CN113521397A

CN113521397A - Preparation method of anti-adhesion membrane for preventing and treating myofascial adhesion and anti-adhesion membrane

Info

Publication number: CN113521397A
Application number: CN202110906851.2A
Authority: CN
Inventors: 刘春杰; 张月明; 白江博; 于昆仑; 田德虎
Original assignee: TANGSHAN WORKER'S HOSPITAL
Current assignee: TANGSHAN WORKER'S HOSPITAL
Priority date: 2021-08-09
Filing date: 2021-08-09
Publication date: 2021-10-22
Anticipated expiration: 2041-08-09
Also published as: CN113521397B

Abstract

The invention relates to the technical field of medical materials, in particular to a preparation method of an anti-adhesion membrane for preventing and treating myofascial adhesion, which comprises the following steps of S1, obtaining fresh amnion; s2, preparing a decellularized amniotic membrane; s3, loading celecoxib; and S4, carrying out freeze-drying treatment. The invention also discloses an anti-adhesion membrane for preventing and treating myofascial adhesion, which is prepared by the method and comprises a decellularized amniotic membrane, wherein a plurality of celecoxib particles are attached to the decellularized amniotic membrane. Has the advantages that: the anti-adhesion membrane prepared by the invention loads celecoxib on the amniotic membrane substrate containing various bioactive substances, can effectively promote endogenous healing of tendons, and reduces adhesion caused by exogenous healing.

Description

Preparation method of anti-adhesion membrane for preventing and treating myofascial adhesion and anti-adhesion membrane

Technical Field

The invention relates to the technical field of medical materials, in particular to an anti-adhesion membrane and a preparation method thereof for preventing and treating myofascial adhesion.

Background

With the development of the industrialization process, the incidence rate of tendon injury is increased year by year. Tendon adhesion of different degrees can occur in about 40% of patients with tendon injury after surgical treatment, which seriously affects the functional rehabilitation of affected fingers, and is one of the problems to be solved urgently in clinic.

The formation mechanism of the adhesion is complex, a large amount of inflammatory factors are released in the inflammatory phase of tendon healing, and the mediated excessive inflammatory reaction is an important reason for the formation of the tendon adhesion, so that a plurality of scholars use non-steroidal anti-inflammatory drugs to inhibit inflammatory cells and factors related to the occurrence of the adhesion and hope to prevent and treat the tendon adhesion by relieving the inflammatory reaction.

Researchers load celecoxib into an anti-adhesion membrane made of polylactic acid polyethylene glycol (PELA), slow drug release increases tendon adhesion inhibition effect, reduces neutrophil infiltration, and achieves good effect of preventing adhesion formation. However, the maximum tensile strength of the tendon tested by the postoperative mechanics shows that the PELA fibromembranous group loaded with celecoxib is obviously lower than that of the control group, which indicates that the released celecoxib partially impairs the healing of the tendon. Therefore, how to optimize the anti-adhesion membrane loaded with non-steroidal anti-inflammatory drugs to mitigate the effect on tendon healing needs further intensive research.

Clinical and basic research of human amniotic membrane has been in history for nearly a hundred years, however, the biological characteristics of amniotic membrane have been completely new and deeply known until recent years, and it is found that human amniotic membrane tissue contains a plurality of cell growth factors, and the factors are important regulatory factors for regulating tendon injury repair and adhesion formation. However, the fresh amnion is not suitable for storage and transportation, has certain immunogenicity and other problems, and is difficult to be widely applied to clinic.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method of an anti-adhesion membrane for preventing and treating myofascial adhesion and the anti-adhesion membrane, which solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of an anti-adhesion membrane for preventing and treating myofascial adhesion comprises the following steps:

s1, performing blunt separation on fresh placenta tissue between amnion and chorion to obtain smooth and semitransparent fresh amnion, soaking in balanced salt solution containing penicillin and streptomycin, and storing in a refrigerator at 4 deg.C;

