Goals of the Thesis
2. Three-dimensional (3-D) adipocyte culture
To study tissue-inherent factors such as cell-cell and cell-matrix interactions, the 2-D culture system is limited and cannot fully capture the relevant complexity of in vivo adipose tissue. In contrast, a 3-D tissue-like model system likely is more appropriate attempting to mimic the in vivo tissue environment as closely as possible. Furthermore, for tissue reconstruction, the use of 3-D substitutes is inevitable.
Chapter 2 Goals of the Thesis
2.1. Investigation of collagens in adipogenesis in vitro
During the last decades, it was shown that the adipocyte function is strongly influenced by the surroundings of the cells [4,5]. Various studies have described the regulation of several extracellular matrix (ECM) components during adipogenic differentiation [6-10].
Nevertheless, the composition of the ECM in adipose tissue as well as its role in adipogenesis has not been fully understood. To study the influence of various ECM components on adipogenic differentiation in vitro, we have previously developed a 3-D adipocyte culture system based on the formation of spheroids [11]. In contrast to scaffold- or gel-based 3-D constructs, this 3-D model system provides a coherent adipose tissue-like context including direct cell-cell and cell-matrix interactions and represents an adequate model for studying the importance of the ECM for adipocyte development. Thereby, the role of collagens in adipogenesis is of major interest since these ECM components are the most abundant molecules in the matrix. However, the role of collagens in adipogenesis has not been investigated in a 3-D tissue-like context so far. As the well organized matrix structure of 3-D adipose tissue seems to modulate adipocyte differentiation, we hypothesized that the inhibition of the collagen synthesis by a prolyl hydroxylase inhibitor has more profound effects in 3-D spheroids compared to 2-D culture. Therefore, we examined the influence of collagens on the adipogenesis of 3T3-L1 cells as well as ADSCs cultured in 3-D spheroids and 2-D culture by using ethyl-3,4-dihydroxybenzoate (EDHB) as an inhibitor of collagen synthesis (Chapter 5).
In general, it is known that the expression of collagens, particularly fibrillar collagens, is regulated during adipose conversion [5,8,10]. However, the organization of the collagen network through interactions of the molecules during adipogenesis is not well characterized.
The so-called fibril-associated collagens with interrupted triple helices (FACIT) are important for the structure of the ECM network. Members of the FACIT family are localized on the surface of major collagen fibrils and act as molecular bridges that arrange the structural integrity of the ECM. Therefore, we supposed that FACITs may be involved in adipocyte differentiation. So far, FACITs have not been investigated during adipocyte development.
One member of the FACIT sub-family is collagen XVI which is mainly expressed in skin and cartilage in humans [12,13]. As collagen XVI mediates anchoring processes and remodelling of the ECM [14,15], we hypothesized that this FACIT collagen is also involved in adipose conversion. Therefore, we investigated the expression of collagen XVI during the in vitro adipogenesis of 3T3-L1 preadipocytes and ADSCs in 2-D and 3-D culture (Chapter 6).
Chapter 2 Goals of the Thesis
- 44 -
2.2. Novel hydrogels for adipose tissue engineering
Besides the benefit of 3-D culture systems in basic research, 3-D adipose substitutes are required for clinical applications to augment soft tissue defects, e.g., after traumatic injuries or tumor resections. However, reviewing currently used adipose engineering strategies, it becomes evident that cell carriers that are more adipose-specific are desirable. Hydrogels represent a promising 3-D scaffold for adipose tissue engineering due to effective cell encapsulation and unrestricted diffusion of nutrients and metabolites. Recently, a novel biodegradable poly(ethylene glycol) (PEG)-based hydrogel has been developed by our group [16]. The gel-forming polymers were functionalized with a synthetic tetrapeptide which enables proteolytic degradation of the hydrogel by cell secreted proteases. Furthermore, the hydrogel provides an elastic microenvironment which mimics the properties of native ECM.
