KR100623220B1 - Biodegradable anti-adhesivie membrane for promoting tissue regeneration and its preparation method - Google Patents

Abstract

The present invention relates to a biodegradable antiadhesion membrane having a tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin and one side thereof having a PEG-PLGA copolymer layer and a method for producing the same.

Biodegradable Adhesion Barrier, Collagen, Atelocollagen, Hyaluronic Acid, Fibronectin, PEG, PLGA

Description

Biodegradable anti-adhesivie membrane for promoting tissue regeneration and its preparation method             

1 is an electron microscope photograph of a mPEG-PLGA membrane in which collagen or collagen-fibronectin is bonded to an ozone-treated PLGA surface. A is a PEG side, B is a collagen-coupled PLGA side, and C is a collagen-fiber. Shows the PLGA side with Ronectin bound

2 is an electron microscope photograph of a bilayer consisting of a porous collagen-hyaluronic acid-fibronectin membrane and an mPEG-PLGA film, where A is a cross-sectional photograph of a double membrane (magnification × 50), and B is an enlarged cross-sectional photograph of a bilayer (porous membrane and adhesion site of mPEG-PLGA film) (magnification x 500).

Figure 3 shows the number of dermal fibroblasts attached to the surface of the collagen-hyaluronic acid membrane, PLGA film, mPEG-PLGA film and Interceed ^TM membrane after 4 hours of cell inoculation.

4 shows a photograph (magnification × 200) of cells attached to collagen-hyaluronic acid membrane, B for PLGA film, C for mPEG-PLGA film, and D: Interceed ^™ .

FIG. 5 is a photograph observed after culturing the membrane containing the cells for 7 days. A is a collagen-hyaluronic acid membrane, B is a histological photograph of H & E staining of cells attached to an mPEG-PLGA film (magnification × 40). )to be.

6A is a collagen-hyaluronic acid membrane, B is an mPEG-PLGA film and C is an Interceed ^™ and a fibro of 30 g / cm 3 (D), 50 g / cm 3 (E) or 100 g / cm 3 (F) Confocal laser microscopy of cells attached to nectin coated collagen-hyaluron-fibronectin membrane.

Figure 7 shows the fluorescence intensity of F-actin in fibroblasts stained with FITC-phalloidin and 30 g / cm 3 and 50, 100 g / cm 3 fibronectin after incubating the membrane containing the medium containing the cells for 4 hours. It is shown that there is a significant difference between the fluorescence intensity of intracellular F-actin attached to the coated membrane (* p <0.05).

8 is a result of decomposition test of a composite double membrane composed of an mPEG-PLGA film and a porous collagen-hyaluronic acid membrane, wherein A is a weight change and B is a graph showing a molecular weight change of the mPEG-PLGA membrane.

9 is a photograph comparing the degree of adhesion 7 days after the injury using the animal abdominal wall injury model, A group treated with mPEG-PLGA membrane bonded to the collagen-fibronectin surface and B did not receive any treatment Control picture.

The present invention relates to an antiadhesion film comprising a tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin, and one side thereof having a layer of PEG-PLGA copolymer, and a method for preparing the same.

When wounds occur after surgery or inflammation, spontaneous wound healing occurs. In the process, excessive fibrous tissue develops abnormal junctions with surrounding tissues. This is called. In general, adhesions occur at a frequency of 67-93% after open surgery, and some of them spontaneously decompose, but in most cases, adhesions exist after wound healing, causing various sequelae. In particular, tissue adhesion is the main cause of ileus (80-90%), and long-term sequelae and pelvic pain after gynecological procedures (Eur. J. Surg. 1997, Supp1577, 32-39).

According to a recent study, uterine adnexal stenosis accounts for at least 20% of infertile women. The main cause of this phenomenon is that exudates from damaged tissues are not absorbed and dissolved, forming fibrous tissues, which then grow together with fibroblasts and eventually collagen deposition occurs.

Various anti-adhesion materials have been studied. Among these, Interceed ^TM (Johnson & Johnson Medical, Inc. Arlinton, TX) and Seprafilm ^TM (Genzyme Cop. Camridge, MA), which use biodegradable polymers as materials, are commercialized and used. In comparison, no significant effect has been reported, and Seprafilm ™ does not show a definite effect because the results vary depending on the experimental model. Poloxamer 407 has the disadvantage of limited use only on dry, bleed-free surfaces, and PRECLUDE ™ Peritoneal Membrane is not biodegradable and requires additional reoperation.

Therefore, in view of the importance of preventing adhesion caused by surgical operation, infection and trauma, there is still a need for the development of a membrane having an excellent adhesion prevention effect and to improve the disadvantages of the conventional adhesion prevention film.

Existing anti-adhesion membranes have been developed with a focus on simply preventing adhesion with surrounding tissues, and studies on membranes that induce wound healing and tissue regeneration at the same time on damaged tissues are insufficient. There is a need for development of membranes with complex functions of prevention as well as wound healing and tissue regeneration.

