KR100455919B1 - The method for inhibition of wound contraction using dermal ring sprint - Google Patents

Abstract

The present invention relates to a method for inhibiting wound contraction of the skin and a method for evaluating the efficacy of a growth factor or a wound treatment drug. The present invention not only minimizes the contraction of the wound, but also delays the rate of natural healing of the wound and refines the wound healing stage, which is advantageous for histological analysis of the mechanism of healing and because there is no obstacle to the movement of cells from the periphery of the wound. Very useful for evaluating pure wound healing mechanisms.

Description

The method for inhibition of wound contraction using dermal ring sprint}

The present invention relates to a method for inhibiting wound contraction of the skin and a method for evaluating the efficacy of a growth factor or a wound therapeutic drug.

The skin is an organ that is responsible for not only maintaining body temperature but also protecting the living body from irritation or contamination from the external environment. Tissue damage that can affect the skin's defenses (wounds) is repaired through a number of complex wound healing stages, including the formation of a scab along with the contraction of the wound. Inflammation, granulation tissue formation, and remodeling of the skin's structure have been restored (Dyson M., Young S., Pendle CL, et al. Comparison of the effects of moist and dry conditions on dermal repair., 1998. J invest Dermatol 91: 434).

In case of small skin loss, it is easily healed by complex tissue reactions with contraction.However, when skin loss such as burns is wide, functional damage due to excessive contraction of scar tissue may appear. Analysis is required (Baur PS, Barratt G., Linares HA, et al. Wound contractions, scar contractures and myofibroblasts: A classical case sturdy., 1978 J Trauma 18 (1): 8-22).

Pigs (immobile skin) and rodents (mobile skin) are used as animals to understand the wound healing mechanism of skin. However, pigs are similar to human skin structure, but there are many difficulties in manipulating experimental animals. This has a disadvantage of entering a lot. On the other hand, rodents can be easily manipulated above all, and can be analyzed according to various conditions. However, because rodent wounds generally form new skin in about 6-7 days with rapid contraction of surrounding tissues, it is difficult to assess the exact wound healing stage within these short periods of time. In addition, it was true that the evaluation of the efficacy of the healing agent after wound injuries could not accurately determine whether the wound was cured by contraction or drug-treated. (Davidson JM Animal models for wound repair., 1998. Arch Dermatol Res 290: S1. And McGrath M. Healing of the open wound.In Rudolph R. (ed): Problems in Aesthetic Surgery.Biological causes and Clinical Solutions. 1986. S. Louis, CV Mosby, 13-48).

As a way to overcome this problem, it has been thought that it can be helpful to understand the wound healing mechanism as well as the various stages of wound healing if it can induce the healing delay by suppressing the wound contraction as much as possible. For this purpose, the administration of drugs such as cytotoxic agents, colchicine, suppression of shrinkage through irradiation, full-thickness or partial skin graft, and skin grafts such as artificial dermal structures Techniques for physically inhibiting wound shrinkage, such as, and bandages or external devices, are used. However, drug-induced contraction suppression techniques may produce different results than expected as a side effect of the drug, and skin grafts, bags or external corrections are very difficult to apply to small animals (mouse or rats) (see Joseph HL, Anderson GL, Barker JH, et al. Inhibition of wound contraction with colchicine and D-penicillamine., 1996.J Sirg Res 61 (1): 197 .; Grillo HC Origin of fibroblasts in wound healing: an autoradiographic study of inhibition of cellular proliferation by local x-irradiation., 1963. Ann Surg 157: 453 .; Boyce ST, Glafkides MC, Foreman TJ, Hansbrough JF Reduced wound contraction after grafting of full-thickness burns with a cllagen and chondroitin-6-sulfate (GAG) .. dermal skin substitute and coverage with biobrane, 1988. J Burn Care Rehabil 9 (4):. 364.; Abercrombie M., James DW, Newcombe JF Wound contraction in rabbit skin, studied by splinting the wound margins, 1960. J Anat 94: 170 .; and Agren MS and Mertz PM Ar e excessive granulation tissue formation and retarded wound contraction due to decreased collagenase activity in wounds in tight-skin mice ?, 1994. Br J Dermatol 131 (3): 337).

The present invention provides a method for inhibiting wound shrinkage of the skin in a simple tool use and relatively easy manner. This can delay and refine the overall wound healing stage, suggesting a very effective means for evaluating the drug for wounds and analyzing the effects of individual or multiple growth factors.

