KR20170012867A - Manufacturing method of thermosensitive anti-adhesion compositions based on micronized acellular dermal matrix - Google Patents

Abstract

The present invention relates to a temperature-sensitive anti-adhesion composition on the basis of human body tissue. More specifically, the present invention relates to a human body tissue-based temperature-sensitive anti-adhesion composition produced by physically mixing particle acellular derma, hyaluronic acid, and temperature-sensitive polymers, and to a method for producing the same. The human body tissue-based temperature-sensitive anti-adhesion composition according to the present invention contains particle acellular derma tissue as a main component, wherein the particle acellular derma tissue is mixed with human body-derived polymers such as hyaluronic acid and temperature-sensitive polymers having biocompatibility. The composition shows excellent biocompatibility, in vivo continuance, tissue attaching properties, and biodegradable properties, and thereby ultimately shows an excellent effect in preventing adhesion. The composition of the present invention is phase-changed to a gel state from a sol state by means of a body temperature, and can be stably coated in tissue. Accordingly, the composition can be conveniently used. Also, after being fed in vivo as a liquid, the composition is maintained in a gel phase, so a physical barrier having thin particle acellular derma tissue is formed and adhesion of a wound part can be effectively controlled. Moreover, according to the present invention, an injection-formulation human body tissue-based anti-adhesion composition can be provided to be suitable for a celoscope operation or a minimum invasion technique which is a recent operation tendency. Also, particle acellular derma tissue and bio-derived or biocompatible polymers are used as a main component, so inflammation, repulsion, or immunoreactions are maximally controlled in vivo. Accordingly, a human body-based anti-adhesion composition having very excellent biocompatibility can be provided.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for manufacturing a particulate acellular dermis-based thermosensitive adhesion preventive composition,

The present invention relates to a thermosensitive adhesion preventive composition based on a human tissue, and more particularly to a thermosensitive adhesion preventive composition based on a human tissue, which is prepared by physically mixing hyaluronic acid and a thermosensible polymer with a particle- Compositions and methods for their preparation.

Adhesion refers to the phenomenon that the surrounding tissues or tissues which are to be separated from each other due to excessive formation of the fibrous tissue during the wound healing process due to inflammation, wound, friction, . Such adhesions can cause serious problems, especially after surgery. Adhesion can occur generally after all kinds of surgery, and due to this phenomenon, the organs or tissues around the surgical site may adhere to each other during the recovery process, resulting in serious clinical sequelae.

The types of sequelae that can be caused by these adhesions vary widely. According to US statistical data, the main symptoms of postoperative adhesions are small intestinal obstruction 49% to 74%, infertility 15% to 20%, chronic osteoporosis 20% to 50% It is known that it reaches 19%.

The adhesion of organs and tissues after surgery is a physiological phenomenon that occurs during proliferation and regeneration of injured tissue cells. However, adhesion of excessive tissue, or unintended adhesion to other tissues or organs, And in some cases it may be a life-threatening factor, such as a need for reattachment of the adhesiolysis.

The adhesion mechanism of the tissue coincides with the general wound healing mechanism. When a wound occurs due to surgery, infection, inflammation, etc., inflammatory reaction mechanism is activated, cytokines are secreted by immune cells, macrophages, platelets, lymphocytes, Mesothelial cells and the like are explosively increased. When thrombin produced by prothrombin coagulates blood and secondarily converts fibrinogen to fibrin, the fibrin is degraded by fibrin degradation after restoring the wound. . If the wound healing mechanism is interrupted for some reason, the extracellular matrix (ECM) is formed through the overproduction of fibrin, and the neovascularization is generated in the ECM, and adhesion progresses gradually for about one week.

There are three main types of anti-adhesion methods currently in use. First, it minimizes tissue damage and foreign matter adherence due to delicate attention and unnecessary procedures at surgery. Second, the pathophysiological process required for inflammatory reaction and adhesion formation is to inhibit drug therapy based on adhesion mechanism. Lastly, anti-adhesion barrier is used to wrap or cover the wound area to prevent contact with the surrounding tissues to prevent adhesion, which is widely used in clinical practice, and its importance is increasing.

Anti-inflammatory agents, anticoagulants, fibrinolytic agents, and antibiotics are drugs that reduce adhesion based on adhesion mechanisms. There is a side effect that the anti-adhesion method through the drug is not yet proved effective in clinical trials, and most of the drugs interfere with the deposition of fibrin during the healing process, so that the wound healing rate is lowered compared with the anti-adhesion effect . Therefore, the use of drugs to prevent adhesion is limited and requires much care.

The adhesion preventive membrane blocks the contact with the wound area by covering the wound area with the physical barrier by covering the part expected to be adhered during the surgical procedure. Suitable materials for the anti-adhesion barrier should be safe and effective, free of inflammatory or immune reactions, free of suture, and active in the blood. In addition, it remains in the healing period of the injured tissue and acts to prevent adhesion. After a certain period of time, it is decomposed or absorbed so that no separate removal is needed. Laparoscopic surgery and laparotomy should be applied to the wound area. Emissions through the body should also be harmless to the human body and be biocompatible.

Currently, the anti-adhesion membrane used in clinical practice can be divided into two types: intra-peritoneal instillator and adhesion barrier. Peritoneal drip agents can be divided into a solution type and a gel type, and the adhesion blocking agent is mainly in the form of a film and a membrane. Adept, Ringer's lactate solution, Dextran 70, CMC solution and so on are available as solutions. In the form of gel, there are products such as Flowgel, Intergel and Guardix. Film and membrane type adhesion inhibitors include products such as Seprafilm, Interceed, Surgiwrap (Ae-gyeong Oh, 2013, Biomater. Res. , 17 (4): 138-145).

Most of the anti-adhesion agents are hyaluronic acid (HA) -based products. In order to increase the half-life of the hyaluronic acid, HA or carboxymethyl cellulose (CMC) (For example, Seprafilm ^TM (Genzyme Corp. Cambridge, Mass., USA), Guardix ^TM (Korea Bioregence Development, Hanmi Pharmaceutical Sales)) have been commercialized and widely used. However, it is difficult to obtain the effect of preventing adhesion as much as expected.

