Yoshimitsu Kuroyanagi

A Cultured Skin Substitute Composed of Fibroblasts and Keratinocytes with a Collagen Matrix: Preliminary Results of Clinical Trials

The cultured skin substitute was created through successive cultivation of fibroblasts and keratinocytes that were combined within a collagen matrix. This collagen matrix was composed of a collagen spongy sheet and a collagen gel. The collagen spongy sheet was designed to produce a honeycomb structure having many holes in which all holes through the sheet were filled with collagen gel. This specific structure thereby allows for the nourishment of the cultured keratinocytes on the surface of the matrix when placed on the graft bed. In this study, autologous cultured skin substitute was applied to a 51-year-old man who had sustained a burn injury. Three sheets of the cultured skin substitute (6 × 9.5 cm) were grafted onto the full-thickness excised wound in the right anterior chest wall. One week after grafting most of the matrix disappeared and stratified keratinocytes were seen to have firmly attached to the underlying tissue. Five weeks after grafting a cornified epidermal layer was seen. Ten months after grafting a mature epidermis and a well-differentiated papillary and reticular dermis replacement were observed. The physical properties and color of this grafted area resemble those of normal skin. In the second test case, autologous cultured skin substitute was applied to a 30-year-old man with a scar remaining after tattoo removal. Eight sheets of the cultured skin substitute (10 × 18 cm) were applied on an excised wound (thickness, 0.02–0.025 in.) of both the fore- and upper arms. The histological appearance of a biopsied skin specimen from the grafted area at 3 months after grafting showed a mature epidermis and a well-differentiated reticular dermis replacement. The regenerated skin at 14 months after grafting showed an excellent result.

Development of a Wound Dressing Composed of Hyaluronic Acid and Collagen Sponge with Epidermal Growth Factor

This study was designed to investigate the effect of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF) on various parameters of wound healing in vitro and in vivo. High-molecular-weight (HMW) HA solution, hydrolyzed low-molecular-weight (LMW) HA solution and heat-denatured Col solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation to the spongy sheet (Type-I dressing). In a similar manner, a spongy sheet containing EGF was prepared (Type-II dressing). The efficacy of these products was firstly evaluated in vitro. Fibroblast proliferation was assessed in culture medium in the presence or absence of a piece of each wound dressing. EGF stimulated cell proliferation after UV irradiation and dry sterilization at 110°C for 1 h. In the second experiment, fibroblasts-embedded Col gels were elevated to the air–liquid interface to create a wound surface model, on which wound dressings were placed and cultured for 1 week. Cell proliferation and the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were investigated. With Type-II dressings, the amounts of VEGF and HGF released from fibroblasts in the Col gel were significantly increased compared with Type-I dressing. Next, the efficacy of these products was evaluated in vivo using Sprague–Dawley (SD) rats. Wound conditions after 1 and 2 weeks of treatment with the wound dressings were evaluated based on the gross and histological appearances. Type-II dressings promoted a decrease in wound size, re-epithelialization and granulation tissue formation associated with angiogenesis. These findings indicate that the combination of HA, Col and EGF promotes wound healing by stimulating fibroblast function.

Development of a Wound Dressing Composed of Hyaluronic Acid Sponge Containing Arginine and Epidermal Growth Factor

