Satoru Takemoto

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.

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.

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/cm2 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/cm2). 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/cm2 bFGF displayed accelerated dermis‐like tissue formation and the CGSs impregnated with 14 µg/cm2 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/cm2 bFGF groups. CGSs impregnated with 7–14 µg/cm2 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. Copyright

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

Abstract 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 &mgr;g/cm2) 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-&mgr;g/cm2 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 patients 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, Dulbeccos 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 β1 (TGF-β1) 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-β1 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.

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

BackgroundA 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 MethodsTwelve 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. ResultsSecondary 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). ConclusionsLaser Doppler imaging could be a useful and noninvasive technique for the evaluation of blood flow to the artificial dermis before grafting.