The composition of the solution type is difficult to be accurately applied on the wound area due to the excessive flow down when applied to the body, and there is a strong tendency to flow out to other parts before the adhesion prevention function is performed because of rapid decomposition in the body. In addition, film or membrane type compositions are poorly adherent when applied to internal organs and have disadvantages of foreign body reactions. Therefore, it is difficult to apply to the minimally invasive method or laparoscopic method, and since the suturing step is essential, secondary adhesion may occur at the suture site at the time of suturing. Gel-type compositions are easily degraded prior to wound healing, resulting in a short half-life in the body, and some non-living body-derived materials may exhibit a foreign body reaction.

Therefore, the anti-adhesion composition having both functionality and ease of use must satisfy four conditions. 1) It should help the wound to regenerate normally during 7 days of wound healing. 2) It should be decomposed, absorbed and removed naturally after the anti-adhesion function. 3) It should be a form that can be injected so as to be suitable for the method or laparoscope.

Polysamer 407 (Pluronic F-127) and Poloxamer 188 (Pluronic F-68), which are representative thermosensitive polymers, are the main components of the composition. In order to satisfy these four conditions, adhesion inhibitors using thermosensitive polymers are being marketed. , Mixed compositions of thermosensitive polymer and sodium alginate or mixed compositions of thermosensitive polymer, gelatin and chitosan are representative examples (Korean Patent Publication Nos. 10-1452041 and 10-1330652). These thermosensitive compositions are present in a solution state at room temperature and are present in the form of a viscous gel near the body temperature, thereby satisfying the convenience of use. However, further long-term monitoring is required for adhesion prevention function, It is true.

Under these circumstances, the present inventors have found that, through thermosensitivity capable of sol-to-gel phase transition, user-friendly ease of use, excellent biocompatibility and persistence in the body, tissue adhesion and biodegradability, And an effort has been made to develop an anti-adhesion composition exhibiting an excellent anti-adhesion effect. As a result, it can be stably applied in the tissue due to phase transition from a sol state to a gel state by body temperature, which is convenient to use, exhibits excellent biocompatibility, persistence in the body, tissue adherence and biodegradability, and can effectively inhibit adhesion , And a human tissue-based thermosensitive adhesion inhibitor of the present invention, which is a combination of hyaluronic acid and a thermosensitive polymer, which is a collagen-based particulate acellular dermis having all of convenience for use and adhesion prevention function as a next generation adhesion inhibitor Thereby completing the composition.

Korea Registered Tokha No. 10-1452041 Korean Patent No. 10-1330652 Korean Patent No. 10-1523878

Ae-gyeong Oh, "Trends of Anti-Adhesion Adjuvant-Review ", Biomater. Res., Vol. 17, No. 4, 138-145, (2013)

An object of the present invention is to provide a human tissue-based thermosensitive adhesion preventive composition prepared by physically mixing granular acellular dermis with hyaluronic acid and a thermosensible polymer, and a method for producing the same.

Another object of the present invention is to provide a method of preventing adhesion, comprising the step of contacting the human tissue-based thermosensitive adhesion-preventive composition to the surface of the local organ of an animal.

In order to achieve the above object, the present invention provides a thermosensitive adhesion preventive composition based on a human body, wherein the granular acellular dermis, hyaluronic acid and thermosensitive polymer are physically mixed. The method for producing a human tissue-based thermosensitive adhesion preventive composition of the present invention comprises the steps of producing a cell-free dermis tissue and disrupting the cell-type dermis tissue in a particle form, physically mixing the granular cell-free dermis tissue with hyaluronic acid and thermosensitive polymer, Followed by sterilization via gamma radiation.

More specifically, the present invention provides a method for preparing a human tissue-based thermosensitive adhesion prevention composition,

(i) removing the epidermal layer from the skin tissue;

(Ii) removing the cells of the dermal layer in the skin tissue from which the skin layer has been removed;

(Iii) lyophilizing the acellular dermis and then pulverizing it into a granular form;

(Iv) physically mixing and hydrating hyaluronic acid and the thermosensible polymer in the particulate acellular dermis; And

(V) sterilizing the composition containing the granular acellular dermis, hyaluronic acid and thermosensitive polymer of step (iv) by irradiating gamma rays;

. ≪ / RTI >

The term "acellular dermis tissue" in the present invention refers to a dermis layer in which isolated dermis has been removed from cells capable of causing an immunological rejection through a chemical treatment process, and collagen and elastin are main components. It is the "particle-type acellular dermis tissue" in the present invention that this acellular dermis is made into a microstructure by granulation. Conventionally, the lyophilized particulate acellular dermis is hydrated by saline or distilled water to be used as a skin substitute and has a merit that it can be injected using a syringe without going through surgery because it can maintain fluid state. For this reason, the particle-type acellular dermis tissue was used not only for the treatment of skin tissue defects due to accidents but also for the treatment of chronic diseases such as diabetic ulcers. As a safe and effective biomaterial in the molding market, particle-type acellular dermal tissue was used as a filler.

In an embodiment of the present invention, the particulate acellular dermis may have a particle size of about 300 to 800 mu m, preferably about 500 mu m. When the particle size of the particle is less than 300 μm, the collagenase (Matrix Metalloproteinase, MMP-1), which is a proteolytic element, accelerates the biodegradation rate of the dermis tissue, If the particle size of the particulate acellular dermis exceeds 800 μm, injection into the body using a syringe may not be performed smoothly. Therefore, it is easy to use since it can be injected into a living body by a syringe without being operated within the particle size range.

In addition, the particle-like acellular dermis tissue is mixed with a thermosensitive polymer and hyaluronic acid and injected into the body to form a particle-like acellular dermis tissue from the wound tissue by the sol-to-gel phase transition And these physical barriers may be present in the body continuously without forming a gel type near the body temperature, and after a long period of time, the particulate acellular dermal tissue may naturally decompose in the body .

In the present invention, the acellular dermis tissue may be a homogeneous dermis tissue, and the process for preparing the acellular dermis tissue may be performed according to the preparation process disclosed in Korean Patent No. 10-1523878.

