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Primary closure of fetal skin in spina bifida protects the spinal cord and improves clinical outcome, but is also associated with postnatal growth malformations and spinal cord tethering. In this study, we evaluated the postnatal effects of prenatally closed full-thickness skin defects in sheep applying collagen scaffolds with and without heparin/vascular endothelial growth factor/fibroblast growth factor 2, focusing on skin regeneration and growth. At 6 months, collagen scaffold functionalized with heparin, VEGF, and FGF2 (COL-HEP/GF) resulted in a 6.9-fold increase of the surface area of the regenerated skin opposed to 1.7 × for collagen only. Epidermal thickness increased 5.7-fold at 1 month, in line with high gene expression of S100 proteins, and decreased to 2.1 at 6 months. Increased adipose tissue and reduced scaffold degradation and number of myofibroblasts were observed for COL-HEP/GF. Gene ontology terms related to extracellular matrix (ECM) organization were enriched for both scaffold treatments. In COL-HEP/GF, ECM gene expression resembled native skin. Expression of hair follicle-related genes in COL-HEP/GF was comparable to native skin, and de novo hair follicle generation was indicated. In conclusion, in utero closure of skin defects using functionalized collagen scaffolds resulted in long-term skin regeneration and growth. Functionalized collagen scaffolds that grow with the child may be useful for prenatal treatment of closure defects like spina bifida. Impact statement Prenatal closure of fetal skin in case of spina bifida prevents damage to the spinal cord. Closure of the defect is challenging and may result in postnatal growth malformations. In this study, the postnatal effects of a prenatally applied collagen scaffold functionalized with heparin and vascular endothelial growth factor (VEGF)/fibroblast growth factor (FGF) were investigated. An increase of the surface area of regenerated skin ("growing with the child") and generation of hair follicles was observed. Gene expression levels resembled those of native skin with respect to the extracellular matrix and hair follicles. Overall, in utero closure of skin defects using heparin/VEGF/FGF functionalized collagen scaffolds results in long-term skin regeneration.

Glycosaminoglycans are important for cell signaling and therefore for proper embryonic development and adult homeostasis. Expressions of genes involved in proteoglycan/glycosaminoglycan (GAG) metabolism and of genes coding for growth factors known to bind GAGs were analyzed during skin development by microarray analysis and real time quantitative PCR. GAG related genes were organized in six categories based on their role in GAG homeostasis, viz. (1) production of precursor molecules, (2) production of core proteins, (3) synthesis of the linkage region, (4) polymerization, (5) modification, and (6) degradation of the GAG chain. In all categories highly dynamic up- and downregulations were observed during skin development, including differential expression of GAG modifying isoenzymes, core proteins, and growth factors. In two mice models, one overexpressing heparanase and one lacking C5 epimerase, differential expression of only few genes was observed. Data show that during skin development a highly dynamic and complex expression of GAG-associated genes occurs. This likely reflects quantitative and qualitative changes in GAGs/proteoglycans, including structural fine tuning, which may be correlated with growth factor handling.

Autologous skin grafts are the gold standard for the treatment of burn wounds. In a number of cases, treatment with autologous tissue is not possible and skin substitutes are used. The outcome, however, is not optimal and improvements are needed. Inspired by scarless healing in early embryonic development, we here set out a strategy for the design and construction of embryonic-like scaffolds for skin tissue engineering. This strategy may serve as a general approach in the construction of embryonic-like scaffolds for other tissues/organ. As a first step, key effector molecules upregulated during embryonic and neonatal skin formation were identified using a comparative gene expressing analysis. A set of 20 effector molecules was identified, from which insulin-like growth factor 2 (IGF2) and sonic hedgehog (SHH) were selected for incorporation into a type I collagen-heparin scaffold. Porous scaffolds were constructed using purified collagen fibrils and 6% covalently bound heparin (to bind and protect the growth factors), and IGF2 and SHH were incorporated either individually (~0.7 and 0.4 µg/mg scaffolds) or in combination (combined ~1.5 µg/mg scaffolds). In addition, scaffolds containing hyaluronan (up to 20 µg/mg scaffold) were prepared, based on the up- or downregulation of genes involved in hyaluronan synthesis/degradation and its suggested role in scarless healing. In conclusion, based on a comprehensive gene expression analysis, a set of effector molecules and matrix molecules was identified and incorporated into porous scaffolds. The scaffolds thus prepared may create an 'embryonic-like' environment for cells to recapitulate embryonic events and for new tissues/organs., (Copyright © 2013 John Wiley & Sons, Ltd.)

