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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 21, 2005 as doi:10.1096/fj.05-3804fje. |
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Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova, Italy
1 Correspondence: Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, Via G. Colombo 3, Padova 35100, Italy. E-mail: g.abatangelo{at}unipd.it
SPECIFIC AIMS
The aim of the present study was to develop an in vitro coculture system that assumed the human dermal-epidermal architecture and included a microcapillary network in a 3-dimensional biomaterial that guaranteed ease of handling in a clinical setting. Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full-thickness skin samples, in scaffolds produced from modified hyaluronic acid. Results were evaluated by histological and immunohistochemical analyses at different time points. In vitro engineered skin obtained with this composite culture developed into a well differentiated upper layer of stratified keratinocytes lining a dermal-like structure, in which fibroblasts, extracellular matrix and a microvascular network were present. Furthermore, the biodegradable fabric produced from hyaluronic acid and used as the scaffolding support for this in vitro constructed skin graft greatly facilitated handling in the peri-operative period.
PRINCIPAL FINDINGS
The present work describes three novel findings: 1) isolation of keratinocyte, fibroblasts, endothelial cells from a single skin biopsy, 2) synthesis of autologous extracellular matrix inside a biodegradable and easy to handle scaffold, and 3) reconstruction of a microcapillary network into the dermal-like layer.
1. Isolation of three different cell types (keratinocytes, fibroblast, and endothelial cells) from a single skin biopsy
Endothelialized skin (ES) was prepared by coculturing three human cell types: keratinocytes, fibroblasts, and endothelial cells, obtained from human full-thickness skin samples, harvested during plastic surgery procedures from an ellipse of skin measuring 3x1 cm.
Keratinocytes and dermal fibroblasts were isolated and cultured according to a modified version of the Rheinwald and Green protocol. Endothelial cells were isolated from the dermal layer. Dermal specimens were immersed in endothelial cell growth medium (Medium 199, Biochrom, AG) supplemented with 20% fetal bovine serum (Biochrom, KG) and minced to release microvascular endothelial cells. The cell suspension was centrifuged and MACS CD105 Micro-Beads were added (Miltenyi Biotec). After incubation for 15 min at 4°C, the Micro-Beads were eluted as described in the manufacturer's instructions. Eluted cells were collected and plated onto collagen coated culture flasks. Micro-Bead immunoseparation of endothelial cells was successful, since 
60% of collected cells were immunohistochemically CD31 positive. The possibility to obtain three different cell types from an unique skin biopsy open new and very promising perspectives in field of skin tissue engineering and its clinical application.
2. Successful enrichment of a biodegradable 3-dimensional scaffold with an autologous extracellular matrix
Biomaterials used in the present study were derived from the total esterification of hyaluronan (synthesized from 80-200 kDa sodium hyaluronate) with benzyl alcohol, and are referred to as HYAFF-11TM. The final product is an uncross-linked linear polymer with an undetermined molecular weight; it is insoluble in aqueous solution yet spontaneously hydrolyzes over time, releasing benzyl alcohol and hyaluronan. HYAFF-11TM was used to create nonwoven meshes of 50 µm-thick fibers, with a specific weight of 100 g/m2 (NW11). These devices were obtained from Fidia Advanced Biopolymers (FAB, Abano Terme, Padova, Italy).
Endothelial/fibroblast cocultures were prepared by first adding a suspension of 2.5 x 105 fibroblasts/cm2 to HYAFF-11TM scaffolds. These were cultured for 1 wk with DMEMc supplemented with ascorbic acid (50 µg/mL), and b-FGF (10 ng/mL). Subsequently, endothelial cells were added to the dermal equivalent at a density of 2.5 x 105 cells/cm2, and cultured for 10 days in M199 medium supplemented with 20% FCS, 2 mM L-glutamine, 50 µg/mL ascorbic acid, and 10 ng/mL b-FGF. Subsequently, keratinocytes cells were seeded onto the endothelialized dermal equivalents at a density of 2 x 105 cells/cm2, and cultured for 1 wk with DMEM and Ham’s F12 in a ratio of 3:1, supplemented with 10% FCS, 0.4 µg/mL hydrocortisone, 5 µg/mL insulin (Sigma), 25 µg/mL adenine, 50 µg/mL ascorbic acid and antibiotics. After 1 wk of submerged culture, endothelialized skin preparations were elevated to the air-liquid interface for an additional 21 days of culture.
