We investigated the effect of basic fibroblast growth factor (bFGF) on the morphogenesis of living skin equivalent (LSE). The thickness of dermal portion of LSE was not suppressed by the addition of bFGF into the medium compared to control. Immuohistochemical staining showed that both keratin 1 and keratin 10 were expressed at upper layer of the epidermis. The expression of keratin 6 and keratin 16, which are known as hyperproliferative keratins, was suppressed by the addition of bFGF compared to control. Alpha smooth muscle actin was strongly expressed at the site of dermo-epidermal junction in the LSEs without bFGF, whereas its expression was markedly suppressed in the LSEs with bFGF. Taking together, the addition of bFGF into the medium might improve the morphogenesis of LSE.

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Although ischemia has been considered the main factor of pressure ulcers, increasing evidence demonstrates a principal role of ischemia-reperfusion (IR). This study assessed the mechanism of pressure ulcer formation using a cutaneous IR injury model by external application of two magnetic plates. In this model, monocyte chemoattractant protein-1 (MCP-1) was remarkably increased, and following infiltration of macrophages and augmented expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) were observed. Therefore, IR cycles were performed in MCP-1 deficient (MCP-1^-/-) mice to evaluate the role of this chemokine in pressure ulcer development. MCP-1^-/- mice showed reduced macrophage infiltration and expression of tumor necrosis factor-α and iNOS during IR cycles leading to attenuated skin injury. MCP-1 played a role in injury during the reperfusion rather than the ischemic period. These findings suggest that recruitment of macrophages from increasing MCP-1 and subsequent release of proinflammatory cytokines and toxic oxygen-derived free radicals induce the development of pressure ulcers during IR cycles.

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A number of studies have reported that basic growth factor (bFGF) promotes the proliferation of chondrocytes, however, the effect of bFGF on elastic cartilage has not yet been reported.
Here we studied the effect of bFGF on the elastic cartilage in rabbit ears (n=3).
In rabbits, bFGF (100μg/ml : Fiblast^® spray) 0.10ml was injected into the subcutaneous tissues of the ear. Specimens of auricular cartilages were obtained 1 day, 3 days, 7 days, 14 days, 30 days, or 90 days after injection. Histological stain (H.E.; Hematoxilin-Eosin EVG ; Elastica van Gieson) and immunochemical stain (S-100 protein, PCNA ; Proliferation cell nuclear antigen) were performed each time.
In the group administered bFGF, perichondrial cells were significantly increased 7 days after bFGF injection, and cartilage matrix was formed in 14 days. Neocartilage cells were increased at 30 days and 90 days. However, adult chondrocytes of auricular cartilage were hardly increased at any time point.
These findings suggested that bFGF is able to stimulate proliferation of elastic perichondrial cells, which differentiate into adult chondrocytes. These effects persisted for 90 days after a single injection of bFGF.

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Myostatin (MSTN) is transiently expressed in the developing skeletal muscle, and negatively regulates muscle growth. A loss of function mutation of the MSTN gene is known to result in excess muscle formation with elevated expression of Wnt4. To examine direct effect of Wnt4 on skeletal muscle formation, Wnt4 cDNA was misexpressed in the presumptive limb field of chick embryos using retrovirus vector. Significant increase in muscle mass was observed in the Wnt4-treated limb compared to the control. The area for fast-type myosin heavy chain-expressing cells showed a significant increase, suggesting the possible involvement of Wnt4 during fast-type muscle formation after MSTN knockout.

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Dermal fibroblasts as well as epidermal keratinocytes show specific characteristics depending on body-sites. We have examined dermal fibroblasts derived from palmo-plantar and trunk skin and oral mucosa, in order to reveal their site-specific characteristics. In this study, we compared the expression of extracellular matrix (ECM) molecules among each body-site fibroblasts after treatment with basic fibroblast growth factor (bFGF). The specific characteristics of dermal fibroblasts were not affected and well-preserved even after bFGF treatment. bFGF decreased expression of fibronectin especially at the early stage of the proliferation, and bFGF might act to promote the proliferation of fibroblasts by inhibiting the expression of ECM. Additionally, bFGF also decreased expression of type I collagen, type III collagen and matrix metalloproteinase (MMP)2 after confluent culture. Meanwhile, those of MMP1 and tissue inhibitor of metalloproteinase 1 were increased. Especially, fibroblasts derived from oral mucosa prominently expressed MMP1 by bFGF. Oral mucosa fibroblasts might have different characteristics of response to bFGF, compared to other skin fibroblasts.