1

As a comparison of wound contractions in the sprint group and the control group,

(A) is a visual observation of the wound shrinkage of the ring sprint loaded group and the control group in the same experimental animal,

(B) is visual observation of wound shrinkage of the ring sprint loaded group and the control group at the same site;

2

As a comparison of wound shrinkage inhibition rate,

Green indicates the shrinkage of the ring sprint loaded group, and red control group;

3 is

Wound constriction Tissue photograph at 7 days, arrow indicates length of wound;

4 is

Three days after the wound, the control group and the sprint group were photographed with white arrows in the 40 × photograph showing the depth of the wound, PMN in the 400 × photograph, white blood cells, and Fibroblast as the fibroblasts. ;

5 is

Tissue photograph after 7 days of wound, 40 × photograph shows that the regenerated epithelium is the regenerated epidermis and its regeneration site is different, and 400 × photograph shows the difference in fibroblast formation;

6 is

In the tissue photograph after 12 days of wounding, the length of the arrow in the 40 × photograph indicates the depth of the wound, and in the 400 × photograph, there is a large difference in the cell spacing;

7 is

Tissue photograph 15 days after the wound, the arrows in the 40 × photographs indicated the epidermis, and the 400 × photographs showed a difference in the appearance of collagen formation;

8 is

Tissue picture of α-SMA staining, with differences in time and site of response;

9 is

The growth factor bFGF efficacy in the wound was evaluated, showing that the effect was maintained.

The present invention relates to a method of inhibiting wound contraction of the skin and a method of evaluating the efficacy of a growth factor or a wound treatment drug.

As a tool used in the method of suppressing wound shrinkage, the ring sprint can be maintained without changing its shape with respect to the wound shrinkage force and does not cause an allergic reaction in contact with the skin, for example, plastic, metal, wood, etc. Is used. In the form of a ring, the diameter needs to be about 2 mm larger than the wound, and the thickness is appropriate for maintaining the shape of the material, which is different for each material, but averages 1.8 mm. The height is about 2.8 mm. The sprint can be in contact with the skin by itself, but is preferably wound with a tape made of natural fibers such as cotton and silk to maximize the skin's allergic reaction. This ring sprint is placed on the mounting site and the ring sprint and the skin and muscle layers are ligated together with a suture. Preferably, the ring sprint is firmly fixed by ligation at 12 to 16 sites. After the ring sprint is fitted, the cut surface is rounded off without damaging the muscle layer in the ring.

In addition, the present invention provides a method for evaluating the efficacy by administering a growth factor or a therapeutic drug for a wound in a state where the contraction of the wound is suppressed as described above.

The following examples are provided to show the effects of the present invention, but the present invention is not limited thereto.

Example

Ring Sprint Mount

5 ml syringes were cut to a size of 5 mm thick. Both cut surfaces were ground with sand paper to make a ring having a flat cut surface having a thickness of 2 mm. The ring is wrapped with cotton cloth tape to prevent the sharp side of the material from being revealed. Sterilized with ethylene oxide gas and stored.

0.1 ml of a mixture of ketamine and xylazine (Ketamine 90 mg / kg, Xylazine 10 mg / kg) was administered intraperitoneally for general anesthesia of Balb / C mice (male, 20 g). The hair of the back of the anesthetized animal was cut with an electric clipper and the hair was removed from the hair root using knit cream (Ildong Pharmaceutical Co., Ltd.). The exposed area was disinfected with 70% ethanol.

A ring sprint was placed on the back of the experimental animal, and the ring was fixed by ligating 14 rings with 2-0 silk suture together. After the wound was induced with an 8 mm biopsy punch, the wound surface was rounded off with scissors so as not to damage the muscle layer.

Control

The ring sprint was placed on the skin of the experimental animal and pressed, and then the wound surface was rounded off with an 8 mm bio punch and scissors.

Experiment 1. Visual observation of wound shrinkage and healing

1) Inhibition of wound shrinkage of the ring sprint mounting portion and the non-mounting portion in the same experimental animal was visually observed on the 3rd and 7th day immediately after the induction of the wound, and is shown in FIG. On the third day, there was almost no change in the area of the wound of the ring sprint attachment site, but it was observed that the non-attachment site was somewhat shrunk. On the 7th day, slight shrinkage was also observed at the ring sprint mounting site, but it was confirmed that the shrinkage rate was significantly lower than that of the wound site at the non-mounting site.

