to break; none of them can be applied.

Comparative example 5

(5) Placing the prefabricated glue into a blast drying box for primary blast drying at 70 ℃ for 2 hours to obtain a primary dried film;

(6) Placing the membrane after primary drying into PBS buffer solution for rinsing for 4 times, 3 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Carrying out secondary air blast drying on the transparent gel at the temperature of 60 ℃ for 1h to obtain a secondary dried film to be cut;

(8) Placing the film after secondary drying on a stainless steel table top, and cutting by using a utility knife or scissors to obtain a semi-finished absorbable medical anti-adhesion film;

(9) Packaging and sterilizing to obtain the finished product of the absorbable medical anti-adhesion film with 11% of the final concentration of the recombinant humanized collagen.

As compared with example 5, films prepared at too high a forced air drying temperature have a tensile strength of 30.247MPa and an elastic modulus of up to 2039.963kgf/cm ² As shown in fig. 3, the rigidity is too high, the flexibility is poor, and the requirements are not met.

Comparative example 6

(4) Preparing a prefabricated glue: transferring the mixed solution obtained in the step 3 into a pre-sterilized polytetrafluoroethylene mould, wherein the height of the solution is 3mm; standing at room temperature to obtain the prefabricated glue;

(5) Placing the prefabricated glue into a vacuum drying box for primary vacuum drying, wherein the temperature is 80 ℃, the vacuum degree is-1.0X100 KPa, and the time is 23 hours, so as to obtain a film after primary drying;

(6) Placing the membrane after primary drying into PBS buffer solution for rinsing for 4 times, 2 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Performing secondary vacuum drying on the transparent gel at 50 ℃ and the vacuum degree of-0.2 multiplied by 100KPa for 6 hours to obtain a secondary dried film to be cut;

Compared with example 2, the edge of the primary dried film produced when the temperature and vacuum degree of the vacuum drying oven are too high is curled and shrunk, as shown in fig. 4, and finally the produced film thickness is uneven, and the film cannot be used.

Example 1

(2) Weighing 4.5ml of 20% recombinant humanized collagen solution, adding 4.86ml of purified water, and stirring; 0.54ml of 10% EDC solution is added and stirred for 2min to obtain a mixed solution with a crosslinking degree of 6%;

(3) Adding 0.1ml of medical grade glycerol into the mixed solution, continuously stirring for 1.5min, and stopping to obtain a mixed solution with the glycerol concentration of 1%;

(4) Preparing a prefabricated glue: transferring the mixed solution into a pre-sterilized polytetrafluoroethylene mould, wherein the height of the solution is 4mm; standing at room temperature to obtain the prefabricated glue;

(5) Placing the prefabricated glue into a vacuum drying box for primary vacuum drying at 45 ℃ and vacuum degree of-0.4X100 KPa for 18 hours to obtain a primary dried film;

(6) Placing the membrane after primary drying into PBS buffer solution for rinsing for 3 times, 1 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Performing secondary vacuum drying on the transparent gel at 45 ℃ and vacuum degree of-0.1 multiplied by 100KPa for 3 hours to obtain a secondary dried film to be cut;

(9) Packaging and sterilizing to obtain the finished product of the absorbable medical anti-adhesion film with 9% of the final concentration of the recombinant humanized collagen.

Example 2

(5) Placing the prefabricated glue into a vacuum drying box for primary vacuum drying, wherein the temperature is 60 ℃, the vacuum degree is-0.6X100 KPa, and the time is 23 hours, so as to obtain a film after primary drying;

Example 3

(2) Weighing 6.5ml of 20% recombinant humanized collagen solution, adding 2.61ml of purified water, and stirring; 0.39ml of 10% EDC solution is added and stirred for 5min to obtain a mixed solution with the crosslinking degree of 3%;

(3) Adding 0.5ml of medical grade glycerol into the mixed solution, continuously stirring for 5min, and stopping to obtain a mixed solution with 5% glycerol concentration;

(5) Placing the prefabricated glue into a vacuum drying box for primary vacuum drying at 65 ℃ and vacuum degree of-0.7X100 KPa for 28 hours to obtain a primary dried film;

(6) Placing the membrane after primary drying into PBS buffer solution for rinsing for 5 times, 2 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Performing secondary vacuum drying on the transparent gel at 55 ℃ and vacuum degree of-0.4X100 KPa for 7h to obtain a film to be cut after secondary drying;

(9) Packaging and sterilizing to obtain the finished product of the absorbable medical anti-adhesion film with 13% of the final concentration of the recombinant humanized collagen.

Example 4

(5) Placing the prefabricated glue into a blast drying box for primary blast drying at 40 ℃ for 3 hours to obtain a primary dried film;

(6) Placing the membrane after primary drying into Phosphate (PB) buffer solution for rinsing for 3 times, wherein the total time is 2 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Carrying out secondary air blast drying on the transparent gel at the temperature of 35 ℃ for 2.5 hours to obtain a film to be cut after secondary drying;

Example 5

(5) Placing the prefabricated glue into a blast drying box for primary blast drying at 55 ℃ for 2 hours to obtain a primary dried film;

(7) Carrying out secondary air blast drying on the transparent gel at the temperature of 45 ℃ for 2 hours to obtain a secondary dried film to be cut;

Example 6

(1) Preparing a raw material solution: purified water was used to prepare a 20% solution of recombinant humanized collagen and a 10% solution of edc, respectively.

