KR20160049459A - Manufacturing method of bio-implant compositions comprising particle-type acellular dermal matrix physically cross-linked with hyaluronic acid via electron beam - Google Patents

Abstract

The present invention provides a method for producing a bio-implant composition. The method comprises the following steps: (i) freezing and drying acellular dermis, and pulverizing the same into particles; (ii) adding and mixing hyaluronic acid and a solvent in the particle-type acellular dermis from the step (i); and (iii) cross-linking the particle-type acellular dermis and the hyaluronic acid by irradiating the mixture from the step (ii) with electron beams. Moreover, the present invention provides a bio-plant composition produced through the method. In vivo maintenance of the bio-implant composition according to the present invention may be longer than that of a simple mixture of acellular dermis and hyaluronic acid, and the bio-implant composition may be used in restoring and molding damaged tissue.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a composition for bio-implantation in which a particle-type acellular dermis and hyaluronic acid are physically crosslinked through an electron beam (manufacturing method of bio-implantoids comprising a particle-type acellular dermal matrix,

The present invention relates to a composition for living body transplantation in which a cell-free allogeneic dermis and hyaluronic acid are physically crosslinked through an electron beam, and more particularly to a composition for physically crosslinking a physical mixture of a granular acellular dermis and hyaluronic acid through an electron beam cross- To a composition and a process for producing the same.

Dermal allogeneic dermis consists of dermis particles around a certain size (~ 500μm), and it can be easily applied to any affected part or injected with a syringe. However, such particle-like cell-free allogeneic dermis is characterized in that it can not be maintained for more than 12 months due to decomposition of protease, especially collagenase, after in vivo injection. For this reason, development of an improved cell-free allogeneic dermis improved in preservation ability in vivo is required.

Fillers are used routinely in the field of plastic surgery as well as for restoration of defective body parts and tissues. The main component of the most widely used filler is hyaluronic acid, which is a water-soluble polysaccharide biopolymer with features such as high viscosity, moisturizing and biocompatibility, and is one of the three major components of the skin, along with collagen and elastin. Hyaluronic acid is degraded in the body by two pathways. First, it is degraded by hyaluronidase. Secondly, it is attached to the cell receptor and is digested into the cell and then degraded by enzyme in the lysosome. will be. Biodegradation of hyaluronic acid in the body is very rapid and is known to be completely degraded within 0.5 to several days. In order to overcome such short biodegradation, hyaluronic acids used as fillers are crosslinked by using various chemical crosslinking agents. When the molecular weight is increased by crosslinking, it is easy to make a desired shape in a precise part, There is an advantage that the action rate of hyaluronidase is reduced. However, there remains a safety problem in the body such as residual crosslinking agent.

Cross-linking is a technique of adding a covalent bond between polypeptides that is the basic structure of a protein, and is used variously to maintain the tertiary structure of the protein or to change the hardness. The crosslinking methods include physical methods and chemical methods. When using a chemical crosslinking agent, consideration should be given to whether efficient crosslinking is carried out, whether the residual crosslinking agent is easily removed through a cleaning process after use, and whether it is harmful to the human body. Physical crosslinking methods physically increase the binding of amino acids, which are constituents of proteins, using ultraviolet (UV), ultra violet, gamma ray, electron beam (E-beam) In particular, E-beam has the advantage of ensuring safety and remaining remnants because it has been proved to have an effect of sterilization in addition to crosslinking effect.

Korean Patent Laid-Open No. 2007-0122315 discloses a composition for injecting human soft tissue fillers containing autologous dermal cells and hyaluronic acid, but discloses a crosslinking method for maintaining a mixture of dermal cells and hyaluronic acid in the body for a long period of time not.

In the present invention, a method for preparing a composition for living body transplantation using physical crosslinking through electron beam by mixing a granular cell-free allogeneic dermis and hyaluronic acid in an appropriate ratio is provided. The composition for living body implantation according to the present invention is a material physically cross-linked with a hyaluronic acid and a granular cell-free allogeneic dermis, and can be maintained in the body for a longer time than a simple mixture of a cell-free allogenic dermis and hyaluronic acid.