2) The wound shrinkage inhibition rate of the ring sprint-mounted group and the control group was visually observed immediately after the wound induction and on the 7th and 15th days, respectively, and is shown in FIG. 1 (B). On day 7, there was a significant difference in wound shrinkage between the ring sprint group and the control group. In the control group, the wound contraction occurred rapidly, and the wound surface was very smooth due to the regenerated skin from about 7 days after the wound. On the other hand, after 15 days, the wound surface was smooth in the animal model, but the wound area was much larger than the control group.

Table 1 below is a comparative measurement of the wound area by day 7.

Comparison of wound shrinkage inhibition rate Right after wound 1 day past 2 days 3 days elapsed Ring Sprint Mount CC Average 8.58 7.84 7.80 7.71 SD 0.35 0.26 0.21 0.17 DV Average 8.28 7.91 7.86 7.81 SD 0.18 0.28 0.22 0.18 Control CC Average 8.65 7.43 6.78 6.27 SD 0.01 0.15 0.21 0.07 DV Average 8.67 7.13 6.68 6.85 SD 0.08 0.74 0.55 0.40

4 days 5 days 6 days past 7 days past Ring Sprint Mount CC Average 7.61 7.48 7.25 7.05 SD 0.35 0.35 0.47 0.53 DV Average 7.64 7.56 7.40 7.17 SD 0.30 0.30 0.30 0.37 Control CC Average 5.65 5.12 4.17 3.56 SD 0.38 0.50 0.78 0.67 DV Average 6.29 5.76 4.72 4.20 SD 0.37 0.45 0.28 0.62

CC: Cranio-Caudal direction (length measurement from head to tail direction)

DV: Dorso-Ventral direction

SD: standard deviation

The results of Table 1 are shown in the graph of FIG.

The wound of the ring sprint mounting group shrinks about 1 to 1.5 mm from the table 1 and the 7th day from FIG. 2, but shows a large difference of 4.5 to 5 mm shrinkage in the control group, so that the method of the present invention has an excellent effect on suppressing wound shrinkage. It was confirmed that there is.

Experiment 2. Histological Analysis of Wound Contraction and Healing Stages

Preparation of Tissue Sections

Ring sprint loaded groups and controls were sacrificed using carbon dioxide on days 3, 7, 12, and 21, and tissues were fixed in 10% neutral formalin buffer solution. After normal tissue treatment, 4 μm thick sections were made and stained with H & E. Tissue photographs of 3, 7, 12 and 15 days after the induction of wounds are shown in FIGS. 4, 3 and 5, 6 and 7, respectively.

Tissue analysis

The arrow in FIG. 3 is the length of the wound and the ring sprint mounting group was much longer than the control group. This is consistent with the appearance of the depth of the wound in the ring sprint mounting group as compared to the control, as shown in the 40 × picture of FIG. 4 and the 40 × picture of FIG. 6. This is because it inhibits the wound shrinkage effect of the method of the present invention visually confirmed.

On the third day of the wound, the white blood cells form a crust in the control group, and in the 400 × photograph of FIG. 4, fibroblasts and leukocytes from the peripheral connective tissue were observed in the subcutaneous tissue. And it was found. On the other hand, in the ring sprint-mounted group, white blood cells form a scab on the wound bed, and many white blood cells were observed in 400 × photographs of FIG. I could confirm the note. On the other hand, in both the ring sprint mounting group and the control group, leukocytes moved from the peripheral connective tissue to the wound site, which was determined as an inflammatory reactor, and the epidermis was regenerated at the wound site.

At the 7th day of wounding, the ring sprint mounting group was covered with the regenerated epidermis only in the periphery of the wound, as shown in 40 × photograph of FIG. In the 400 × photographs, the control group was observed to be densely packed with fibroblasts in the center as well as around the wound, indicating that it was already granulation tissue formation. On the other hand, the ring sprint-mounted group was able to see the regenerated epidermis only at the periphery of the wound, and observed it moving to the center of the wound. Leukocytes, fibroblasts, and blood vessels were also observed. However, fibroblasts are densely observed only under the regenerated epidermis, so granulomatous tissues are formed in the periphery of the wound, but the core of the wound is still covered. We can see that the steps are broken down.