Another promising approach of this gel system is the possibility to functionalize the hydrogel with adhesion molecules, hormones or growth factors to promote the formation of a coherent adipose tissue. The aim of this study was to investigate the suitability of this novel biointeractive PEG-based hydrogel for adipose tissue engineering. To this end, the hydrogels were seeded and cultured with 3T3-L1 preadipocytes to study the influence of substrate stiffness, adhesiveness, and degradability on cell proliferation and adipogenic differentiation in vitro (Chapter 7).
Chapter 2 Goals of the Thesis
References
1. Hasengschwandtner F. Phosphatidylcholine treatment to induce lipolysis.
J.Cosmet.Dermatol. 2005; 4: 308-313.
2. Rittes P G. The use of phosphatidylcholine for correction of lower lid bulging due to prominent fat pads. Dermatol.Surg. 2001; 27: 391-392.
3. Zuk P A, Zhu M, Mizuno H, Huang J, Futrell J W, Katz A J, Benhaim P, Lorenz H P, Hedrick M H. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Engineering 2001; 7: 211-228.
4. Ibrahimi A, Bonino F, Bardon S, Ailhaud G, Dani C. Essential role of collagens for terminal differentiation of preadipocytes. Biochem.Biophys.Res.Commun. 1992; 187:
1314-1322.
5. Nakajima I, Muroya S, Tanabe R, Chikuni K. Extracellular matrix development during differentiation into adipocytes with a unique increase in type V and VI collagen. Biol.Cell 2002; 94: 197-203.
6. Mariman E C, Wang P. Adipocyte extracellular matrix composition, dynamics and role in obesity. Cell Mol.Life Sci. 2010.
7. Kuri-Harcuch W, Arguello C, Marsch-Moreno M. Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture. Differentiation 1984; 28: 173-178.
8. Aratani Y, Kitagawa Y. Enhanced synthesis and secretion of type IV collagen and entactin during adipose conversion of 3T3-L1 cells and production of unorthodox laminin complex. J.Biol.Chem. 1988; 263: 16163-16169.
9. Ono M, Aratani Y, Kitagawa I, Kitagawa Y. Ascorbic-Acid Phosphate Stimulates Type-Iv Collagen-Synthesis and Accelerates Adipose Conversion of 3T3-L1 Cells.
Experimental Cell Research 1990; 187: 309-314.
10. Weiner F R, Shah A, Smith P J, Rubin C S, Zern M A. Regulation of collagen gene expression in 3T3-L1 cells. Effects of adipocyte differentiation and tumor necrosis factor alpha. Biochemistry 1989; 28: 4094-4099.
11. Weiser, B. Adipose Tissue Engineering - Precultivation Strategies towards Clinical Applications & A Novel 3-D Model of Adipogenesis for Basic Research. Ph.D. thesis 2008. University of Regensburg, Regensburg, Germany.
12. Grassel S, Unsold C, Schacke H, Bruckner-Tuderman L, Bruckner P. Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis. Matrix Biol. 1999; 18: 309-317.
13. Kassner A, Hansen U, Miosge N, Reinhardt D P, Aigner T, Bruckner-Tuderman L, Bruckner P, Grassel S. Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils. Matrix Biol. 2003; 22: 131-143.
14. Senner V, Ratzinger S, Mertsch S, Grassel S, Paulus W. Collagen XVI expression is upregulated in glioblastomas and promotes tumor cell adhesion. FEBS Lett. 2008;
582: 3293-3300.
15. Eble J A, Kassner A, Niland S, Morgelin M, Grifka J, Grassel S. Collagen XVI harbors an integrin alpha1 beta1 recognition site in its C-terminal domains.
J.Biol.Chem. 2006; 281: 25745-25756.
16. Brandl F, Hammer N, Blunk T, Tessmar J, Goepferich A. Biodegradable hydrogels for time-controlled release of tethered peptides or proteins. Biomacromolecules. 2010; 11:
496-504.