s2, rinsing fresh human amniotic membrane with Sucrose Phosphate Synthase (SPS) solution, then crosslinking with glutaraldehyde, placing the crosslinked amniotic membrane into 0.5% Sodium Dodecyl Sulfate (SDS) to shake for 24h, digesting with trypsin for 4h, thoroughly rinsing, freeze drying, subpackaging, sterilizing with ethylene oxide, cutting the acellular amniotic membrane for later use;

s3, accurately weighing 100mg of celecoxib, grinding solid particles, and completely dissolving the powder in 10mL of absolute ethyl alcohol to prepare a celecoxib mother solution with the concentration of 10 mg/mL;

s4, dissolving the prepared celecoxib mother solution in PBS, and preparing a load celecoxib acellular amniotic membrane matrix anti-adhesion membrane loaded with different concentrations with the amniotic membrane;

s5, placing the celecoxib-loaded acellular amniotic membrane matrix anti-adhesion membrane in a working chamber of a freeze dryer, pre-freezing for a plurality of hours at low temperature, starting a condensation chamber for refrigeration, starting a vacuum pump, and performing vacuum freeze drying;

s6, sterilizing the freeze-dried amnion by cobalt 60 irradiation.

Further, the size of the decellularized amniotic membrane cut in the step of S2 is 1.0cm × 0.5 cm.

Further, the concentration of the loaded celecoxib loaded on the cellular amniotic membrane substrate anti-adhesion membrane in the step S4 is selected from 5%, 10% or 15%.

Further, in the step S5, the temperature in the freeze dryer is-10 ℃ to-56 ℃ and the humidity is 20% or less.

An anti-adhesion membrane for preventing and treating myofascial adhesion comprises a decellularized amniotic membrane, wherein a plurality of celecoxib particles are attached to the decellularized amniotic membrane.

Further, the size of the decellularized amniotic membrane is 1.0cm × 0.5 cm.

Compared with the prior art, the invention has the following beneficial effects: the prepared anti-adhesion membrane loads celecoxib on the amniotic membrane substrate containing various bioactive substances, can effectively promote endogenous healing of tendons and reduce adhesion caused by exogenous healing; according to the invention, the acellular amniotic membrane treated by enzyme is a semipermeable membrane, so that cell components in the amniotic membrane are effectively removed, a base membrane layer, a fiber mesh structure of a compact layer and a plurality of bioactive substances are reserved, the tensile resistance of the amniotic membrane is enhanced by abundant collagen fibers, and a sufficient three-dimensional space structure is provided for loading celecoxib by a three-dimensional porous structure; the acellular amniotic membrane for preventing the adhesion membrane prepared by the invention contains various bioactive substances, can effectively promote endogenous healing of the tendon, and the celecoxib loaded on the acellular amniotic membrane can relieve the adhesion caused by inflammatory reaction and exogenous healing obstruction.

Drawings

FIG. 1 is a schematic view of the structure of an anti-adhesion membrane

FIG. 2 is a schematic view showing the appearance of an anti-blocking film

FIG. 3 is a light microscope picture before and after decellularization of fresh amnion

FIG. 4 is a scanning electron micrograph of fresh amnion before and after decellularization

FIG. 5 is a statistical chart of drug release test of celecoxib

FIG. 6 is a statistical chart of residual DNA content determination after fresh amniotic membrane decellularization

FIG. 7 is a scanning electron micrograph of a specimen after the affected part of an animal is repaired

