Development of novel wound dressing composed of hyaluronic acid and collagen sponge containing epidermal growth factor and vitamin C derivative.

This study was designed to investigate the potential of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) spongy sheet containing epidermal growth factor (EGF) and vitamin C derivative (VC). High-molecular-weight HA aqueous solution, hydrolyzed low-molecular-weight 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 of the spongy sheet (C-wound dressing). In a similar manner, three types of spongy sheet containing EGF (EGF-wound dressing), containing VC (VC-wound dressing) or containing EGF and VC (EGF·VC-wound dressing) were prepared by freeze-drying the mixed solution containing the specified components. Cytokine production by fibroblasts was assessed in a wound surface model using a fibroblast-incorporating Col gel sheet (cultured dermal substitute; CDS). CDS was elevated to the air-medium interface, onto which each wound dressing was placed and cultured for 7 days. Fibroblasts in CDS covered with EGF-wound dressing released 3.6 times more VEGF and 3.0 times more HGF, as compared with the C-wound dressing. Fibroblasts in CDS covered with EGF·VC-wound dressing released 4.2 times more VEGF and 6.0 times more HGF, as compared with the C-wound dressing. The efficacy of these wound dressings was evaluated in animal tests using diabetic mice. Each wound dressing was applied to a full-thickness skin defect on the dorsal area measuring 1.5 × 2.0 cm. After 1 week of application, wound conditions were evaluated histologically. The EGF·VC-wound dressing more effectively promoted granulation tissue formation associated with angiogenesis, as compared with other wound dressings.

Potential of wound dressing composed of hyaluronic acid containing epidermal growth factor to enhance cytokine production by fibroblasts.

This study aimed to investigate the potential of a wound dressing composed of hyaluronic acid (HA) containing epidermal growth factor (EGF) to enhance cytokine production by fibroblasts. The present wound dressing has a two-layered spongy structure: an upper layer composed of crosslinked high-molecular-weight HA, and a lower layer composed of low-molecular-weight HA containing arginine (Arg) and vitamin C derivative (VC) with or without EGF. Human fibroblast-embedded collagen gel sheet (cultured dermal substitute: CDS) was elevated to the interface between the air and culture medium to create a wound surface model onto which each wound dressing was placed, which was followed by culture for 7 days. The EGF dressing (with EGF, Arg, VC) significantly enhanced the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CDS as compared to the EGF-free dressing (with Arg, VC). To evaluate if this enhanced production of VEGF and HGF achieved with the EGF dressing is sustained, a second experiment was conducted using a wound surface model. Each wound dressing was placed on the CDS in the wound surface model. Culture was then performed for 3 days (first period), after which each dressing was placed on another CDS for a further 3-day culture period (second period). The EGF dressing enhanced the production of VEGF and HGF by CDS during the first and second periods as compared to the corresponding production when using the EGF-free dressing. These results suggest that EGF can be maintained in the hydrated layer of a wound dressing composed of crosslinked high-molecular-weight HA.

Clinical trial of allogeneic cultured dermal substitutes for intractable skin ulcers.

The effect of allogeneic cultured dermal substitute (CDS) on wound healing was evaluated in 9 intractable skin ulcers in 5 patients who had failed to improve despite conventional topical treatment with basic fibroblast growth factor (bFGF) for more than 2 months. In general, the topical application of bFGF is effective in facilitating wound healing. However, skin regeneration was very slow in the present 9 cases. In this study, to improve the condition of these wounds, allogeneic CDS was applied once a week for 2 months. The wound healing process was evaluated, focusing on the reduction ratio of wound size through the granulation tissue formation associated with epithelialization. In all 9 cases, the wound size was successfully decreased after the application of CDS, and ulcers were completely resurfaced in 2 cases. In all cases, except the 2 cases showing complete wound closure, the mean wound size decreased to 33.3% of the original size, i.e., a mean reduction ratio of 33.3%. The present results indicate that allogeneic CDS can promote wound healing of intractable skin ulcers that fail to improve despite treatment with bFGF.