The process of obtaining the acellular dermis tissue can be roughly divided into two steps. In the first step, the skin layer is removed from the prepared tissue, and the epidermal layer and the dermal layer are separated. In an embodiment of the present invention, the skin layer can be separated from the dermal layer using proteolytic enzymes. Generally, various proteolytic enzymes are used to separate the dermal layer and the epidermal layer. When the enzyme is used, if the concentration is too low or the treatment time is too short, the separation will not be performed well. If the concentration is too high or the treatment time is too long, the cell or tissue will be damaged. Therefore, it should be treated according to appropriate concentration and time. Enzymes used to separate the dermis and epidermis include the neutral proteases disaspase, tramolysin, and trypsin. The dermal layer and the epidermal layer can be separated by treating with a 1.0 unit / ml of a disperse at 37 DEG C for 60 to 120 minutes. Alternatively, treatment with tamolysin at a concentration of 200 占 퐂 / ml for 30 minutes at 37 占 폚 can separate the dermal layer and the epidermal layer. The use of tamoxifen reduces the risk of basement membrane damage compared with the use of distearate. Another method is to separate the two layers of tissue by changing the ionic strength of the solution. This method also depends on the conditions such as ionic strength, treatment time, and treatment temperature. Treatment with 1 mol or more of sodium chloride solution at 37 ° C for 14 to 32 hours can separate the dermal layer and the epidermal layer. Bacteria and fungi can not grow in a solution of 1 moles or more chloride sodium chloride, so the risk of microbial contamination can be reduced. Or 20 mM of ethylenediaminetetraacetic acid (EDTA) at 37 DEG C for 14 to 32 hours to separate the dermal layer and the epidermal layer. EDTA can reduce tissue damage by proteolytic enzymes because EDTA acts as a protease inhibitor. Therefore, treatment with 1 to 5 mM of EDTA in 1 mol of sodium chloride solution can minimize microbial contamination and tissue damage caused by enzymes, and can separate the dermal layer and the epidermal layer.

In the second step, the skin layer is removed as described above, and then the cells of the dermal layer are removed. The immune response is mainly caused by membrane proteins present in the cell membrane. Therefore, removal of the cells can minimize the immune response. We use a method to selectively remove cells without damage to the tissue using the difference in physical and chemical properties between cells and extracellular epilepsy. The main component of the cell membrane is phospholipid, and various surfactants can be used to remove cells without damaging the tissue. In an embodiment of the present invention, the cells of the dermal layer may be removed by a surfactant or ultrasonication. For this purpose, ionic surfactants such as sodium dodecyl sulfate (SDS), or ionic surfactants such as Triton X-100, Tween 20, Tween 40, Twin 60, Twin 80, Nonidetip- (NP-40), and the like are used.

When the dermal layer is treated at room temperature for 30 to 120 minutes with an SDS solution at a concentration of 0.2 to 1% at room temperature, cells can be removed without damaging the tissue. Or treatment with a Tween 20 solution at a concentration of 0.1 to 2.0% for 30 to 180 minutes at room temperature, or treatment with a Triton X-100 or Nonidtip-40 solution at a concentration of 0.2 to 2% at 22 to 37 ° C for 30 to 180 minutes Cells can be removed without tissue damage.

In addition to the above chemical methods, the cells can be removed by a physical method. Ultrasonic waves of 10 to 100 kHz for 5 to 60 minutes can be used to remove the cells. Alternatively, the combination of a surfactant and ultrasonic waves can also remove cells without damaging the tissue. In addition, using a solvent (TNBP) and a surfactant, cell removal and virus removal can be performed at the same time.

The term "hyaluronic acid (HA)" in the present invention is a biosynthetic natural substance that exists in many skin, articular fluid, cartilage and the like in animals and the like and is a mucopolysaccharide having hydrophilic properties because it has many hydroxyl groups. It binds with water and becomes gel state. It is involved in lubrication of joints and flexibility of skin. It is also highly viscous and plays an important role in preventing invasion of bacteria or penetration of toxins into skin. If the skin lacks hyaluronic acid, the skin becomes dry, elastic and wrinkled. Because of this, hyaluronic acid can be used as a filler to prevent wrinkles or to fill the area that has been turned off. Hyaluronic acid, expressed in microorganisms, has a size of 1,100,000 Daltons (1,100 kDa) or 3,000,000 Daltons (3,000 kDa), and physical methods such as ultraviolet radiation, radiation and electron beam, or 1,4-butanediol diglycidyl ether The physical and physiological properties are different according to cross-linking through the chemical method used.

The term "thermosensitive polymer" in the present invention is a polymer capable of sol-to-gel phase transition which exists in a solution state at room temperature and exists in a viscous gel state at a temperature close to the temperature, Polymer of any kind or composition can be included in the scope of the present invention. In an embodiment of the present invention, the thermosensitive polymer is a polyethylene oxide (PEO) -polypropylene oxide (PPO) -polyethylene oxide (PPO) having a temperature-sensitive sol- PEO) triblock copolymer. The polyethylene oxide (PEO) -polypropylene oxide (PPO) -polyethylene oxide (PEO) triblock copolymer is generally referred to as a poloxamer group. The molecular weight of PEO, which is a hydrophilic polymer, PPO, which is a hydrophobic polymer, It exhibits various temperature sensitivity and physical properties depending on the content. The polyethylene oxide (PEO) -polypropylene oxide (PPO) -polyethylene oxide (PEO) triblock copolymer is produced and supplied by BASF, Germany. Specifically, Poloxamer 407 (Pluronic F-127, molecular weight: 9840 to 14600 Da) or Poloxamer 188 (Pluronic F-68, molecular weight 7680 to 9510 Da), and preferably Poloxamer 407 (Pluronic F-127, molecular weight 9840 to 14600 Da).

The present invention also includes a step of sterilizing the composition obtained by physically mixing the particulate acellular dermis tissue with hyaluronic acid and the thermosensible polymer and hydrating the composition, by irradiating gamma rays.