Accordingly, the present inventors prepare an anti-adhesion film having a PEG-PLGA copolymer on one side of a tissue adhesion layer containing collagen, hyaluronic acid, fibronectin, and the like, and applying the membrane to a surgical site. In this case, PEG, a hydrophilic polymer arranged on the outer surface of the anti-adhesion membrane, effectively prevents adhesion of peripheral cells from the damaged tissue, and collagen, hyaluronic acid, and fibrofectin present on the inner surface of the anti-adhesion membrane may be effectively separated from the damaged tissue. It was confirmed that promoting wound healing and tissue regeneration while improving adhesion, and completed the present invention for a biodegradable anti-adhesion film.

One object of the present invention is to provide a tissue adhesion layer comprising collagen and fibronectin and an anti-adhesion film having one side thereof having a layer of PEG-PLGA copolymer.

It is a further object of the present invention to provide a tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin and an anti-adhesion film having one side thereof with a layer of PEG-PLGA copolymer.

Another object of the present invention is to prepare a PEG-PLGA copolymer; Preparing a mixed solution containing collagen and fibronectin; And it provides a method for producing an anti-adhesion film comprising the step of crosslinking the mixture with a PEG-PLGA copolymer.

Another object of the present invention is to prepare a PEG-PLGA copolymer; Preparing a mixed solution containing collagen; Lyophilizing the mixed solution and binding to PEG-PLGA copolymer to prepare a membrane; And it provides a method for producing an anti-adhesion film comprising the step of coating the membrane with fibronectin.

In one embodiment, the present invention relates to a tissue adhesion layer comprising collagen and fibronectin, and to an adhesion membrane having one side thereof with a layer of PEG-PLGA copolymer.

In the present invention, "adhesion" refers to the growth of undesirable tissue that occurs between adjacent body tissues, between tissues and internal organs, often a variety of heart surgery, orthopedic, cerebral neurosurgery, abdominal surgery, obstetrics and gynecology It is caused by surgical operations, mechanical trauma, chemicals, and local infections. The adhesion causes pain or dysfunction and is not only a major cause of pleurisy, peritonitis, intestinal obstruction, etc., but in severe cases, surgery to detach the adhesion is required separately, so prevention of adhesion is very important.

In the present invention, "anti-adhesion film" means a film that acts as a physical barrier for separating tissues from each other in the wound regeneration process, and covers or wraps an application site (affected or injured area) that may be adherent with an anti-adhesion film. During the healing period, the wound is isolated from the surrounding tissue to prevent the adhesion. Such anti-adhesion membranes are commercialized and the most used adhesion membranes include, for example, hyaluronic acid and carboxymethyl cellulose mixed membranes.

The anti-adhesion film of the present invention is characterized in that the tissue adhesion layer comprising collagen and fibronectin and one side thereof has a PEG-PLGA copolymer layer, the membrane having different properties on both sides of the membrane.

The tissue adhesion layer is a layer containing collagen and fibronectin on the side facing the damaged tissue.

Collagen is preferred as a medical material because it has excellent biocompatibility and tissue adhesion, low antigenicity, excellent sutureability, functions to promote differentiation and proliferation of cells, and has a characteristic of being completely degraded and absorbed in vivo. . Any type of collagen extracted from nature or recombinantly produced may be used, and the natural collagen may be used in various organs such as skin, bone, cartilage, tendons and organs of mammals such as cattle, pigs, rabbits, sheep and mice. From connective tissue, it can be extracted and purified by acid solubilization, alkali solubilization, neutral solubilization, enzyme solubilization and the like. To date, Type I to XIX have been found, of which Type I to V collagen is the most used. In the present invention, all types of collagen may be used alone or in combination of one or more components, but are preferably type I atelocollagen. Atelocollagen is a collagen obtained by removing telopeptides of the N- and C-terminus of collagen, which act as antigenicity, and can be obtained by pepsin digestion. Is suitable as.

The present invention used fibronectin with collagen. Fibronectin is a substance present as an insoluble glycoprotein dimer that acts as a linker in the extracellular matrix (ECM) or a soluble disulfide linked dimer found in plasma. Tissue regeneration, embryogenesis, It is known to be involved in processes such as blood clotting and cell migration / adhesion. Indeed, through specific examples of the present invention, it was found that fibronectin induces reorganization of the actin skeleton important for cell migration. In the crude direct adhesion layer of the membrane of the present invention, fibronectin can be used together with collagen to induce more appropriate adhesion with damaged tissue and to obtain the effect of tissue regeneration. The fibronectin can be obtained from various biological species and tissues.

One aspect of the tissue adhesion layer having excellent tissue regeneration effect including collagen and fibronectin of the anti-adhesion film of the present invention has a PEG-PLGA copolymer layer.