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A preparation method of an anti-adhesion membrane for preventing and treating myofascial adhesion,

s1, obtaining fresh amnion: fresh placenta tissue, performing blunt separation between amnion and chorion to obtain smooth and semitransparent fresh amnion, soaking in balanced salt solution containing penicillin and streptomycin, and storing in refrigerator at 4 deg.C;

s2, preparation of the decellularized amniotic membrane: rinsing fresh human amniotic membrane with Sucrose Phosphate Synthetase (SPS) solution, crosslinking with glutaraldehyde, placing the crosslinked amniotic membrane into 0.5% Sodium Dodecyl Sulfate (SDS) and shaking for 24 hr, digesting with trypsin for 4 hr, thoroughly rinsing, freeze drying, packaging, sterilizing with ethylene oxide, and cutting into 1.0cm × 0.5 cm;

s3, loading celecoxib: accurately weighing 100mg of celecoxib, grinding solid particles, completely dissolving the powder in 10mL of absolute ethyl alcohol to prepare a celecoxib mother solution with the concentration of 10mg/mL, dissolving the celecoxib mother solution in a certain amount of PBS, and preparing a loaded celecoxib acellular amniotic membrane matrix anti-adhesion membrane with the loading concentration of 5% and 10% with the amniotic membrane;

s4, freeze-drying treatment: placing the celecoxib-loaded acellular amniotic membrane matrix anti-adhesion membrane in a working chamber of a freeze dryer, pre-freezing for a plurality of hours at low temperature, starting a condensation chamber for refrigeration, enabling the temperature of a cold trap to reach-10 ℃ to-56 ℃, starting a vacuum pump, carrying out vacuum freeze drying, reducing the water content to below 20%, and carrying out cobalt 60 irradiation sterilization on the freeze-dried amniotic membrane.

Example 2

An anti-adhesion membrane for preventing and treating myofascial adhesion comprises a decellularized amniotic membrane, wherein a plurality of celecoxib particles are attached to the decellularized amniotic membrane, and the size of the decellularized amniotic membrane is 1.0cm multiplied by 0.5 cm.

The acellular amniotic membrane contains various bioactive substances and can effectively promote endogenous healing of tendons, celecoxib can relieve adhesion caused by inflammatory reaction blocking exogenous healing, the combination of the celecoxib and the inflammatory reaction improves the treatment effect of anti-adhesion membranes, and the time for postoperative recovery is shortened.

Example 3

Drug release test of celecoxib:

accurately weighing 100mg of celecoxib, grinding solid particles, and completely dissolving the powder in 10mL of absolute ethyl alcohol to prepare a celecoxib mother solution with the concentration of 10 mg/mL. Then dissolving the celecoxib in PBS to prepare a series of celecoxib standard solutions with known concentrations, wherein the concentrations of the celecoxib standard solutions are 0 to 0.1 mg/mL. The absorbance of the celecoxib standard solution of known concentration above was measured at 249nm using a UV-vis spectrophotometer (UV-2550, Shimadzu, Japan). Furthermore, the concentration (x) of the celecoxib is used as an abscissa, and the absorbance (y) is used as an ordinate, so as to carry out plotting, and thus the solution standard curve equation of the celecoxib is obtained as follows: y is 1.65x +0.0343, and the correlation coefficient is r2 is 0.9998.

The PCL/AM nano-fiber composite membrane with the celecoxib content of 5%, 8% and 10% is cut into 20mm multiplied by 20mm sheets respectively, the sheets are accurately weighed, the weight is about 50mg, and then three groups of parallel samples are respectively set up. The three groups of samples are respectively placed into a centrifuge tube, 20mL of PBS is added, and then the centrifuge tube is placed into an air bath oscillator (Taihang Medical Apparatus Co., Jiangsu, China) to be kept at a constant temperature (37 ℃) and oscillated at a constant speed (100 rmp). At predetermined time points, 5mL of the release solution was removed from the centrifuge tube at a time, and 5mL of fresh PBS was added to maintain the total volume. The absorption intensity of the taken-out release liquid was measured at 249nm with an ultraviolet-visible spectrophotometer, and the cumulative celecoxib release amount was calculated from the standard curve. The cumulative release ratio of the medicine loaded by the PCL/AM nano-fiber composite membrane is the ratio of the cumulative release amount of the medicine to the initial medicine addition amount, and the specific calculation method is as follows: the cumulative drug release percentage is Mt/M ∞ 100%, where Mt represents the cumulative amount of ibuprofen released in the sample at time t and M ∞ is the initial amount of ibuprofen added to the sample.