In the present invention, the mixing may physically uniformly mix and hydrate the particulate acellular dermis tissue, hyaluronic acid, and thermosensible polymer without causing a chemical reaction. In the embodiment of the present invention, a syringe filled with the particulate acellular dermis tissue, hyaluronic acid and thermosensitive polymer, and a syringe filled with sterilized water are connected to each other using a connector, and advancing and retracting the plunger to approximately mix the packings. Further, the mixed fillers were physically remixed at a magnetic rate of 60 rpm to 800 rpm and a rate of revolution of 1,800 rpm to 2,200 rpm, preferably at a magnetic rate of 400 rpm and a revolution rate of 2,000 rpm using a mixer .

In the present invention, sterilization can completely kill deadly infective bacteria, parasites and insects by sterilizing the composition without raising the temperature by irradiating gamma rays with strong permeability without using heat or chemicals. In the embodiment of the present invention, the irradiation dose of the gamma ray may be 15 kGy to 40 kGy, preferably 25 kGy, in which the dermal tissue is not destroyed by gamma irradiation. In addition, the irradiation time of the gamma ray may be 37,000 to 40,000 seconds, preferably 37,467 seconds.

The present invention also relates to a method for producing a thermosensitive adhesion preventive composition based on human tissues as described above, (vi) a method for producing a thermosensitive adhesion preventive composition comprising the steps of: (vi) platelet rich plasma, and mixtures thereof. < / RTI > More specifically, the particulate acellular dermis mixed with the hyaluronic acid and the thermosensible polymer of the present invention can be mixed with sterilized water, physiological saline, or PRP extracted from blood (platelet rich plasma). Inflammation, immune response and the like can be avoided through the use of the solution.

In addition, the present invention provides a human tissue-based thermosensitive adhesion preventive composition prepared by the above method. More specifically, the present invention provides a thermosensitive adhesion preventive composition based on a human tissue, comprising hyaluronic acid and a thermosensible polymer added to a particulate acellular dermis tissue, and mixed by physical stirring to hydrate. The above-mentioned human tissue-based thermosensitive adhesion preventive composition is applied as a solution in a wound state due to surgery or laparoscopic and endoscopic surgery, and is applied as an anti-adhesion agent having a property of inhibiting or preventing adhesion of a wound site by gelatinization by body temperature . The composition according to the present invention is a composition in which hyaluronic acid and thermosensitive polymer are physically mixed with a particulate acellular dermis tissue, more specifically, a composition comprising a particulate acellular dermis tissue having a particle size injectable into a syringe without surgery, Sensitive adhesive polymer which is a polymer capable of sol-to-gel phase transition by hyaluronic acid and a thermosensitive polymer is mixed through physical agitation. In an embodiment of the invention, the anti-adhesion composition is in the form of a viscous gel near the body temperature (Fig. 2) and decomposes spontaneously over time at a temperature similar to body temperature (Fig. 3) (FIG. 4). In addition, it was confirmed that the composition was injected into the abdomen of the experimental animal by injection, and the adhesion preventive membrane was maintained in the body after one week (FIG. 5). In addition, it was confirmed that the composition had excellent adhesion-inhibiting ability as compared with a commercial adhesion inhibitor (FIGS. 8, 9 and 10). Therefore, the anti-adhesion composition of the present invention is in a sol state outside the body and is in a state of gel state in the body, so that it can be easily introduced into organ or tissue of a complicated structure, does not stay in the body for a long time, is harmless to human body, (At least seven days or more), and thus, it is useful as an anti-adhesion agent to prevent adhesion of wound sites due to surgery.

The human tissue-based thermosensitive adhesion preventive composition of the present invention comprises the above particulate acellular dermis tissue, hyaluronic acid and thermosensible polymer as main components, and the three main components are mixed by physical stirring without adding a cross-linking agent. The content of the particulate acellular dermis tissue, hyaluronic acid, and thermosensible polymer as main components relative to the whole composition is suitably determined in consideration of degradation characteristics in vivo, formulation characteristics of the anti-adhesion agent, site characteristics (tissue, organ, area, etc.) . Specifically, the content of the particulate acellular dermis in the whole composition may be 1.0 wt% to 6.0 wt%, preferably 3.0 wt% to 4.0 wt%. The content of the thermosensitive polymer may be 13 wt% to 25 wt%, preferably 15 wt% to 18 wt% of the total composition, and the content of hyaluronic acid may be 0.1 wt% to 0.5 wt% %, Preferably 0.16 wt% to 0.33 wt%. The anti-adhesion composition of the present invention, which is mixed with the above-mentioned contents relative to the total composition, is a thermosensitive composition for prevention of adhesion based on human tissues. It is a liquid composition in order to prevent adhesion of tissues after surgical operation. It can be applied rapidly and shows a change in properties from a liquid phase to a gel phase within a short period of time after application, and it is possible to prevent adhesion of particles to a wound site by forming a physical barrier in a gel state. In addition, the anti-adhesion compositions of the present invention, which have been injected into the wound site and mixed with the above-mentioned contents relative to the total composition, can be naturally biodegraded or absorbed and removed after an average adhesion occurrence period (about 7 days) has elapsed.

The solvent contained in the human tissue-based thermosensitive adhesion preventive composition of the present invention is not particularly limited so long as it is a physical type of thermosensitive adhesion preventive composition comprising a particulate acellular dermis tissue, hyaluronic acid and thermosensitive polymer, As the solvent capable of mixing and hydrating through stirring, there may be used a solution selected from the group consisting of distilled water, water for injection, physiological saline solution, natural peritoneal fluid and artificial peritoneal fluid having a composition of natural peritoneal fluid.

The human tissue-based thermosensitive adhesion preventive composition of the present invention may further contain a drug having an independent pharmacological effect. Such a drug may be one that controls the gelation property of the composition applied to a wound site or acts as an active ingredient in wound healing. Such drugs that can be used include antithrombogenic agents such as heparin or tissue plasminogen activators, aspirin, ibuprofen, ketoprofen or other nonsteroidal antiinflammatory drugs, hormone chemostatic factors, analgesics Or anesthetics.

The human tissue-based thermosensitive adhesion preventive composition of the present invention can be applied to open surgery and laparoscopic surgery, and can be applied not only to conventional peritoneal surgery, bladder or gynecologic surgery, spinal surgery, but also to cardiac surgery, rectal surgery, And can be applied to general surgical fields such as plastic surgery.

