Efficacy of the dual controlled release of HGF and bFGF impregnated with a collagen/gelatin scaffold.

BACKGROUND: We previously developed collagen/gelatin sponges (CGS) able to sustain and release basic fibroblast growth factor (bFGF) and reported that this CGS impregnated with bFGF promoted dermis-like tissue formation. We herein confirmed the single-sustained release of hepatocyte growth factor (HGF) and the dual sustained release of HGF and bFGF from CGSs, and explored its efficacy using a murine model of skin defects. MATERIALS AND METHODS: The sustained release of HGF alone and both HGF and bFGF from CGSs were evaluated in vitro. CGSs (8 mm in diameter) impregnated with normal saline solution (NSS) (NSS group), HGF solution (10 or 50 µg/cm2) (HGF-L or HGF-H group), bFGF solution (7 µg/cm2) (bFGF group), or HGF (10 µg/cm2) and bFGF (7 µg/cm2) solution (HGF + bFGF group) were implanted into full-thickness skin defects on the backs of mice. The wound area, neoepithelium length, dermis-like tissue formation and newly formed capillaries were evaluated. RESULTS: The single release of HGF and the dual release of HGF and bFGF from CGSs were confirmed. At week 1, the wound closure and neoepithelium length were promoted in the HGF-L group compared with the NSS group. At week 2, the wound closure, neoepithelium length, dermis-like tissue formation and newly formed capillary formation were promoted in the bFGF and HGF + bFGF groups compared with the NSS and HGF-H groups. Newly formed capillary formation was superior in the HGF + bFGF group compared with the bFGF group. CONCLUSIONS: The dual release of HGF and bFGF from CGS was a promising treatment for full-thickness skin defects.

Development of a novel bioabsorbable implant that is substituted by adipose tissue in vivo.

Recently, adipose tissue has been regenerated by combining scaffolds, growth factors, and/or adipose-tissue-derived stromal cells. However, the safety of growth factors and adipose-tissue-derived stromal cells has not been confirmed in cancer patients. We reported the regeneration of adipose tissue in the internal space of a polypropylene mesh containing a collagen sponge (CS), without using any growth factors or cells. We herein explored the formation of adipose tissue, using the bioabsorbable implant containing CS, in rats. We prepared the implants without and with CS, using threads of either poly-l-lactide-co-ε-caprolactone or poly-l-lactic acid (PLLA), and measured their strengths. The procedure was performed in the rat inguinal region. In the control group, no operative procedure was performed. In the sham-operation group, skin incision without implantation was performed. The other groups received CS alone and the 2 implants with and without CS. The areas of formed tissue and adipose tissue inside the implants and the remnants of CS were evaluated. All implants maintained the internal space before implantation. At 6 and 12 months after implantation, the internal space was maintained and the formation of adipose tissue was promoted in the 2 PLLA groups. At 6 months, the internal space was maintained, and more adipose tissue was formed in the PLLA-with-CS group than in the PLLA group. Porcine collagen was absorbed within 3 months. The PLLA implant with CS is a novel bioabsorbable implant that is replaced with autologous adipose tissue after implantation.

A Comparison of Conventional Collagen Sponge and Collagen-Gelatin Sponge in Wound Healing.

The objective of this study was to compare the effectiveness of the collagen-gelatin sponge (CGS) with that of the collagen sponge (CS) in dermis-like tissue regeneration. CGS, which achieves the sustained release of basic fibroblast growth factor (bFGF), is a promising material in wound healing. In the present study, we evaluated and compared CGSs and conventional CSs. We prepared 8 mm full-thickness skin defects on the backs of rats. Either CGSs or CSs were impregnated with normal saline solution (NSS) or 7 µg/cm(2) of bFGF solution and implanted into the defects. At 1 and 2 weeks after implantation, tissue specimens were obtained from the rats of each group (n = 3, total n = 24). The wound area, neoepithelial length, dermis-like tissue area, and the number and area of capillaries were evaluated at 1 and 2 weeks after implantation. There were no significant differences in the CGS without bFGF and CS groups. Significant improvements were observed in the neoepithelial length, the dermis-like tissue area, and the number of newly formed capillaries in the group of rats that received CGSs impregnated with bFGF. The effects on epithelialization, granulation, and vascularization of wound healing demonstrated that, as a scaffold, CGSs are equal or superior to conventional CSs.

Efficacy of gelatin gel sheets in sustaining the release of basic fibroblast growth factor for murine skin defects.

