DERMAL LAYER FOR GRAFTING HAVING IMPROVED GRAFT SURVIVAL RATE AND METHOD FOR PRODUCING SAME

Abstract

The present invention relates to a dermal layer which is for grafting and has an improved graft survival rate, and a method for producing the same, wherein the dermal layer for grafting can be produced by filling a filling solution, including a DNA fragment mixture and chitosan, into an acellular dermal matrix from which cells have been removed. It was observed that the dermal layer for grafting produced in this manner, due to the filling solution filled therein and including a DNA fragment mixture and chitosan, increases the rate at which cells flow in from the tissue surrounding the graft and are fixed, and thereby alleviates an initial inflammatory reaction and promotes blending with the surrounding tissue.

Claims

1. A dermal layer for grafting, produced by applying a loading solution comprising a DNA fragment mixture and a chitosan on to an acellular dermal matrix, wherein the DNA fragment mixture is from fish testis or sperm, wherein the DNA fragment mixture and chitosan is mixed in a weight ratio of 20:1-10,000:1, and wherein the dermal layer has an improved graft survival rate.

2. The dermal layer for grafting of claim 1, wherein the fish is of the family Salmonidae.

3. The dermal layer for grafting of claim 1, wherein the DNA fragment mixture has a molecular weight of 50 kDa to 10,000 kDa.

4. The dermal layer for grafting of claim 1, wherein the chitosan has a molecular weight of 3 kDa to 1,000 kDa.

5. The dermal layer for grafting of claim 1, wherein the DNA fragment mixture is in an amount of 0.01% by weight to 3% by weight, based on the total weight of the loading solution.

6. The dermal layer for grafting of claim 1, wherein the chitosan is in an amount of 1×10.sup.−6% by weight to 0.15% by weight, based on the total weight of the loading solution.

7. The dermal layer for grafting of claim 1, wherein the acellular dermal matrix is a biologically inactivated collagen-based tissue supplement material that has been obtained by removing cells and impurities from a dermal matrix and subjecting the dermal matrix to virus inactivation and then to lyophilization.

8. The dermal layer for grafting of claim 1, wherein the dermal matrix is an autograft, allograft, or heterograft.

9. An agent for treating skin injury, comprising the dermal layer for grafting of claim 1.

10. The agent of claim 9, wherein the skin injury is caused by at least one selected from the group consisting of abrasion, incision, laceration, skin avulsion, skin bruise, puncture wound, and surgical skin excision.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1 shows results determining whether or not a loading solution containing a DNA fragment mixture and chitosan is loaded. No violet regions stained by hematoxylin are found in either control 1, which is an acellular dermal matrix not loaded with a loading solution (A), or control 2, which is an acellular dermal matrix immersed in a loading solution (B) whereas multiple portions stained violet are observed among red stains of collagens in the dermal layer for grafting according to the present invention (C). Black arrows indicate regions which are stained violet by hematoxylin and into which the loading solution is loaded.

[0069] FIG. 2 shows a size change in the dermal layer with time after the dermal layer for grafting is implanted. The grafted acellular dermal matrices did not change in size with time for control 1 in which an acellular dermal matrix had been immersed in PBS (A), control 2 in which an acellular dermal matrix had been immersed in loading solution 1-1 for about 30 min (B), or control 3 in which 0.005% by weight of chitosan had been loaded (C). In contrast, a dermal layer for grafting having a loading solution 1-1 loaded thereto according to the present invention exhibited a behavior similar to those of the controls for three weeks, but became small as the edges of the grafted dermal layer were degraded from week 4 after grafting (D).

[0070] FIG. 3 shows a degree of inflammation induced by the dermal layer for grafting according to the present invention. The implantation of the dermal layer for grafting according to the present invention (B) was observed to cause a reduced initial inflammation reaction, compared to that of control 1 (A), as measured in terms of thicknesses of fibrous tissue capsules induced by inflammation.

[0071] FIG. 4 shows cell survival rates after the implantation of a dermal layer for grafting according to the present invention. A greater population of cells survived upon the implantation of the dermal layer for grafting according to the present invention (B) than control 1 (A).

MODE FOR CARRYING OUT THE INVENTION

[0072] Hereinafter, particular embodiments of the present invention will be concretely explained. However, the present invention is not limited to the Examples given below, but can be embodied in other forms. Rather, they are provided to make the contents introduced herein thorough and perfect and to deliver the spirit of the present invention sufficiently.

Example 1: Production of Dermal Layer for Grafting Having Improved Graft Survival Rate

Example 1-1. Preparation of Loading Solution Containing DNA Fragment Mixture and Chitosan

[0073] For use in producing a dermal layer for grafting having an improved graft survival rate according to the present invention, a loading solution containing a DNA fragment mixture and chitosan was prepared.