The thermosensitive adhesion preventive composition of the present invention can be applied to various forms such as a tube type, a cream type, a syringe type and a spray type depending on the application to be used. The composition can be applied to a wound tissue by injecting, Can be neglected.

The application amount of the body tissue-based thermosensitive adhesion preventive composition of the present invention may be varied depending on the polymer composition, molecular weight, concentration, required adhesion prevention period, internal body action site, patient condition, and the like.

The present invention also provides a method of preventing adhesion, comprising contacting a tissue-based thermosensitive adhesion preventative composition of the present invention to the surface of the topical organ of an animal.

In the present invention, "contacting" may mean treatment with an anti-adhesion composition on the surface of the local organs for the purpose of preventing adhesion of the surface of the local organ, such as by spraying, applying, spraying or attaching.

In the present invention, the "local organ surface" may be all or a part of an organ or tissue surface that has undergone surgery such as surgical operation or the like, in which inflammation has occurred for some reason and adhesion has to be prevented. There are no particular limitations on the site or type of the organ, but it is preferable that the organ is an animal other than the animal (mouse), rat, hamster, rabbit, and other mammals including the human. Examples thereof include gastrointestinal organs such as stomach, small intestine, large intestine, reproductive organs such as uterus and ovary, respiratory organs such as heart and lungs, organs such as muscles, May be a reproductive organs such as uterus, ovary, etc. The state of the surface of the local organs is also not particularly limited.

The operation contents of the organ are not particularly limited. In addition, the surface of the local organs may be a direct object of surgery, and may not be a direct object of surgery, but may be a scarred area as a result of surgery.

In the method of bringing the anti-adhesion composition into contact with the surface of the local organs, the form of the anti-adhesion composition and the ratio of the active ingredient can be appropriately selected according to the type of the organs, tissues, The amount of contact with the surface may be such as to cover the surface.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The thermosensitive adhesion preventive composition based on human tissue according to the present invention is a composition containing as a main component a particulate acellular dermis tissue in which a biomedical polymer such as hyaluronic acid and a biocompatible thermosensitive polymer are mixed, It exhibits persistence in the body, tissue adhesion and biodegradability, and ultimately exhibits excellent adhesion prevention effect. The composition of the present invention is phase-transformed from a sol state to a gel state by body temperature and can be applied stably in the tissue, so that it is convenient to use. In addition, the composition is injected into the body in a liquid phase, It is possible to form a thin physical barrier and effectively inhibit the occurrence of adhesion at the wound site.

In addition, according to the present invention, it is possible to provide a composition for preventing adhesion of human tissues based on injectable form suitable for minimally invasive or laparoscopic surgery, which is a recent trend of surgery, It is possible to provide a composition for preventing adhesion on the basis of a human body which is excellent in biocompatibility by suppressing inflammation reaction, foreign body reaction, immune reaction or the like in vivo as much as possible.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram (a) of the antiadherence composition of the present invention and a diagrammatic view (b) of an action mechanism of the antiadhesive composition. FIG.
FIG. 2 is a photograph showing temperature-dependent change in properties of the anti-adhesion composition of the present invention at a room temperature (RT) and a temperature of 37 ° C. FIG. Here GDX-SG ^® is a thermosensitive adhesion inhibitor commercial product Dix (Guardix-SG ^®).
3 is a graph showing the results of an in vitro gel stability test of the antiadherence composition of the present invention. Here GDX-SG ^® is a thermosensitive adhesion inhibitor commercial product Dix (Guardix-SG ^®).
4 is a graph showing the results of the concentration-dependent cytotoxicity test of the anti-adhesion composition of the present invention.
FIG. 5 is a photograph showing the result of the maintenance test of the adhesion preventive membrane in the body as compared with the Guardix-SG ( ^R ) treatment group, which is a commercial product for the sale of a temperature sensitive adhesion inhibitor, in order to evaluate the retention ability of the adhesion preventive composition of the present invention.
FIG. 6 is a photograph showing a process of establishing a cohesive animal model for evaluating the adhesion inhibiting ability of the anti-adhesion composition of the present invention. FIG.
FIG. 7 is a photograph showing the criteria for evaluating the degree of adhesion according to an animal experiment in four stages. The standard of the four-step evaluation grade according to the degree of tissue adhesion is 0, 1, 2 and 3, and the larger the number, the greater the adhesion.
8 is a graph showing the results of the adhesion area evaluation as compared with the Guardix-SG ( ^R ) treatment group as a commercial control for the evaluation of adhesion inhibition ability of the anti-adhesion composition of the present invention Graph. Here, control is a negative control group, and means anti-adhesion inhibitor-free treatment group.
9 is a graph showing the results of the adhesion strength evaluation as compared with the Guardix-SG ( ^R ) treated group as a commercial control for the evaluation of adhesion inhibition ability of the anti-adhesion composition of the present invention Graph. Here, control is a negative control group, and means anti-adhesion inhibitor-free treatment group.
10 shows the adhesion grade evaluation results as compared with the Guardix-SG ( ^R ) treated group as a commercial control for the evaluation of adhesion inhibition ability of the anti-adhesion composition of the present invention Graph. Here, control is a negative control group, and means anti-adhesion inhibitor-free treatment group.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: According to the present invention Temperature Sensitivity  Preparation of anti-adhesion composition

Example  1-1: Particle type Acellular cell  Manufacture of dermal tissue

The skin tissue (collected from the donated tissue from a tissue bank for the purpose of treatment of a patient for non-profit purposes) was treated with a concentration of 1.0 units / ml of the neutral protease, Dispase, and incubated at 37 < 0 & After stirring for 3 minutes, the epidermis was removed by separating the dermal layer and the epidermal layer by washing three times with sterilized distilled water. The tissue from which the epidermis layer was removed was treated with a 1% Triton X-100 solution at 30 ° C for 100 minutes to remove the cells of the dermal layer. After washing with sterilized distilled water more than 3 times, the treated materials used in the process were removed and free cell-free allogeneic dermis was frozen at a temperature of -40 ° C or lower for 2 hours or more, then placed in a freeze dryer and dried for 12 hours or longer. Respectively. The homogenous dermis is cut by a surgical knife to a size of about 1 cm in width and about 1 cm in size, and then 100 g of allogeneic dermis is put into a microfibrillator that does not break the tertiary structure of the same kind of dermis to prevent denaturation of collagen and elastin The dermis of the cell-free allogeneic dermis was passed through a 500-μm-sieve sieve having the most favorable adherence rate in vivo in a sterile state.