Korean Patent Publication No. 2007-0122315

An object of the present invention is to provide a method for manufacturing a composition for living body transplantation, which comprises physically mixing a particle-type acellular allogeneic dermis and hyaluronic acid to hydrate and then cross-linking through an electron beam.

Another object of the present invention is to provide a composition for in vivo transplantation prepared by the above method.

In order to achieve the above object, the present invention provides a composition for living body transplantation in which a particle-like acellular dermis and hyaluronic acid are physically cross-linked. A method for producing a composition for living body implantation of the present invention is a method for producing a body-grafted dermal tissue, which comprises producing a cell-free dermis tissue and disintegrating the same in a particle form, physically mixing and hydrating the dermal cell-free dermal tissue with hyaluronic acid, .

More specifically, the method for producing a composition for a living body,

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

(Ii) adding and mixing hyaluronic acid and a hydrating solution to the particulate acellular dermis of step (i); And

(Iii) cross-linking the particle-type acellular dermis and hyaluronic acid by irradiating the mixture of step (ii) with an electron beam.

As used herein, the term " acellular dermis " refers to a dermis layer that removes cells capable of causing an immunological rejection through a chemical treatment of isolated dermis, and is composed mainly of collagen and elastin. It is the " particle-type acellular dermis " in the present invention that this acellular dermis is made into a fine structure by granulation. The acellular dermis or the particulate acellular dermis may be prepared using known methods well known to those skilled in the art. In the present invention, the acellular dermis tissue may be a human-derived allogeneic or animal-derived dermis tissue, and the process of preparing the acellular dermis tissue may include the steps of preparing the same kind of acellular dermis tissue as follows Describes:

There is a risk of tissue damage due to hypoxia, degradation by autolytic enzymes, and damage of extracellular matrix by proteolytic enzymes when the tissues of the donated body are transported separately from the body. In addition, physical damage may occur due to the osmotic pressure of the carrier solution. In addition, there is always a risk of contamination by microorganisms such as bacteria and fungi. Therefore, it is necessary to add a substance that can prevent degradation by hypoxia, decomposition by autolytic enzyme, degradation by protein and decomposition enzyme, and add antibiotic and antibiotic which can prevent microorganism contamination. shall. Appropriate buffer solutions should be included to prevent tissue damage by osmotic pressure. The osmotic pressure of the tissue transport solution should have an osmotic pressure of about 260 to 320 mOsm / kg, which is the plasma osmotic pressure. In the commercial medium, which is widely used for animal cell culture, osmotic pressure is about 260 ~ 320 mOsm / kg, which is similar to plasma osmotic pressure. Therefore, a commercial medium is used as a base solution and various components are added to it.

Antibiotics such as penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, vancomycin, etc., alone or in combination, are added to prevent contamination of bacteria and fungi, and amphotericin-b, nystatin, The same antimicrobial agent is added alone or in combination. An enzyme inhibitor should be added to prevent tissue damage caused by various degrading enzymes.

Enzyme inhibitors include N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis '-Tetraacetic acid (EGTA) and the like, protease inhibitors such as lupeptin, apoproteinin and the like. In addition, tissue must be transported in a manner that minimizes physical damage.

Most enzyme reactions are highly influenced by temperature and are most active around the human body temperature of 37 ° C, thus transporting tissues at a low temperature of about 4 ° C. Generally, ice cubes are used to transport ice cubes. If the transport solution carries tissue to a frost-free temperature, ice crystals can damage the tissue and should be avoided.

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 treatment with 1.0 units / ml of distearate at 37 ° C for 60 to 120 minutes. Alternatively, treatment with tamolysin at a concentration of 200 [mu] g / ml for 30 minutes at 37 [deg.] C 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 ethylenediaminetetraacetic acid (EDTA) solution 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 molar 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 0.2 to 1% SDS solution at room temperature, the cells can be removed without damaging the tissue. Or Tween 20 solution at a concentration of 0.1 to 2.0% at room temperature for 30 to 180 minutes, or treated at a concentration of 0.2 to 2% with a Triton X-100 or Nonidt P-40 solution at 22 to 37 ° C for 30 to 180 minutes You can also remove cells without damaging the tissue.