On day 12 of the wound, in the 400 × photograph of FIG. 6, the control group showed that fibroblasts in the majority of the wound decreased numerically and existed at a certain distance, and the shape of the nucleus began to appear irregular and entered the initial remodeling phase. Could know. On the other hand, in the ring sprint mounting group, the number of fibroblasts decreased and constant direction was observed at the periphery of the wound, but the number of leukocytes was present at the center of the wound and the healing stage was delayed.

By day 15 of the wound, in the 40 × photograph of FIG. 7, the epidermis of the ring sprint mounting group was covered with a smooth and uniform thickness, while the thickness was uneven in the control group. The 400 × photographs showed that the fibroblasts of the control group had already matured and showed various forms of nuclei, and collagen components were thickly clustered together to form bundles. On the other hand, in the ring sprint loaded group, the fibroblasts began to bend at a moderate interval and the shape of the nucleus bent in some cells. In addition, the collagen components secreted by the fibroblasts appeared to be very finely aggregated, and white blood cells were still present, which was an immature reconstructor.

From the above results, in the ring sprint mounting group, the healing process is delayed, and several healing stages are observed in one wound, thereby confirming that the healing stage is subdivided.

Experiment 3. Evaluation of Wound Contraction Mechanism

Tissue staining

To confirm the contraction caused by fibroblasts known as another mechanism of wound contraction, tissue sections made in the same manner as in Experiment 2 were subjected to immunohistochemistry for α-smooth muscle actin (α-SMA). Stained. Tissue photographs of days 4, 6 and 8 of the sprint loaded group and the control group are shown in FIG. 8.

Tissue analysis

In FIG. 8, the ring sprint mounting group showed a positive reaction only under the regenerated epidermis of the wound periphery on the 7th day, but the control group showed a strong positive reaction before the wound site. The ring sprint group showed positive reactions in the center of the wound and under the epidermal layer on days 12 and 21, but the control group showed negative reactions after 12 days.

The control group showed strong positive reactions throughout the wound on the 7th day of the wound, and the contraction was actively progressing, and it was confirmed that it was a granulation tissue formation phase. Since then, however, the reaction was no longer positive, and it was completely past the granulation tissue formation stage of the wound healing stage. On the other hand, the ring sprint-mounted group showed α-SMA positive reaction only under the epidermal layer on the periphery of the wound on the 7th day of the wound, but showed α-SMA positive reaction on the center of the wound by the 12th day. In addition, on day 21 of the wound, a slight positive reaction of α-SMA was found just below the epidermis in the center of the wound, indicating that the early granulation phase was not completed.

By this method, the contraction step of the fibroblasts was significantly delayed as well as the healing step in the wound was further confirmed.

Table 2 summarizes the observation results of the tissues after the wound induction.

Tissue observation of wound contractions and healing stages Ring Sprint Mount Control 3 days elapsed Inflammatory reaction epidermis regenerates at the periphery of wounds. Leukocyte infiltration from connective tissue.Difficult to identify fibroblasts in subcutaneous tissue.Skin formation by tissue fluid and leukocytes.α-SMA (negative) Inflammatory epidermis regenerates around the wound. Leukocyte infiltration from connective tissue. Distribution of fibroblasts and leukocytes in subcutaneous tissue. Α-SMA (negative). 7 days past Inflammatory Reactor / Granulation Tissue Formation Wound Peripheral-Epidermal formation, very densely distributed fibroblasts and leukocytes, ejected blood present under the epidermis, α-SMA positive.Wound center-Initiation of fibroblasts, formation of leukocytes in the upper layer , α-SMA negative. Granulation Tissue Formation Wound Peripheral-Separation of epidermis and dermis, tight distribution of fibroblasts, active blood vessel formation.Central wounds-Epidermal formation, difficulty in distinguishing from dermis, active blood vessel formation.Positive in most α-SMA wounds. 12 days past Granulation Tissue Formation / Initial Reconstructor Incomplete epidermal formation. Leukocytes, macrophages and fibroblasts present. Active vascularization. Fibroblasts have no directivity, high density, and few fibrous cells in areas other than the wound periphery. Nurtured in the center of the wound. Cell layers of immature reconstituted epidermis about 7 to 8 layers. Fibrous cells and ECM maintain regular intervals. Fibrous cells show constant orientation. White blood cells, macrophages decrease in number. Α-SMA negative. 15 days past Immature reconstructor epidermis completely covers the wound; reconstructed at the periphery of the wound and the central fibrous cells show a constant orientation and a long rectangle; the nucleus is uniform and the fine collagen is tightly distributed; micropositive in the center of α-SMA wound. The cell layer of the mature reconstituted epidermis is about 4 to 5 layers. White blood cells, macrophages rarely observed. Fibrous cells are regularly arranged. Very irregular cell nucleus and nuclear membrane. Thick collagen bundle distribution. Α-SMA negative. 21 days passed Numerical reduction and constant orientation of fibroblasts. Rarely white blood cells present. Well-developed collagen fibers. Α-SMA weak positive. Epidermal cell layer about 1 to 3 layers. Fibrous cells and collagen fiber development. Α-SMA negative.

