Abstract
BackgroundThe seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Alternatively, adipose tissue-derived microvascular fragments (ad-MVF) may serve as vascularization units. In contrast to SVF single cells, they represent a mixture of intact arteriolar, capillary and venular vessel segments. Therefore, we herein hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF single cells-based approach.ResultsSVF single cells and ad-MVF were enzymatically isolated from epididymal fat pads of green fluorescent protein (GFP)+ donor mice to assess their viability and cellular composition using fluorescence microscopy and flow cytometry. Moreover, collagen-glycosaminoglycan matrices (Integra®) were seeded with identical amounts of the isolates and implanted into full-thickness skin defects within dorsal skinfold chambers of GFP− recipient mice for the intravital fluorescent microscopic, histological and immunohistochemical analysis of implant vascularization and incorporation throughout an observation period of 2 weeks. Non-seeded matrices served as controls. While both isolates contained a comparable fraction of endothelial cells, perivascular cells, adipocytes and stem cells, ad-MVF exhibited a significantly higher viability. After in vivo implantation, the vascularization of ad-MVF-seeded scaffolds was improved when compared to SVF-seeded ones, as indicated by a significantly higher functional microvessel density. This was associated with an enhanced cellular infiltration, collagen content and density of CD31+/GFP+ microvessels particularly in the center of the implants, demonstrating a better incorporation into the surrounding host tissue. In contrast, non-seeded matrices exhibited a poor vascularization, incorporation and epithelialization over time.ConclusionsThe present study demonstrates that ad-MVF are highly potent vascularization units that markedly accelerate and improve scaffold vascularization when compared to the SVF.
Highlights
The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering
Cellular composition and activity of SVF single cells and adipose tissue-derived microvascular fragments (ad-MVF) SVF single cells and ad-MVF were isolated from the bilateral epididymal fat pads of transgenic green fluorescent protein (GFP)+ C57BL/6 mice (Fig. 1a-c)
Additional flow cytometric analyses showed a comparable cellular composition of SVF single cells and ad-MVF (Table 1). They contained a mixture of CD31+ endothelial cells, α-smooth muscle actin (SMA)+ perivascular cells, adipocyte-specific adhesion molecule (ASAM)+ adipocytes as well as cells positive for the stromal/stem cell surface markers CD29, CD90 and CD117 (Table 1)
Summary
The seeding of scaffolds with the stromal vascular fraction (SVF) of adipose tissue is a common prevascularization strategy in tissue engineering. Blood vessels do consist of one specific cell type but exhibit a complex composition with an inner endothelial lining and surrounding vessel wall-stabilizing cell layers Taking this into account, the stromal vascular fraction (SVF) of adipose tissue is frequently used to induce the formation of microvascular networks [10, 14]. Digestion of adipose tissue for 45–60 min exclusively results in SVF single cells [19, 20], whereas a shorter digestion time of only 10 min provides a mixture of single cells and ad-MVF [21] These ad-MVF still represent intact vessel segments and, exhibit the unique feature of rapidly reassembling into new microvascular networks after transplantation [18]. We hypothesized that the ad-MVF-based prevascularization of scaffolds is superior to the conventional SVF-based approach
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