[0074] A DNA fragment mixture was dissolved at 60° C. for 2 hours or longer in a 190 mM sodium phosphate dibasic dodecahydrate buffer with the aid of a heat stirrer to give a DNA fragment stock solution. Chitosan was dissolved at room temperature for 3 hours or longer in 90 mM acetic acid to give a chitosan stock solution.

[0075] The DNA fragment stock solution and the chitosan stock solution prepared above were mixed with each other and stirred for 2 hours at 70° C. in a heat stirrer. In this regard, the DNA fragment mixture and the chitosan were mixed according to the conditions given for the concentration and mix ratio in Table 1, below. The loading solution containing the DNA fragment mixture and chitosan thus prepared were sterilized using an autoclave.

TABLE-US-00001 TABLE 1 Final Concentration Mixture Ratio (% by weight) (weight ratio) DNA DNA fragment fragment Configuration mixture Chitosan mixture Chitosan Loading solution 1-1 0.5  0.005  100 1 Loading solution 1-2  0.01   0.0005   20 1 Loading solution 1-3  0.01    0.000001 10000 1 Loading solution 1-4 3    0.15   20 1 Loading solution 1-5 3     0.0003 10000 1

Example 1-2: Production of Dermal Layer for Grafting Having Graft Survival Rate

[0076] For use in producing a dermal layer for grafting having an improved graft survival rate according to the present invention, an acellular dermal matrix (tradename: Nature-Derma) was purchased from HansBiomed (Seoul, Korea). The commercial product is a collagen-based tissue supplement material that is prepared by removing cells and impurities from swine dermis through chemical treatment processes and inactivating viruses, followed by lyophilization and which is used for compensating for defected or injured bones, cartilages, etc. upon treatment in internal medicine or surgery or upon operation.

[0077] After the acellular dermal matrix purchased from HansBiomed was immobilized to a vacuum drug loading device, loading solution 1-1 was loaded to the acellular dermal matrix to produce a dermal layer for grafting according to the present invention. Loading solution 1-1 was identified to exhibit the best sense of use among the loading solutions comprising DNA fragment mixtures and chitosan, prepared in Example 1-1, as measured by inventor's inspection with the naked eye and a loading test.

Example 2: Identification of Loading of Loading Solution Containing DNA Fragment Mixture and Chitosan

[0078] H&E (hematoxylin and eosin) staining was conducted to determine whether the acellular dermal matrix was loaded with a loading solution containing a DNA fragment mixture and chitosan. In the H&E staining procedure, hematoxylin binds to negatively charged nucleic acids to express a violet color while a red color appears as eosin binds to positively charged intracellular proteins. Collagen, which is the main ingredient of the acellular dermal matrix, is bound with eosin and stained red.

[0079] Among the dermal layers for grafting prepared in Example 1-2, the dermal layer loaded with loading solution 1-1 of Table 1 was subjected to H&E staining. In this regard, H&E staining was also conducted on controls including an acellular dermal matrix that was not loaded with a loading solution (control 1), and an acellular dermal matrix immersed for about one hour in a loading solution (control 2). The results are shown in FIG. 1.

[0080] As shown in FIG. 1, eosin stained collagen red in the acellular dermal matrix that had not been loaded with a loading solution containing a DNA fragment mixture and chitosan (control 1) (A) and the acellular dermal matrix immersed in loading solution (control 2) (B) whereas violet stains of hematoxylin were not observed in any of them. In contrast, violets stains (black arrows) were observed among the collagen stained red in the dermal layer for grafting, produced in Example 1-2 (C).

[0081] It is understood through the data that a loading solution cannot be introduced into the acellular dermal matrix by simple immersion in the loading solution. Therefore, it was recognized that a method using a vacuum drug loading device is most efficient as a method for loading an acellular dermal matrix with a loading solution containing DNA fragment mixture and chitosan.

Experimental Example 1: Preparation of Experimental Animal

[0082] For use in animal experiments, Sprague-Dawley lineage male rats, each being 7 weeks old and weighing 230-280 g, were purchased from YoungBio (Sungnam City, Korea). Solid diets for experimental animals were purchased (Harlan laboratories, Inc., USA) and placed in a feeder to which the experimental animals were given free access. Drinking water was filtered through a filter water flowing sterilizer and exposed to UV radiation before the animals were allowed to have free access to the drinking water in an automatic water feeder. A breeding chamber for the animals was maintained at a temperature of 23±3° C., a relative humidity of 55±15% under a light/dark cycle of 12/12 hours with a light intensity of 150˜300 Lux.

Experimental Example 2: Identification of Graft Survival Rate of Dermal Layer for Grafting

Experimental Example 2-1: Implantation of Dermal Layer for Grafting

[0083] The dermal layer for grafting produced in Example 1-2 was immersed for an additional 30 min in the loading solution having the same concentration before use. An acellular dermal matrix that had not been loaded with a loading solution was immersed in PBS (phosphate buffer saline) (control 1) and in loading solution 1-1 for 30 min (control 2). An acellular dermal matrix was loaded with 0.005% by weight chitosan (control 3).