Hyaluronic acid (HA) has the ability to promote wound healing. Epidermal growth factor (EGF) is able to promote the proliferation of various cell types, in addition to epidermal cells. A novel wound dressing was designed using high-molecular-weight hyaluronic acid (HMW-HA) and low-molecular-weight hyaluronic acid (LMW-HA). Spongy sheets composed of cross-linked high-molecular-weight hyaluronic acid (c-HMW-HA) were prepared by freeze-drying an aqueous solution of HMW-HA containing a crosslinking agent. Each spongy sheet was immersed into an aqueous solution of LMW-HA containing arginine (Arg) alone or both Arg and epidermal growth factor (EGF), and were then freeze-dried to prepare two types of product. One was a wound dressing composed of c-HMW-HA sponge containing LMW-HA and Arg (c-HMW-HA/LMW-HA + Arg; Group I). The other was a wound dressing composed of c-HMW-HA sponge containing LMW-HA, Arg and EGF (c-HMW-HA/LMW-HA + Arg + EGF; Group II). The efficacy of these products was evaluated in animal tests using rats. In the first experiment, each wound dressing was applied to a full-thickness skin defect with a diameter of 35 mm in the abdominal region of Sprague–Dawley (SD) rats, leaving an intact skin island measuring 15 mm in diameter in the central area of this skin defect. Commercially available polyurethane film dressing was then applied to each wound dressing as a covering material. In the control group, the wound surface was covered with polyurethane film dressing alone. Both wound dressings (Group I and Group II) potently decreased the size of the full-thickness skin defect and increased the size of the intact skin island, when compared with the control group. The wound dressing in Group II showed particularly potent activity in increasing the distance of epithelization from the intact skin island. This suggests that EGF release from the spongy sheet serves to promote epithelization. The wound dressing in Group II enhanced early-stage inflammation after 1 week, as compared with the other two groups. In the second experiment, each wound dressing was applied to a full-thickness skin defect measuring 35 mm in diameter in the abdominal region of SD rats, after removing necrotic skin caused by dermal burns. Polyurethane film dressing was applied to each wound dressing as a covering material. In the control group, the wound surface was covered with polyurethane film dressing alone. Both wound dressings (Group I and Group II) potently decreased the size of the full-thickness skin defect and increased epithelization from the wound margin, as compared with the control group. The wound dressing in Group II was found to enhance early-stage inflammation after 1 week, as compared with the other two groups. The findings in both experiments indicate that the wound dressing composed of HA-based spongy sheets containing Arg and EGF potently promotes wound healing by inducing moderate inflammation. The release of EGF in the early stages of wound healing induces moderate inflammation. This suggests that wound healing is facilitated directly by topical application of EGF, and indirectly by cytokines derived from inflammatory cells stimulated by EGF.

Effect of a collagen matrix containing epidermal growth factor on wound contraction

Excessive wound contraction is known to lead to pathological wound contracture. Using a rabbit model, we applied a bovine type I collagen matrix sponge as a dermal substitute and human epidermal growth factor to full‐thickness excisional wounds. Wound contraction was assessed 14 and 28 days after wounding. It was found that both collagen matrix and epidermal growth factor significantly inhibited wound contraction (p < 0.001) in all wounds treated with collagen matrix alone or treated with 0.1 and 1 µg of epidermal growth factor 28 days after wounding. Interestingly, the combination of collagen matrix with epidermal growth factor strongly inhibited wound contraction over matrix alone (p < 0.01 on day 28). Histological analyses showed a regular horizontal arrangement of collagen fibers in the dermis under wounds treated with these substances but not under untreated wounds. Furthermore, using a fibroblast‐populated collagen gel, the direct inhibitory effect of epidermal growth factor on gel contraction by fibroblasts was also observed. Collagen gels without stimulation contracted to 29.5 ± 0.6% of their original size, as determined 6 days after culturing. At 3 days or more, epidermal growth factor inhibited collagen gel contraction by fibroblasts (after 6 days: 34.2 ± 1.8%, p > 0.05; 36.5 ± 2.8%, p < 0.05; and 39.8 ± 2.1%, p < 0.001 at 1, 10, and 100 ng/ml of epidermal growth factor, respectively). In conclusion, collagen matrix and epidermal growth factor, particularly in combination, may be useful in the prevention of wound contracture.

