- Email: [email protected]
Biointegration of Permacol™ paste as a novel dermal substitute in full-thickness wounds
ECSAPS 2006 Abstracts and improved after 4 weeks, which were stained positive both by antibody KL-1 and HLA-1. Conclusion: This study gives evidence that our isolation technique allows culturing of ORSCs. The SE which we restructured with fibroblasts and OSRCs in vivo is able to form histological structures similar to human dermis and epidermis in 2 weeks.
O38. Role of inos and no in adipose tissue engineering K. Hemmrich a,b, G.P.L. Thomas a, C. Gummersbach b, K. Abberton a, E.W. Thompson a, N. Pallua b, W.A. Morrison a a Bernard O’Brien Institute of Microsurgery, Melbourne, Victoria, Australia b Department of Plastic Surgery and Hand Surgery e Burn Center, University Hospital of the Aachen University of Technology, Aachen, Germany Description of aims: The yeast-derivate Zymosan-A (Zy) induces fat formation in a Matrigel-filled chamber, implanted around a vessel in a murine tissue engineering model. To identify the key cytokines that guide cell recruitment and fat formation in the chamber, this study analyzed the role of inducible nitric oxide synthase (iNOS) in Zymosan-mediated fat formation. Additionally, human adipose tissue precursor cells were treated with the iNOS-derived product nitric oxide (NO) and assessed for their differentiation potential. Methods: Matrigel-filled bilateral chambers (42 ml) were placed around the right and left superficial epigastric vessels of mice. On one side, Matrigel was suspended with Zy (0.01 mg/chamber). Aminoguanidine (2 mg/chamber), an iNOS inhibitor, was added on the same side as Zy. Constructs were harvested at 6 weeks and percentage of adipose tissue (vol %) was determined. Additionally, preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured until confluence and differentiated to fat cells. To evaluate the influence of the iNOS product NO on preadipocyte differentiation, DETA-NO was added at various concentrations. Differentiation was measured by analysis of the enzyme glycerophosphate dehydrogenase (GPDH). Results: In the in vivo mouse model, Zy induced new adipose tissue formation as expected. The adipose tissue (vol%) in the chambers was 40% 7% on the Zy side and 41% 4% contralateral. Aminoguanidine reduced fat volume to 24% 7.6% ipsilateral and 26% 8.5% on the opposite site (p < 0.05). As revealed by GPDH levels, NO in combination with the conventionally used differentiation-inducing factors significantly enhanced maturation of precursor cells to adipocytes (p < 0.001). Conclusions and significance: iNOS is a crucial factor to generate adipose tissue formation in vivo. The in vitro results underline the adipogenic properties of NO. These findings are encouraging for applying NO-donors during transplantation of preadipocytes in a three-dimensional setting or even cell-free to accelerate and optimize adipose tissue engineering. O39. Biointegration of Permacolä paste as a novel dermal substitute in full-thickness wounds R.V. Shevchenko a, J.R. Sharpe a, P.D. Sibbons b, S.E. James a a Blond McIndoe Centre, Queen Victoria Hospital, East Grinstead, Sussex RH19 3DZ, UK b Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, UK Insufficient skin for autologous grafts makes treatment of fullthickness extensive burns difficult. Poor long-term results from application of cultured keratinocyte sheets or suspensions to such wounds are thought to be due to the absence of a dermis. The porcine collagen-based biomaterial Permacolä in sheet form is useful as a stable implant in reconstructive surgery, but its use as a dermal substitute has been hindered by slow cell penetration and
S13 vascularisation. Here, a paste formulation was evaluated as a dermal substitute in full-thickness wounds to encourage cellular infiltration. Biopsy and full-thickness wound-chamber models were done in the Large White pig under local ethics committee approval and government licence. Biopsy model allowed 24 samples of 8 mm diameter and the chamber model allowed 6 samples of 4 cm diameter per pig. Permacolä was applied as a paste to the wound bed and covered by a split-thickness skin graft or a silicone dressing, with suitable control wounds in parallel. Biopsies taken at specific time points. were assessed for biointegration and neovascularisation using histological and immunofluorescent analysis. Permacolä paste accepted host dermal mesenchymal and epidermal cells. It was well penetrated by cells on day 2, and was accepted by the host tissue without causing inflammation, indicating successful biointegration. Host cellular infiltration of Permacolä paste was much superior to Permacolä sheet and very similar to Integraâ Paste was visible within the tissue samples and intact up to day 28, indicating presence of a stable biomaterial. Early neovascularisation was noted at day 4, and functional microvessels with circulating blood cells were discovered at day 7 and confirmed by immunostaining for vascular endothelium. This was comparable to Integraâ, gold standard dermal substitute. This pilot study indicates that a paste formulation of Permacolä successfully biointegrated into full-thickness wounds and may act as an alternative to current dermal substitutes.