Example 4

Determination of residual DNA content:

1. and (3) treating and digesting the decellularized amniotic membrane: freeze-drying amnion before and after decellularization, weighing 5mg, putting into a 1.5ml centrifuge tube, adding 180 μ l Buffer ATL and 20 μ l protein kinase K, oscillating, mixing uniformly, and incubating at 56 ℃ until the tissue is completely cracked. After tissue lysis, 200. mu.l Buffer AL and 200. mu.l ethanol were added in sequence, and mixed by shaking. The mixture was transferred to a DNeasy Mini filter column in a 2ml centrifuge tube using a pipette gun, centrifuged at 6000 Xg (8000rpm) for 1min and the filtrate and centrifuge tube discarded. The column was placed in a new 2ml collection tube, 500. mu.l Buffer AW1 was added, 6000 Xg was centrifuged for 1min, and the filtrate and collection tube were discarded. The cartridge was placed in a new 2ml collection tube and 500. mu.l Buffer AW2, 20000 Xg (14000rpm) was added. Centrifuging for 3min, and discarding filtrate and collecting tube. The filter column was transferred to a new 1.5ml or 2ml centrifuge tube. Mu.l of Buffer AE was added to the center of the filter membrane to elute the DNA, and incubated at room temperature (15 ℃ to 25 ℃) for 1 minute. Centrifuge at 6000 Xg for 1 min.

Measurement of tissue DNA by PicoGreen dsDNA quantification kit: calf thymus DNAsndard (100. mu.g/ml) was diluted 50-fold with 1 XTE to make 2. mu.g/ml. The following table was formulated to determine the DNA standard curve:

add 100. mu.l of standard DNA solution or sample to be tested to 3.96 well plate and 100. mu.l of PicoGreen working solution, and leave in dark for 5 minutes at room temperature. And (4) performing measurement on the machine, and recording the concentration fluorescence value of each hole. And calculating the content of the DNA contained in the sample according to the standard curve.

4. Statistical analysis: the data are processed by SPSS 24.0 statistical software

Representing, the mean comparison of the two samples, using a t test, P<A difference of 0.05 is significant.

5. As a result: the normal amniotic membrane DNA is 351.5 plus or minus 9.23ug/ml, the decellularized amniotic membrane DNA is 15.66 plus or minus 0.82ug/ml after freeze-drying, and the P is less than 0.0001 by adopting two independent sample t tests, and has significant difference. Compared with the normal amniotic membrane composition, the content of DNA is removed by 95.6%.

Example 5

Animal experiments:

four adult New Zealand white rabbits were treated with pentobarbital sodium (30mg/kg) by intravenous injection at the ear margins, fixed in prone position, and under aseptic conditions, 1.5cm incisions were made between the 3 rd metatarsophalangeal joint of the hind foot to the proximal interphalangeal joint, the tendon sheaths were excised, the tendon of the deep flexor of the toe was exposed, and the transverse incision was performed with a modified Kessler method, which was performed strictly in accordance with the standards of the national laboratory animal administration and use regulations.

(I) the experimental individuals use a PCL/AM-8% celecoxib nanofiber composite membrane to wrap the repair area.

After 4 weeks of operation, the implanted part is rechecked, the wound is well healed, and no infection or ulcer appears. And cutting a specimen, and performing HE staining.

And (3) detecting results under an electron microscope: the circumference of the tendon is smooth, collagen fibers in the tendon show good repair, adhesion tissues are hardly formed, and the PCL/AM-8% celecoxib nano-fiber composite membrane is clearly visible.