PLGA is a copolymer of polylactide (PLA) and polyglycolide, and is a representative biodegradable polymer that is gradually converted into a non-toxic decomposition product after a certain time in vivo. In the present invention, the PLGA layer serves as a physical support for the membrane including collagen and fibronectin. Since the anti-adhesion film is no longer present after the wound is treated, use a biodegradable polymer. At this time, the biodegradation rate of the membrane can be adjusted according to the purpose according to the ratio of lactide and glycotide, the preparation method of the suspension. In addition, the PLGA layer of the anti-adhesion film of the present invention is characterized by being used by copolymerizing with PEG without using PLGA alone.

PEG (polyethylene glycol) is a hydrophilic substance confirmed biocompatibility. It is well known that the hydrophilic molecules prevent the adsorption of blood proteins to prevent the formation of blood clots. In the present invention, the surface of the membrane is modified by using a hydrophilic polymer, PEG, to prevent adhesion to blood and surrounding tissues. It was. PEG is preferably mPEG (methoxy PEG).

The biodegradable polymer of the coalescing membrane is most preferably PLGA, but may include all biodegradable polymers having the property of enhancing the mechanical strength of the membrane and preventing the adhesion and degrading. Such polymers include, for example, polylactide (PLA), polyglycolide (PGA) or copolymers thereof poly (lactide-co-glycolide (PLGA)). Polyesters such as poly (lactide-co-glycolide) -glucose, PLGA-glucose, star polymers, polyorthoesters and polyanhydrides (Polyanhydride), polydioxanone (polydioxanone), polyhydroxybutyric acid (polyhydroxybutyric acid), polycaprolactone (Polycaprolactone), polyalkylcarbonate (Polyalkylcarbonate) and the like, but is not limited thereto. The biodegradable polymer may be used alone or by polymerizing two or more components.

Hydrophilic polymers copolymerized with biodegradable polymers are most preferred PEG, but in addition polyaniline, polythiophene, poly (aniline-co-o-anthranilic acid) (Poly (Aniline-Co-O) -Anthranilic Acid)). At this time, the hydrophilic polymer may be used alone or by polymerizing two or more components.

The tissue adhesion layer comprising collagen and hyaluronic acid may be in the form of a multilayer as well as a single mixed layer. Each multilayer may have the same or different composition, and various modifications in the composition, arrangement, etc. of the multilayers are included within the scope of the present invention.

That is, the anti-adhesion film having a PEG-PLGA copolymer layer on one side of the tissue adhesive layer including collagen and fibronectin of the present invention is a non-uniform film characterized by both sides of the membrane for function, and the tissue adhesive film faces the damaged tissue. It is possible to induce effective tissue regeneration through proper adhesion with damaged tissues, and the copolymer layer surrounding one side of the tissue adhesive film plays a physical role of the membrane and the membrane surface is modified with PEG to surround the damaged tissue. Adhesion can be effectively prevented.

The membrane may comprise hyaluronic acid.

In another aspect, the present invention relates to a tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin, and to one side thereof, an anti-adhesion film having a layer of PEG-PLGA copolymer.

Hyaluronic acid is a complex polysaccharide consisting of amino acids and uronic acid. It is a major mucopolysaccharide together with chondroitin sulfate and is rich in ECM of various tissues such as skin and cartilage. Hyaluronic acid is present in biological organisms and plays an important role in a variety of key functions such as substrate structure stabilization, water balance, lubrication, cell migration and differentiation (Turley EA, et al. J Biol. Chem 2002277: 4589-92) Due to this biological property, hyaluronic acid, together with components such as collagen, has been used for tissue engineering applications as a biosupport material (Miralles G, et al. J Biomed Mater Res 2001, 57: 268-78). Collagen membranes containing hyaluronic acid have been shown to promote the proliferation and adhesion of fibroblasts when compared to polyurethane membranes or cell culture dishes (Park SN, et al., Biomaterial 2003; 24: 1631-41). The hyaluronic acid can be obtained from various species and tissues.

One aspect of the tissue adhesion layer having good tissue regeneration effect, including collagen, hyaluronic acid and fibronectin, has a PEG-PLGA copolymer layer.

The tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin may be in the form of multiple layers with the same or different composition as well as a single mixed layer. Various modifications in the composition, arrangement, etc. of each multilayer are included in the scope of the present invention. In a preferred embodiment of the present invention, a membrane composed of collagen-hyaluronic acid is coated with fibronectin to prepare a bilayer.

The size, thickness and decomposition rate of the anti-adhesion film can be easily adjusted by those skilled in the art.

The anti-adhesion membrane may be used for various purposes, and is preferably used in surgical procedures, and there is no limitation in the type of such surgical operations, but abdominal surgery (for example, intestine, cavities, sactomy, and dissolution of abdominal adhesions) , Kidney, after school, urethral and prostate surgery), obstetrics and gynecology surgery (tubal dissection, fallopian tubes, ovarian detachment, removal of endometriosis, malformation, myoma resection, hysterectomy, etc.) Resection, flexion tendon surgery, spinal fusion, joint replacement, etc.), thoracic surgery (bypass anastomosis, cardiac valve replacement), and the like.