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Basic fibroblast growth factor (bFGF,FGF-2) is a potent mitogen for a wide variety of cells, such as fibroblasts, keratinocytes and endothelial cells. bFGF also induces the differentiation, angiogenesis and chemotaxis of a variety of cells. In wound healing, bFGF particularly stimulates the formation of mature granulation tissue and plays a key role in angiogenesis and re-epithelization. Recombinant human bFGF has been widely used as a progressive therapeutic drug for skin ulcers in Japan since 2001. In the future, bFGF will be used in the regeneration of bone tissues and alveolar bones. In the present study, we analyzed the proliferation of cultured human dermal fibroblasts. We found that bFGF stimulated the proliferation of the cells in a dose-dependent manner in conditions with low-dose (1%) bovine serum, however, bFGF did not stimulate the proliferation of cells without serum. We suggest that bFGF and unknown molecules in serum have cooperative effects in the activation of proliferation.

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Human recombinant basic fibroblast growth factor (bFGF) has been available for the treatment for non-healing skin ulcer, however, there still exist unknown biological effects on wound healing. Fibroblast-collagen matrix has been used as a model system to study how cells organize connective tissue. Previous our works showed that PI3K, Rac and Rho kinase are involved in bFGF-stimulated collagen matrix contraction. Recent clinical reports have pointed out bFGF promotes scarless wound healing. The mechanisms, however, are still unclear. The current studies were carried out to elucidate the mechanisms. It has been reported that transforming growth factor-β1 (TGF-β1) was required to activate fibroblasts to express the myofibroblast phenotype in vitro, i.e., increased expression of α-smooth muscle actin (αSMA). In the present study, we employed this technique to obtain myofibroblasts.
Levels of cellular αSMA increased after 2-4 days of TGF-β1 treatment alone and were consistently elevated after 5 days. However, levels of cellular αSMA increased 4-6 days after costimulation of bFGF and TGF-β1. Although spontaneous contraction was seen in stressed myofibroblast-collagen matrix, bFGF could cancel the gel contraction. There were not significant differences on cell spreading in myofibroblast-collagen gel, however, the number of cells was decreased when they were stimulated with bFGF. bFGF stimulation for myofibroblasts as well as fibroblasts caused transient Rac and Rho activation. Levels of diphosphorylated myosin light chain (MLC) were highest in fibroblasts 30 mins after bFGF stimulation. In contrast, basal level of diphosphorylated MLC was elevated in myofibroblasts without bFGF stimulation. After bFGF stimulation, levels of diphosphorylated MLC were further elevated up to 60 mins. bFGF promoted apoptosis in myofibroblasts but not in fibroblasts even if two different inhibitors, LY294002 for phosphatidylinositol-3-Kinase and Akt inhibitor, were present.
The present study implicates that the down regulation of PI3K to Akt pathway is involved in bFGF-promoted myofibroblast apoptosis. These results suggest that bFGF can promote scarless wound healing consequent upon induction of apoptosis in myofibroblasts.

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Epidermolytic hyperkeratosis is the characteristic histological change in certain disorders of keratin mutation, such as bullous congenital ichthyosiform erythroderma (BCIE), Vörner type palmoplantar keratoderma (PPK), and ichthyosis bullosa Siemens. Inflamation in histological aspects has not been fully investigated, while clinically, inflammatory changes such as erythema are often observed. Inflammatory cells in the upper dermis contained many mast cells stained with toluidin blue stain. bFGF is one of the chemotactic factors against mast cells. We stained skin samples from patients of BCIE and Vörner type PKK, which revealed staining of basal layer compared to suprabasal staining in healthy subjects. Basic FGF has positive effects on keratinocyte proliferation, vessel proliferation as well as wound healing, which could explain the histological changes in BCIE and Vörner type PKK.

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Repeated cutaneous injections of bleomycin (50μg/day) into the dorsal skin of mice induced cutaneous sclerosis. When 6mm-size rounded full-thickness wounds were prepared, wound closure in bleomycin-induced sclerotic skin was delayed in comparison with normal skin wounds. We then compared the stimulatory effect of basic fibroblast growth factor (bFGF) on the healing of wounds prepared in bleomycin-injected mice and normal control mice. bFGF (1μg/cm²) administered once after wounding slightly stimulated wound healing in both mice models. mRNA levels of TGFβ1 and Col1A1 were determined by quantitative PCR method using tissues extracted from the bleomycin-injected skin and normal control skin. The expression level of TGFβ1 was not different between the two groups, while that of Col1A1 in bleomycin-injected skin was 100 times higher than the normal control skin. These results confirmed the clinical utility of Fibrast spray in the treatment of chronic skin ulcers.

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During the wound healing process, inflammatory cell infiltration promotes the wound healing by producing various cytokines and growth factors. Wound healing is delayed in mice lacking CD19, positive response regulator of B cells. This delayed wound healing was normalized by treatment with basic fibroblast growth factor. This indicates that basic fibroblast growth factor plays an important and critical role in the wound healing process.

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