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Notes: Currently, skin cells or tissues intended for therapeutical or cosmetic applications are typically cultured using standard in vitro culture techniques. The avian chorioallantoic membrane (CAM) has been traditionally used for many years to assess angiogenesis of exogenous substances. Avian embryos have also been used to generate viruses for vaccine development. Most of the uses of the avian CAM cultures are typically conducted in ovo. However, xenobiotic cultures of ex ovo avian embryos and explanted organs, tissues or cells from human or other mammalian origin have not been used in the past with the explicit purpose of achieving the appropriate extent of expansion, differentiation or vascularization required for therapeutic or cosmetic implantation into humans or animals. Here, a novel method of xenobiotic skin culture was developed that can be employed for wound healing studies or for tissue engineering applications to sustain the culture of live skin explants through the benefit of a xenobiotic vascularization. The method uses fertile Japanese quail (Cortunix cortunix japonica) eggs. The eggs were incubated for at least 3 days in ovo using a static egg incubator. On day 3 the egg is opened under sterile conditions through the air chamber and placed in a cylindrical culture dish slightly larger than the overall diameter of the egg yolk. The embryo was then cultured at 37 °C for an additional 24 to 48 hours until a well-vascularized CAM covers the entire surface of the culture dish. Next, fragments of skin were placed directly over the CAM. The cultures were continued for 8 days and then the skin explants were transferred to new CAMs. These changes, every 8 days, were done at least five times allowing the skin to be maintained in culture for several weeks without apparent damage in viability. The skin fragments remained viable and capable of reepithelialization when wounded. Skin explants from both mouse and human were successfully cultured in this manner. Because immunological response cannot occur in the embryonic culture environment, it was possible to xenotransplant skin fragments without risk of rejection. The ensuing embryonic vessels developed into the xenotransplanted skin and generated a permeable vascular network that provided oxygen and nutrients necessary to ensure viability and promote proliferation and differentiation. Viability of the explants was evaluated by immunohisto chemical methods using proliferation and apoptosis markers (Caspase-3, BrdU, and PCNA). Vascularization was assessed using species-specific anti endothelial antibodies (anti-PECAM human and mouse and QH1 and QH2 monoclonal antibodies). The new method described herein has the distinct advantage of sustaining live, functional skin cultures by means of a rich vascular supply that provides cells with nutrients and oxygen.

Notes: Angiogenesis is a dynamic process that requires interaction of proangiogenic and antiangiogenic factors. Several methodologies have been used to evaluate this process. The quail chorioallantoic membrane (CAM) is a suitable model to explore angiogenesis in vivo. Recently, the morphometric parameters of fractal dimension (Df) and grid intersection (ρ) are used to quantify angiogenesis in CAM. However, more information is necessary to understand angiogenic mechanisms. New methods to measure additional parameters such as the average blood vessel thickness, tortuousity, total branch length and complexity (junction node number), are described herein. For the analysis, CAM images were obtained from ex ovo culture of E8-E9 quail embryos. MatLab®(The MathWorks, Natick, MA) software was used for the analyses of branches nodes, thickness and length of the forming branches. Conventional morphometry was done using IPLab (Scanalytics, Fairfax, VA). To validate the new parameters, leptin, VEGF and neutralizing antibodies were evaluated. Leptin and VEGF treated CAM had increased complexity and vascular densities (Df = 1.5 ± 0.005; ρ = 12.3 ± 0.689) and (Df = 1.6 ± 0.006; ρ = 14.3 ± 0.465), respectively, compared to PBS control (Df = 1.3 ± 0.035; ρ = 8.6 ± 0.439). There was a corresponding decrease antileptin and anti-VEGF antibodies. The node-structural map applied to CAM images reveled 53% increase in the number of one-branch nodes and 50% increase in three-branch nodes with leptin compared to controls. Additionally, the total branch length was 30% greater with leptin along with a decrease in vessel thickness, The analyses allowed quantification of branching, tortuousity, thickness, and length of the new blood vessels. Together with the conventional morphometric parameters, these new analyses will help understand the mechanisms exerted by angiogenesis-modulating molecule.