KR20070095494A - Semi-solid gel composition for adipose tissue regeneration - Google Patents

Abstract

A semi-solid gel composition for adipose tissue regeneration is provided to obtain a composition for adipose tissue regeneration by mixing collagen in a semi-solid gel state, washed adipose tissue and other several materials. To form a semi-solid gel composition for adipose tissue regeneration, an adipose tissue that is obtained from a human body or an animal and washed and 0.01-5% of gel type collagen are mixed in a volume ratio of 1:8 to 8:1, and an adipose generation facilitating solution, a serum containing growth factor, and an additive containing culture medium are mixed in the aforesaid mixture in a volume ratio of 1:10 to 10:1 (v/v). The washed adipose tissue is obtained by washing the adipose tissue at least three times by adding a 1X~10XPBS(Phosphate-Buffered Saline) solution to which 0.01~1% BSA(Bovine Serum Albumin) is added.

Description

Semi-Solid Gel Composition For Adipose Tissue Regeneration

1 is a photograph showing the result of injecting a semi-solid gel composition for adipose tissue repair according to the present invention in nude mice,

Figure 2 is a graph showing a volume trend line compared for each group using a semi-solid gel composition for adipose tissue repair according to the present invention,

Figure 3 is a photograph showing the results of the injection size of the semi-solid gel composition for adipose tissue repair according to the present invention,

Figure 4 is a photograph showing the tissue staining results corresponding to the control group and experimental groups 1 and 2 of the semi-solid gel composition for adipose tissue repair according to the present invention.

The present invention relates to a semi-solid gel composition for repairing adipose tissue, and more particularly, collagen, adipogenesis-promoting solution, serum, and medium in adipose tissue washed with adipose tissue washing solution. ) Is a semi-solid gel composition for adipose tissue repair.

In general, collagen is synthesized and secreted from fibroblasts, chondrocytes, osteoblasts, smooth muscle cells, etc., and constitutes about 25% of the weight of all proteins in the body. The main function of collagen is structural proteins, skeletal proteins to form the organ structure of the body. In addition, the basement membrane of the renal glomerulus is ultrafiltration of urine, which acts as a sieve that passes small molecules but does not pass high molecular weight molecules such as proteins. It has also been found to be involved in the proliferation and differentiation of cells through specific interactions with other extracellular matrix components.

Collagen plays a role in maintaining tissue structure, but it does not always maintain a constant shape but is continuously degraded and reconstituted by an enzyme called collagenase, and it is usually by a protease. Does not decompose. On the other hand, in tissues such as bone, the metabolism is so slow that it takes about 10 years for collagen to be replaced with new collagen.

Currently, there are more than 15 types of collagen, and they are numbered according to the order in which they were found. Type I collagen was first discovered and is the largest in quantity. Collagen other than type I has a repeating structure of glycine-X-Y in part and has a triple helix structure. The next most studied type I collagen is type II, III and IV collagen. Type II is found in cartilage and type III is present in connective tissue. Type IV forms a sheet-like knit structure that is not fibrous and is the only collagen component found in the basement membrane.

The method of separating collagen from connective tissue is to take small amounts of animal skin and soak it in dilute acetic acid for 2 or 3 days in a refrigerator to obtain a viscous collagen solution. Gelling. [Reference ; Ming-Thau Sheu, Ju-Chun Huang, Geng-Chang yeh, Hsiu-O Ho, Characterization of collagen matrices for cell culture, Biomaterials 22, 1713-1719, 2001; Nimni ME, Cheung DT, Steates B, Kodama M, Sheikkh. Collagen, vol. 3, pp 1-37, Eds., Nimni ME ,; biotechnology. Boca Raton, CRC Press, 1988] In the case of type I collagen, it is relatively easy to isolate a large amount, control the sol-gel potential, and provide a good environment for cell function expression, so it is widely used as a cell support in tissue engineering. do.

In addition, the dermis (Alloderm) using a cadaver allograft as a substitute for the dermis has been developed and applied in clinical practice, and a product called Integra, which substitutes the dermis as a complex of bovine collagen and chondroitin sulfate, has been developed. It is used for burn treatment. In addition, a lot of research has been attempted to develop a kind of complex artificial dermis combining a scaffold and cultured cells obtained from living bodies. In addition, many strains of collagen sponge have been developed, and maximizing engraftment and proliferation of fibroblasts by controlling the pore size of the skeleton using various matrix proteins or hyaluronic acid. Investigations have been reported. Recently, commercialized dermagrafts have been commercialized by culturing human neonatal fibroblasts in a polyglactin acid Vicryl mesh.