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 non-saturated dermal tissue is finally obtained as a granular cell-free dermis after lyophilization and particle-type grinding.

The particulate acellular dermis may have a particle size of 300-800 μm, preferably about 500 μm. 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.

The particulate acellular dermis of step (ii) and hyaluronic acid may be mixed at a weight ratio of 20 to 50: 1, and may be mixed at a weight ratio of 30 to 40: 1, and mixed at a weight ratio of 35: But is not limited thereto.

As used herein, the term " hyaluronic acid " is a biosynthetic natural substance that exists in many skin, articular fluid, and cartilage of 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 hyaluronic acid may be contained in an amount of 0.01 to 1.0% by weight of the whole composition. In an embodiment of the present invention, the content of the hyaluronic acid may be 0.01 to 0.5 wt% of the total composition. And preferably 0.03% by weight, which is generally the concentration of hyaluronic acid present in the body. When the content of hyaluronic acid exceeds 0.5% by weight of the total composition, the particulate acellular dermal tissue composition to which a high concentration of hyaluronic acid is bound can be reversed to the physical properties of hyaluronic acid rather than the physical properties of the dermal tissue, It may be difficult to inject into the body through a syringe, and there is a possibility that the compositions may flow down after injection. On the other hand, it is difficult to expect that the particle-type acellular dermal tissue composition to which low-concentration hyaluronic acid is bound less than 0.01% by weight does not increase the preservability of the body.

The hyaluronic acid may be a low molecular weight hyaluronic acid having a molecular weight of 1.0 to 2.0 MDa or a polymer hyaluronic acid having a molecular weight of 3.0 to 4.0 MDa and is preferably a polymer hyaluronic acid having a molecular weight of 3.0 to 3.5 MDa, It may be, but is not limited to, a polymeric hyaluronic acid having a molecular weight of 3.3 MDa.

The hydrating solution of step (ii) may be selected from the group consisting of sterilized water, physiological saline solution, and mixtures thereof. However, the hydrating solution is not limited thereto, and any known hydrating solution capable of hydrating hyaluronic acid Can be used.

The irradiation dose of the electron beam is preferably 15-35 kGy, but is not limited thereto. The present invention also includes physically cross-linking the hyaluronic acid-mixed particulate acellular dermis tissue by irradiating an electron beam. When an electron beam is irradiated, hyaluronic acid and the particulate acellular dermal tissue can be permanently crosslinked. In the embodiment of the present invention, when the irradiation dose of the electron beam is 15-35 kGy and the irradiation intensity of the electron beam is too low to be less than 15 kGy, crosslinking may not occur. If the intensity exceeds 35 kGy and the intensity is too high, The dermal tissue may be destroyed.

The present invention also relates to a method for producing a composition for a living body implantation, comprising the steps of: (iv) dissolving a granular acellular dermis crosslinked with hyaluronic acid of step (iii) in sterilized water, physiological saline, ), And mixtures thereof. The method may further comprise: More specifically, the hyaluronic acid-bound granular acellular dermis of the present invention can be mixed with sterilized water, physiological saline, or PRP (platelet rich plasma) extracted from blood. Inflammation, immune response and the like can be avoided through the use of the solution.

The mixing ratio of the hydration solution and the dermis may range from 1: 1 to 1: 3.

The composition for biotransplantation may be a composition for tissue repair.

Conventionally, electron beams have been used for sterilization in the production of compositions for living body transplantation. In sterilization, collagen fibers (collagen fibers-collagen fibers) are occasionally formed between the collagen fibers of the acellular dermis. However, chemical cross-linking agents were mostly used for cross-linking between acellular dermis and hyaluronic acid. In contrast, the present invention shows that the particle-type acellular dermis and hyaluronic acid are cross-linked by an electron beam, and show excellent cross-linking effect without using a chemical cross-linking agent.