The anti-adhesion film of the present invention may contain a drug. In particular, since the drug is encapsulated in the biodegradable polymer membrane, the drug is continuously released according to the decomposition of the polymer. Therefore, it is effective for the administration of drugs that must be administered for a certain period or maintain a constant blood concentration, and the drug is locally applied to the surgical site for a certain period of time. I can deliver it. The loading of the drug may be carried out before or after the membrane preparation or the membrane preparation, and the drug that can be supported is not particularly limited, but antithrombotic agents (eg, heparin, tissue plasminogen activator, etc.), anti-inflammatory agents (Eg, aspirin, ibuprofen, ketoprofen, etc.), chemostatic factors, analgesics, anesthetics, protein and peptide drugs such as hormones, and the like.

In another aspect, the present invention provides a method for preparing PEG-PLGA copolymer; Preparing a mixed solution containing collagen and fibronectin; And it relates to a method for producing an anti-adhesion film comprising the step of crosslinking the mixture with a PEG-PLGA copolymer.

The PEG-PLGA copolymer may be prepared by physical crosslinking by radiation, electron beam, ultraviolet light or heat, and by chemical crosslinking by treatment with a crosslinking agent such as aldehydes, epoxys, carbodiimides, isocyanates, tannins, and chromium. . In a specific embodiment of the present invention it is copolymerized by a method of heat treatment at 130 ℃. In this case, PEG of various molecular weights can be used. For example, the molecular weight of PEG can be adjusted to 200 to 6000 and the molecular weight of mPEG can be adjusted to 550, 2000, 6000. In addition, PLGA having various ratios of lactide and glycoside may be used. In a specific embodiment of the present invention, PLGA having a ratio of 75:25 is used.

The thickness of the PEG-PLGA copolymer membrane and the size of the pores are variously adjustable depending on the appropriate rate of decomposition and the purpose of use, preferably about 10 to 100 μm thick, 1-10 μm pore size, more preferably about It has a thickness of 40 to 60 μm, a pore size of 2 to 6 μm, most preferably a thickness of 50 μm and a pore size of 3 to 5 μm.

Hyaluronic acid may be included when preparing a mixed solution containing collagen and fibronectin. Homogenize fibronectin in the collagen suspension or the collagen-hyaluronic acid suspension and prepare to pH 7.4. At this time, the addition ratio of collagen, hyaluronic acid and fibronectin can be variously controlled.

The method of crosslinking the mixed solution with the PEG-PLGA copolymer is not particularly limited, but in a specific embodiment of the present invention, ozone treatment or EDC-NHS was used.

The anti-adhesion film comprising collagen and fibronectin having a layer of PEG-PLGA copolymer on one side or the collagen, hyaluronic acid and fibronectin having a layer of PEG-PLGA copolymer on one side An anti-adhesion film can be produced effectively.

In another aspect, the present invention provides a method for preparing PEG-PLGA copolymer; Preparing a mixed solution containing collagen; Lyophilizing the mixed solution and binding to PEG-PLGA copolymer to prepare a membrane; And it relates to a method for producing an anti-adhesion film comprising the step of coating the membrane with fibronectin.

 PEG-PLGA copolymers can be prepared in the same manner as above.

When the mixed solution containing collagen is prepared, the mixed solution may include hyaluronic acid. After crosslinking the mixed solution, freeze-drying was performed, and chloroform was sprayed onto the surface of the prepared PEG-PLGA copolymer and the mPEG-PLGA membrane, and then the lyophilized collagen membrane or collagen-hyaluronic acid membrane was combined to prepare a membrane. Treatment of fibronectin with the prepared membrane can produce an anti-adhesion membrane having a double membrane. At this time, the addition ratio of collagen, hyaluronic acid and fibronectin can be variously controlled.

Hereinafter, the specific method of this invention is demonstrated in detail by an Example. However, the following Examples are merely to illustrate the invention, the present invention is not limited to the following Examples.

Example 1 Preparation of Collagen-Fibronectin Surface-bound mPEG-PLGA Membrane Using Ozone Treatment

To add 75:25 molar ratio of lactide and glycoside to the PYREX ampule, methoxy PEG was used to synthesize the mPEG-PLGA copolymer. (mPEG, Mw. 550, Sigma-Aldrich, St. Louis, Mo., USA) was added. Lactide and glycosides were purchased from Boehringer Ingelheim (Ingelheim, German). Tin catalyst (stannous octate, Sigma Chemical Co., St. Louis, MO, USA) was added after dissolving in toluene. The ampoule was removed from residual solvents such as toluene and water under reduced pressure, the top of the ampoule was sealed, and the ampoule was heated at 130 ° C. for 12 hours.