Treatment of intractable skin ulcers caused by vascular insufficiency with allogeneic cultured dermal substitute: a report of eight cases.

Chronic leg ulcers have various causes and can be difficult to treat, although topical treatments, including basic fibroblast growth factor and PGE1, have been used. We applied an allogeneic cultured dermal substitute (CDS) to eight patients with intractable ulcers. The patients had various underlying diseases, including diabetes mellitus, systemic lupus erythematosus, antiphospholipid syndrome, necrobiosis lipoidica, stasis dermatitis, livedo vasculopathy, and rheumatoid arthritis. The CDS was prepared by seeding cultured human fibroblasts on a spongy matrix consisting of hyaluronic acid and atelocollagen. Good clinical results were achieved, as demonstrated by reepithelization, healthy granulation tissue formation, and a subsequent decrease in wound size. Daily dressing changes became unnecessary when the allogeneic CDS was used. Based on these results, we suggest that CDS may be useful for the treatment of intractable skin ulcers.

Response of intractable skin ulcers in recessive dystrophic epidermolysis bullosa patients to an allogeneic cultured dermal substitute.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder caused by mutations in the COL7A1 gene, which encodes collagen VII (COL7). Skin ulcers in RDEB patients are sometimes slow to heal. We describe here the therapeutic response of intractable skin ulcers in two patients with generalized RDEB to treatment with an allogeneic cultured dermal substitute (CDS). Skin ulcers in both patients epithelialized by 3-4 weeks after this treatment. Immunohistochemical studies demonstrated that the COL7 expression level remained reduced with respect to the control skin and that it did not differ significantly between graft-treated and untreated areas. Electron microscopy showed aberrant anchoring fibrils beneath the lamina densa of both specimens. In conclusion, CDS is a promising modality for treatment of intractable skin ulcers in patients with RDEB, even though it does not appear to increase COL7 expression.

A case of lower-extremity deep burn wounds with periosteal necrosis successfully treated by use of allogenic cultured dermal substitute.

In patients with burns, bone exposure accompanies serious problems which occasionally lead to amputation. We present a case of an 82-year-old woman who sustained 22% of total body surface area flame burns on her bilateral lower extremities with bone exposure. Despite fascial excision and mesh skin graft, muscles, bones, and tendons were widely exposed on her right leg. The wound was infected by methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. To promote wound healing, we applied an allogeneic cultured dermal substitute (CDS) to the wound surface once weekly, resulting in healthy granulation except for the exposed bone area of the right anterior tibia. We then shaved the cortex of the exposed bone surface until bone marrow bleeding, and grafted mesh skin in combination with CDS. Finally, all wounds healed without osteomyelitis. The use of CDS to treat deep burns exposing bone surface may expand reconstructive options for extremities that otherwise might have been amputated.

Optimum preservation for autologous cultured dermal substitutes.

The application of autologous cultured dermal substitute (CDS) is a promising procedure for improving burn scar contracture. Preparation of CDS requires a period of about 3 weeks before the date of surgery. When the date of surgery is postponed, CDS must be preserved under optimum conditions. This study was thus designed to investigate these conditions. CDS was preserved in culture medium for 2 weeks at 37 degrees C, 25 degrees C, or 4 degrees C in dishes sealed with tape. During this period, culture medium was exchanged every week. CDS fibroblast activity was then examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell activity was best retained at 37 degrees C, as compared with the other 2 temperatures. The amount of vascular endothelial growth factor (VEGF) released from CDS was measured by enzyme-linked immunosorbent assay. At 37 degrees C, the amount of VEGF was markedly higher. In contrast, the amount of VEGF decreased at the other 2 temperatures. The results of both MTT assay and enzyme-linked immunosorbent assay indicate that preservation of CDS should be carried out at 37 degrees C.

Effective treatment of intractable skin ulcers using allogeneic cultured dermal substitutes in patients with systemic lupus erythematosus.