O40. Synthesis and initial evaluation of a pro-angiogenic synthetic dermal replacement C. Baldwin, N. Patel, L. Clayton, L. Irvine, J.F. Dye The Raft Institute, Leopold Muller Building, Mount Vernon Hospital, Northwood, HA6 2RN, UK Despite huge improvements in burns care, there remains a real need for a true skin substitute, that is immediately available, provides a barrier to infection and becomes integrated into the host either permanently or long enough to prevent hypertrophic scarring. Integration of Integra, the most commonly used dermal substitute today, remains a significant problem. Key to integration is angiogenesis, we describe the development of a second generation of skin substitute designed to promote vessel growth into it. Fibrin has long been considered as a source material for wound healing, with recognised angiogenic properties. Direct comparisons of fibrin with other potential bio-polymers, notably collagen-1, to support endothelial migration in 3D gel assays, prompted us to renewed investigation of its use as a scaffold material. An iterative approach to scaffold development has been taken, ultimately using human placental microvascular endothelial cell infiltration as a selector of successful formulations. A foam mixing method was used to create a scaffold structure from test materials (fibrinogen, thrombin, alginate). Different cross-link stabilisation protocols were evaluated (vacuum heat, glutaraldehyde, EDC, EDC + NHS, lysine, BH4) with a proteolytic degradation assay. Endothelial and fibroblast toxicity assays demonstrated quenching of crosslink agents. In vitro proof of concept has been demonstrated with significant endothelial cell migration into stabilised fibrin matrix over 48 hr, in contrast to Integra. Immunhistochemistry has demonstrated that stabilised fibrin matrices support endothelial cell proliferation and differentiation (vWF, VE-cadherin, CD133 & VEGF expression). This work has provided us with a prototype stable fibrin-based synthetic dermal substitute ready for in vivo evaluation (porcine full thickness wound model). Industrial and pharmaceutical links already established aim to allow the necessary development of this formulation to produce a clinical grade novel second generation dermal scaffold specifically designed to integrate ready for phase 1 clinical trial within 24 months.
O38. Role of inos and no in adipose tissue engineering K. Hemmrich a,b, G.P.L. Thomas a, C. Gummersbach b, K. Abberton a, E.W. Thompson a, N. Pallua b, W.A. Morrison a a Bernard O’Brien Institute of Microsurgery, Melbourne, Victoria, Australia b Department of Plastic Surgery and Hand Surgery e Burn Center, University Hospital of the Aachen University of Technology, Aachen, Germany Description of aims: The yeast-derivate Zymosan-A (Zy) induces fat formation in a Matrigel-filled chamber, implanted around a vessel in a murine tissue engineering model. To identify the key cytokines that guide cell recruitment and fat formation in the chamber, this study analyzed the role of inducible nitric oxide synthase (iNOS) in Zymosan-mediated fat formation. Additionally, human adipose tissue precursor cells were treated with the iNOS-derived product nitric oxide (NO) and assessed for their differentiation potential. Methods: Matrigel-filled bilateral chambers (42 ml) were placed around the right and left superficial epigastric vessels of mice. On one side, Matrigel was suspended with Zy (0.01 mg/chamber). Aminoguanidine (2 mg/chamber), an iNOS inhibitor, was added on the same side as Zy. Constructs were harvested at 6 weeks and percentage of adipose tissue (vol %) was determined. Additionally, preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured until confluence and differentiated to fat cells. To evaluate the influence of the iNOS product NO on preadipocyte differentiation, DETA-NO was added at various concentrations. Differentiation was measured by analysis of the enzyme glycerophosphate dehydrogenase (GPDH). Results: In the in vivo mouse model, Zy induced new adipose tissue formation as expected. The adipose tissue (vol%) in the chambers was 40% 7% on the Zy side and 41% 4% contralateral. Aminoguanidine reduced fat volume to 24% 7.6% ipsilateral and 26% 8.5% on the opposite site (p < 0.05). As revealed by GPDH levels, NO in combination with the conventionally used differentiation-inducing factors significantly enhanced maturation of precursor cells to adipocytes (p < 0.001). Conclusions and significance: iNOS is a crucial factor to generate adipose tissue formation in vivo. The in vitro results underline the adipogenic properties of NO. These findings are encouraging for applying NO-donors during transplantation of preadipocytes in a three-dimensional setting or