After the reaction was terminated and the ampoules were removed, the polymerized copolymer was dissolved in methylene chloride (MC) and precipitated in excess methanol to remove unreacted monomers and catalysts.

The purified polymer was dried under reduced pressure, a specific ratio of mPEG-PLGA copolymer and PLGA were dissolved in MC and an appropriate amount of dimethyl sulfoxide (DMSO) and cast into a glass mold to prepare an mPEG-PLGA membrane. After drying the membrane for 12 hours at room temperature, the membrane was left to soak for 5 hours in an ultrapure water bath at 22 ° C. Take it out and dry at room temperature for 10 hours. The thickness of the membrane produced is 50 μm and the pore size is 3-5 μm.

1% type Ι atelocollagen (RBC I, Regenmed, Seoul, Korea) suspension or 20% (w / w) hyaluronic acid (sodium salt, Mw = 120,000-150,000, Hanwha group R & E center, Tae Chun, Korea) A locollagen mixture was prepared. Fibronectin (from bovine plasma, Sigma-Aldrich, St. Louis, MO, USA) was added to the collagen suspension or collagen-hyaluronic acid mixture at a concentration of 50 μg / ml, homogenized at 4 ° C and 8000 rpm for 3 minutes, and then Sodium (NaOH) was used to adjust the pH to 7.4.

For ozone treatment of porous PEG-PLGA membrane surface, fix the PLGA face up to the dish bottom and place it in the chamber, then ozone generator at 4L / hr, oxygen pressure 1 bar, 60 V It was exposed to ozone generated for 45 minutes by passing through. After ozone treatment, oxygen purification was performed to remove unreacted ozone.

The PEG-PLGA membrane was fixed at the bottom of the dish, and the collagen- (hyaluronic acid) -fibronectin mixture was poured on the ozone treated side and reacted at 4 ° C. for about 24 hours. After washing with distilled water to remove unreacted protein, and freeze-dried for long-term storage it was stored refrigerated.

Samples dried for microscopic observation were coated with gold / Pt in an ion sputter (ion sputter (E1010, HITACHI, Tokyo, Japan)) and differential scanning microscope (S-800, HITACHI, Tokyo, Japan) Observed by. 1 is an electron microscope photograph of an mPEG-PLGA membrane in which collagen-fibronectin is bound to only one side.

Example 2 Preparation of Collagen-Fibronectin Surface-Bound mPEG-PLGA Membrane Using EDC-NHS as Crosslinking Agent

A 75:25 molar ratio of lactide and glycosides were placed in a PYREX ampoule and mPEG was added to synthesize the mPEG-PLGA copolymer. Tin catalyst was added after dissolving in toluene. The ampoule was removed from residual solvent such as toluene and water under reduced pressure, the upper part of the ampoule was sealed and heated at 130 ° C. for 12 hours.

After the reaction was terminated and the ampoules were removed, the polymerized copolymer was dissolved in MC and precipitated in excess methanol to remove unreacted monomers and catalysts. The purified polymer was dried under reduced pressure.

The mPEG-PLGA membrane was prepared by dissolving a specific ratio of mPEG-PLGA copolymer and PLGA in MC and an appropriate amount of DMSO and casting it in a glass mold. After drying the membrane for 12 hours at room temperature, the membrane was left to soak for 5 hours in an ultrapure water bath at 22 ° C. Take it out and dry at room temperature for 10 hours. The thickness of the membrane produced is 50 μm and the pore size is 3-5 μm.

A 1% type I atelocollagen suspension or 20% (w / w) hyaluronic acid / atelocollagen mixture was prepared. Fibronectin was added to the collagen suspension or the collagen-hyaluronic acid mixture at a concentration of 50 μg / ml, homogenized at 4 ° C. and 8000 rpm for 3 minutes, and adjusted to pH 7.4 using sodium hydroxide.

Secure the PLGA side of the porous PEG-PLGA membrane to the bottom of the dish and seal the membrane around (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (molar ratio) = 2: 1) buffer (0.1 MES, 0.3M NaCl, pH 6.5) was poured on it, stirred for 15 minutes, and then poured into the collagen-hyaluronic acid-fibronectin mixture and reacted at 4 DEG C for about 12 hours. Membranes were washed with distilled water, lyophilized for long term storage, and stored for a long time.

Samples dried for microscopic observation were coated with gold / platinum in ion sputters (E1010, HITACHI, Tokyo, Japan) and observed with a differential scanning microscope (S-800, HITACHI, Tokyo, Japan).

Example 3 Preparation of Composite Bilayer of Porous Collagen-Haluronic Acid-Fibronectin Membrane and mPEG-PLGA Film

Add a 75:25 molar ratio of lactide and glycoside to the PYREX ampoule. mPEG was added to synthesize the mPEG-PLGA copolymer. Tin catalyst was added after dissolving in toluene. The ampoule was removed from residual solvents such as toluene and water under reduced pressure, the top of the ampoule was sealed, and the ampoule was heated at 130 ° C. for 12 hours.