CLINICAL EVALUATION OF AN ALLOGENEIC CULTURED DERMAL SUBSTITUTE COMPOSED OF FIBROBLASTS WITHIN A SPONGY COLLAGEN MATRIX

We have developed an allogeneic cultured dermal substitute (CDS) that was prepared by plating fibroblasts on to a spongy collagen matrix and culturing them for 7 to 10 days. The matrix was freeze-dried from a 1% aqueous solution of bovine-hide atelocollagen. This study was designed to evaluate the efficacy of promoting epithelialisation clinically on 26 donor-site wounds for split-thickness skin grafts. One half of a wound was covered with an allogeneic CDS and the other half side was covered with a commercially-available freeze-dried porcine dermis (FPD). Both macroscopically and histologically the epithelialisation on the area of the donor site that was covered with allogeneic CDS was more rapid than that covered with FPD. In a representative donor-site wound covered with allogeneic CDS, there was a stratified structure of epithelial cells on the underlying connective tissue on day 5, and the epithelium had matured by day 12. When covered with FPD a stratified structure of epithelial cells was noted on day 8, and the epithelium had matured by day 15. We conclude that allogeneic CDS provides a good environment for epithelialisation.

Development of a new wound dressing with antimicrobial delivery capability

A bilaminar wound dressing composed of an outer membrane and an inner three‐dimensional matrix of a fabric or a sponge may be considered to constitute an ideal structure that promotes wound healing: the outer membrane prevents body fluid loss, controls water evaporation, and protects the wound surface from bacterial invasion, and the inner matrix encourages adherence by tissue growth into the matrix. Using this concept, we developed a biosynthetic wound dressing with a drug delivery capability. This medicated wound dressing is composed of a spongy sheet of a chitosane derivative and collagen mixture that is laminated to an antimicrobial‐impregnated polyurethane membrane. In this study, a gentamycin sulfate—impregnated wound dressing was prepared and evaluated. The antimicrobial efficacy of this wound dressing was examined on an agar plate seeded with Pseudomonas aeruginosa. Also, the cytotoxicity of an antimicrobial released from this wound dressing was examined in an in vitro system with cultured skin substitutes. Both in vitro tests have shown that this wound dressing is capable of suppressing bacterial growth and minimizing cellular damage. In addition, in the treatment of wounds inflicted on rats and rabbits, this wound dressing was shown to be efficacious in covering full‐thickness and split‐thickness skin defects. Finally, the efficacy of this wound dressing was evaluated in a nonrandomized open‐label study of 31 clinical cases. In 31 cases treated with this wound dressing, good or excellent wound healing was achieved.

Characterization of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and collagen combined with fibroblasts

Abstract The authors have developed an allogeneic cultured dermal substitute (CDS) through cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelocollagen (Col). The Col spongy layer is essential for attachment and proliferation of fibroblasts on the two-layered spongy matrix. The HA spongy layer is necessary for maintaining the moisture environment on the wound surface. The optimal weight ratio of HA/Col is determined by considering the following characteristics: mechanical properties for handling, cell viability after thawing, potency of vascular endothelial growth factor (VEGF) release after thawing, efficacy of wound healing, and manufacturing cost. This study is designed to investigate the physical properties for handling, the growth behavior of fibroblasts on the spongy matrix, and the quantitative analysis of VEGF released from fibroblasts in the fresh or cryopreserved CDS. The results of this study suggest that a CDS composed of Col spongy matrix alone has the highest potency in regard to the release of VEGF. However, taking into account the manufacturing cost, coupled with the potency of VEGF release, a two-layered sponge of HA and Col with a weight ratio of 5/2 is very promising for commercial application.

Allogeneic cultured dermal substitute composed of spongy collagen containing fibroblasts: evaluation in animal test