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., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cutaneous wounding often leads to contraction and scarring, which may result in a range of functional, cosmetic, and psychological complications. Tissue-engineered skin substitutes are being developed to enhance restoration of the skin and improve the quality of wound healing. The aim of this review is to provide researchers in the field of tissue engineering an overview of the methods that are currently used to clinically evaluate skin wound healing, and methods that are used to evaluate tissue-engineered constructs in animal models. Clinically, the quality of wound healing is assessed by noninvasive subjective scar assessment scales and objective techniques to measure individual scar features. Alternatively, invasive technologies are used. In animal models, most tissue-engineered skin constructs studied are at least evaluated macroscopically and by using conventional histology (hematoxylin-eosin staining). Planimetry and immunohistochemistry are also often applied. An overview of antibodies used is provided. In addition, some studies used methods to assess gene expression levels and mRNA location, transillumination for blood vessel observation, in situ/in vivo imaging, electron microscopy, mechanical strength assessment, and microbiological sampling. A more systematic evaluation of tissue-engineered skin constructs in animal models is recommended to enhance the comparison of different constructs, thereby accelerating the trajectory to application in human patients. This would be further enhanced by the embracement of more clinically relevant objective evaluation methods. In addition, fundamental knowledge on construct-mediated wound healing may be increased by new developments in, for example, gene expression analysis and noninvasive imaging.

In spina bifida the neural tube fails to close during the embryonic period and it is thought that prolonged exposure of the unprotected spinal cord to the amniotic fluid during pregnancy causes additional neural damage. Intra-uterine repair might protect the neural tissue from exposure to amniotic fluid and might reduce additional neural damage. Biodegradable collagen scaffolds may be useful in case of fetal therapy for spina bifida, but biochemical properties need to be studied. The aim of this study was to investigate whether biodegradable collagen scaffolds can be used to treat full-thickness fetal skin defects. We hypothesized that the pro-angiogenic growth factors VEGF and FGF2 would enhance vascularization, epidermialization and lead to improved wound healing. To investigate the effect of these two growth factors, a fetal sheep model for skin defects was used. Compared to wounds treated with bare collagen scaffolds, wounds treated with growth factor-loaded scaffolds showed excessive formation of capillaries and less myofibroblasts were present in these wounds, leading to less contraction. This study has demonstrated that collagen scaffolds can be used to treat fetal skin defects and that the combination of collagen scaffolds with VEGF and FGF2 had a beneficial effect on wound healing., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Large-scale in vivo evaluation of biomaterials is time-consuming and limited by ethical considerations. The availability of a library of biomaterials would allow a fast and rational in vitro selection of those biomaterials to be evaluated in vivo. For this reason, we developed an array of 48 different, molecularly-defined films based on native fibrillar collagen. The films differed in the type and amount of extracellular matrix components (type I/IV collagens, fibrous/solubilised elastin, glycosaminoglycans, heparin, chondroitin sulfate or dermatan sulfate), method of preparation (homogenisation) and method and extent of crosslinking (carbodiimide (EDC/NHS) or glutaraldehyde). The array was evaluated by studying morphology, proliferation and differentiation of primary human keratinocytes/fibroblasts. Major differences were observed. Only a small selection of films (especially those containing elastin fibres) specifically stimulated the proliferation of keratinocytes, but not fibroblasts. Such films may be the biomaterials of choice for in vivo evaluation for skin tissue engineering and regenerative medicine.

Objective: To compare the tissue reactions to implanted collagen scaffolds in the palate and the skin of rats., Design: Crosslinked collagen scaffolds were implanted submucoperiosteally in the palate, and subcutaneously on the skull and on the back of 25 rats and evaluated after up to 16 weeks. On H&E-stained sections, the cell density and the number of giant cells within the scaffolds were determined. Blood vessels, inflammatory cells, and myofibroblasts were detected by immunohistochemistry., Results: A faster ingrowth of myofibroblasts and blood vessels in the palate was found during the first week compared with the skin. A more severe inflammatory response was initially found in the back skin. Furthermore, about twice as much giant cells were present in these scaffolds., Conclusion: The oral environment seems to promote the ingrowth of myofibroblasts and blood vessels into the scaffolds. Mechanical stimuli seem to enhance the initial inflammatory response. Overall, the scaffolds were gradually integrated within the host tissue, eliciting only a transient inflammatory response. The scaffolds were biocompatible and are promising for future applications in oral surgery.