After the reaction was terminated and the ampoules were removed, the polymerized copolymer was dissolved in MC and precipitated in excess methanol to remove unreacted monomers and catalysts. The purified polymer was dried under reduced pressure. The mPEG-PLGA membrane was prepared by dissolving mPEG-PLGA in MC and casting it into a glass mold, and drying the membrane at room temperature for 24 hours.

A 20% (w / w) hyaluronic acid / atelocollagen mixture was prepared by mixing a 1% type I atelocollagen suspension with a 1% hyaluronic acid solution. Homogenized at 4 ° C., 8000 rpm for 3 minutes and adjusted to pH 7.4 with NaOH. The mixed solution was attached to a dish and frozen at -70 ° C. for at least 5 hours, and then freeze-dried. The lyophilized porous membrane was crosslinked by soaking in a 95% ethanol solution containing 50 mM EDC at room temperature for 24 hr. After washing with distilled water and freeze-dried again.

Chloroform was sprayed onto the surface of a previously prepared mPEG-PLGA membrane using a glass injector, and a freeze-dried collagen-hyaluronic acid membrane was attached in a short time, and the bilayer was dried for at least 24 hours. To prepare the fibronectin-containing membrane, 1% fibronectin phosphate buffer solution was added to the collagen-hyaluronic acid membrane surface at a capacity of 30, 50 or 100 g / cm 3, and left for 5 hours at 37 ° C. in a high humidity environment.

Unbound fibronectin was removed by rinsing the fibronectin-coated composite bilayer in phosphate buffer for 5 minutes. The membrane was washed with distilled water and lyophilized for long term storage. Samples dried for microscopic observation were coated with gold / platinum in ion sputters (E1010, HITACHI, Tokyo, Japan) and observed with a differential scanning microscope (S-800, HITACHI, Tokyo, Japan).

FIG. 2 is a photograph of a cross section of a composite double membrane composed of a PEG-PLGA film and a porous collagen-hyaluronic acid-fibronectin membrane, using a scanning electron microscope (SEM). The collagen-hyaluronic acid-fibronectin porous membrane was observed to maintain the pore structure resulting from ice crystals formed by water molecules evenly distributed in the dispersion even after binding to the PEG-PLGA film.

Example 4 Cell Attachment and Proliferation Tests on mPEG-PLGA Membrane and Collagen-Haluronic Acid-Fibronectin Membrane

Human dermal fibroblasts were obtained from Yonsei Medical Center Plastic Surgery with permission from The Medical Material Control and Management Committee of Yonsei University. Existing methods [Park SN, et al. 1% penicillin / streptomycin / amphotericin-ratio (penicillin) in 175 cm 2 tissue culture flasks (NUNC, Roskilde, Denmark) after obtaining cells from primary culture using Biomaterial 2003; 24: 1631-41]. The cells were cultured using DMEM (JBI Inc, Daejeoun, Korea) growth medium containing / streptomycin / amphotericin-B) and 10% fetal bovine serum (JBI Inc, Daejeoun, Korea). The culture was carried out in an incubator maintained at 5% CO ₂ and 95% air environment. Fibroblasts between 5 and 7 passages were used for this experiment.

100 μl of medium containing 2 × 10 ⁵ cells was treated with mPEG-PLGA film, PLGA film, collagen-HA membrane with a diameter of 10 mm, and Interceed ^TM (Ethicon, Inc. NJ, USA) Sufficient medium was added to the surface after 1 hour. Media with and without serum were used to together observe the effect of serum on cell adhesion.

The number of attached cells was determined after 4 hours of application of cell dispersion to 3- (4,5-dimethylthiazolyl) -2,5-diphenyltetrazolium bromide (MTT) assay (Sung HW, et al. J Biomed Mater Res 2001; 55: 538-46; Chevally B, et al. J Biomed Mater Res 2000; 49: 448-59) and measured by ELISA reader (Spectra Max 340, Molecular Device Inc. CA, USA). The measured values were converted into cell numbers using a calibration curve of the cells prepared in advance and the absorbance values.

Membrane-attached membranes were washed with PBS, fixed with 2% (w / v) glutaraldehyde and re-washed with PBS for microscopic observation of the adhered membranes. It was coated with gold / platinum (E1010, HITACHI, Tokyo, Japan) and observed with a differential scanning microscope (S-800, HITACHI, Tokyo, Japan).

In order to examine cell proliferation in different types of membranes, each type of membranes to which the medium containing 10 ⁵ cells was added was incubated for 7 days and the number of proliferated cells was measured by MTT assay. In addition, after 7 days, the sample was fixed with 10% neutral-buffered formalin solution, embedded in paraffin wax, cut into 3 μm thickness, and then hematoxylin-eosin (haematoxylin). -eosin (HE)) and observed under a microscope.

