gms | German Medical Science

Objectives: In adipose tissue engineering for reconstructive and plastic surgerythe survival and the achievable size of engineered adipose constructs are crucially defined by the extent of vascularization, representing a key and limiting factor. For overcoming this hurdle decellularized jejunal segments represent a potential scaffolding system. The pre-existing vascular structures in these biological extracellular matrix (ECM) segments can be repopulated with endothelial cells, while the lumen provides a large volume for adipogenesis. This combination potentially fosters the development of large-scale vascularized tissue constructs. Therefore, objectives of this study were a) to reconstruct the capillary structures of decellularized porcine jejunal segments with human microvascular endothelial cells (hMVEC), b) to seed human adipose-derived stem cells (hASC) into the lumen of the porcine-derived jejunal ECM constructs, and c) to induce adipogenic differentiation of the hASCs in this co-culture system.

Materials and Methods: Human ASCs were isolated from subcutaneous adipose tissue of healthy female donors obtained in lipoaspiration procedures. Human MVECs were obtained from Lonza (Basel, Switzerland). For the reconstruction of the vasculature of porcine decellularized jejunum (length of segments 6–8 cm), corresponding cell numbers of hMVECs (passage 3; 1,5*10⁶ cells/cm) were seeded into the capillary network. Culture of the seeded hMVECs alone was performed in a medium based on VascuLife (CellSystems, Troisdorf, Germany) in a flow-through bioreactor system with a physiological pulse rate. After a culturing period of two weeks, the co-culture with hASCs (passage 3) was started. The lumen of the jejunum was seeded with 1,5*10⁶ hASCs per centimeter. From this time point on, the constructs were cultured in a previously established co-culture medium based on PBM-2 (Lonza) and VascuLife in a 1:1 ratio with an ongoing physiological pulse rate. After one further week of expansion in co-culture, adipogenic induction was initiated and continued for three weeks.Histology, immunohistochemistry, whole mount staining, MTT assay, and qRT-PCR were used to investigate segments of the cultured construct at different time points.

Results: Successful repopulation of the vascular structures with hMVECs was demonstrated by immunohistochemical staining for CD31 and vWF (various time points), and a MTT assay for living cells (day 14 and 42). Substantial adipogenesis of hASCs was observed within the jejunal lumen between day 21 and 42 in the adipogenically induced constructs. Using histological staining with oil red O on cryosections and a fluorescent BODIPY dye on whole mount stains, an increasing number of lipid droplets and their cumulative enlargement were detected. On the gene expression level, an increase of key adipogenic transcription factors like PPARγ and C/EBPα and of adipogenic markers such as aP2 could be observed over time. In comparison to adipogenically induced constucts, distinctly weaker adipogenesis was observed in control samples. Furthermore, the development of major extracellular matrix components of adipose tissue within adipogenically induced segments could be demonstrated on cryosections, e.g., by immunohistochemical staining for laminin. Remarkably, in induced jejunal segments, close proximity of endothelial cell structures and adipogenically differentiating hASCs was observed, as detected using immunohistochemical and whole mount staining for CD31 and lipid droplets.

Conclusion: In conclusion, the presented work represents a novel approach to the engineering of vascularized adipose tissue in vitro. It could be shown that the capillary structures of decellularized jejunal segments were successfully reseeded with microvascular endothelial cells (MVEC) utilizing a custom-made flow-through bioreactor system. Furthermore, in this six week culture experiment, adipogenesis of hASCs seeded into the jejunal lumen could be demonstrated. In ongoing work, the culture conditions will be optimized for vascularization and adipogenic differentiation within the adipose construct.

Acknowledgements: The authors wish to acknowledge financial support from the Bavarian Research Foundation (Bayerische Forschungsstiftung), Grant No. AZ-1044-12 FORMOsA, to H.W. and T.B.