In relation to adipose tissue, collagen is sometimes used as a substitute for soft tissue reconstruction. For example, these substances are largely made by processing acellular dermal tissue or extracellular matrix (Tissue matrix) and biological stability. It can be divided into a synthetic polymer having a. They are mainly used as a substitute for adipose tissue and may be useful when used in small amounts, but they also serve to simply fill in the deficiency rather than being functional reinforcement.

In addition, although some studies have been attempted to reconstruct complex tissues that combine cultured cells collected from animals with collagen skeletons, it is expected that it will take a long time to be applied to clinical applications. The adipose tissue transplantation, which is currently applied in clinical practice, is reconstructing the tissue with a basic technique that replaces only adipose tissue with saline solution. Such adipose tissue graft also has the disadvantage that volume is reduced by more than 40 ?? 60% within a year and has to be re-operated due to problems such as fibrosis of the tissue.

The present invention is to solve the above problems, unlike the conventional sponge-type scaffold made of a mixture of several substances that can multiply the cells in collagen and washed adipose tissue, other adipose tissue in the form of semi-solid gel It is an object of the present invention to provide a semi-solid gel composition for adipose tissue repair which is much more effective as a composition for adipose tissue repair than conventional adipose tissue transplantation.

Features of the present invention for achieving the above object,

Adipose tissue obtained from a human body or an animal and washed with water and 0.01 to 5% gel type collagen are mixed at a volume ratio (v / v) of 1: 8 to 8: 1. It is characterized in that the additive containing the serum and the medium containing the mixture in a volume ratio (v / v) of 1:10 ~ 10: 1.

Herein, the washed adipose tissue obtained from a human body or an animal is a 1X-10X Phosphate-Buffered Saline (PBS) solution added with 0.01-1% BSA (Bovine Serum Albumin), which is a 5-15 times the weight of adipose tissue. The adipose tissue is washed at least three times by addition.

Here, the 1X-10X PBS solution to which the water washing solution 0.01 ~ 1% BSA was added was 0.144-1.44g of KH ₂ PO ₄ , NaCl 9.000-90.00g, Na ₂ HPO ₄ · 7H ₂ O 0.795 in 900 ml of purified water. Dissolve ~ 7.950g, adjust the pH to 7.2 ~ 7.8, make final volume 1000ml with purified water, and then filter, add antibiotic gentamicin (gentamicin) to final concentration of 100㎍ / ㎖ Prepare 1X PBS solution, dissolve 0.05-5g BSA in 400ml 1X PBS solution to make 500ml final volume with 1X ~ 10X PBS solution and filter.

Here, the 0.01 ~ 5% gel type of collagen is filtered from 100 ml of 0.01 ~ 0.5% collagen obtained by the enzyme treatment and salt treatment from pig tendon (Tendon) to make the filtered collagen into a semi-solid gel form In order to neutralize the pH with 6.5-7.5 with 1N NaOH, the collagen was left at 20-30 ° C. for 1-15 hours, and then centrifuged at 5,000-15,000 g for 15-60 minutes to remove the upper layer. After taking down the precipitate (Pellet) in the bottom layer, it is prepared by adding 10ml of Physiological Phosphate-Buffer to it and maintained at 4 ° C until use.

Here, the fat production promoting solution is 20 to 200 g of refined soybean oil (Soybean oil), 10 to 25 g of glycerol (glycerol), 5 to 12 g of egg phosphate (Egg phosphatides), 0.03 to 0.3 g of sodium oleate (Sodium oleate) It is dissolved in 100 ml of distilled water and filtered.

Here, the medium is 8 ~ 11g DMEM (Dulbecco`s Minimum Essential Medium) powder, NaHNO <sub>3</sub> (Sodium Bicarbonate) 2000 ~ 2400 mg, HEPES (N-2-Hydroxyethylpiperazine-N`-2-Ethane in 800 ml of purified water Sulfonic Acid) After dissolving 2200 ~ 2400mg, adjust the pH of the dissolved solution to 6.8 ~ 7.8, prepare final volume to 1000ml with purified water, 8 ~ 11g F-12 powder to 800ml of purified water, NaHNO ₃ After dissolving 1000-1200 mg, the pH of the dissolved solution was adjusted to 6.8-7.8, and the final volume was prepared to be 1000 ml with purified water, and the solutions prepared above were mixed at a volume ratio of 1: 1 (v / v). And filter.

