Kentaro Kubo

Spongy matrix of hyaluronic acid and collagen as a cultured dermal substitute: evaluation in an animal test

Abstract The authors have developed a two-layered spongy matrix composed of hyaluronic acid (HA) and atelo-collagen (Col) as a cultured dermal substitute (CDS). This spongy matrix was applied to a full-thickness skin defect on the dorsum of Sprague-Dawley rats, and the wound conditions were observed over a period of 2 weeks. A piece of two-layered spongy matrix was applied to the wound, over which a commercially available polyurethane film dressing was applied. Both the macroscopic and the histological evaluations indicate that the two-layered spongy matrix is able to prepare a highly vascularized granulation tissue at an early stage. These findings suggest that this type of spongy matrix would be useful as a CDS.

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.

Development of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and atelo-collagen combined with fibroblasts: fundamental evaluation

We have developed an allogeneic cultured dermal substitute (CDS) by cultivating fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The HA sponge was designed to have a honeycomb structure with many holes (0.5 mm diameter) separated by a distance of 4 mm. Part of the Col sponge was able to penetrate into these holes, and the resulting anchoring structure allows binding of a HA spongy layer with a Col spongy layer. The preparation of the CDS consists of two steps: (i) attachment of cells to the Col surface of the hydrated 2-layered spongy matrix and (ii) proliferation of cells on this sponge immersed in culture medium. The aim of the present study was to assess properties of fresh and cryopreserved CDS. Fibroblasts seeded on the Col surface of the 2-layered spongy matrix attached, proliferated and released vascular endothelial growth factor (VEGF) and fibronectin. The amount of VEGF released from cryopreserved CDS after thawing slowly in an incubator at 37°C and re-cultivation for 1 week was about 300 pg/ml. After thawing quickly in a water bath at 37°C and re-cultivation for 1 week, the amount of VEGF released was about 600 pg/ml. These findings indicate that the cryopreserved CDS maintained its ability to release a significant amount of VEGF. Retention of the therapeutic properties of CDS after cryopreservation is important for clinical use.

Effects of vascular endothelial growth factor released from cultured dermal substitute on proliferation of vascular endothelial cells in vitro

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen. CDS can be cryopreserved and transported to other hospitals in a frozen state. The present study was designed to analyze the amounts of vascular endothelial growth factor (VEGF) released from fibroblasts in fresh and cryopreserved CDS and to investigate the effects of this VEGF on proliferation of vascular endothelial cells in vitro. The culture medium used in preparing CDS (fresh CDS culture medium sample) was collected and stored at −30°C for the quantitative analysis of VEGF. After thawing cryopreserved CDS, it was recultured in a culture medium for 1 week. The culture medium used was collected and stored at −30°C for quantitative analysis of VEGF. The amounts of VEGF released from the fresh and cryopreserved CDS into the culture medium were about 610 pg/ml and 640 pg/ml, respectively. This finding suggests that the cryopreserved CDS retains its ability to release VEGF. Immunohistological analysis indicated that some of the VEGF adhered to the matrix. Human vascular endothelial cells were cultured in medium mixed with the fresh or cryopreserved CDS culture medium sample. Proliferation of vascular endothelial cells was enhanced by increasing the concentration of both CDS culture medium samples. When antihuman VEGF antibody was added to the culture medium, the proliferative activity of vascular endothelial cells was reduced. These findings confirm that VEGF released from CDS promotes proliferation of vascular endothelial cells.

An allogeneic cultured dermal substitute suitable for treating intractable skin ulcers and large skin defects prior to autologous skin grafting: three case reports.

Intractable skin ulcers that arise as secondary lesions from disease and full‐thickness skin defects that result from skin tumor excision often need autologous skin grafting to close the wound. We developed an allogeneic cultured dermal substitute (CDS) to shorten the time needed to prepare a wound bed suitable for autologous skin grafting. The CDS was prepared by plating normal human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelo‐collagen. The allogeneic CDS was then placed on the rinsed wound surface. This procedure was repeated twice a week for up to five weeks, until the wounds were closed by autologous skin grafting. In all three cases, after CDS treatment for two to five weeks, the wound conditions became suitable for skin grafting; these conditions had not been improved by conventional topical treatments, including topical basic fibroblast growth factor (bFGF). Healthy granulation tissue developed rapidly, concomitant with wound size reduction. The present results indicate that CDS is an excellent biological wound dressing for improving wound conditions so that they are suitable for subsequent autologous skin grafting as well as for shortening the treatment duration for skin ulcers and full‐thickness skin defects.

Skin regeneration for children with burn scar contracture using autologous cultured dermal substitutes and superthin auto-skin grafts: preliminary clinical study.

We have evaluated a novel treatment of burn scar contracture in children. This method involves the application of an autologous cultured dermal substitute (CDS), followed by a graft of superthin split-thickness skin. In the first operation, the autologous CDS was applied to the skin defect that had occurred after releasing the scar contracture. In the second operation, a superthin thickness skin graft (4∼6/1000 inches) was applied 5∼12 days after the first operation. The autologous CDS was applied to 10 sites of 5 children. On 8 sites, the skin grafts were contracted to some extent at an early stage. However, these skin grafts were stretched gradually to a range from 60% to 100% of an original size. At 2 sites, the skin grafts had stretched from 110% to 130% of the original size. This strategy may be useful for the treatment of burn scar contracture in children.

Clinical study with allogeneic cultured dermal substitutes for chronic leg ulcers

Background  Clinical studies of the use of allogeneic cultured dermal substitutes (CDSs) have been conducted in 30 medical centers across Japan with the support of the Millennium Project of the Ministry of Health, Labor and Welfare. The CDS is prepared by plating cultured fibroblasts on a spongy matrix made from hyaluronic acid and atelo‐collagen. The aim of the present clinical study was to evaluate an allogeneic CDS as cell therapy in which cytokines are released to promote wound healing.