BACKGROUND: Gelatin has been used as a material sustaining the release of basic fibroblast growth factor (bFGF), which promotes fibroblast proliferation and capillary formation and accelerates wound healing. In the application of these materials, bFGF is impregnated immediately before application, and it is difficult to conform the shape to the wound. In this study, we prepared a pliable and plastic gelatin gel sheet (GGS) that sustains bFGF and conforms to the shape of the wound as a result of cross-linking just before application. In addition, we examined the sustained release profile of bFGF from GGS and its effect on wound healing in murine skin defects. MATERIALS AND METHODS: A 13-wt% gelatin solution was mixed with bFGF before cross-linking with 1% glutaraldehyde solution. GGSs impregnated with 7 µg/cm(2) of bFGF were incubated in phosphate-buffered saline and collagenase solution, and GGS degradation and bFGF release were evaluated. In the murine experiments, GGSs treated without bFGF and GGSs impregnated with 1, 3.5, 7, or 14 µg/cm(2) of bFGF were applied to full-thickness skin defects created on the backs of C57BL/6JJcl mice, and the wound closure, epithelial length, extent of granulation tissue and capillary formation were compared. RESULTS: bFGF was released according to the degradation of GGS in phosphate-buffered saline, and the remaining bFGF was released in collagenase solution. In the animal studies, epithelialization was accelerated in the GGSs treated with 1 and 3.5 µg/cm(2) of bFGF, and granulation tissue formation and angiogenesis were promoted based on the amount of bFGF impregnated into the GGS. CONCLUSIONS: GGS impregnated with bFGF is capable of sustaining the release of bFGF, with consequent accelerated epithelialization, granulation tissue formation, and angiogenesis in vivo. GGS is a novel and promising wound dressing that sustains bFGF and can be adapted to the shape of various wounds in the treatment of both acute and chronic wounds.

Efficacy of gelatin gel sheets sustaining epidermal growth factor for murine skin defects.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in wound healing. However, EGF must be applied daily due to rapid inactivation in vivo. We investigated the sustained release of EGF from gelatin gel sheets (GGSs) and the efficacy of GGSs impregnated with EGF for promoting wound healing. MATERIALS AND METHODS: GGSs impregnated with EGF were prepared by cross-linking via glutaraldehyde to gelatin solution containing EGF. The sustained release of EGF and the bioactivity of released EGF were evaluated. Then, three kinds of GGSs containing NSS (normal saline solution; NSS group), 2.5 µg of EGF (EGF-L group), or 25 µg of EGF (EGF-H group) were applied to full-thickness skin defects created on the backs of mice. The wounds covered with polyurethane film without GGS were used as a control (PUF group). The wound area, neoepithelium length, regenerated granulation tissue, and newly formed capillaries were evaluated. RESULTS: EGF was sustained and released from GGS as it degraded. The bioactivity of released EGF was confirmed. EGF-L group promoted the neoepithelium length, regenerated granulation tissue, and newly formed capillaries compared with those in the PUF and NSS groups. The area of regenerated granulation tissue in the NSS group (week 1: 2.6 + 0.2 mm(2), week 2: 2.8 + 0.3 mm(2)) was larger than that in the PUF group (week 1: 0.6 + 0.1 mm(2), week 2: 1.0 + 0.1 mm(2)). The area of newly formed capillaries in the EGF-L group (9967 + 1903 µm(2)) was larger than that of the EGF-H group (3485 + 1050 µm(2)). CONCLUSIONS: GGSs impregnated with EGF-L showed promising results regarding wound healing.

Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor.

We have developed a collagen/gelatin sponge (CGS) that can provide a sustained release of basic fibroblast growth factor (bFGF). In our previous study, it was shown that CGS impregnated with the appropriate dosage of bFGF accelerates dermis-like tissue formation two or three times earlier than an existing collagen sponge. In this study, adipogenesis was evaluated using CGSs disseminated with adipose tissue-derived stem cells (ASCs). Human ASCs were primarily isolated from human adipose tissue that was obtained during breast cancer surgery with informed consent at Kyoto University Hospital. ASCs were isolated from collagenase digests of adipose tissue. ASCs were labelled with PKH26. CGSs (8 mm diameter × 3 mm thickness) were impregnated with bFGF (0.1, 1, 7, 14 µg/cm(2) ) or normal saline solution. Then the labelled cells were disseminated (passage 3) on CGSs at a seeding density of 1 × 10(5) cells/cm(2) and implanted into the back subcutis of nude mice. Six weeks after implantation, adipogenesis at the administered site was evaluated. Immunohistological staining with von Willebrand factor (vWf) was performed to evaluate newly formed capillaries. Newly formed adipose tissue was observed macroscopically and histologically in all groups. The weight and area of regenerated adipose tissue were largest in the 1 µg/cm(2) bFGF group. Under a fluorescent microscope, newly formed adipose tissue in the bFGF-administered group was PKH-positive. These findings show that ASCs differentiated and formed adipose tissue. In this study, we showed that our CGSs impregnated with bFGF could be used as scaffolds with ASCs for adipogenesis.