Figure 3 compares the number of cells attached to the collagen-hyaluronic acid membrane, PEG-PLGA film, PLGA film and Interceed ^TM membrane in serum-containing medium and serum-free medium conditions. Fibroblasts were observed to adhere more to the collagen-hyaluronic acid membrane than to Interceed ^™ or PEG-PLGA films. mPEG-PLGA film showed the lowest cell adhesion and no increase in adherent cell number was observed due to the addition of serum in both mPEG-PLGA and PLGA membranes.

Differential scanning microscopy showed that the fibroblasts on the collagen-hyaluronic acid membrane were all evenly distributed and in a spread form (FIG. 4A). Cells attached to the mPEG-PLGA film or the Interceed ^™ matrix were observed to be clustered and not spread well (FIGS. 4B and C). The reason why mPEG-PLGA has anti-adhesion ability is that the reduction of interfacial free energy (Park KD, et al. Biomaterials 1998; 19: 851-9) due to end-grafted PEG chains prevents surface adhesion of protein and cells. Because. Surface hydrophilicity resulting from PEGylation may also inhibit cell adhesion [van Wachem PB, et al. Biomaterials 1985; 6: 403-08. In the case of Interceed ^TM , when the medium containing the cells was added, it was observed that the medium was acidified through the color change of phenol red contained in the medium.

The proliferation of each fibroblast on the PEG-PLGA film and the collagen-hyaluronic acid membrane was observed by H & E staining. 5A shows that fibroblasts penetrate deep into the collagen-hyaluronic acid network at increased density. On the other hand, a very small number of single layer fibroblasts were observed on the PEG-PLGA film surface (FIG. 5B).

Example 5 Confocal Laser Scanning Microscopy

The staining intensity of fibrous actin in fibroblasts attached on the collagen-hyaluronic acid membrane, the collagen-hyaluronic acid-fibronectin membrane, mPEG-PLGA film and PLGA film prepared by the method of Example 3 was measured after 4 hours of culture. An SS, et al. Am J Physiol Cell Physiol 2002; 283: C797-801.

Attached cells were fixed with 3.7% paraformaldehyde-phosphate buffered saline containing 0.1% Triton X100 for 10 minutes at room temperature, pH7.2. Cells were washed twice with phosphate buffered saline and treated with 0.2% Triton X 100-phosphate buffered saline for 10 minutes.

Cells were stained with paloidine (5 g / ml) with fluorescent isothiocionate (FITC) attached with blocking solution overnight at room temperature in a dark room and cells were washed strongly with phosphate buffered saline to stain fibrous actin. . Propidium iodide (PI) solution is added for nuclear staining.

Intracellular fibrous actin staining intensity is calculated using MetaMorph software. Mean pixel intensity (digital representation of mean fluorescence) was calculated for at least five cytoplasm of each cell. It does not include areas that contain edges or nuclear contours of cells. In each experiment, fibrous actin staining intensities of at least 10 different cells were averaged. Increasing the staining intensity of fibrous actin means increasing the reorganization of actin [An SS, et al. Am J Physiol Cell Physiol 2002; 283: C797-801. Significant differences were determined by ANOVA, with P <0.05 being valid.

Confocal laser scanning microscopy showed that the polymerization of actin microfibers in fibroblasts was increased in collagen-hyaluronic acid membranes and especially in fibronectin-coated collagen-hyaluronic acid membranes compared to Interceed ^TM , mPEG-PLGA membranes. It was found (FIG. 6). Actin cables present in cells spread well on the fibronectin coated collagen-hyaluronic acid membrane and the uncoated collagen-hyaluronic acid membrane were formed in association with the focal adhesion of the cell periphery.

In the picture, green indicates F-actin stained with FITC-phalloidine and red indicates cell nuclei stained with PI. Actin fibers in non-muscular cells are important for the movements involved in cell structure and adhesion spot formation, and it has already been reported that the attachment of cells and extracellular matrix leads to reorganization of the actin skeleton.

The intensity of actin staining of cells attached to the fibronectin coated and uncoated membranes was measured to assess the effect of the fibronectin coated collagen-hyaluronic acid membrane on actin fiber reorganization. In Fig. 7, the average pixel intensity of the fibroblasts of the collagen-hyaluronic acid membrane was 1933.6 and the membranes coated with fibronectin of 50 or 100 g / cm 3 slightly increased to 2031.8 and 2054.7. This means that the reorganization of the actin microfibers in the fibronectin coated membrane is increased.

Example 6 Biodegradability Test

The degradation rate of the membrane was examined by measuring the mass of the sample and the molecular weight of mPEG-PLGA. The mPEG-PLGA / collagen-hyaluronic acid constituent membrane was immersed in 5 ml of phosphate buffer containing 2% penicillin / streptomycin / amphotericin-B agonist and incubated at 37 rpm at 20 rpm. The samples were collected, lyophilized and weighed on days 3, 7, 14 after incubation. mPEG-PLGA copolymer was analyzed by gel permeation chromatography (GPC).

