Development of a reconstructed human skin model for angiogenesis

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5–7 days when placed on well‐vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small‐vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue‐engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells. (WOUND REP REG 2003;11:275–284)

[1]  D. Supp,et al.  Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor. , 2000, The Journal of investigative dermatology.

[2]  M. Nozaki,et al.  Effect of Cultured Endothelial Cells on Angiogenesis in Vivo , 1998, Plastic and reconstructive surgery.

[3]  R. Auerbach,et al.  Expression of organ-specific antigens on capillary endothelial cells. , 1985, Microvascular research.

[4]  R. Swerlick,et al.  HMEC-1: establishment of an immortalized human microvascular endothelial cell line. , 1992, The Journal of investigative dermatology.

[5]  S. Macneil,et al.  Keratinocytes contract human dermal extracellular matrix and reduce soluble fibronectin production by fibroblasts in a skin composite model. , 1997, British journal of plastic surgery.

[6]  S. Mac Neil,et al.  Development of autologous human dermal–epidermal composites based on sterilized human allodermis for clinical use , 1999, The British journal of dermatology.

[7]  M. Balasubramani,et al.  Skin substitutes: a review. , 2001, Burns : journal of the International Society for Burn Injuries.

[8]  M. Gerritsen,et al.  Sites of Prostaglandin Synthesis in the Bovine Heart and Isolated Bovine Coronary Microvessels , 1981, Circulation research.

[9]  Lucie Germain,et al.  In vitro reconstruction of a human capillary‐like network in a tissue‐engineered skin equivalent , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  H. Green,et al.  Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma , 1975, Cell.

[11]  H Green,et al.  Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. , 1975, Cell.

[12]  K. Bensch,et al.  Isolation and growth of endothelial cells from the microvessels of the newborn human foreskin in cell culture. , 1980, The Journal of investigative dermatology.

[13]  M. M. Ghosh,et al.  A Comparison of Methodologies for the Preparation of Human Epidermal‐Dermal Composites , 1997, Annals of plastic surgery.

[14]  H. Hechtman,et al.  Heterogeneity of intimal and microvessel endothelial cell barriers in vitro. , 1986, Microvascular research.

[15]  S. Jimenez,et al.  Isolation and characterization of microvascular endothelial cells from the adult human dermis and from skin biopsies of patients with systemic sclerosis. , 1994, Laboratory investigation; a journal of technical methods and pathology.

[16]  M A Karasek.  Microvascular endothelial cell culture. , 1989, The Journal of investigative dermatology.

[17]  D. Greenhalgh,et al.  Skin anatomy and antigen expression after burn wound closure with composite grafts of cultured skin cells and biopolymers. , 1993, Plastic and reconstructive surgery.

[18]  M. Detmar,et al.  A simple immunomagnetic protocol for the selective isolation and long-term culture of human dermal microvascular endothelial cells. , 1998, Experimental cell research.

[19]  S. Kumar,et al.  Heterogeneity in endothelial cells from large vessels and microvessels. , 1987, Differentiation; research in biological diversity.

[20]  J. Sandy,et al.  Psychological outcomes amongst cleft patients and their families. , 1997, British journal of plastic surgery.

[21]  R. Hebbel,et al.  A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions: isolation by panning and stimulation with vascular endothelial growth factor. , 1997, Experimental cell research.

[22]  S. Boyce,et al.  The 1999 clinical research award. Cultured skin substitutes combined with Integra Artificial Skin to replace native skin autograft and allograft for the closure of excised full-thickness burns. , 1999, The Journal of burn care & rehabilitation.