Stem cell-based adipose tissue engineering

In reconstructive and plastic surgery, there exists a continuously increasing demand for adipose tissue surrogates to replace fat tissue following tumor resections, complex trauma and congenital abnormalities as well as to augment tissues for cosmetic purposes. However, the optimal approach to adipose tissue replacement and reconstruction still remains elusive after more than a decade of research. The fast growing field of tissue engineering may provide alternative strategies that improve upon the conventional surgical options. Recently, promising new therapy strategies based on tissue engineering techniques that combine de novo adipogenesis and cell-based therapeutic approaches using adipocytes and preadipocytes have been developed. Stem cells derived from adult bone marrow, also referred to as mesenchymal stem cells (MSCs), represent a promising alternative cell source for soft tissue engineering. However, no study on tissue engineered fat exists using MSCs. In this thesis, the potential of MSCs for adipose tissue engineering applications was investigated. Thereby, the growth factor basic fibroblast growth factor (bFGF) was found to strongly enhance the hormonal cocktail-induced adipogenesis of MSCs in 2-D cell culture. This was determined on the histological and molecular level employing Oil Red O staining, FACS analysis, glycerol-3-phosphate dehydrogenase (GPDH) activity measurement, and RT-PCR of the genes glucose transporter 4 (GLUT4) and peroxisome proliferator-activated receptor gamma (PPAR gamma). This adipogenic culture protocol could be successfully transferred to a 3-D culture system using poly(lactic-co-glycolic acid) (PLGA) scaffolds as a cell carrier. In presence of bFGF, the constructs partially resembled characteristics of native fat such as unilocular adipocytes as determined by osmium tetroxide histology and scanning electron microscopy and the expression of adipocyte-specific genes and proteins including GLUT4, PPAR gamma, and GPDH. In addition, biomimetic polymers were utilized for the generation of vascularized constructs by a novel principle based on the instant surface modification of polymeric scaffolds with the growth factor bFGF. Recently, a new generation of polymers, also termed biomimetic polymers, has been designed in order to control the cellular behavior on the molecular level. In our laboratory, poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymers have been synthesized to which peptides and proteins can be covalently bound by an instant surface modification. Furthermore, a technique has been recently developed to process these polymers into 3-D scaffolds. As a first step towards the proof of the feasibility of the tethering of bFGF to scaffolds made from a PEG-PLA derivative, the interactions of bFGF with PEG-PLA (adsorption, desorption) derivatives were investigated. In the next study, the tethering of bFGF to the scaffolds was characterized in detail. The maximum amount of bFGF which could be covalently immobilized to the scaffolds under appropriate conditions was determined. The stability of the linkage of bFGF to the scaffolds was determined in an in vivo experiment. Radiolabeled bFGF could be anchored for at least three weeks within the scaffolds. And last but not least, the bioactivity and the angiogenic effect of tethered bFGF was assessed in a further in vivo study. Remarkably, only tethered bFGF strongly induced angiogenesis within the scaffolds, whereas adsorbed bFGF remained ineffective. In conclusion, this thesis shows the potential of MSCs for adipose tissue engineering applications for the first time. Basic FGF was found to be a suitable growth factor for the enhancement of the adipogenesis of MSCs in 2-D and 3-D cell culture and to strongly improve the generation of engineered adipose tissue-like constructs. In addition, the delivery of bFGF by tethering to 3-D scaffolds allows for the vascularization of tissue constructs in vivo. This system represents a novel �off-the-shelf� product for tissue engineering applications. In future studies, a combination of these two strategies may lead to mature vascularized engineered adipose tissue