[0084] The rats in Experimental Example 1 were subjected to inhalation anesthesia with isoflurane and sterilized with povidone and ethanol at a back region to which a graft would be applied. After sterilization, the back region of the rat was incised along the spine and the epidermis was raised with forceps to secure an implantation space into which the dermal layer for grafting according to the present invention or the controls prepared above were then inserted, followed by suturing the incision. In this regard, one of the controls and the dermal layer for grafting according to the present invention were subcutaneously implanted into the left back and the right back with reference to the spine, respectively. Each of the rats that underwent surgery was placed in one cage and kept in a stable condition.

Experimental Example 2-2: Identification of Graft Survival Rate by Observation with Naked Eye

[0085] After implantation in Experimental Example 2-1, the rats were sacrificed one by one each week. Tissues were separated from the grafted regions and monitored for a change in the dermal layer for grafting with the naked eye. The results are given in Table 2 and depicted in FIG. 2. Table 2 shows a change in the size of the dermal layer for grafting 4 weeks after implantation and expresses the post-implantation sizes in percentages (%) relative to the size of the dermal layer upon implantation.

TABLE-US-00002 0 Size of Dermal Layer for Condition Grafting (%) Control 1 Immersion in PBS 100 Control 2 Immersion in  98 loading solution 1-1 Control 3 Loaded with 0.005%  94 by weight chitosan Loading Loaded with solution 1-1 loading solution 1-1  64

[0086] When grafted, as shown in Table 2 and FIG. 2, control 1 (acellular dermal matrix immersed in PBS) (A), control 2 (acellular dermal layer immersed in loading solution 1-1) (B), and control 3 (acellular dermal matrix loaded with 0.005% by weight chitosan) (C) showed almost no change in size with time. In contrast, the dermal layer for grafting according to the present invention did not change in size for three weeks, but became small as the edge of the grafted dermal layer became degraded from week 4 after grafting.

[0087] The results indicate that a graft survival rate is not improved in either the case where a loading solution is absorbed to only the surface of an acellular dermal matrix by immersion therein, like control 2, or the case where a matrix is loaded with a solution containing chitosan alone, like control 3. Only the dermal layer for grafting produced by loading an acellular dermal matrix with a loading solution containing a DNA fragment mixture and chitosan exhibited an increased graft survival rate.

Experimental Example 2-3: Identification of Cell Survival Rate and Biocompatibility Through Tissue Staining

[0088] The dermal layer for grafting according to the present invention that was loaded with loading solution 1-1 in Experimental Example 2-1 and the acellular dermal matrix immersed in PBS (control 1) were grafted. Tissues were harvested on weeks 1 and 4 after grafting and stained with H&E to determine biocompatibility and survival rates.

[0089] The tissues harvested on weeks 1 and 4 were fixed with formalin, subjected to a series of dehydration, and then embedded in paraffin to construct a paraffin block. The paraffin block was sectioned into 5 μm thick pieces. The tissue pieces were reacted for 1 second with Mayer's hematoxylin solution (Sigma, USA) and washed for 10 min with flowing water. Thereafter, a reaction with an eosin solution (Sigma, USA) was conducted for 3 seconds. After completion of the staining, the tissues were subjected to dehydration and sealed with Permount (Fischer scientific, USA). Histopathological changes of the H&E stained tissue pieces were observed under a microscope and the results are depicted in FIGS. 3 and 4. FIG. 3 shows results of biocompatibility identified by observing fibrous tissue capsules formed by inflammation and FIG. 4 shows results of cell survival rate identified by monitoring the number of cells in the grafted dermal layer.

[0090] As shown in FIG. 3, fibrous tissue capsules attributed to inflammation were thin for the dermal layer for grafting according to the present invention (B), compared to control 1 (A), on week 1 after grafting. The fibrous tissue capsules became thin for both control 1 (A) and the dermal layer for grafting according to the present invention (B) on week 4.

[0091] For cell survival rates, as shown in FIG. 4, lots of empty spaces were observed among collagens in control 1 (A) and the dermal layer for grafting according to the present invention on week 1 after grafting. In contrast, the empty space was filled with cells on week 4 after grafting. Particularly, a greater population of cells were counted for the dermal layer for grafting according to the present invention (B) than control 1 (A).

[0092] From these results, it is understood that the loading solution containing the DNA fragment and chitosan, loaded into the dermal layer for grafting according to the present invention, alleviates initial inflammation and allows cells to rapidly move from adjacent tissues, whereby fusion between the dermal layer and the adjacent tissue can be promoted to increase the cell survival rate.