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A major obstacle to 3-dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the model's vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.20+/-3.14% compared with 3.59+/-2.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.

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a b s t r a c tHistioconductive approaches to soft-tissue defects use scaffolds seeded with lineage-and tissue-specific progenitors to generate tissue which should reside in equilibrium with adjacent tissue. Scaffolds guide histiogenesis by ensuring cell-cell and cell-matrix interactions. Hyaluronic acid-based (HA) preadipocyte-seeded scaffolds were evaluated for their adipo-conductive potential and efficacy in humans. Preadipocytes were isolated from lipoaspirate material and seeded on HA scaffolds. The cellular biohybrid (ADIPOGRAFT Ò ) and an acellular control scaffold (HYAFF Ò 11) were implanted subcutaneously. At specific time points (2, 8 and 16 weeks) explants were analyzed histopathologically with immunohistochemistry. No adverse tissue effects occurred. Volume loss and consistent degradation of the HYAFF Ò 11 scaffolds compared to the ADIPOGRAFT Ò group indicated progressive tissue integration. No consistent histological differences between both groups were observed. By 8 weeks all void spaces within the scaffolds were filled with cells with pronounced matrix deposition in the ADIPOGRAFT Ò bio-hybrids. Here we show that HA scaffolds were stable cell carriers and had the potential to generate volumeretaining tissue. However, no adipogenic differentiation was observed within the preadipocyte-seeded scaffolds.

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We have recently shown that Matrigel-filled chambers containing fibroblast growth factor-2 (FGF2) and placed around an epigastric pedicle in the mouse were highly adipogenic. Contact of this construct with pre-existing tissue or a free adipose graft was required. To further investigate the mechanisms underpinning formation of new adipose tissue, we seeded these chambers with human adipose biopsies and human adipose-derived cell populations in severe combined immunodeficient mice and assessed the origin of the resultant adipose tissue after 6 weeks using species-specific probes. The tissues were negative for human-specific vimentin labeling, suggesting that the fat originates from the murine host rather than the human graft. This was supported by the strong presence of mouse-specific Cot-1 deoxyribonucleic acid labeling, and the absence of human Cot-1 labeling in the new fat. Even chambers seeded with FGF2/ Matrigel containing cultured human stromal-vascular fraction (SVF) labeled strongly only for human vimentin in cells that did not have a mature adipocyte phenotype; the newly formed fat tissue was negative for human vimentin. These findings indicate that grafts placed in the chamber have an inductive function for neo-adipogenesis, rather than supplying adipocyte-precursor cells to generate the new fat tissue, and preliminary observations implicate the SVF in producing inductive factors. This surprising finding opens the door for refinement of current adipose tissue-engineering approaches.