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.

Efficacy of the controlled release of concentrated platelet lysate from a collagen/gelatin scaffold for dermis-like tissue regeneration.

INTRODUCTION: A collagen/gelatin scaffold (CGS) can provide a sustained release of basic fibroblast growth factor (bFGF), which promotes wound healing. However, bFGF is approved for clinical use in Japan and China only. One potential alternative to bFGF is platelet lysate (PL), a safe and easily attainable source of a wide range of growth factors necessary for tissue repair. In the present study, we investigated the use of PL with CGS to repair wounds and identified the optimal concentration of PL for wound healing. MATERIALS AND METHODS: We generated PL from concentrated platelets harvested from individual healthy donors. We measured growth factors in PL. Transforming growth factor (TGF)-ß1, platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), and bFGF were selected because they were the major growth factors contained in platelets and showed the greatest ability to enhance the maturation of newly formed blood vessels. Pieces of CGS impregnated with PL solution (×1, ×2, ×3, or ×4 concentrated) or normal saline solution (NSS) were implanted into full-thickness skin defects on the backs of mice. We evaluated the wound area, neoepithelium length, and total area of newly formed capillaries in the implanted CGS. RESULTS: Our release experiments revealed that PDGF-BB and TGF-ß1 were released from CGS incubated with collagenase in a sustained manner. CGS impregnated with concentrated PL was more effective than CGS impregnated with NSS in all evaluated items. The ×2 concentrated PL accelerated wound healing and enhanced cell proliferation and vessel growth in granulation tissue. CONCLUSION: Our findings indicate that CGS can bind to TGF-ß1 and PDGF-BB and release these growth factors in a sustained manner. ×2 concentrated PL-impregnated CGS accelerates the formation of dermis-like tissue.

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.

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.

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.

Incorporation of basic fibroblast growth factor into preconfluent cultured skin substitute to accelerate neovascularisation and skin reconstruction after transplantation.

Cultured skin substitutes (CSS) with both epidermal and dermal components seem to be ideal, but they have not been widely used clinically, partly because it takes several weeks to produce them. Decreasing the number of seeding cells may reduce the period required for production, but it still takes a long time before the cells become confluent and neovascularisation is completed in CSS after grafting. As we have already succeeded in reducing the number of seeded keratinocytes in this study, we first attempted to reduce the number of seeded fibroblasts. Consequently, preconfluent CSS with 10 x 10(3) cells/cm2 of fibroblasts combined with 100 x 10(3) cells/cm2 of keratinocytes could be successfully grafted on to full-thickness wounds. bFGF-impregnated gelatin microspheres were then added to the preconfluent CSS before grafting. Incorporation of bFGF significantly accelerated neovascularisation and increased epidermal thickness, cellular components, and thickness of the dermis. The incorporation of bFGF makes CSS a potential therapeutic approach for management of skin wounds.

Viability and function of autologous and allogeneic fibroblasts seeded in dermal substitutes after implantation.

BACKGROUND: Fibroblast-seeded collagen sponges have been used for the treatment of skin defects and skin ulcers. However, the viability of the fibroblasts after implantation is still unknown. The objective of this study was to investigate the viability and distribution of autologous and allogeneic fibroblasts after implantation and to clarify which type is more effective for wound healing. MATERIALS AND METHODS: Skin samples of Hartley guinea pigs were retrieved and autologous fibroblasts were isolated and cultured. Fibroblasts isolated from the skin of a Strain2 guinea pig were used as allogeneic fibroblasts. Three full-thickness wounds were created on the backs of guinea pigs and an acellular collagen sponge, a collagen sponge seeded with autologous fibroblasts, and a collagen sponge seeded with allogeneic fibroblasts were transplanted. Before implantation, fibroblasts were labeled with PKH26. The guinea pigs were sacrificed 1, 2, and 3 weeks after implantation. The epithelization and contraction of the wounds were assessed, and the viability and distribution of the seeded fibroblasts were observed in cross sections. RESULTS: Three weeks after implantation, the PKH26-labeled autologous and allogeneic fibroblasts remained viable. In the wounds covered with the autologous fibroblast-seeded collagen sponge, the epithelization was fastest, and the percent wound contraction was smallest. In contrast, in the wounds covered with allogeneic fibroblasts, the epithelization was slowest and the percent contraction was largest. CONCLUSION: The allogeneic fibroblasts seeded in the collagen sponge survived and remained viable on the grafted area, but did not accelerate wound healing.