Skin ulcers in systemic lupus erythematosus (SLE) patients are non-healing or intractable, because various factors or complications, including vasculitis and immunosuppressants, impair wound healing. In the present study, we applied cultured dermal substitutes (CDSs) to 3 cases of SLE skin ulcers because various systemic or topical therapies were ineffective. CDSs are prepared by culturing human fibroblasts on two-layered spongy matrices of hyaluronic acid and atelo-collagen, and they effectively promote the healing of severe skin defects. After using CDSs in the 3 cases, healthy granulation tissues formed within 6 weeks, and skin grafts were successfully performed. These results indicate that allogeneic CDSs provide new therapeutic alternatives as topical therapies for intractable skin ulcers in SLE.

Standardization for mass production of allogeneic cultured dermal substitute by measuring the amount of VEGF, bFGF, HGF, TGF-beta, and IL-8.

Fibroblasts were isolated from a piece of donated skin measuring about 1 x 1 cm and were proliferated over nine successive cultivations. Fibroblasts obtained from the fourth cultivation were cryopreserved in 10 cryotubes as master cells. Fibroblasts obtained from the fifth to ninth cultivations were cryopreserved in 49 cryotubes in total as working cells. At the end of each procedure in the fourth to eighth cultivations, about three-quarters of the proliferated fibroblasts were cryopreserved, and the remaining one-quarter were proliferated in the next cultivation. All fibroblasts obtained from the ninth (final) procedure were cryopreserved as the last working cells. Two cryotubes of the last cryopreserved working cells (ninth cultivation) were thawed and proliferated. Cultured dermal substitute (CDS) was prepared by performing ten successive cultivations. After each cultivation, about three-quarters of the resulting fibroblasts were used to prepare CDS, and the remaining one-quarter were proliferated in the next cultivation for 1 week. Finally, all fibroblasts produced by the tenth (final) procedure were used to prepare CDS. In each of the first to ninth cultivations for CDS preparation, the density of fibroblasts remained relatively constant, and the levels of cytokines, i.e., vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor-beta 1, and interleukin-8 released from CDS remained relatively constant. In total, 47 sheets of CDS measuring 10 x 10 cm were prepared. The present findings indicate that about 1000 sheets of CDS measuring 10 x 10 cm can be prepared using all the working cells prepared from a piece of donated skin measuring 1 x 1 cm.

Application of cultured dermal substitute for amelioration of maxillary bone growth suppression after cleft palate operation in rats.

The growth and development of maxillary bone and dentition is often impaired in patients who have undergone the cleft palate operation (push back method). Wound contraction exerts adverse effects on maxillary bone growth. The present study investigates the ability of cultured dermal substitute (CDS) to ameliorate maxillary bone growth suppression in experimental animals. We prepared CDS by incorporating rat fibroblasts into a matrix comprising a spongy hyaluronic acid (HA) sheet combined with collagen gel. The number of fibroblasts in the CDS was adjusted to 1.0 x 10(5) cells/cm(2). Wistar rats were randomly assigned to one of the following groups: I, control (no operation); II, surgically exposed bone without matrix or CDS; III, surgery with matrix; IV, surgery with CDS. Under pentobarbital anesthesia, about 2 x 4 mm of the mucosa and periosteum was surgically removed from the left half of the palate. A silicone sheet was placed on the matrix or CDS and affixed with superglue. The palatal width was measured 9 weeks later in skull preparations as the distance between the cheek side cusps of the second molar. The palates in group IV were significantly wider than those in groups II and III, and did not significantly differ from that in the control group. These findings indicated that CDS has the ability to promote wound healing and reduce scar formation through the synergic effects of fibroblasts and the matrix, and thereby to ameliorate indirectly the growth of maxillary bone.

Comparative evaluation of re-epithelialization promoted by fresh or cryopreserved cultured dermal substitute.