Example  1-2: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Preparation of anti-adhesion composition

Poloxamer 407 (Pluronic F-127, PF-127) and hyaluronic acid (HA) were added to the sterilized water, and the mixture was physically mixed and mixed Solution.

Specifically, two 50 mL syringes were prepared. One syringe contained the granular acellular dermis tissue prepared in Example 1-1, Poloxamer 407 (Pluronic F-127, PF-127) and hyaluronic acid (Hyaluronic acid, HA) were filled in a weight ratio, and the other syringe was filled with sterilized water. The two prepared syringes were connected using a connector, and the plungers were advanced and reversed left and right at a temperature of 4 ° C to mix the fillings roughly. The mixed packings were remixed at a rotation rate of 400 rpm and a revolution rate of 2,000 rpm for 10 minutes using an AR-250 planetary mixer (manufactured by Thinky Corporation) at a temperature of 20 ° C.

The thus prepared mixed solution was incubated at 4 ° C for 24 hours to prepare a thermosensitive adhesion preventive composition. Poloxamer 407 (Pluronic F-127, PF-127) and hyaluronic acid (HA) were mixed at a concentration as shown in Table 1 according to the above preparation method, Thereby preparing a thermosensitive adhesion preventative composition. After the preparation, the thermosensitive adhesion-preventive composition was sterilized by irradiation with a gamma ray of 25 kGy for 37,467 seconds.

Further, according to the above manufacturing method, as a control group, Poloxamer 407 (Pluronic F-127, PF-127) and / or hyaluronic acid (HA) A composition without particle-type acellular dermis was prepared.

Sample No. PF-127 (wt%) HA (wt%) Particle-type acellular dermis (wt%) Sample # 1 15.71 0.17 1.45 Sample # 2 15.50 0.16 2.85 Sample # 3 16.74 0.16 1.43 Sample # 4 17.75 0.16 1.41 Sample # 5 15.71 0.16 5.42

Comparative Example PF-127 (wt%) HA (wt%) One 15.71 - 2 15.71 0.16

Example  2: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Of the adhesion preventive composition Gelation  And in vitro gel stability test

Example  2-1: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Confirmation of gel formation of the anti-adhesion composition

It was confirmed whether the particle-type acellular dermis-based thermosensitive adhesion preventive composition prepared in Example 1-2 exhibited a sol-gel phase transition according to the temperature change.

Specifically, the anti-adhesion preventive composition samples # 1, # 2, and # 5 prepared in Example 1-2 were prepared, and 2 mL of each sample was placed in a glass vial, sealed, And the mixture was gelated in a hot water bath for 30 minutes. At this time, as a control, thermosensitive adhesion inhibitor commercial product, the Dix (Guardix-SG ^®, Geneva welsa) and particulate acellular dermis Comparative Example 1 that does not include (PF-127 15,71% by weight; control # 1) and compared A sample of Example 2 (15.71% by weight of PF-127 / 0.16% by weight of hyaluronic acid; control # 2) was used.

As a result of gelation for 30 minutes at 37 ° C in a hot water bath, as shown in FIG. 2, sample samples in a sol state at room temperature (RT) were in a viscous gel state at 37 ° C. Phase transition. Furthermore, it was confirmed that even in the case of Sample Sample # 5 in which the particulate acellular dermis tissue was contained in a predetermined weight ratio or more, the sol-gel phase transition was achieved without decreasing the temperature sensitivity of the entire composition due to the inherent viscosity of the dermis.

As a result, it was confirmed that the temperature-sensitive adhesion preventive composition based on the particle-type acellular dermis of the present invention exhibited a sol-gel phase transition according to the temperature change, and the presence of the gel- Proved to be able to satisfy gender.

Example  2-2: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Evaluation of Gel Stability of Anti-

In order to confirm the gel stability of the particle-type acellular dermis-based thermosensitive adhesion preventive composition which was gelated in a warm water bath at 37 ° C. in Example 2-1, the volume of the anti-adhesion composition remaining in gel state with time Were measured.

Specifically, the anti-adhesion preventive composition sample samples # 1 to # 4 prepared in Example 1-2 were prepared, and 2 mL of each sample was placed in a glass vial, sealed and sealed at 37 ° C in a hot water bath And then gelated for 30 minutes. After gelling, sterilized water at 37 ° C was slowly added to each vial in 1 mL increments and sealed again to measure the volume of the anti-adhesion composition remaining in the gel state every 24 hours. The temperature of the hot water exchanger is 37 ℃, rpm was maintained at 50 rpm determine the gel stability over a period of 4 days, it was used as a control group the Dix (Guardix-SG ^®, Geneva welsa) thermosensitive adhesion inhibitor commercial product.

As a result, as shown in FIG. 3, the gelled, particulate acellular dermis-based thermosensitive adhesion preventive composition of the present invention, like Guardix-SG ( ^R ), which is a product for use as a control agent for temperature sensitive anti- And it was confirmed that it dissolves naturally.

Through this, it was confirmed that the gelled particle-type acellular dermis-based thermosensitive adhesion preventive composition of the present invention was decomposed naturally at 37 ° C., which is similar to body temperature, so that it can be decomposed, absorbed and removed naturally after the anti- And has functionality as an anti-adhesion agent.

Example  3: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Cytotoxicity test of anti-adhesion composition

To confirm the cytotoxicity of the particle-type acellular dermis-based thermosensitive adhesion preventive composition prepared in Example 1-2, the eluate obtained from the sample of the anti-adhesion composition kept at 37 ° C for 24 hours was used The cytotoxicity of the anti-adhesion composition was confirmed by a cell viability measurement technique (MTT assay). At this time, the eluate obtained from the sample of the anti-adhesion composition was filtered using an eluent not containing the particulate acellular dermis filtered with a 0.2 μm syringe filter and an eluent containing the particulate acellular dermis before filtering Respectively.

