Immediate evaluation of neovascularization in a grafted bilayered artificial dermis using laser Doppler imaging.

BACKGROUND: A bilayered artificial dermis is widely applied for skin defects. Its collagen sponge is biodegraded and replaced with dermis-like tissue after application. There is no reliable method for quantitatively evaluating the blood flow of artificial dermis. In this study, we used laser Doppler imaging to evaluate the perfusion of artificial dermis. MATERIALS AND METHODS: Twelve patients treated with artificial dermis and secondary skin grafting were included. We measured the perfusion unit just after application of artificial dermis, 1 week after, and before skin grafting. RESULTS: Secondary skin grafts of 6 patients took completely, and the others showed partial necrosis. Laser Doppler imaging could detect blood flow in the artificial dermis, and a significant difference was observed in perfusion units between the "complete take" group and "partial necrosis" group before grafting (P < 0.05). CONCLUSIONS: Laser Doppler imaging could be a useful and noninvasive technique for the evaluation of blood flow to the artificial dermis before grafting.

An exploratory clinical study on the safety and efficacy of an autologous fibroblast-seeded artificial skin cultured with animal product-free medium in patients with diabetic foot ulcers.

Cultured dermal substitutes have been used for the treatment of chronic skin ulcers; however, the biological risks of animal-derived materials in the culture process such as foetal bovine serum (FBS) have been reported. In this study, we prepared an autologous fibroblast-seeded artificial dermis (AFD) using animal-product-free medium supplemented with 2% patient autologous serum and without any animal-derived materials such as trypsin in the culturing process. We applied the AFD in five patients with diabetic ulcers and investigated its safety and efficacy. As the primary endpoint, we defined 'wound bed improvement' according to the percentage of granulation area to the whole wound area on day 21, and 60% or higher was regarded as improved. The mean age of the patients was 60·6 years and the mean duration of the ulcer was 22·6 months. In the evaluation of the primary endpoint, the 'wound bed' was improved in all patients [proportion of improvement: 100%, 95% confidence interval (CI): 48% to 100%]. Three patients had complete wound healing within 12 weeks after application and two patients had >80% wound healing at 12 weeks. Side effects were not serious. Our AFD may be a safe and effective treatment of diabetic ulcers.

Efficacy of novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor for dermis-like tissue regeneration.

We have developed collagen/gelatin sponges (CGS) with a gelatin concentration of 10 wt% to sustain the release of basic fibroblast growth factor (bFGF). The objective of this study is to elucidate the efficacy of CGS impregnated with different concentrations of bFGF, using mouse skin defects. CGSs impregnated with normal saline solution (NSS) or bFGF solution (1, 7, 14, or 50 µg/cm) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, and total area of newly formed capillaries in CGS were evaluated. The group of CGS with 7-µg/cm bFGF was significantly superior to the NSS group in all evaluated items. CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation 2 or 3 times earlier than existing artificial dermis. The combination of CGS and bFGF could solve the problem of the existing artificial dermis and be very promising for the treatment of skin defects.

The utilization of animal product-free media and autologous serum in an autologous dermal substitute culture.

BACKGROUND: Cultured dermal substitutes are used for the treatment of skin ulcers. However, the biological risks of fetal bovine serum (FBS) in the culture process have been reported. The use of the patient's autologous serum (AS) is another possibility, but the amount available is limited. In this study, we examined whether animal product-free media (HFDM-1) supplemented with 2% AS could support the growth of autologous fibroblasts in primary culture and their dissemination to dermal substitutes. MATERIALS AND METHODS: We cultured autologous fibroblasts using HFDM-1 with 2% AS, Dulbecco's modified eagle medium (DMEM) with 10% FBS, and DMEM with 10% human serum (HS). Then, we disseminated and cultured the cells for 10 d. The fibroblast proliferation and concentrations of vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGF-ß1) in each medium, as well as the deposition of human type I collagen into dermal substitutes were examined. RESULTS: The number of fibroblasts cultured in HFDM-1 with AS was highest. After seeding, the number of fibroblasts cultured in DMEM with HS was higher than those in DMEM with FBS and HFDM-1 with AS, but no significant difference was found between these two media. The VEGF concentration in DMEM with HS was also larger, but no significant difference was found between two other media. No significant difference was observed in TGF-ß1 concentration or the deposition of collagen. CONCLUSIONS: This study shows that HFDM-1 with 2% AS can be used to produce cultured dermal substitutes instead of DMEM with 10% FBS.

Collagen-gelatin scaffold impregnated with bFGF accelerates palatal wound healing of palatal mucosa in dogs.