The authors developed a cultured dermal substitute (CDS) composed of a spongy collagen containing cultured fibroblasts. The cultured fibroblasts derived from Sprague Dawley rat skin were seeded on a spongy collagen at a density of 5 x 10(5) cells cm(-2) and cultured for 7 days. This CDS was applied to the debrided wound of full-thickness burn which was inflicted experimentally on the dorsum of Wister rat, and then the wound conditions were observed over a period of 2 weeks. The comparative study was conducted using an acellular spongy collagen as well as a commercially available temporary wound dressing, Biobrane, since a different type of cultured dermal substitute, Dermagraft-TC, is composed of Biobrane, whose inner site is combined with cultured fibroblasts. Each covering material was applied on the debrided wound area and exchanged by new one 1 week later. When the debrided wound was covered with Biobrane, a small portion of necrotic tissue was observed 1 week after application, and the granulation tissue formation was greatly delayed. This wound area showed a poor granulation tissue even 2 weeks later. On the contrary, when covered with an acellular spongy collagen, no necrotic tissue was observed. This wound area showed a more or less irregular granulation tissue at 1 week and then a healthy granulation tissue 2 weeks later. This preliminary comparative study suggests that an acellular spongy collagen is able to function as a more suitable matrix for CDS, compared with Biobrane. The wound area covered with a CDS assumed a moist, shiny, and hyperaemic appearance 1 week after application showing a healthy granulation tissue. The macroscopic evaluations indicate that the CDS is able to prepare a healthy granulation tissue at an earlier stage, compared with the acellular spongy collagen. In addition, the histologic views demonstrate that the CDS is able to prepare a thicker and denser granulation tissue, compared with the acellular spongy collagen. Although the fate of cultured fibroblasts in the CDS on the wound surface within 1 week is not clear, these findings suggest that fibroblasts in CDS are able to provide excellent conditions for wound healing.

Development of new wound dressing composed of spongy collagen sheet containing dibutyryl cyclic AMP

Although cyclic AMP has been considered to regulate cell proliferation, the mechanism of this function is largely unknown. Recent studies suggest that cyclic AMP promotes the proliferation of skin cells in a dose-dependent manner. An ointment containing dibutyryl cyclic AMP has been used in the treatment of skin ulcers and found to be effective in promoting tissue repair. To search more efficacious wound management, the authors developed a new wound dressing composed of a spongy atelo-collagen sheet containing dibutyryl cyclic AMP. This wound dressing was evaluated in two types of animal tests. One is the application of the wound dressing to a full-thickness skin defect in order to evaluate the granulation tissue formation and the wound size reduction. The wound dressing was found to promote the granulation tissue formation and naturally reduce the wound size. The other test was the application of the wound dressing to the full-thickness skin defect, leaving behind a skin island in a central portion, in order to evaluate the epithelialization. This skin island left in a full-thickness skin defect was extremely enlarged. The enlargement of the skin island seems to be related to the epithelialization from the margin of the skin island as well as by the expansion of a skin island induced by contraction of the developed granulation tissue in the surrounding wound area. These results suggest that an atelo-collagen spongy sheet containing dibutyryl cyclic AMP is effective in promoting the granulation tissue formation and epithelialization.

Evaluation of a Wound Dressing Composed of Hyaluronic Acid and Collagen Sponge Containing Epidermal Growth Factor in Diabetic Mice

Abstract This study investigated the effect of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF) on wound healing in diabetic mice. High-molecular-weight (HMW) HA aqueous solution, hydrolyzed low-molecular-weight (LMW) HA aqueous solution and heat-denatured Col aqueous solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation to the spongy sheet (Type-I wound dressing). In a similar manner, a spongy sheet containing EGF (Type-II wound dressing) was prepared by freeze-drying the mixed solution of HMW-HA, LMW-HA and Col containing EGF. The efficacy of these products was evaluated in type-II diabetic BKS.Cg-+Leprdb/+Leprdb (db/db) mice. Wound dressings were applied to a full-thickness, dorsal skin defect measuring 1.5 cm × 2.0 cm, showing adipose tissue. In the control group, a commercially available artificial dermis composed of collagen spongy sheet (TERUDERMIS®) was used. A commercially available polyurethane film dressing (Bioclusive®) was applied over each wound dressing. After 1 week of application, wound conditions were evaluated based on their gross and histological appearances. Type-I and -II wound dressings promoted a decrease in wound size associated with angiogenesis and granulation tissue formation, compared with the artificial dermis. In particular, Type-II wound dressings promoted sufficient re-epithelialization. These findings indicate that the combination of HA, Col and EGF promotes wound healing by stimulating cell activity including cell migration and proliferation on the adipose tissue in a diabetic wound. Type-I and -II wound dressings would be useful to prepare a well-vascularized wound bed acceptable for split-thickness auto-skin grafting.