Specifically, L-929 cells, which are fibroblasts of monolayer culture, were treated with Trypsin-EDTA (Trypsin-EDTA) to adjust the cell concentration to 10 ⁴ per 0.1 mL, and the cells were plated in 96-well plates in 0.1 mL increments And cultured for 24 hours. After 24 hours of incubation, the wells containing the monolayer cultured cells were diluted with the culture medium to a concentration of 0, 20, 40, 60, 80, 100% Not filterd and Filterd were each dispensed in 0.1 mL increments. The eluate of the sample for preventing adhesion-preventing composition was treated in a concentration-dependent manner as described above, and then cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. After 48 hours, 5 mg / mL MTT (3, (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) reagent was diluted 1: 100 in the culture medium and treated with 0.1 mL And cultured for 5 hours. After 5 hours, the culture of each well of the 96 well plate was removed, and 0.1 mL of DMSO (dimethyl sulfoxide) was added to each well to dissolve the cells. Then, the 96-well plate was placed in a spectrophotometer, and the absorbance was measured at a wavelength of 540 nm.

As a result, as shown in FIG. 4, it was confirmed that the cell death killing effect according to the concentration of the eluate of the sample of the anti-adhesion composition of the present invention did not appear.

Thus, it was confirmed that the sample suspension of the sample of the temperature-sensitive adhesion preventive composition based on the particle-type acellular dermis of the present invention showed a cell survival rate of not less than 90% in a concentration-dependent manner, thereby demonstrating that it can be safely applied to the human body as an anti- Respectively.

Example  4: Particle type Acellular cell  Dermis-based Temperature Sensitivity  In-vivo adhesion preventive membrane maintenance experiment of anti-adhesion composition

To evaluate the retentivity of the anti-adhesion agent for temperature-sensitive adhesion based on the particle-type acellular dermis-based anti-adhesion agent prepared in Example 1-2, an animal experiment was conducted using ICR mice at 6 weeks of age.

Specifically, 6-week-old ICR mice were anesthetized with abdominal aesthetic and epilated at the site where the anti-adhesion composition was injected. The epilated area was sterilized with alcohol and 0.5 mL of the anti-adhesion composition sample # 2 was injected slowly into the abdomen of the mouse by subcutaneous injection using a syringe. After the injection, the syringe was slowly removed from the injection section for about 1 minute until the composition became gelled in the abdomen. After removal of the syringe, dressings were applied with iodine and antibiotic ointment, and the wound dressing was finally attached and periodically managed. After 1 week and 4 weeks, mice injected in each group were sacrificed to confirm that the injected anti-adhesion composition well formed the physical barriers necessary to prevent adhesion. In this case, a thermosensitive adhesion agent were commercial products compared to the control group using the Dix (Guardix-SG ^®, Geneva welsa).

As a result, as shown in FIG. 5, the particle-type acellular dermis-based thermosensitive adhesion preventive composition of the present invention was also applied to experimental animals, like Guardix-SG ^® , a product for use as a control for the temperature sensitive antiadhesive agent , It was confirmed that the animal body was gelated by body temperature. In addition, after one week of application of the anti-adhesion composition, it was confirmed that the retention capacity of the body was maintained. As a result, in the case of the control group, Guardix-SG ^® , the injected amount did not maintain the anti- out, whereas in the case of the sample to which the adhesion preventive composition sample sample # 2 was injected, it was confirmed that the adhesion preventive film was maintained in the body. In addition, in the case of the sample to which the anti-adhesion composition sample # 2 was injected, it was confirmed that the anti-adhesion effect was maintained even after 4 weeks.

Through this, it is confirmed that the composition for preventing temperature-sensitive adhesion of a particle-type acellular dermis-based anti-adhesion agent of the present invention injected into the abdomen of an experimental animal is gelated through body temperature, so that a formulation suitable for minimally invasive method or laparoscopic method is possible . Further, by confirming that the adhesion preventive membrane is maintained in the body even after 1 week and 4 weeks after the composition is applied to the experimental animals, the function as an anti-adhesion agent that helps the wound to be normally regenerated for a wound healing period of at least 7 days Proved to be fully equipped.

Example  5: Particle type Acellular cell  Dermis-based Temperature Sensitivity  Anti-adhesion test of adhesion preventive composition

Animal experiments were conducted to confirm the ability of the particle-type acellular dermis-based thermosensitive adhesion preventive composition prepared in Example 1-2 to inhibit adhesion to the body. At this time, 7-week-old SD rats (Sprague-Dawley rats) were used as the experimental animals. An animal adhesion model was established through the procedure shown in FIG. 6, and the particle- To evaluate the adhesion inhibition ability in the body.

Specifically, in order to establish an animal adhesion model to observe the anti-adhesion effect by observing macroscopic findings at the time of autopsy, 7-week old SD rats were anesthetized by intraperitoneal anesthesia, and then the abdominal operation site was removed and sterilized with iodine And then lined up along the midline of the abdomen about 4-5 cm. After laparotomy, the peritoneal epithelium was removed from the peritoneum about 2 cm away from the incision using a surgical blade (1 × 1 ㎠ area) and the surface of the cecum, Using a surgical knife, rupture of the vas deferens caused rubbing to the extent that slight bleeding occurred.

As described above, the peritoneal membrane and the cecal surface of rats were artificially wounded to cause forced adhesion, and the sample of the thermosensitive adhesion preventive composition based on the granular type acellular dermis, prepared in Example 1-2, was applied to the abrasion- Lt; RTI ID = 0.0 > ml < / RTI > Thereafter, the peritoneum and abdominal wall were sutured, treated with iodine and antibiotic ointment, and treated with analgesics and antibiotics on a daily basis. At this time, was the group treated with the test sample in test group # 2, a thermosensitive adhesion inhibitor commercial product, the adhesion Dix animal model group (Guardix-SG ^®, Geneva welsa) processing 1 ㎖ was applied as a positive control. On the other hand, an animal adhesion model group (untreated group) not treated with an adhesion inhibitor was used as a negative control group, and a total of 12 rats in each group were used.