Here, the additive is added to the fat production promoting solution, the growth factor-containing serum and medium in a volume ratio of 1: 1: 1 (v / v).

Hereinafter, the configuration of the semi-solid gel composition for adipose tissue repair according to the present invention will be described in detail with reference to the accompanying drawings.

In the following description of the present invention, if it is determined that specific descriptions of related known functions or configurations may unnecessarily obscure the subject matter of the present invention, the detailed description thereof will be omitted. Terms to be described later are terms defined in consideration of functions in the present invention, and may be changed according to intentions or customs of users or operators. Therefore, the definition should be made based on the contents throughout the specification.

Figure 1 is a photograph showing the result of injecting a semi-solid gel composition for adipose tissue repair according to the present invention in nude mice, Figure 2 is compared with each group using a semi-solid gel composition for adipose tissue repair according to the present invention Figure 3 is a graph showing the volume trend line, Figure 3 is a photograph showing the results of the injection size of the semi-solid gel composition for adipose tissue repair according to the present invention, Figure 4 is a semi-solid gel composition for adipose tissue repair according to the present invention It is a photograph showing the tissue staining results corresponding to the control group and the experimental groups 1 and 2.

The semi-solid gel composition for adipose tissue repair according to the present invention is obtained by washing with adipose tissue obtained from a human body or an animal and washing the adipose tissue with 0.01 to 5% of the gel type collagen in a volume ratio (v / v) of 1: 8 to 8: 1. The mixture is mixed with the adipogenic solution, the serum containing growth factors, and the medium containing the additives in a volume ratio (v / v) of 1:10 to 10: 1 with respect to the mixture. Here, the additive is added to the fat production promoting solution, the growth factor-containing serum and medium in a volume ratio of 1: 1: 1 (v / v).

At this time, the washed adipose tissue obtained from the human body or animal is added to the 1X ~ 10X Phosphate-Buffered Saline (PBS) solution with 0.01-1% BSA (Bovine Serum Albumin), which is a 5-15 times the weight of the adipose tissue weight. Clean the adipose tissue at least three times. Here, 1X ~ 10X PBS (Phosphate-Buffered Saline) solution to which 0.01-1% BSA (Bovine Serum Albumin) was added to the washed water, 0.144 ~ 1.44g of KH ₂ PO ₄ , 9.000-90.00g, Dissolve 0.795 ~ 7.950g of Na ₂ HPO ₄ · 7H ₂ O to adjust the pH to 7.2 ~ 7.8, make the final volume 1000ml with purified water, and then filter, and the final concentration of antibiotic gentamicin (gentamicin) in the filtered solution. Is added to 100 ㎍ / ㎖ to prepare a 1X PBS solution, dissolved 0.05 ~ 5g BSA in 400ml 1X PBS solution to make a final volume of 500ml with 1X ~ 10X PBS solution and filtered.

In addition, 0.01 ~ 5% gel type collagen was filtered from 100 ml of 0.01 ~ 0.5% collagen obtained by enzyme treatment and salt treatment from pig tendon (Tendon), 1N NaOH to make the filtered collagen into semi-solid gel form Neutralize the pH to 6.5 ~ 7.5 and leave the collagen at 20 ~ 30 ℃ for 1 ~ 15 hours, then centrifuge at 5,000 ~ 15,000g for 15 ~ 60 minutes to remove the upper layer and go to the lower layer. After taking the precipitate (Pellet), it is prepared by adding 10ml of Physiological Phosphate-Buffer to it and maintained at 4 ℃ until use.

In addition, the fat production promoting solution is 20 ~ 200g of refined soybean oil, 10-25g of glycerol, 5-12g of egg phosphatides, 0.03 ~ 0.3g of sodium oleate, 100ml of distilled water Dissolved in and filtered.

On the other hand, the medium was dissolved in DMEM powder 8-11g, NaHNO ₃ (Sodium Bicarbonate) 2000 ~ 2400mg, HEPES (N-2-Hydroxyethylpiperazine-N`-2-Ethane Sulfonic Acid) 2200 ~ 2400mg in 800ml of purified water. The pH of the dissolved solution was adjusted to 6.8-7.8, and the final volume was prepared to be 1000 ml with purified water. After dissolving 8-11 g of F-12 powder and 1000-1200 mg of NaHNO3 in 800 ml of purified water, the dissolved solution was dissolved. The pH of the solution was adjusted to 6.8-7.8, and the final volume was prepared to be 1000 ml with purified water, and the solutions prepared above were mixed and mixed at a volume ratio of 1: 1 (v / v).