Allogeneic cultured dermal substitute (CDS) was prepared by plating cultured fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen, followed by culturing for 1 week. The resulting fresh CDS was then cryopreserved in a freezer at -152 degrees C in accordance with conventional procedures. Fresh CDS was rinsed thoroughly with lactated Ringer's solution to remove fetal bovine serum (FBS) and was then used in the clinical study, whereas cryopreserved CDS was thawed and then rinsed with lactated Ringer's solution to remove cryoprotectant and FBS. The present study was designed to compare the efficacy of fresh and cryopreserved CDS in promoting reepithelialization at donor sites leaving behind split-thickness skin autografts. Fourteen donor sites were used for this comparative study. There were no differences in the period of complete re-epithelialization between the fresh and cryopreserved CDS. The results of this comparative study thus suggest that cryopreserved CDS is able to maintain the same level of potency to promote re-epithelialization as fresh CDS. This indicates that, although the release of growth factors is suppressed to some extent during the course of cryopreserving, thawing, and rinsing procedures, it is still sufficient to promote re-epithelialization.

Root coverage with cultured gingival dermal substitute composed of gingival fibroblasts and matrix: a case series.

Cultured gingival dermal substitute (CGDS), composed of gingival fibroblasts and matrix and fabricated using tissue-engineering techniques, has been used for root coverage procedures. Fourteen sites from four patients with > or = 2 mm of Miller Class I or II facial gingival tissue recession were treated. The autologous CGDS sheet, prepared prior to surgical treatment, was grafted over the teeth with gingival recession and then covered with a coronally positioned flap. Vertical and horizontal recession was measured at baseline (prior to the surgical procedure) and 13 to 40 weeks (average: 30.7 +/- 9.6 weeks) after surgery. The average vertical and horizontal root coverage after surgery was 79.1% +/- 25.7% and 75.2% +/- 31.4%, respectively. Moreover, there was a significant increase of keratinized and attached gingival tissue at the final clinical evaluation compared with preoperative measurements (P < .05). These results demonstrate CGDS as a promising grafting material for use with root coverage procedures in periodontal therapy.

Case report: a case of pyoderma gangrenosum with intractable leg ulcers treated by allogeneic cultured dermal substitutes.

Pyoderma gangrenosum (PG) is an idiopathic, inflammatory, ulcerative disease of undetermined cause. Both topical and systemic treatments of corticosteroids and cyclosporine are commonly used for the ulcers of PG, but these ulcers are often intractable despite treatment. We employed allogeneic cultured dermal substitutes (CDS) in a patient with intractable ulcers due to PG. The CDS was prepared by cultured human fibroblasts on two-layered sponges of extracellular matrix such as hyaluronic acid and atelo-collagen. In the present case, re-epithelization and healthy granulation were induced by the CDS without increasing the dosage of systemic prednisolone. Then the subsequent autologous skin graft was successfully performed. This indicates that CDS is one of the useful tools for the treatment of intractable ulcers in patients with PG.

Intractable venous leg ulcer treated successfully with allogeneic cultured dermal substitute.

Venous leg ulcers are resistant to various treatments, including autologous skin grafting. We applied an allogeneic cultured dermal substitute in a patient with such a wound, and the wound improved, healthy granulation tissue formed, and the size of the wound was reduced.

[Regenerative medicine for skin].

Tissue engineering is moving rapidly from fundamental research to commercial applications. The first product is an autologous cultured epidermal substitute. Other representative products are allogeneic cultured dermal substitute and allogeneic cultured skin substitute. Surgical closure with auto-skin grafting is the gold standard for treatment of victims with extensive deep skin defects. Therefore, the tissue engineers have to develop the cultured skin substitutes, taking into account the successful application of auto-skin grafting, Kuroyanagi developed an allogeneic cultured dermal substitute, which was composed of a 2-layered spongy matrix of hyaluronic acid and atelo-collagen containing fibroblasts. This product is able to release a number of biologically active substances that are necessary for wound healing. A multi-center clinical study on the use of this product has been performed at 30 hospitals across Japan as the Regenerating Medical Millennium Project of the Ministry of Health, Labor and Welfare.