(5) Placing the prefabricated glue into a blast drying box for primary blast drying at 65 ℃ for 1.5 hours to obtain a primary dried film;

(6) Placing the membrane after primary drying into PBS buffer solution for rinsing for 3 times, 5 min/time; the membrane absorbs water to form transparent gel, and the transparent gel is placed in a drying plate;

(7) Carrying out secondary air blast drying on the transparent gel at the temperature of 55 ℃ for 1h to obtain a secondary dried film to be cut;

Characterization example

1. Tensile Strength test

The absorbable medical anti-blocking films prepared in each example were cut into 1cm×5cm strips, respectively, and then tested for tensile strength and deformation rate using a medical packaging property tester (MED-01). The results are shown in Table 1 below, and FIG. 1 is a stress-strain graph thereof, with samples 1-6 corresponding to the antiblocking films prepared in examples 1-6, respectively.

TABLE 1

Name of the name	Average force value (N)	Tensile strength (MPa)	Modulus of elasticity (kgf/cm) ² )
				Example 1	1.064	1.552	7.076
Example 2	3.103	6.380	20.669
				Example 3	16.898	18.969	150.109
Example 4	2.174	3.612	12.748
				Example 5	6.334	11.732	42.563
Example 6	26.686	18.899	766.063

From the above data, it is found that the absorbable medical anti-blocking film has a tensile strength of 1 to 20MPa and an elastic modulus of 7 to 800kgf/cm ² Indicating good flexibility and resistance to deformation.

2. In vitro degradation test

The absorbable medical anti-adhesion films prepared in each example were cut into 2cm×2cm, placed in 10ml PBS respectively, placed in a 37℃incubator, and the time to start degradation and complete degradation was observed and recorded. The results are shown in Table 2 below.

TABLE 2

Sample name	Degradation (Tian)	Complete degradation (Tian)
			Example 1	4	8
Example 2	5	10
			Example 3	6	13
Example 4	5	9
			Example 5	5	12
Example 6	6	14

The results show that the in vitro degradation time of the absorbable medical anti-adhesion membrane is gradually prolonged along with the increase of the content of the recombinant humanized collagen and the increase of the crosslinking degree. Therefore, the degradation time of the absorbable medical anti-adhesion membrane can be controlled by adjusting the content and the crosslinking degree of the recombinant humanized collagen.

3. Water absorption

Experiments were performed according to GB/T1034-2008 for water absorption. Respectively taking a certain amount of the absorbable medical anti-adhesion film prepared in the above example, placing in a 50 ℃ oven for drying until the quality is no longer changed, and accurately weighing to obtain m ₁ . Placing the dried anti-adhesion film in a container filled with a proper amount of purified water for about 24 hours, removing superfluous water on the surface, weighing the mass of the fully expanded anti-adhesion film, and marking the mass as m ₂ . Finally, the anti-adhesion film is dried in a drying oven at 50 ℃ again until the quality is unchanged, and the quality of the anti-adhesion film which is weighed and dried to constant weight is recorded as m ₃ . According to the formula, the water absorption mass fraction c= (m ₂ -m ₁ )/m ₃ The water absorption was calculated as x 100%, and the result is shown in fig. 2, sample 1 being the anti-blocking film prepared in example 5 and sample 2 being the anti-blocking film prepared in comparative example 5.

The result shows that the water absorption rate of the absorbable medical anti-adhesion film is 1300-2100%, and the absorbable medical anti-adhesion film has strong water absorption capacity.

4. Cytotoxicity of cells

Part 5 according to medical device biological evaluation: in vitro cytotoxicity assays GB/T16886.5-2017 and part 12: sample preparation and reference materials GB/T16886.12-2017 were tested. L-929 mouse fibroblasts are selected for experiments, and the experimental principle is as follows: succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of the formazan can be measured at 570nm wavelength by an enzyme-labeled instrument, and the MTT crystallization forming amount is proportional to the cell number within a certain cell number range. The number of living cells was judged based on the measured absorbance value (OD value), and the greater the OD value, the stronger the cell activity.

(1) Sample preparation and Experimental methods

(1.1) test articles were placed in DMEM medium without 10% serum (FBS)Leaching the raw materials according to the ratio of 0.1g to 3.2ml at 37 ℃ and 5 percent CO ₂ Incubate under conditions for 24 hours. A blank (DMEM medium, supplemented with 10% FBS) and a positive control (5% DMSO) were prepared under the same conditions. At the time of the experiment, the test group was supplemented with 10% FBS.

