Department of Biochemistry 280, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Acta Biomater. 2011 Mar;7(3):1063-71. doi: 10.1016/j.actbio.2010.10.011. Epub 2010 Oct 18.
Skin substitutes are of great benefit in the treatment of patients with full thickness wounds, but there is a need for improvement with respect to wound closure with minimal contraction, early vascularisation, and elastin formation. In this study we designed and developed an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes. The epidermal layer was prepared using type I collagen, heparin and fibroblast growth factor 7 (FGF7), while the porous dermal layer was prepared using type I collagen, solubilised elastin, dermatan sulfate, heparin, fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). The construct was biochemically and morphologically characterised and evaluated in vivo using a rat full thickness wound model. The results were compared with the commercial skin substitute IntegraDRT and untreated wounds. The double-layered construct was prepared according to the design specifications. The epidermal layer was about 40 μm thick, containing 9% heparin and 0.2 μg FGF7 mg per layer, localised at the periphery. The dermal layer was 2.5 mm thick, had rounded pores and contained 10% dermatan sulfate+heparin, and 0.7 μg FGF2+VEGF mg per layer. The double-layered skin construct was implanted in a skin defect and on day 7, 14, 28 and 112 the (remaining) wound area was photographed, excised and (immuno) histologically evaluated. The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison with IntegraDRT and/or the untreated wound. On day 14 the double-layered skin construct also had the fewest myofibroblasts present. On day 112 the double-layered skin construct contained more elastic fibres than IntegraDRT and the untreated wound. Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly any were found in IntegraDRT. The results provide new opportunities for the application of acellular skin constructs in the treatment of surgical wounds.
皮肤替代物在治疗全层创面患者方面具有重要意义，但在实现最小收缩、早期血管化和弹性蛋白形成的创面闭合方面仍需要改进。在这项研究中，我们设计并开发了一种无细胞双层皮肤构建体，使用基质分子和生长因子来针对特定的生物学过程。表皮层使用 I 型胶原、肝素和成纤维细胞生长因子 7（FGF7）制备，而多孔真皮层使用 I 型胶原、可溶性弹性蛋白、硫酸皮肤素、肝素、成纤维细胞生长因子 2（FGF2）和血管内皮生长因子（VEGF）制备。该构建体进行了生化和形态学表征，并在大鼠全层创面模型中进行了体内评估。结果与商业皮肤替代物 IntegraDRT 和未处理的创面进行了比较。双层构建体是根据设计规格制备的。表皮层约 40 μm 厚，每层层含有 9%的肝素和 0.2 μg FGF7，位于周边。真皮层 2.5 mm 厚，有圆形孔，每层层含有 10%硫酸皮肤素+肝素和 0.7 μg FGF2+VEGF。双层皮肤构建体被植入皮肤缺损处，在第 7、14、28 和 112 天拍摄（剩余）创面面积，切除并进行（免疫）组织学评估。与 IntegraDRT 和/或未处理的创面相比，双层皮肤构建体在早期时间点显示出更多的细胞内流、显著更少的收缩和增加的血管形成。在第 14 天，双层皮肤构建体中也存在最少的肌成纤维细胞。在第 112 天，双层皮肤构建体中含有比 IntegraDRT 和未处理的创面更多的弹性纤维。在双层皮肤构建体和未处理的创面中发现了类似于毛囊和皮脂腺的结构，但在 IntegraDRT 中几乎没有发现。结果为无细胞皮肤构建体在治疗外科创面中的应用提供了新的机会。