Experiment 4. Evaluation of Fibroblast Growth Factor (bFGF) Efficacy

Preparation of Experiment Group

200 ng of basic fibroblast growth factor (bFGF) was administered to the wound site of the ring sprint mounting group and then wounded with Opsite, Smith & Nephew Medical LTD. As a control group, PBS (phosphate buffer solution) was once administered to the wound site of the ring sprint mounting group and then covered with an opsite. On the 2nd, 4th, 6th, and 8th days, 4 μm-thick sections were made by tissue treatment as in Experiment 2 and stained with H & E. Tissue photograph results are shown in FIG. 9.

Observation of the experimental group

Since the wound was covered with the opsite, there was almost no contraction of the wound. Therefore, the visual difference with the control group could not be distinguished, but the histological analysis showed a significant difference.

On the second day of wounding, macrophages and leukocytes began to infiltrate submucosa in the bFGF-administered group, and a group of leukocytes existed just below the opsite, whereas in the control group, the number of leukocytes in the bFGF-treated group was compared to the bFGF-treated group. I wrote it down.

On day 4, the leukocyte barrier was shown in the bFGF-administered group of FIG. 9 and the number of fibroblasts was increased. At the periphery of the wound, the epidermis began to regenerate and underneath it increased the number of fibroblasts and blood vessels. In addition, the upper part of the submucosa of the skin showed thin basophilic staining and the number of white blood cells was increased. On the other hand, only the leukocyte infiltration was confirmed in the control photograph. In the periphery of the wound, epidermal regeneration was observed and fibroblasts were increased, but the submucosa of the center of the wound was still small in number of white blood cells.

On the 6th day, the fibroblasts of the dermis showed high density and high density in the bFGF-administered group of FIG. 9. Epidermal cell layers in the periphery of the wound were 7 to 9 layers, and no desmosome between the cells was observed. In contrast, the control group showed infiltration of fibroblasts but no directionality, and the number of fibroblasts and leukocytes was less than that of the bFGF group.

On the 8th day, the extracellular matrix (ECM) component of the fibroblasts was well developed in the bFGF-administered group, and the well-developed fibroblasts had a certain orientation. Fibroblasts had a higher density under the regenerated epidermis at the periphery of the wound, and one or two prominent nucleolus were present in the large, rounded nucleus, and the cells showed constant orientation. But in the center of the wound, fibroblasts still had less invasion. On the other hand, the control photograph showed an increase in the number of fibroblasts, but the ECM component was poor and no mature fibroblasts could be identified.

From the above experimental results, it was confirmed that even if the wound contraction is suppressed by the method of the present invention, the effect of growth factors can be effectively exerted. This shows that the wound shrinkage suppression method of the present invention only delays the wound shrinkage suppression and the healing rate, and maintains the healing mechanism of the wound itself, and thus can be an excellent means for analyzing the effect of pure growth factors.

The present invention minimizes wound shrinkage of the skin, and also delays the rate of natural healing of the wound and refines the stage of wound healing, which is advantageous for histological analysis of the healing mechanism, and there are no obstacles to the movement of cells from the periphery of the wound. This is very useful for evaluating the pure wound healing mechanism of growth factors or drugs.

Claims (5)

A method of suppressing wound shrinkage of the skin by attaching a ring sprint to the skin of an animal other than a human, and then inducing a wound in the ring sprint. The method of claim 1, wherein the ring sprint, skin and muscle layers are ligated together with a suture to mount a ring sprint to inhibit wound shrinkage of the skin. The method of claim 1 or 2, wherein the ring sprint is used in the form of wrapping with natural fibers. The method of claim 1 wherein the animal is a rodent. A method of assessing the efficacy of a growth factor or a wound treatment drug by mounting the ring sprint on the skin of an animal other than a human, and then inducing a wound in the ring sprint and administering a growth factor or therapeutic drug for the wound.