Therefore, when the method of the present invention for preparing a bio-implantable material is used, a cross-linking agent for chemically cross-linking a granular acellular dermis with hyaluronic acid is not used. . In addition, since the composition for living body produced by the above method is excellent in bioavailability even after cross-linking using only the electron beam, which is a physical method, without using a chemical crosslinking agent, even after 10 weeks of the composition injection, the composition is useful as a composition for living body transplantation Can be used.

The present invention also provides a composition for in vivo transplantation prepared by the above method.

The composition for biotransplantation may be a composition for tissue repair, more specifically, a composition for graft-less acellular dermis tissue and a composition for cross-linking hyaluronic acid through electron beam cross-linking, but is not limited thereto.

The composition for bio-implantation can be used as a wound healing agent for repairing skin and tissues, and as a biomaterial.

The biomaterial for tissue repair according to the present invention is a composition for physically cross-linking a particulate acellular dermis tissue having a particle size injectable by a syringe without surgery and a polymer hyaluronic acid having enhanced bioavailability. Therefore, acute inflammatory reaction such as pain and swelling fever that may appear at the time of transplantation is small, and the bioavailability is long due to less effect of proteolytic degradation. Therefore, it can be utilized as a biological material for damaged tissue repair. In addition, it can be used as a biomaterial for tissue repair due to wound in dermatology, image surgeon and general surgery, and can be used as a filler for various purposes such as eye recession in breast surgery and breast reconstruction in breast cancer treatment.

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 composition for living body implantation containing a particle-like acellular allogeneic dermis physically cross-linked with hyaluronic acid according to the present invention has an improved in vivo storage ability as compared with a composition for living body implantation containing only a conventional particle-type acellular allogeneic dermis. The composition for living body implantation according to the present invention acts as a scaffolder in vivo to function as a physiological space in which cells of a recipient migrate and can grow and capillaries are formed due to angiogenesis, And thus can be used as an implantable tissue for restoration and molding of damaged tissue. In addition, since the composition for living body implantation according to the present invention is excellent in preservation in the body without using a chemical crosslinking agent, the composition can be effectively used as a composition for living body implantation without causing safety problems such as residue of a crosslinking agent in the body.

FIG. 1 is an SEM (scanning electron microscope) photograph of an electron beam E-beam dose for confirming the effect on an acellular dermis when an electron beam is irradiated.
2 is a graph showing Fourier transform infrared spectroscopy (FT-IR) results for confirming the degree of physical crosslinking of the acellular dermis during electron beam irradiation.
FIG. 3 is a schematic diagram showing the intra-abdominal injection position of nude mice injected with a composition of hyaluronic acid and a particle-type acellular allogenic dermis mixture and an electron beam.
4 is a graph showing changes in volume with time after injection of nude mouse intraperitoneal implants.
FIG. 5 is a photograph showing a nude mouse opened and a composition obtained after 10 weeks from the injection of the composition. FIG.
FIG. 6 is a graph showing a result of volume measurement of a composition extracted from a nude mouse 10 weeks after the injection of the composition. FIG.
Figure 7 shows H & E staining (haematoxylin and eosin staining) for histological analysis of compositions extracted from nude mice 10 weeks after the injection of the composition:
A, particle-free acellular allogenic dermis (MegaFill, MF, control);
B, a composition of particle-free acellular allogenic dermis and 1.4 MDa hyaluronic acid (MF + 1.4 HA);
C, electron beam crosslinked particle-like cell-free allogeneic dermis-1.4 MDa hyaluronic acid (MF-1.4 HA E-beam) composition;
D, a composition of particle-free acellular allograft-3. 3 MDa hyaluronic acid (MF-3.3 HA); And
E, electron-beam crosslinked particle-like cell homogenous dermis -3.3 MDa hyaluronic acid E-beam (MF-3.3HA E-beam) composition.
8 is a diagram showing the histological changes with time of the electron beam crosslinked acellular dermis inserted into the experimental pigs:
A, cell infiltration over time (H & E staining);
B, changes in collagen production over time (MT staining); And
C, change of elastin production with time (VG staining).
FIG. 9 is a graph showing the time-dependent change in angiogenesis of an electron beam cross-linked acellular dermis inserted into a laboratory pig;
A, blood vessel staining results; And
B, a bar graph showing the results of blood vessel staining.
FIG. 10 is a graph showing the expression of extracellular matrix factors expressed by an electron beam crosslinked acellular dermis inserted into a laboratory pig by time, using qRT-PCR.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for illustrating the present invention only, and the scope of the present invention is not limited by these examples.