Wounds of the peritoneum cause an inflammatory response from the mesenteric surface, and increased permeability of blood vessels in the traumatic tissues drains mesenteric blood effusion. This outflow solidifies in as little as 3 hours. Usually, most of the fibrous adhesions dissolve within the first few days of development. If they last three days or more, fibroblast proliferation will occur within them, causing bond formation. Therefore, the mechanical barrier should remain in place for several days and should be absorbed in the treatment of the peritoneum that persists during the critical period of mesenteric re-epithelialization [Gomel V, et al. J Reprod Med 1996; 41: 35-41.

After exposure to 37 ° C phosphate buffer solution, the weight of the synthetic film continuously decreased (Fig. 8), and after two weeks, it was found that about 30% of the initial mass was decomposed. The molecular weight of mPEG-PLGA decreased very rapidly within the first three days and then gradually decreased as in FIG. 8B.

Rapid degradation was caused by the high hydration of the membrane and the swelling properties of the ethylene glycol flakes of mPEG-PLGA [Garcia JT, et al. J Microencapsulation 1999 16: 83-94. Hydrophilic fragments of PEG are more easily lost than fragments of PLGA [Jiang HL, et al. Preparation, Polym Int 1999; 48: 47-52], after which the polymer becomes more hydrophobic, which leads to a sequential decrease in degradation rate after initial degradation.

Example 7 In Vivo Effect Validation Test Using Rat Inner Abdominal Wall Injury Model

Thirty female Sprague-Dawley rats were paralyzed by intramuscular administration of a combination of ketamine hydrochloride (125 mg / kg) and xylazine (12 mg / kg). After laparotomy by a centerline incision, 2 cm × 1 cm of damage was formed in the anterior abdominal wall with a scalpel.

The fused side of the collagen-fibronectin-fused mPEG-PLGA membrane (3 cm × 2 cm) prepared in Example 1 was allowed to contact the damaged inner wall. Seprafilm ™ (3 cm × 2 cm, Genzyme, USA) was applied to the peritoneal injury site of rats and rats without any treatment were used as controls. Animal testing procedures were managed in accordance with the guidance and control of Yonsei University's use and care of animals. Incisions of all animals were closed. At 7 days after surgery, rats were euthanized to examine the incidence and adhesion of adhesions.

The mPEG-PLGA membrane surface-treated with collagen-fibronectin significantly reduced the adhesion of the injury site. No adhesion was found in about 70% of animals treated with the membrane, compared to 40% of the control group not treated with any membrane. This is similar to Seprafilm ™, which is a conventional membrane, and there was no significant difference between the Seprafilm ™ treated group and the prepared mPEG-PLGA membrane treated group.

Figure 9 shows the results of confirming the degree of adhesion of the abdominal wall injuries and untreated areas to which the collagen-fibronectin surface-treated mPEG-PLGA surface was applied one week after surgery.

The biodegradable anti-adhesion membrane prepared by the present invention can suppress the adhesion of other peripheral tissues to the wound site of the abdominal intestine or abdominal wall of the abdominal incision surgery patient, thereby removing the causes of intestinal obstruction, long-term sequelae, and pelvic pain. At the same time, unlike other anti-adhesion membranes, the inner surface of the membrane combines fibronectin, which is involved in cell adhesion and regulates cell proliferation and migration, and extracellular matrix components such as collagen and hyaluronic acid, which can promote tissue regeneration. It can increase the healing speed of damaged areas.

Claims (11)

A biodegradable anti-adhesion membrane comprising a tissue adhesion layer comprising collagen and fibronectin and one side thereof having a layer of polyethylene glycol-polylactide-co-glycolide (PEG-PLGA) copolymer. The biodegradable anti-adhesion membrane of claim 1, wherein the collagen is type I atelocollagen. The biodegradable antifouling membrane according to claim 1, wherein the PEG is mPEG (methoxy PEG). A biodegradable anti-adhesion membrane comprising a tissue adhesion layer comprising collagen, hyaluronic acid and fibronectin and one side thereof having a layer of PEG-PLGA copolymer. 5. The biodegradable antifouling membrane according to claim 4, wherein the collagen is type I atelocollagen. The biodegradable antifouling membrane according to claim 4, wherein the PEG is mPEG. Preparing a PEG-PLGA copolymer; Preparing a mixed solution containing collagen and fibronectin; And cross-linking the mixed solution with a PEG-PLGA copolymer. 8. The method of claim 7, wherein the mixed solution further comprises hyaluronic acid. The method according to claim 7, which is crosslinked using ozone treatment or EDC (1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide) -NHS (N-hydroxysuccinimide). Preparing a PEG-PLGA copolymer; Preparing a mixed solution containing collagen; Lyophilizing the mixed solution and binding to PEG-PLGA copolymer to prepare a membrane; And coating the membrane with fibronectin. The method of claim 10, wherein the mixed solution further comprises hyaluronic acid.

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