(1.2) operating in an ultra clean bench in the whole process, and ensuring the aseptic operation process. L-929 cells were cultured in DMEM medium (10% FBS, 1% Streptomyces lividans) at 37℃and 5% CO ₂ Culturing under the condition, digesting the cells grown to logarithmic phase with 0.25% trypsin (containing EDTA), centrifuging the cell suspension (1000 rpm,5 min), discarding the supernatant, resuspending the cells with MEM medium, and counting to obtain 1×10 ⁵ Individual/ml cell suspension.

(1.3) 100. Mu.l of the cell suspension per well was inoculated into a 96-well plate and cultured in a cell incubator (37 ℃,5% CO) ₂ ，>90% humidity), and observing the cell morphology under a mirror.

(1.4) after 24 hours of incubation, cells grew to about 70% on the wall, the original medium in the 96-well plates was discarded, and 100. Mu.l of each of the extract solutions (final concentrations: 100%, 75%, 50%, 25%) and the blank control sample and the positive control sample were added to the corresponding wells of the 96-well plates. The 96-well plate was placed in a cell incubator (37 ℃,5% CO) ₂ ，>90% humidity) for 24 hours, 3 duplicate wells were placed in each group.

(1.5) after 24 hours of incubation, the 96-well plate was removed, the cell morphology was observed under a microscope, and then the liquid was removed, 50. Mu.l of MTT (final concentration of 1 mg/ml) was added to each well, and the mixture was placed at 37℃and 5% CO was added ₂ Culturing in an incubator. After 2 hours, the supernatant was removed, 100. Mu.l of DMSO was added to each well to dissolve crystals, and the absorbance at 490nm was measured on a microplate reader to calculate cytotoxicity.

2. Statistical method

Mean ± standard deviation (x ± s);

cell viability% = test (or positive and negative) sample set OD ₅₇₀ Blank OD ₅₇₀ ×100％。

3. Cell morphology description

TABLE 3 cell morphology scoring grade criteria

4. Evaluation criteria

1) The sample leaching solution with the concentration of 50% has the same or higher cell activity compared with the sample leaching solution with the concentration of 100%, otherwise the test is repeated;

2) The lower the percentage of cell viability, the greater the potential cytotoxicity;

3) If the cell activity is less than 70% of the blank group, the sample is proved to have potential cytotoxicity;

4) Cell viability under sample leach liquor was achieved with 100% leach liquor concentration.

5) If the negative control observes a cytotoxic effect or the positive control does not cause a cytotoxic effect, the test does not meet the acceptance criteria.

5. Test results

(1) Cell morphology results

TABLE 4 Table 4

(2) MTT results

TABLE 5

6. Conclusion(s)

Under the test conditions, the anti-adhesion film prepared in the example has no potential cytotoxicity to L-929 cells.

Claims

1. The preparation method of the degradation-controllable absorbable medical anti-adhesion film is characterized by comprising the following specific steps of:

2. The method according to claim 1, wherein in the step (1), the mass concentration of the recombinant humanized collagen solution is 10% to 20%, and the mass concentration of the water-soluble carbodiimide-based solution is 5% to 15%.

3. The process according to claim 1, wherein in the step (1) or (2), the stirring speed is 100 to 500rpm and the stirring time is 1 to 5 minutes.

4. The method according to claim 1, wherein in the step (1), the recombinant humanized collagen has a molecular weight of 56-112 Kda, and the water-soluble carbodiimide is selected from dicyclohexylcarbodiimide, N' -diisopropylcarbodiimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

5. The method according to claim 1, wherein in the step (3), the height of the mixed solution transferred into the mold is 1.5mm to 4mm, and the mold is made of stainless steel, polytetrafluoroethylene or glass.

6. The method according to claim 1, wherein the first drying in step (4) and the second drying in step (6) are performed in the same manner, and are vacuum drying or air drying; when vacuum drying is adopted, the first drying parameters are as follows: the temperature is 45-65 ℃, the vacuum degree is-0.4X100 KPa to-0.7X100 KPa, and the time is 18-28 h; the second drying parameters were: vacuum drying parameters: the temperature is 45-55 ℃, the vacuum degree is-0.1X100 KPa to-0.4X100 KPa, and the time is 3-7 h; when adopting the forced air drying, the first drying parameter is: the temperature is 40-65 ℃ and the time is 1.5-3 h; the second drying parameters were: the temperature is 35-55 ℃ and the time is 1-2.5 h.

7. The preparation method according to claim 1, wherein in the step (5), the PBS buffer is prepared from 0.05M to 1M sodium dihydrogen phosphate solution and 0.05M to 1M disodium hydrogen phosphate solution according to a volume ratio of 1 to 7:3 to 9, and the pH value is 6.5 to 7.9.

8. The preparation method according to claim 1, wherein the PBS buffer is prepared from a 0.1M sodium dihydrogen phosphate solution and a 0.1M disodium hydrogen phosphate solution according to a volume ratio of 3: 7.

9. The method according to claim 1, wherein in the step (5), each rinsing time is 1 to 5 minutes, and the rinsing times are 3 to 5 times; in the step (7), the sterilization mode is radiation sterilization, and the dosage is 10-20 KGy.

10. An absorbable medical anti-blocking film prepared by the preparation method of any one of claims 1 to 9.