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only intended to illustrate the invention, so the scope of the invention is not to be construed as limited by these embodiments.

Example 1-Example of Composition of Gel for Adipose Tissue Repair Using Liposuction of Adipose Tissue

The procedure for preparing a gel composition for adipose tissue repair using adipose tissue (liposuction) on a day from a person is as follows.

First, in order to weigh the liposuctioned adipose tissue that has been moved to the refrigerated state at -20 to 4 ° C, the weight of the container containing no tissue of the 50 ml centrifuge tube is measured, and the tissue is contained. The total weight of the container is measured while the weight of the container is subtracted.

After weighing the tissue, add the adipose tissue washing solution 5 to 15 times the tissue weight, and close the 50 ml centrifuge tube cap and shake gently to wash the tissue. The washing process is repeated three times in total.

The prepared adipose tissue is put in one syringe, and the other syringe is mixed with 0.01 ~ 5% gel-type collagen at 1: 8-8: 1 (v / v), but mixed evenly using a syringe connector. Be sure to

At this time, the adipogenic solution, serum, and medium, which is a substance that induces cell proliferation in adipose tissue, are mixed together in a volume ratio of 1: 1: 1 (v / v / v), and the mixture of adipose tissue and collagen is mixed. It is mixed in a volume ratio (v / v) of 1:10 to 10: 1 to make a gel composition.

The result of injecting the adipose tissue repair gel composition into each experimental group is shown in FIG. 1.

Example 2 Example of Nude Mouse Experiment Using Gel Composition for Tissue Repair

After making a gel composition for adipose tissue restoration using liposuction adipose tissue, only the adipose tissue washed with saline, which is a method currently used in clinical use, was used as a control, and the adipose tissue washing solution in Experiment 1 The experiment was performed by dividing the adipose tissue and collagen washed with 8: 1 (v / v) and the experimental group 2 with the adipose tissue and collagen washed with adipose tissue washing solution 1: 8 (v / v).

To carry out this experiment, 5 females of BALB c / Nu 8 weeks old and 5 females were used in each group, and the scapula of nude mice in each control group and experimental group 1 and 2 were injected with a total volume of 1 ml to 1, 2, 3.5 Samples were taken at the expense of mass size every 7, week and 11 weeks, and at the expense of nude mice every sampling day.

In addition, the average volume is calculated using the sample injection size of each group, and a graph of the volume trend graph shows how much the volume of the adipose tissue repair gel composition is effective compared to the method currently used in the clinic. In order to see the proliferation and expression of adipose tissue, tissue staining, Oil-red O staining, was performed to interpret the results.

Table 1 below shows the composition of each group.

Table 1 Semi-solid Gel Composition for Adipose Tissue Repair in Control and Experimental Groups

Adipose tissue Fat Producing Solution serum badge Control Saline flushing adipose tissue X X X Experimental group 1 Adipose tissue washed with adipose tissue washing solution ○ ○ ○ Experiment group 2 Adipose tissue washed with adipose tissue washing solution ○ ○ ○

       The volume trendline compared to each group using the adipose tissue repair gel composition is shown in FIG. 2, and the results of the mass size of the adipose tissue repair gel composition are shown in FIG. 3. The tissue staining results corresponding to the control group and the experimental groups 1 and 2 of the composition are shown in FIG. 4.

      Therefore, current adipose tissue grafts are filled with adipose tissue, washed with saline, and filled with defects. It is true that there are various problems such as fibrosis, tissue necrosis caused by physical damage, and the like.

Moreover, the problem of having to reduce the volume and repeat the procedure several times puts a lot of economic and physical burden on the part of the patient.

Therefore, the present invention makes the fat cells in the adipose tissue as active as possible by washing the adipose tissue using the adipose tissue washing solution, and using the semi-solid gel form collagen to increase the engraftment and the proliferation of the cells. In addition, the injectable type (Injectable type) is characterized by a very simple in terms of treatment.

In addition, new adipose tissue transplantation is possible by supplying growth factors of activated cells in adipose tissue to generate blood vessels and eventually coalescing with the original adipose tissue.