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.

[Advances in regenerative medicine for skin].

A typical engineered product is autologous cultured epidermal substitute, which is composed of stratified keratinocytes (in USA). There are two types of allogeneic cultured dermal substitute (CDS) composed of fibroblasts combined with a synthetic scaffold (in USA). Another product is allogeneic cultured skin substitute, which is composed of keratinocytes and fibroblasts combined with a collagen gel (in USA). Recently, however, the commercialization of these allogeneic products has been discontinued. There seem to be some problems in the design of these products. Kuroyanagi et al. developed an allogeneic CDS composed of a fibroblast combined with a 2-layered spongy matrix of hyaluronic acid and collagen. A multicenter clinical study on the use of allogeneic CDS was performed in 30 hospitals across Japan as the Regenerating Medical Millennium Project of the Ministry of Health, Labor and Welfare. The results of clinical study suggest that this type of allogeneic CDS can effectively promote the healing of full-thickness severe skin defects, such as chronic ulcers and burn injuries.

Digital gangrene associated with idiopathic hypereosinophilia: treatment with allogeneic cultured dermal substitute (CDS).

In the present case study, the patient was a 65-year-old man who suddenly developed purpuric and necrotic lesions with severe pain in his fingers and toes. Laboratory investigations revealed marked eosinophilia (77.9%), but there was no evidence to support a diagnosis of parasitic infections, allergic disease, neoplasm or connective tissue disorder. The histopathological findings did not show any distinct vasculitis, but there were obliterative changes of the arterioles. The digital gangrene gradually progressed and was unresponsive to corticosteroid therapy. The patient eventually underwent amputation of the distal phalanges. We applied allogeneic cultured dermal substitute (CDS) to the skin defect. The allogeneic CDS was prepared by culturing fibroblasts on a two-layered sponge of hyaluronic acid and atelo-collagen. This CDS is able to release a number of cytokines including VEGF. The present case had a good clinical result.

Clinical evaluation of allogeneic cultured dermal substitutes for intractable skin ulcers after tumor resection.

Clinical research on allogeneic cultured dermal substitute (CDS), which was newly developed at the R&D Center for Artificial Skin of Kitasato University, has been carried out in medical centers across Japan with the support of the Millennium Project of the Ministry of Health, Labor and Welfare of Japan. Allogeneic CDS was prepared by cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelo-collagen. This paper reports the clinical results of application of allogeneic CDS in 12 patients with full-thickness skin defects after surgical resection of skin tumors. In 9 of 10 patients, healthy granulation tissue developed immediately, allowing us to perform split-thickness skin grafts at an early stage. In two cases, allogeneic CDS was used to cover an expanded mesh skin graft that had been applied to treat a large ulcer, and rapid epithelization was observed. No patient developed local infection nor local tumor recurrence after treatment with CDS. The spongy matrix itself as well as the vascular endothelial growth factor (VEGF) released by the allogeneic CDS seemed to be beneficial for the treatment of intractable skin ulcers. Allogeneic CDS functions as an excellent biological dressing, and could dramatically change the treatment of intractable skin ulcers.

[Regenerative medicine for skin].

Regenerative tissue engineering is moving rapidly from the fundamental research to the commercial applications. A number of skin substitutes have been produced by in vitro culture techniques. The U.S. Food and Drug Administration has already approved allogeneic skin substitutes. Recently, the Japanese Ministry of Health, Labor and Welfare published the guide-line for regenerative medicine. R & D Center for Artificial Skin of Kitasato University is now the heart of the Regenerating Medical Millennium Project (skin department) of the Ministry of Health, Labor and Welfare since 2000. This center established the banking system for multi-center's clinical study in 27 hospitals across Japan, using the allogeneic cultured dermal substitute composed of hyaluronic acid and collagen spongy matrix with fibroblasts.