And processing the sample of sample # 2 in the wound area group as described above, the Dix (Guardix-SG ^®, Geneva welsa) positive control treated, and the treatment the rats of the negative control not treated with adhesion agent both after 7 days Sacrificed and blinded experiments were performed to observe the effect of preventing cecum and peritoneal adhesion of rats in each group.

Adhesion was assessed using a grading system of 4 grades (0, 1, 2, 3, greater number of adhesions) depending on degree of tissue adhesion. The adhesion grade criteria are as shown in Table 3 and FIG.

Grade Adhesion grading scale (Hooker score) 0 No adhesion One Adhesion is also separated by light force
(Gentle blunt dissection required to free adhesions) 2 Strong force must be applied for adhesion separation
(Aggressive blunt dissection required to free adhesions) 3 Very difficult to separate due to very strong adhesion
(Sharp dissection required to free adhesions)

After 7 days of induction of adhesion, all rats in each group were exposed to each other and visually observed by a blinded method in which the control group (positive control group and negative control group) could not be distinguished from the test group, and the adhesion area, Adhesion grade and the like to examine the degree of adhesion prevention effect. As a result, the particle-type acellular dermis-based thermosensitive adhesion preventive composition of the present invention prepared in Example 1-2 (sample sample # (FIG. 8), adhesion strength (FIG. 9) and degree of adhesion (FIG. 10) in comparison with the control group (Guardix-SG ^® and negative control group It was confirmed that the adhesion was more effectively prevented.

The average value of the specific, bound in an area evaluation, a negative control (non-treatment group) and positive control (Guardix-SG ^®) The average value inde 0.5 ㎠ and 0.4 ㎠ each hand, the test group (test sample # 2) is a 0.1 ㎠ Respectively. In addition, the average rating of even, negative control yieoteuna (untreated group), and a positive average level of the control group (Guardix-SG ^®) are each 2.7 rating and 1.7 rating, the experimental group (Sample Sample # 2) to the adhesion rating is 1.4 Rating Respectively. In addition, the adhesion strength average values of in the evaluation result for the adhesion strength, the negative control (non-treatment group) and positive control (Guardix-SG ^®) was was respectively 2.5 and 1.3, whereas, the experimental group (Sample Sample # 2) is 1.1 degree of adhesion of It was evaluated to have strength. The evaluation criteria for adhesion strength are shown in Table 4 below. All the numerical results were used for statistical analysis using T-test and the significance was evaluated with a 95% confidence interval.

Classification Adhesion strength ( Adhesion surface  Classification by declining intensity at separation) One Adhesion is also separated by light force 2 Adhesion must be removed with moderate force 3 Strong force must be applied for adhesion separation 4 Very difficult to separate due to very strong adhesion

Positive one over the result, the particulate acellular experimental group treated with the dermis based thermosensitive adhesion prevention composition (Sample Sample # 2) of the invention process the commercial adhesion of the Dix (Guardix-SG ^®, Geneva welsa) inhibitor The adhesion can be prevented more effectively in the adhesion area, the adhesion strength, the degree of adhesion, and the like compared with the negative control without the control and the anti-adhesion agent. Thus, the wound can be normally regenerated during the wound healing period of at least 7 days The results of this study are as follows.

Claims (17)

(i) removing the epidermal layer from the skin tissue;
(Ii) removing the cells of the dermal layer in the skin tissue from which the skin layer has been removed;
(Iii) lyophilizing the acellular dermis and then pulverizing it into a granular form;
(Iv) physically mixing and hydrating hyaluronic acid and the thermosensible polymer in the particulate acellular dermis; And
(V) sterilizing the composition containing the granular acellular dermis, hyaluronic acid and thermosensitive polymer of step (iv) by irradiating gamma rays;
Based on the total weight of the composition.
The method of claim 1, wherein the thermosensitive polymer is a polyethylene oxide (PEO) -polypropylene oxide (PPO) -polyethylene oxide (PEO) triblock copolymer having a temperature sensitivity that exhibits a sol- Wherein the thermosensitive adhesion preventive composition is a thermosensitive adhesive composition.
The method according to claim 1, wherein the content of the thermosensitive polymer is 13 to 25% by weight of the total composition.
The method according to claim 1, wherein the particle-type acellular dermis has a particle size of 300 to 800 탆.
The method of claim 1, wherein the content of the particulate acellular dermis is in the range of 1.0 to 6.0% by weight of the total composition.
The method of claim 1, wherein the hyaluronic acid content is 0.1 to 0.5 wt% of the total composition.
The method of claim 1, wherein the skin layer is separated from the dermal layer using proteolytic enzymes.
The method of claim 1, wherein the cells of the dermal layer are removed by a surfactant or ultrasonic treatment.
The method of claim 1, wherein the irradiation dose of the gamma ray is 15 kGy to 40 kGy.
The method of claim 1, further comprising: (vi) mixing the hyaluronic acid and the thermosensible polymer-mixed particulate acellular dermis with at least one selected from sterilized water, physiological saline, platelet rich plasma (PRP) ;
Wherein the thermosensitive adhesion preventive composition further comprises a thermosetting adhesive composition.
A human tissue-based thermosensitive adhesion preventative composition prepared by the method of any one of claims 1 to 10.
12. The composition of claim 11, wherein the composition comprises an aqueous solution selected from the group consisting of distilled water, water for injection, physiological saline solution, natural peritoneal solution and natural peritoneal solution.
12. The composition of claim 11, further comprising one or more drugs selected from the group consisting of antithrombogenic agents, non-steroidal anti-inflammatory drugs, hormone chemostatic agents, analgesics and anesthetics, .
12. The composition of claim 11, which is applied after open surgery, laparoscopic surgery, peritoneal surgery, bladder surgery, gynecologic surgery, spinal surgery, cardiac surgery, rectal surgery, dental surgery or cosmetic surgery, .
12. The composition of claim 11, wherein the composition is formulated as a tubular, cream, syringe, or spray.
[12] The composition of claim 11, wherein the composition has a property of gelation upon treatment at a surgical site to prevent adhesion of tissues.
18. A method of preventing adhesion, comprising contacting a human tissue-based thermosensitive adhesion preventative composition according to any one of claims 11 to 16 to a surface of a topical organ of an animal other than a human.

Images