According to the present invention, the semi-solid gel composition for adipose tissue repair, which is configured as described above, unlike the conventional sponge-type scaffold, some of the cells that can proliferate the cells in the collagen, the washed adipose tissue, and other adipose tissue in the form of a semi-solid gel It is a composition for repairing adipose tissue made of a mixture of substances, which may be much more effective than conventional adipose tissue transplantation.

As those skilled in the art would realize, the described embodiments may be modified in various ways, all without departing from the spirit or scope of the present invention. It is to be understood, however, that the invention is not to be limited to the specific forms mentioned in the description, but rather includes all modifications, equivalents, and substitutions within the spirit and scope of the invention as defined by the appended claims. It must be understood.

Claims (7)

Adipose tissue obtained from a human body or an animal and washed with water and 0.01 to 5% gel type collagen are mixed at a volume ratio (v / v) of 1: 8 to 8: 1. Semi-solid gel composition for adipose tissue repair, characterized in that the mixture containing the serum and the medium containing the additives in a volume ratio (v / v) of 1:10 to 10: 1. The method of claim 1, Washed adipose tissue obtained from the human body or animals, Washing adipose tissue at least three times by adding 1X ~ 10X Phosphate-Buffered Saline (PBS) solution to which 0.01-1% BSA (Bovine Serum Albumin) was added, which is 5-15 times the weight of adipose tissue. Semi-solid gel composition for fat tissue repair. The method of claim 2, The 1X-10X PBS solution to which 0.01-1% BSA which is the said water washing solution is added, After dissolving 0.144 ~ 1.44g of KH ₂ PO ₄ , 9.000 ~ 90.00g of NaCl, 0.795 ~ 7.950g of Na ₂ HPO ₄ · 7H ₂ O in 900ml of purified water, adjust the pH to 7.2 ~ 8.8 After filtering, and adding the antibiotic gentamicin (gentamicin) to the final concentration of 100 ㎍ / ㎖ to prepare a 1X PBS solution, dissolved 0.05 ~ 5g BSA in 400ml 1X PBS solution to 1X ~ Semi-solid gel composition for adipose tissue repair, characterized in that the final volume to 500ml with 10X PBS solution and then filtered. The method of claim 1, The collagen of the gel type of the said 0.01-5%, 100 ml of 0.01 ~ 0.5% collagen obtained by enzyme treatment and salt treatment from pig tendon (Tendon) and neutralized with 1N NaOH to 6.5 ~ 7.5 in order to make the filtered collagen into semi-solid gel form, The collagen was left at 20 to 30 ° C for 1 to 15 hours, then centrifuged at 5,000 to 15,000 g for 15 to 60 minutes to remove the upper layer, and the precipitate was settled in the lower layer. Semi-solid gel composition for repairing adipose tissue, which is prepared by adding 10 ml of a buffer solution (Physiological Phosphate-Buffer) and maintaining it at 4 ° C. until use. The method of claim 1, The fat production promoting solution, Purified soybean oil 20 ~ 200g, glycerol 10 ~ 25g, egg yolk phosphatides 5 ~ 12g, sodium oleate 0.03 ~ 0.3g dissolved in 100ml of distilled water and filtered Semi-solid gel composition for fat tissue repair. The method of claim 1, The badge, DMEM (Dulbecco`s Minimum Essential Medium) Powder 8 ~ 11g in 800ml of purified water, NaHNO <sub>3</sub> (Sodium Bicarbonate) 2000 ~ 2400mg, HEPES (N-2-Hydroxyethylpiperazine-N`-2-Ethane Sulfonic Acid) 2200 ~ 2400mg After dissolving the solution, adjust the pH of the dissolved solution to 6.8-7.8, prepare the final volume to be 1000ml with purified water, Dissolve 8-11 g of F-12 powder and 1000-1200 mg of NaHNO _{3 in} 800 ml of purified water, adjust the pH of the dissolved solution to 6.8-7.8, and prepare the final volume to 1000 ml with purified water. Semi-solid gel composition for adipose tissue repair, characterized in that the solution prepared by mixing each of the volume ratio 1: 1 (v / v) by filtration. The method of claim 1, The additive is A semi-solid gel composition for repairing adipose tissue, comprising adding an adipogenesis promoting solution, a serum containing growth factor, and a medium in a volume ratio of 1: 1: 1 (v / v).

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