Comparison between endothelial progenitor cells and human umbilical vein endothelial cells on neovascularization in an adipogenesis mouse model
Introduction
In reconstructive and plastic surgery, there is a tremendous demand for biologically functional adipose tissue aiming at restoring contour defects after soft tissue removal. Standard approaches for the reconstruction of soft tissues include alloplastic implants and autologous tissue flaps which have the disadvantages of foreign body reaction and donor site morbidity, respectively. Autologous free fat grafting represents a minimally invasive alternative to this but results in unpredictable graft resorption, especially in the long term (Kaufman et al., 2007), most likely due to insufficient vascularization.
Tissue engineering of adipose tissue equivalents by means of a suitable cell population in combination with an adequate scaffold would provide a strategy to perform de novo adipogenesis and angiogenesis (Tanzi and Fare, 2009). Adipose-derived stem cells (ASCs) are considered to be such a suitable cell population. ASCs can be easily harvested from human fat tissue and have the potential to differentiate into lineages of adipogenic, osteogenic, chondrogenic and myogenic cells (Zuk et al., 2001). Several in vivo studies reported the formation of human adipose tissue after the implantation of human ASCs in different scaffolds with or without the addition of growth factors (Kimura et al., 2003, Patrick et al., 1999, Torio-Padron et al., 2007, von Heimburg et al., 2001) which were also characterized by ingrowth of host vasculature.
ASCs are known to secrete several proangiogenic growth factors (Kilroy et al., 2007, Rehman et al., 2004) and therefore the use of ASCs may improve angiogenesis, because adipogenesis is regulated by factors that also drive angiogenesis (Christiaens and Lijnen, 2010). However, it is still one of the main challenges in adipose tissue engineering to provide a sufficient and functional vasculature. As oxygen diffusion is limited to a distance of about 200 μm (Malda et al., 2007), long-term survival of tissue engineered adipose tissue relies on rapid development of blood vessels throughout the tissue graft. Although ASCs stimulate angiogenesis by migration of endogenous endothelial cells, for adipose constructs of larger size this vascularization is not rapid enough and an earlier vascular supply from the center throughout the whole graft with connections to host vasculature is a crucial requirement for the long-term survival of the graft. Therefore, formation of a capillary network by co-implantation with endothelial cells within a construct might be beneficial for tissue engineering applications. Recently, we and others conducted studies in which human ASCs were cocultured with human endothelial cells to mimic the environment found in vivo. In vitro, we found a stimulatory effect of ASCs on endothelial tube formation (Borges et al., 2007, Strassburg et al., 2013b) as well as the formation of functional blood vessels in short-term semi-in vivo studies on the chorioallantoic membrane (Strassburg et al., 2013a).
Some in vivo studies coimplanted ASCs with human umbilical vein endothelial cells (HUVECs) in different scaffolds (Frerich et al., 2012, Koike et al., 2004, Verseijden et al., 2010) or with human endothelial progenitor cells (EPCs) in Matrigel (Melero-Martin et al., 2007, Melero-Martin et al., 2008). Even if these studies showed promising results in regard to adipose tissue formation and formation of stable long lasting blood vessels, neither HUVECs nor Matrigel are suitable for clinical applications. Therefore, a coimplantation of ASCs with an easy accessible autologous cell population such as endothelial progenitor cells from human peripheral blood in a clinically applicable biomaterial would be of desire.
In the present study, we investigated the potential of human ASCs and EPCs (and HUVECs as positive control) for the formation of vascularized human adipose tissue. To test this, ASCs and EPCs/HUVECs were suspended alone or in combination in fibrin and subcutaneously injected into the back of athymic nude mice. After 1, 3 and 6 months, neovascularization and adipogenesis were evaluated by histology over time. Our results show that only coimplanted HUVECs gave rise to a functional human neovasculature, which was associated with human-derived perivascular cells and that (co)implanted ASCs effectively support adipogenesis.
Section snippets
Cell culture
All cell culture was maintained at 37 °C with 5% CO2 and 20% oxygen in a humidified environment with medium change of 2–3 times a week.
Human adipose-derived stem cells (ASCs; 2 females, 1 male, mean age: 34 years) were isolated from human subcutaneous fat tissue by collagenase-II digestion and erythrocytes lysis (17 mM tris(hydroxymethyl)aminomethane, 16 mM ammonium chloride) as described in detail before (Torio-Padron et al., 2007). ASCs were cultured in EGM-2 Bullet Kit (Lonza) supplemented with
Results
Characteristics and thus suitability of utilized ASCs and EPCs are extensively described in our former publications (Strassburg et al., 2013a, Strassburg et al., 2013b).
A total of 72 mice were operated without complications. The animals were healthy during the experimental period and no sign of inflammation or any infection was noted at the implant sites.
Discussion
Several in vivo approaches are described in the literature to induce neovascularization of tissue engineered adipose tissue equivalents, for example by the application of angiogenic growth factors (Kawaguchi et al., 1998, Masuda et al., 2004, Tabata et al., 2000), scaffold design (Stosich et al., 2007), integration of a vascular pedicle (Dolderer et al., 2007, Stillaert et al., 2007, Walton et al., 2004) or cell based strategies by the addition of endothelial cells (Borges et al., 2003).
In the
Disclosure statement
No competing financial or personal interests exist.
Acknowledgment
The authors thank Birgit Scherer for excellent technical assistance. This work was supported by funding through the Deutsche Forschungsgemeinschaft (TO 614/2-1).
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Both authors contributed equally.