Example  1. Hyaluronic acid and Acellular cell  Determination of electron beam dose for physical crosslinking of dermis

To investigate the effect of E-beam irradiation on the acellular dermis, an electron beam was irradiated to the acellular dermis by the dose and SEM (scanning electron microscope) was measured.

As a result of SEM measurement, it was observed that the acellular dermis irradiated with an electron beam of 25 to 30 kGy showed a stable collagen fiber structure and a higher density of dermis than an electron beam without irradiation. However, when irradiated with an electron beam of 50 kGy or more, most of the collagen fiber structure was broken and the density of the dermis was found to be very low (FIG. 1).

Further, FT-IR measurement was performed to confirm the degree of physical crosslinking of the acellular dermis during electron beam irradiation.

As a result of the FT-IR measurement, the amide Ⅱ peak was shifted with the increase of the electron beam dose in the amide I, Ⅱ and Ⅲ spectra which can identify the collagen structure, (Fig. 2). ≪ tb > < TABLE >

Therefore, through the results of FIGS. 1 and 2, it can be concluded that when the electron beam is irradiated for physical crosslinking between hyaluronic acid and acellular dermis, the dose is 15-35 kGy.

Example 2 Animal Injection Experiment of the Composition According to the Invention

Example 2-1. Preparation of composition

To inject into nude mice, a mixture of hyaluronic acid and a particulate acellular allogenic dermis and a composition crosslinked through an electron beam were prepared.

Specifically, skin tissue (collected from a donated body for the purpose of treatment of a non-profit patient from a tissue bank) was treated with a concentration of 1.0 units / ml of the neutral protease, Distase, and incubated at 60 ° C For 120 minutes and then washed three times with sterilized distilled water to separate the dermal layer and the epidermal layer to remove the epidermal layer. The tissues from which the epidermal layer was removed were treated with 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 was 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 was placed in a microfibrillator that did not break the tertiary structure of the allogeneic dermis and the denaturation of collagen and elastin The sample was pulverized for 3 minutes at a rotation speed of 5,000 rpm, which is an optimum condition for preventing clotting, and a dermal cell-free dermis was prepared from the dermis of a 500 μm-diameter sieve having the most favorable adherence rate in vivo in a sterile condition Respectively.

Then, a mixture in which the particle-like dermal cell dermal tissue and hyaluronic acid and hydration solution were mixed was prepared. The hyaluronic acid used in the preparation of the mixture was a small molecule of 1.4 MDa (1.4 HA) and a polymer of 3.3 MDa (3.3 HA). The mixing ratio of the particle-type acellular allogeneic dermis to physiological saline was 1: 1.5 (v / v), and the concentration of hyaluronic acid was mixed with 0.5 wt% of the total composition. The mixing ratio of the particle type acellular dermis to hyaluronic acid was 35: 1 (w / w). In addition, a cross-linked composition was prepared by irradiating an electron beam (E-beam) of 30 kGy for physical cross-linking between hyaluronic acid and a particle-like acellular allogeneic dermis.

(MF-1.4 HA, MF-3.3 HA), an electron beam cross-linked particle dendritic cell dermis-hyaluronic acid composition (MF-1.4 HA E-beam and MF-3.3 HA E-beam).

Example 2-2. Injection of composition into nude mice

Nude mice were anesthetized by injecting zolazepam into the abdominal cavity of nude mice and then fixed to the nude mice. Each composition prepared in Example 2-1 was injected into a dorsum of nude mice by 300 쨉 l using an 18-gauge needle. The injection positions of the respective compositions are shown in Fig.

