Development of an bioactive preadipocyte loaded fibroin biohybrid for the augmentation of soft tissue defects.

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This proposal originated at the DFG Nachwuchsakademie Zahnmedizin 2010 in Ulm and represents the second stage of the Nachwuchsakademie program.Defects of subcutaneous adipose tissue in the facial area with a loss of the isolating layer to the deeper laying structures cannot be reconstructed adequately without the use of suitable soft tissue transplants. Nowadays, autologous adipose tissue obtained by liposuction is commonly used, even though it has known shortcomings: the adipose cells are fully differentiated or damaged by the removal technique and are therefore prone to ischemic damage. The transplants shrink and harden or are even lost completely over time. Neovascularisation and nutritive supply are crucial factors for the survival of the transplanted adipose cells. The proposed project aims to develop an alternative to these conventional techniques by utilizing a novel scaffold material consisting of fibroin that presents the growth factors vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2). These growth factors promote the differentiation of adipocytes and vascularization of the transplant. The use of adipose tissue precursor cells (preadipocytes), which are capable of cell division, is advantageous to the use of differentiated adipocytes, mainly due to their higher tolerance for ischaemia. It is planned to construct specifically designed, bioactive three-dimensional scaffolds consisting of fibroin, that will be seeded with autologous preadipocytes and examined regarding their applicability as transplants. Fibroin, a silk protein, is a well-studied and validated biomaterial, that does not cause foreign body reactions or wound healing disorders. For the evaluation of fibroin as a carrier material for preadipocytes, scaffold materials in different three-dimensional structures and with different presentation densities of VEGF and FGF-2 are seeded with human preadipocytes. Subsequently, the adipogenic differentiation is monitored for 21 days by determining the expression levels of specific markers for adipogenic differentiation (PPAR-gamma, Glut4, FABP4, ACS, GPDH) on the transcriptional level (real time PCR) as well as on the protein level (ELISA). Successful differentiation will be verified in functional enzymatic and metabolic assays (GPDH activity, lipolysis and glucose-uptake assay) as well as immunohistologically (PPAR-gamma, Glut4, FABP4, ACS, GDPH, vWF, CD31) and morphologically (Oil-Red-O-staining). If differentiated adipose tissue transplants are successfully generated, an in vivo evaluation of the transplants in a follow-up project is planned.