even cell-free to accelerate and optimize adipose tissue engineering. O39. Biointegration of Permacolä paste as a novel dermal substitute in full-thickness wounds R.V. Shevchenko a, J.R. Sharpe a, P.D. Sibbons b, S.E. James a a Blond McIndoe Centre, Queen Victoria Hospital, East Grinstead, Sussex RH19 3DZ, UK b Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, UK Insufficient skin for autologous grafts makes treatment of fullthickness extensive burns difficult. Poor long-term results from application of cultured keratinocyte sheets or suspensions to such wounds are thought to be due to the absence of a dermis. The porcine collagen-based biomaterial Permacolä in sheet form is useful as a stable implant in reconstructive surgery, but its use as a dermal substitute has been hindered by slow cell penetration and
S13 vascularisation. Here, a paste formulation was evaluated as a dermal substitute in full-thickness wounds to encourage cellular infiltration. Biopsy and full-thickness wound-chamber models were done in the Large White pig under local ethics committee approval and government licence. Biopsy model allowed 24 samples of 8 mm diameter and the chamber model allowed 6 samples of 4 cm diameter per pig. Permacolä was applied as a paste to the wound bed and covered by a split-thickness skin graft or a silicone dressing, with suitable control wounds in parallel. Biopsies taken at specific time points. were assessed for biointegration and neovascularisation using histological and immunofluorescent analysis. Permacolä paste accepted host dermal mesenchymal and epidermal cells. It was well penetrated by cells on day 2, and was accepted by the host tissue without causing inflammation, indicating successful biointegration. Host cellular infiltration of Permacolä paste was much superior to Permacolä sheet and very similar to Integraâ Paste was visible within the tissue samples and intact up to day 28, indicating presence of a stable biomaterial. Early neovascularisation was noted at day 4, and functional microvessels with circulating blood cells were discovered at day 7 and confirmed by immunostaining for vascular endothelium. This was comparable to Integraâ, gold standard dermal substitute. This pilot study indicates that a paste formulation of Permacolä successfully biointegrated into full-thickness wounds and may act as an alternative to current dermal substitutes.
O40. Synthesis and initial evaluation of a pro-angiogenic synthetic dermal replacement C. Baldwin, N. Patel, L. Clayton, L. Irvine, J.F. Dye The Raft Institute, Leopold Muller Building, Mount Vernon Hospital, Northwood, HA6 2RN, UK Despite huge improvements in burns care, there remains a real need for a true skin substitute, that is immediately available, provides a barrier to infection and becomes integrated into the host either permanently or long enough to prevent hypertrophic scarring. Integration of Integra, the most commonly used dermal substitute today, remains a significant problem. Key to integration is angiogenesis, we describe the development of a second generation of skin substitute designed to promote vessel growth into it. Fibrin has long been considered as a source material for wound healing, with recognised angiogenic properties. Direct comparisons of fibrin with other potential bio-polymers, notably collagen-1, to support endothelial migration in 3D gel assays, prompted us to renewed investigation of its use as a scaffold material. An iterative approach to scaffold development has been taken, ultimately using human placental microvascular endothelial cell infiltration as a selector of successful formulations. A foam mixing method was used to create a scaffold structure from test materials (fibrinogen, thrombin, alginate). Different cross-link stabilisation protocols were evaluated (vacuum heat, glutaraldehyde, EDC, EDC + NHS, lysine, BH4) with a proteolytic degradation assay. Endothelial and fibroblast toxicity assays demonstrated quenching of crosslink agents. In vitro proof of concept has been demonstrated with significant endothelial cell migration into stabilised fibrin matrix over 48 hr, in contrast to Integra. Immunhistochemistry has demonstrated that stabilised fibrin matrices support endothelial cell proliferation and differentiation (vWF, VE-cadherin, CD133 & VEGF expression). This work has provided us with a prototype stable fibrin-based synthetic dermal substitute ready for in vivo evaluation (porcine full thickness wound model). Industrial and pharmaceutical links already established aim to allow the necessary development of this formulation to produce a clinical grade novel second generation dermal scaffold specifically designed to integrate ready for phase 1 clinical trial within 24 months.