Examples 2-3. Observation of volume changes of compositions injected into nude mice over time

To examine the volume change with time of the compositions injected in Example 2-2, the volumes of the injected sites were measured from immediately after the injection to 10 weeks. The measurement method is shown in FIG. 4 by calculating the volume of the entire composition grafted by the calculation formula of 0.5 × long axis × short axis ² by measuring long axis and short axis using a digital caliper.

As a result, the volume reduction rate of the control group (MF) was the highest in all 10 weeks, and the saturation point of the volume reduction analyzed by the trend line was also ahead of the other experimental groups. In contrast, in the experimental group, the volume reduction rate of MF-1.4 HA group without electron beam irradiation was lower than that of control group until 6th week, but from 8th week, saturation point of volume reduction started to appear, In the MF-1.4 HA group irradiated with electron beam and the MF-3.3 HA group irradiated with no electron beam showed similar volume reduction rates as a whole, the MF-1.4 HA group irradiated with electron beams showed no saturation point of volume reduction, The MF-3.3 HA group, which did not irradiate the electron beam, showed the saturation point of volume reduction from the 8th to the 10th week. Finally, the 10th-volume reduction rate of the MF-3.3 HA group Which was lower than the 10th volume reduction rate. Finally, in the MF-3.3 HA group irradiated with the electron beam, the volume reduction rate was the lowest at 30% or less and the saturation point of the volume reduction was not observed at all (FIG. 4).

Therefore, compared with the case where the granular cell-free allogeneic dermis was injected singly from immediately after the injection of the composition until the 10th week, the hyaluronic acid (HA) was mixed with the hyaluronic acid of the polymer, It was confirmed that the physically cross-linked composition had enhanced bioavailability.

Examples 2-4. After 10 weeks of injection of the composition, analysis of the injected composition

When the composition was injected into the nude mice in Example 2-2, after 10 weeks, the nude mice were opened. Fig. 5 shows a state in which a nude mouse is opened and a state in which a nude mouse is extracted.

The volume of the composition extracted from the nude mouse was measured. As a result, after 10 weeks, the particle-type acellular dermis-low molecular weight hyaluronic acid (MF-1.4 HA) and the granular acicular dermis-low molecular weight hyaluronic acid (MF-1.4 HA E-beam) and hyaluronic acid (MF-3.3 HA) of the granular acicular dermis-polymer were degraded and showed a volume similar to that of the control (FIG.

Therefore, it was confirmed that the composition (MF-3.3 HA E-beam) in which the hyaluronic acid of the particle-type acellular dermis-polymer was crosslinked by the electron beam had a higher final bioavailability after 10 weeks than the composition not crosslinked.

The histochemical analysis of the extracted composition by staining with H & E (hematocylin and eosin) is shown in FIG. (A), a composition (B) of a granular cell-free allogeneic dermis and 1.4 MDa hyaluronic acid (MF + 1.4 HA), an electron beam crosslinked granular cellless homolog (D) of a composition (C) of a dermis-1.4 MDa hyaluronic acid (MF-1.4 HA E-beam), a particle type noncellular allogenic dermis -3.3 MDa hyaluronic acid (MF-3.3 HA) (E) of a cell allograft dermis-3. 3 MDa hyaluronic acid E-beam (MF-3.3 HA E-beam).

As a result of the analysis, in the control group, the interface between the collagen structures was observed to be wider than that of the experimental groups, and fibroblasts were introduced into the internal space (FIG. 7A). In the case of the mixture of HA, collagen was surrounded by HA (Fig. 7B-E).

Particularly, in the case of the mixture of the particle-type acellular dermis-polymer hyaluronic acid (MF-3.3 HA E-beam) crosslinked with the electron beam, a collagen arrangement having a higher density than the other conditions was observed due to the crosslinking effect, Extracellular matrix (ECM), such as collagen, was produced by self - organization of fibroblasts by migration and proliferation inside the mixture.