The general observation shows that no adhesion tissue is formed after the PCL/AM-8% celecoxib nanofiber composite membrane wraps the tendon, and the affected part of the New Zealand white rabbit is free from infection and ulcer under the action of reducing inflammatory reaction of celecoxib.

And (II) covering the repair area by using amnion for the first control individual.

After 4 weeks of operation, the wound healed well with no infection or ulceration. Gross observation revealed that the amniotic membrane was indistinguishable and a small amount of fibrous tissue adhered between the tendon and the surrounding tissue. And cutting a specimen, and performing HE staining.

And (3) detecting results under an electron microscope: pathological section tendon is smooth and well healed.

And (III) coating the repair area by using the PELA fiber membrane loaded with celecoxib in a second control individual.

The wound healed better around the post-operative period, with no infection or ulceration. A small amount of adhesion occurred between the tendon and the surrounding tissue. And cutting a specimen, and performing HE staining.

And (3) detecting results under an electron microscope: the case section shows that the wound is well healed.

(IV) control individuals three did not use anti-adhesion membrane to report repair area.

After 4 weeks, the specimen is cut out, HE staining is carried out, and the dense adherent tissues around the repaired tendon are generally observed and found to be separated by a sharp instrument. And cutting a specimen, and performing HE staining.

And (3) detecting results under an electron microscope: the pathological section finds that the tendon is not healed well.

Comparing the experimental results, the compact tissue adhesion appears at the wound without being coated by the anti-adhesion membrane, and the tendon recovery effect is poor; the tendon part coated with the amnion has a small amount of tissue adhesion, and the tendon is well healed; a wound part wrapped by the PELA fiber membrane loaded with celecoxib is slightly adhered, so that the wound is well healed; the tendon part coated by the PCL/AM-8% celecoxib nanofiber composite membrane has no adhesion tissue, and the affected part has no infection or ulcer and is well healed.

The amnion can provide cell growth factors for tendon injury, but the amnion is difficult to store, so that the problems of immunogenicity and the like are easy to occur clinically, and the wound infection is caused; the drug is slowly released to the wound from the interior of the PELA membrane loaded with the celecoxib, so that the inhibition effect of tendon adhesion is increased, the neutrophil infiltration is reduced, but the tensile mechanical property of the repaired tendon is poor, and the celecoxib damages the healing of the tendon; the PCL/AM-8% celecoxib nanofiber composite membrane reserves the base layer membrane layer of the amnion, and celecoxib is attached to the base layer membrane layer, so that a bioactive substance is provided for damaged tendons, and inflammatory reaction is relieved through the celecoxib.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of an anti-adhesion membrane for preventing and treating myofascial adhesion is characterized by comprising the following steps:

s6, sterilizing the freeze-dried amnion by cobalt 60 irradiation.

2. The method for preparing an anti-adhesion membrane for preventing and treating sarcolemma adhesion as claimed in claim 1, wherein: the size of the decellularized amniotic membrane cut in the step S2 is 1.0cm × 0.5 cm.

3. The method for preparing an anti-adhesion membrane for preventing and treating sarcolemma adhesion as claimed in claim 1, wherein: the concentration of the loaded celecoxib loaded cell amniotic membrane matrix anti-adhesion membrane in the step S4 is selected from 5% and 10%.

4. The method for preparing an anti-adhesion membrane for preventing and treating sarcolemma adhesion as claimed in claim 1, wherein: s5, the temperature in the freeze dryer is-10 to-56 ℃, and the humidity is below 20%.

5. An anti-adhesion membrane for preventing and treating sarcolemma adhesion, which is prepared by the preparation method of claim 1, characterized in that: comprises a decellularized amniotic membrane, wherein celecoxib particles are attached to the decellularized amniotic membrane.

6. An anti-adhesion membrane for the control of myofacial adhesions, as claimed in claim 5, where: the size of the decellularized amniotic membrane is 1.0cm multiplied by 0.5 cm.