Inosculation of Tissue‐Engineered Capillaries with the Host's Vasculature in a Reconstructed Skin Transplanted on Mice

The major limitation for the application of an autologous in vitro tissue‐engineered reconstructed skin (RS) for the treatment of burnt patients is the delayed vascularization of its relatively thick dermal avascular component, which may lead to graft necrosis. We have developed a human endothelialized reconstructed skin (ERS), combining keratinocytes, fibroblasts and endothelial cells (EC) in a collagen sponge. This skin substitute then spontaneously forms a network of capillary‐like structures (CLS) in vitro. After transplantation to nude mice, we demonstrated that CLS containing mouse blood were observed underneath the epidermis in the ERS in less than 4 days, a delay comparable to our human skin control. In comparison, a 14‐day period was necessary to achieve a similar result with the non‐endothelialized RS. Furthermore, no mouse blood vessels were ever observed close to the epidermis before 14 days in the ERS and the RS. We thus concluded that the early vascularization observed in the ERS was most probably the result of inosculation of the CLS network with the host's capillaries, rather than neovascularization, which is a slower process. These results open exciting possibilities for the clinical application of many other tissue‐engineered organs requiring a rapid vascularization.

[1]  J. Isner,et al.  Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. , 1999, Circulation research.

[2]  F A Auger,et al.  In vitro construction of a human blood vessel from cultured vascular cells: a morphologic study. , 1993, Journal of vascular surgery.

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

[4]  O. Damour,et al.  In vitro reconstructed skin models for wound coverage in deep burns , 1997, The British journal of dermatology.

[5]  J. Ricotta,et al.  Use of Dermagraft, a Cultured Human Dermis, to Treat Diabetic Foot Ulcers , 1996, Diabetes Care.

[6]  R. Hoffman,et al.  Expression of prostate-specific antigen in human prostate specimens in in vitro three-dimensional histoculture , 1993, In Vitro Cellular & Developmental Biology - Animal.

[7]  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.

[8]  D J Mooney,et al.  Up-Regulation of Bcl-2 in microvascular endothelial cells enhances intratumoral angiogenesis and accelerates tumor growth. , 2001, Cancer research.

[9]  François A. Auger,et al.  Skin equivalent produced with human collagen , 1995, In Vitro Cellular & Developmental Biology - Animal.

[10]  David J Mooney,et al.  Engineering and Characterization of Functional Human Microvessels in Immunodeficient Mice , 2001, Laboratory Investigation.

[11]  D. Young,et al.  Species-specific in situ hybridization with fluorochrome-labeled DNA probes to study vascularization of human skin grafts on athymic mice. , 1996, The Journal of burn care & rehabilitation.

[12]  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.

[13]  J. Converse,et al.  Distribution of diphospho‐pyridine nucleotide diaphorase in rat skin autografts and homografts , 1962 .

[14]  P. Hewett,et al.  Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue , 1993, In Vitro Cellular & Developmental Biology - Animal.

[15]  N C Krejci,et al.  In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. , 1991, The Journal of investigative dermatology.

[16]  S. Boyce,et al.  Principles and practices for treatment of cutaneous wounds with cultured skin substitutes. , 2002, American journal of surgery.

[17]  L. Bordenave,et al.  Optimization of use of UEA-1 magnetic beads for endothelial cell isolation , 1996, Cell Biology and Toxicology.

[18]  R. Rieben,et al.  Endothelial Cell Protection by Dextran Sulfate: A Novel Strategy to Prevent Acute Vascular Rejection in Xenotransplantation , 2004, American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.

[19]  T. Ono,et al.  Analysis of the Vascularity of an Atelocollagen Sponge Substitute Dermis in the Human , 2001, The Journal of dermatology.

[20]  N. Fusenig,et al.  Formation of epidermis after reimplantation of pure primary epidermal cell cultures from perinatal mouse skin. , 1974, Journal of the National Cancer Institute.

[21]  J. Haller,et al.  Studies of the Origin of the Vasculature in Free Skin Grafts , 1967, Annals of surgery.

[22]  D. Hartmann,et al.  An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse. , 1987, Transplantation.

[23]  D. J. Wainwright.  Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. , 1995, Burns : journal of the International Society for Burn Injuries.

[24]  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.

[25]  O Damour,et al.  Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice. , 2001, Matrix biology : journal of the International Society for Matrix Biology.

[26]  J. Pober,et al.  Engraftment of a vascularized human skin equivalent , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  E. Butcher,et al.  Vascular morphogenesis and differentiation after adoptive transfer of human endothelial cells to immunodeficient mice. , 2002, The American journal of pathology.

[28]  I. Sussman,et al.  Long-term culture of human endothelial cells , 1983, In Vitro.

[29]  Dai Fukumura,et al.  Tissue engineering: Creation of long-lasting blood vessels , 2004, Nature.

[30]  L. Germain,et al.  Use of in vitro reconstructed skin To cover skin flap donor site. , 1997, The Journal of surgical research.

[31]  F A Auger,et al.  Improvement of human keratinocyte isolation and culture using thermolysin. , 1993, Burns : journal of the International Society for Burn Injuries.

[32]  G Tellides,et al.  In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Converse,et al.  Inosculation of vessels of skin graft and host bed: a fortuitous encounter. , 1975, British journal of plastic surgery.

[34]  J. Hansbrough.  Current status of skin replacements for coverage of extensive burn wounds. , 1990, The Journal of trauma.

[35]  R. Penny,et al.  Human dermal microvascular endothelial cells: An improved method for tissue culture and a description of some singular properties in culture , 1985, In Vitro Cellular & Developmental Biology.

[36]  D. Supp,et al.  Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[37]  M. Boyer,et al.  Isolation of endothelial cells and their progenitor cells from human peripheral blood. , 2000, Journal of vascular surgery.

[38]  O. Damour,et al.  Optimization of thickness, pore size and mechanical properties of a biomaterial designed for deep burn coverage. , 1994, Clinical materials.

[39]  R. Wilson,et al.  VASCULARIZATION OF SKIN GRAFTS. , 1964, Surgical forum.

[40]  Takayuki Asahara,et al.  Isolation of Putative Progenitor Endothelial Cells for Angiogenesis , 1997, Science.

[41]  O Damour,et al.  A tissue‐engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary‐like tube formation in vitro , 2003, The British journal of dermatology.

[42]  S. Boyce,et al.  Cultured skin substitutes: a review. , 1996, Tissue engineering.