Examples 2-5. Animal experiments with experimental pigs (micropigs)

An animal experiment using an experimental pig was conducted to verify that the electron beam irradiation dose of Example 1 did not impair the physiological function of the composition crosslinked with the electron beam. Two male Yorkshire pigs weighing 20 kg were anesthetized with zoletil (5 mg / kg, Virbac, Carros, France) and rumpun (2 mg / kg, Bayer, Seoul, Korea) . After respiratory anesthesia with isoflurane (IsoFlo, Abbott Lab, Abbott Park, Ill., USA), the experimental pigs were placed on a sterile surgical table and the surgical site was cleaned with iodine solution. In Example 1, crosslinked acellular dermis was prepared with an electron beam irradiation dose of 25 kGy, and each of the 16 insertion sites was marked on the back region of the experimental pig. Insertion space is created a space of about 3x3 cm2 to the subcutaneous layer depth from the display position, and then inserts the cell-free dermis crosslinked to each of the insertion space, and antibiotics, Te the bushes (Tegaderm ^TM, 3M, MN, USA) Postoperatively. The inserted cell-free dermis was recovered from experimental pigs after 1, 2, 3, and 6 months, respectively, and a paraffin tissue slide was prepared to confirm cell inflow, neovascularization, collagen production, and elastin production. The time-dependent changes of extracellular matrix factors such as COL1A1, TGF-beta1, MMP-1, MMP-2, MMP-9 and TIMP-1 were quantified and verified by qRT-PCR.

As a result, it was confirmed that host cells were stably introduced into the inside of the acellular dermis crosslinked with the electron beam for up to 6 months except for the initial weak inflammatory reaction (Fig. 8A) The results showed that the density of collagen and elastin also increased over 6 months (BC in FIG. 8). The formation of new blood vessels into the acellular dermis crosslinked with an electron beam was confirmed by blood vessel staining from the samples from 1 month to 6 months (Fig. 9A), and the results were quantified and displayed as a bar graph. (Fig. 9B). The angiogenesis was observed to increase rapidly between 2 months and 3 months and then to be maintained thereafter. The changes in extracellular matrix factors with time were also analyzed as quantitative results (FIG. 10), and most of the remaining factors except MMP-2 were rapidly expressed around 2 months after insertion. These results indicate that the extracellular matrix factors that are delayed by 1 ~ 2 months are involved in the remodeling of the extracellular matrix of the insertion site, which means that the physiological function of the acellular dermis crosslinked with the electron beam is not impaired.

Thus, specific details of the present invention have been described in detail. This specific description is only a preferred embodiment, and the scope of the present invention is not limited by the embodiments described. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims (10)

(I) lyophilizing the acellular dermis and then pulverizing it into a granular form;
(Ii) adding and mixing hyaluronic acid and a hydrating solution to the particulate acellular dermis of step (i); And
(Iii) cross-linking the particle-type acellular dermis and hyaluronic acid by irradiating the mixture of step (ii) with an electron beam.
The method of claim 1, wherein the particle-type acellular dermis has a particle size of 300-800 μm.
[3] The method of claim 1, wherein the particulate acellular dermis of step (ii) and hyaluronic acid are mixed at a weight ratio of 20 to 50: 1.
The method according to claim 1, wherein the hyaluronic acid of step (ii) is contained in an amount of 0.01-1.0 wt% of the total composition.
The method according to claim 1, wherein the hydrating solution of step (ii) is selected from the group consisting of sterilized water, physiological saline, and mixtures thereof.
The method according to claim 1, wherein the irradiation dose of the electron beam in step (iii) is 15-35 kGy.
[3] The method according to claim 1, further comprising: (iv) separating the granular acellular dermis of hyaluronic acid crosslinked in step (iii) from the group consisting of sterilized water, physiological saline, platelet rich plasma extracted from blood, And mixing the resultant mixture with a selected hydration solution.
8. The method of claim 7, wherein the mixing ratio of the hydration solution to the dermis ranges from 1: 1 to 1: 3.
9. The method according to any one of claims 1 to 8, wherein the composition for biotransplantation is a composition for tissue repair.
A composition for in vivo implantation produced by the method of any one of claims 1 to 9.

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