Histological analysis of endothelialized skin stained with haematoxylin and eosin demonstrated that cultured cells were organized into dermal and epidermal tissue compartments. After 14 and 21 days of culture at the air-liquid interface, the epithelial layer became progressively stratiform, with a basal layer, including cubic perpendicularly oriented cells and a superficial layer of flattened cells. Beneath the epithelial layer, the scaffold was populated by fibroblasts, which were distributed both on top and within the 3-dimensional construct. The demarcation line between epidermis and dermis was also clearly evident, and this junction was further verified by the presence of type VII collagen and laminin, typical molecules of the basal lamina. Moreover, flattened cells resembling endothelial cells (see below) adhered to the biomaterial fibers and delimited the ring structures in the dermal layer.
3. A well-defined microcapillary network was integrated in the endothelialized skin
CD31 staining of endothelialized skin constructs demonstrated that endothelial cells seeded onto the dermal equivalent had, after 7 days of culture, adhered and proliferated along the entire surface of the dermal layer, and began to infiltrate within the scaffold structure. Subsequently, endothelial cells migrated, forming capillary-like ring structures in the newly synthesized extracellular matrix. After 21 days of coculture at the air-liquid interface, endothelial cells attached to the mesh fibers. Moreover, in the interstitial spaces of the scaffold, endothelial cells were organized into ring aggregates, similar to capillary structures, in which a lumen was clearly evident. Immunolocalization of endothelial cells using anti-vWF antibodies after 21 days of coculture at the air-liquid interface was also employed, and demonstrated that endothelial cells formed ring structures in the interstitial spaces of the scaffold. Moreover, endothelial cells and fibroblasts produced adhesive extracellular matrix molecules as demonstrated by positive immunostaining of collagen IV, VII and laminin that was localized both along the epidermal-dermal junction and around the capillary-like structures (see Fig. 1
).
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CONCLUSIONS AND SIGNIFICANCE
These findings confirm that with coculture of human keratinocytes, fibroblasts, and endothelial cells, it was possible to create a two-layered skin construct provided with a microcapillary network. A well-orchestrated basic skin structure was generated in which a differentiated epidermal layer covered an underlying endothelialized dermal structure. This technique allows for the creation of a vascularized skin equivalent that can be easily detached from the culture dish with conventional surgical instruments, thus facilitating its implant at the wound site. An additional advantage of the here-described endothelialized skin substitute involves the biomaterial used. Thus far, the materials used for in vitro reconstruction of human skin with autologous or homologous cells enriched by microcapillary structures were derived from synthetic polymers or from heterologous collagen. These reconstructed skin equivalents are fragile and difficult to handle in the clinical setting. Furthermore, allogeneic collagen can promote undesirable immunologic reactions. To overcome these immunological and handling difficulties, we employed a scaffold made from a hyaluronic acid-derived biopolymer that confers both mechanical stability and high biocompatibility.
Last, these results highlight the biological performance of degradable HYAFF-11 scaffolds, which have been shown to be highly suitable for deposition of the autologous extracellular matrix critical for both keratinocyte and endothelial cell differentiation. The findings presented in our paper constitute a further advancement toward the realization of a skin substitute in which autologous extracellular matrix and a microcapillary network within the dermal stratum maximize the authenticity of the engineered tissue. Furthermore, the exceptional handling properties of the scaffolds tested should reflect greater facility of handling in the clinical setting, particularly with regard to what has been evaluated clinically up to now. These physiological and practical improvements should facilitate the take of a graft, particularly in full thickness wounds.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3805fje; doi: 10.1096/fj.05-3805fje.
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