BACKGROUND: We have developed a collagen-gelatin sponge (CGS) as a scaffold capable of the sustained release of bFGF to improve the healing process of the existing collagen scaffold. The aim of this study was to evaluate the efficacy of CGS impregnated with basic fibroblast growth factor (bFGF) in palatal wound healing in beagles. MATERIALS AND METHODS: Four standardized 6 mm diameter full-thickness wounds were made in the palate of each dog and covered with CGS impregnated with normal saline or bFGF at concentrations of 1 µg/cm2, 7 µg/cm2 and 14 µg/cm2. One and 2 wk after surgery, the wound area, neoepithelium length, thickness, area of regenerated submucosal tissue, and the number and total area of neoformed capillaries were evaluated. RESULTS: Two weeks after implantation, wounds treated with bFGF 7 µg/cm2 and 14 µg/cm2 were completely epithelized, while the length of the neoformed epithelium was significantly longer in the 7 µg/cm2 group. Groups impregnated with bFGF 7 µg/cm2 and 14 µg/cm2 showed promoted regeneration of submucosal tissue 2 wk later. The number and area of neoformed capillaries were significantly higher in the bFGF 7 µg/cm2 group than in other groups. We conclude that palatal wound healing in the bFGF 7 µg/cm2 group was promoted with good neovascularization and showed less contracture than other groups. CONCLUSIONS: Our new collagen-gelatin scaffold, CGS, impregnated with bFGF, could be a promising treatment to accelerate the regeneration of palatal mucosa.

Resurfacing of the donor defect with a second toe plantar flag flap after free first toe pulp flap.

A second toe plantar flap was attached to the donor site after a free first toe pulp flap. Twenty-two days postoperatively, the wound had almost healed. Although the whole defect is impossible to cover with this flap, partial resurfacing will reduce the healing time and morbidity from recurrent ulceration.

An acquired giant vascular tumour of the breast.

We present a rare case of an acquired giant vascular tumour of the breast that was diagnosed as angiomatosis. It was characterised by the mixture of blood-filled haemangiomatous, and apparently empty lymphangiomatous, vascular channels. The breast remnant returned to a normal configuration and contour without breast reduction.

Giant basal cell carcinoma: improvement in the quality of life after extensive resection.

We describe a rare case of giant basal cell carcinoma which invaded the orbital tissue and the anterior skull base. Though the eyeball in the right orbit was preserved with the tumour at the patient's request, the improvement in the quality of the patient's life was achieved.

Evaluation of a novel collagen-gelatin scaffold for achieving the sustained release of basic fibroblast growth factor in a diabetic mouse model.

The objective of this study was to evaluate the ability of a scaffold, collagen-gelatin sponge (CGS), to release basic fibroblast growth factor (bFGF) in a sustained manner, using a pressure-induced decubitus ulcer model involving genetically diabetic mice. We confirmed that CGSs impregnated with a bFGF concentration of up to 50 µg/cm(2) were able to sustain the release of bFGF throughout their biodegradation. We prepared decubitus ulcers on diabetic mice. After debriding the ulcers, we implanted CGSs (diameter 8 mm) impregnated with normal saline solution (NSS) or bFGF solution (7, 14, 28 or 50 µg/cm(2)). At 1 and 2 weeks after implantation, the mice were sacrificed and tissue specimens were obtained. The wound area, neoepithelium length and numbers and total area of newly formed capillaries were evaluated. The CGSs impregnated with NSS became infected and degraded, whereas the CGSs impregnated with 7 or 14 µg/cm(2) bFGF displayed accelerated dermis-like tissue formation and the CGSs impregnated with 14 µg/cm(2) bFGF produced significant improvements in the remaining wound area, neoepithelium length and numbers and total area of newly formed capillaries compared with the NSS group. No significant difference was observed between the NSS and 50 µg/cm(2) bFGF groups. CGSs impregnated with 7-14 µg/cm(2) bFGF accelerated wound healing, and an excess amount of bFGF did not increase the wound-healing efficacy of the CGSs. Our CGS is a scaffold that can release positively charged growth factors such as bFGF in a sustained manner and shows promise as a scaffold for skin regeneration.

Novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor: clinical trial for chronic skin ulcers.

Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in healthcare. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), which is capable of sustained release of basic fibroblast growth factor (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Patients with chronic skin ulcers that had not healed in at least 4 weeks were treated with CGS impregnated with bFGF at 7 or 14 µg/cm(2) after debridement, and the wound bed improvement was assessed 14 days after application. Wound bed improvement was defined as a granulated and epithelialized area on day 14 with a proportion to the baseline wound area after debridement of 50% or higher. The wound area, the wound area on day 14, and the granulation area on day 14 were independently measured by blinded reviewers in a central review using digital images of wounds taken with a calibrator. Patients were followed up until 28 days after application to observe the adverse reactions related to the application of CGS. From May 2010 to June 2011, 17 patients were enrolled and, in 16 patients, the wound bed improved. Among the randomized patients in step 2, no significant difference was seen between the low-dose group and the high-dose group. No serious adverse reactions were observed. Adverse reactions with a clear causal relationship to the study treatment were mild and patients quickly recovered from them. This study is the first-in-man clinical trial of CGS and showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. This combination therapy could be a promising therapy for chronic skin ulcers.

An exploratory clinical trial for combination wound therapy with a novel medical matrix and fibroblast growth factor in patients with chronic skin ulcers: a study protocol.

BACKGROUND: Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in health care. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), that is capable of the sustained release of basic fibroblast growth factor (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Methods/ DESIGN: Seventeen patients (≥ 20 years of age) with chronic skin ulcers that have not healed by conventional therapy for at least 4 weeks are being recruited. Patients will be applied with CGS impregnated with bFGF of 7 µg/cm(2) or 14 µg/cm(2) after debridement, and the wound bed improvement will be assessed 14 days after application. "Wound bed improvement" is defined as a granulated and epithelialized area on Day 14 in proportion to the baseline wound area after debridement of 50% or higher. Patients will be followed up until 28 days after application to observe the adverse events related to the application of CGS. CONCLUSION: This study has been designed to address the safety and efficacy of CGS impregnated with bFGF. If successful, this intervention may be an alternative to bioengineered skin substitutes and lead to substantial and important changes in the management of chronic skin ulcers such as diabetic ulcers and venous ulcers.

Nicotine at a low concentration promotes wound healing.

BACKGROUND: The adverse effects of smoking on wound healing of the skin are known clinically. Recently, an endogenous cholinergic pathway for angiogenesis mediated by endothelial nicotinic acetylcholine receptors was discovered. The objective of this study was to investigate the appropriate concentration of nicotine at which angiogenesis and wound healing are accelerated in a murine excisional wound model. MATERIALS AND METHODS: Full-thickness skin defects (8 mm) were created on the dorsum of C57BL mice and a silicone sheet (8 mm) was sutured. PBS (10 microL), bFGF (1 microg), nicotine (10(-1) M, 10(-3) M, 10(-4) M, 10(-7) M, and 10(-10)M), and both bFGF and 10(-4) M nicotine were topically injected for 7 days. Mice were sacrificed on day 8, and the wound area, the neoepithelium length, and the area of newly formed capillaries were assessed. RESULTS: The wound area was significantly decreased in the wound treated with bFGF, with 10(-4) M nicotine, and with both bFGF and 10(-4) M nicotine. The length of the epithelium was significantly longer and the area of capillaries was also increased significantly in these three groups. The wound area, the length of the epithelium, and the area of capillaries in the group treated with both bFGF and 10(-4) M nicotine were significantly different from those in the 10(-4) M nicotine-treated group. CONCLUSIONS: In this study, nicotine at a low concentration accelerated angiogenesis and promoted wound healing; these effects of nicotine were synergistic with bFGF.

Preparation of collagen/gelatin sponge scaffold for sustained release of bFGF.

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.

In vivo culturing of a bilayered dermal substitute with adipo-stromal cells.

BACKGROUND: Skin grafting is an important procedure to cover skin defects. Recently, cultured epidermal sheets and bilayered cultured skin have been used clinically, but they lack subcutaneous tissue. The objective of this study was to produce a bilayered dermal substitute with adipose tissue simultaneously in vivo. MATERIALS AND METHODS: We disseminated adipo-stromal cells on one side of a collagen sponge at a density of 1,0 x 10(5)cells/cm(2) and incubated overnight. Then, we turned over the sponge and disseminated dermal fibroblasts and keratinocytes at a density of 1,0 x 10(6)cells/cm(2) on the other side of the sponge. Finally, we cultured this for 1 wk and implanted it on the backs of severe combined immunodeficiency mice with or without basic FGF. RESULTS: Six weeks after implantation, specimens were harvested. Macroscopically, the formed tissue in the bFGF-administered group was thick, and the epidermal component, the dermal component, and adipose tissue were formed in the cross section. The thickness of newly formed tissue in bFGF-administered group was significantly greater than that in the group without bFGF administration. The area of the newly formed capillaries in the bFGF-administered group was significantly larger than that in the group without bFGF administration. CONCLUSIONS: We could produce a thick composite tissue in vivo, combining three kinds of human cells, collagen scaffold, and bFGF. This composite graft was thicker than the bilayered dermal substitute and could be a substitute for a skin flap.