Claudia Fischbach1, Thilo Spruß2, Barbara Weiser1, Achim Göpferich1, Torsten Blunk1
1Department of Pharmaceutical Technology, University of Regensburg, 93040 Regensburg, Germany
2Department of Pharmaceutical Chemistry, University of Regensburg, 93040 Regensburg, Germany
To be submitted to Exp. Cell Res.
To develop therapeutic approaches for the prevention and treatment of obesity, a thorough understanding of adipose tissue development is required. Although the potentially large impact of cell-cell and cell-extracellular matrix interactions on adipogenesis still remains to be clarified, the presently used preadipocyte culture models lack a tissue-like context and are, therefore, unsuitable for addressing such interactions. Herein, we report that tissue engineering allows for the reorganization of the 3T3-L1 cell line into fat-like tissues that are histologically comparable to native fat. After cell-seeding of 3-D polymeric scaffolds, the resulting constructs were hormonally stimulated and differentiated for either 9, 21, or 35 days in vitro. Histological investigation revealed that only long-term culture yielded tissues exhibiting the typical composition of unilocular signet ring adipocytes enveloped by a basement membrane. Analysis of triglyceride storage strongly supported the microscopic observations. Despite apparent dissimilarities in tissue coherence, the constructs did not vary substantially with regard to gene expression (mRNA and protein level) and lipolysis.
However, tissue formation did increase leptin secretion. In vivo, only the implantation of differentiated 3-D constructs was demonstrated to yield vascularized fat-pads.
This study demonstrated for the first time the development of 3T3-L1 into fat-pads in vitro and in vivo. The presented model system is suggested as a useful tool to address tissue-inherent interactions under both standardized in vitro and physiological conditions.
Introduction
Although obesity is known to represent a major risk factor for metabolically related disorders, such as cardiovascular diseases and type 2 diabetes mellitus, its prevalence has dramatically increased over the last decades [1-3]. Until a few years ago, adipose tissue was commonly viewed as a passive depot for the storage of energy and obesity was accepted to result from excess calorie storage when energy intake exceeds nutritional requirements.
However, the link between the regulation of adipose tissue mass and metabolic syndromes remained elusive until it was discovered that adipose tissue additionally operates as an endocrine organ secreting a variety of signaling molecules [4,5]. Meanwhile, adipocytes are convincingly recognized to target physiological and pathological processes by releasing factors known to have an impact on immunological responses, vascular function, and appetite regulation [1]. Because there is increasing evidence that obesity is not only due to an increase in adipocyte cell size (hypertrophy) but also caused by de novo differentiation of adipocytes (hyperplasia) [6,7], many studies have been conducted with the aim of gaining thorough insight into how adipose conversion is regulated. By elucidating the factors and signaling pathways critical for adipose differentiation, they distinctly improved the existent knowledge.
However, the mechanisms underlying adipogenesis are not fully clarified yet and, thus, require further examination. For instance, even though a few studies indicated a pivotal role for cell-cell and cell-extracellular matrix (ECM) interactions, the respective aspects have not yet been comprehensively addressed [1].
The use of 3T3-L1 cells, an extensively used model system for analysis of adipocyte differentiation in vitro, may prove beneficial to facilitate investigations into interactions among adipose cells and their ECM. Indeed, conventional 2-D cell culture of those cells does not feature the typical 3-D cell-cell and cell-ECM interactions present within real fat and, thus, only partly reflects physiological conditions. In order to enable studies on adipose tissue formation within an environment better resembling in vivo conditions, we recently established a 3-D model system consisting of 3T3-L1 cells and polyglycolic acid (PGA) polymeric fiber meshes [8]. By evaluating various tissue engineering strategies, we determined the appropriate culture conditions suitable for generating coherent fat-like constructs. The tissues accordingly developed were analyzed extensively and demonstrated typical characteristics of adipose tissue, e.g. triglyceride storage, appropriate adipocyte gene expression, and functionality. However, histological examination showed that they lacked large signet ring cells and, thus, only faintly resembled natural fat. Apparently, the time span investigated (9
days of differentiation) allowed for formation of the mature adipose phenotype, however, it was not adequate to complete cellular reorganization into appropriate tissue structures, which are necessary for addressing the tissue-inherent interactions. As the size of the adipocytes increases during triglyceride accumulation, the detection of undersized cells prompted us to presume that an insufficient cultivation period led to restriction of the fat cell size and, in turn, to the observed differences in tissue coherence.
Therefore, the focus of this study was to cultivate the cell-polymer constructs over prolonged periods of time to generate 3-D fat-like constructs consisting of unilocular signet ring cells in vitro. Subsequently, the correlation between the properties of the engineered tissues and the culture period were investigated. Finally, the tissue development under in vivo conditions was assessed. Specifically, adipose tissue constructs were generated in vitro and harvested at day 9, 21, and 35 after hormonal induction of adipogenesis. Afterwards, they were thoroughly analyzed in terms of appropriate adipose tissue formation. For this purpose, construct appearance was assessed macroscopically as well as by histological staining for cellularity and laminin, a characteristic component of the ECM synthesized by adipocytes.
Furthermore, lipid accumulation was determined by measurement of intracellular triglyceride content and the activity of glycerol-3-phosphate dehydrogenase (GPDH). Appropriate gene expression was analyzed for various typical fat-cell genes by means of RT-PCR. Additionally, leptin, a peptide hormone secreted by mature adipocytes, was quantitatively investigated with ELISA. The functionality of the constructs cultivated for different periods was studied by investigating their lipolysis rates. To examine the development of the engineered tissues in vivo, they were implanted subcutaneously (s.c.) into nude mice. Until now, 3T3-L1 cells have not been shown to give rise to mature fat pads in vivo [9,10], in contrast to other preadipose cell lines such as 3T3-F442A. Because the previously reported studies were performed by s.c. injection of a 3T3-L1 single cell suspension, we aimed at investigating if the implantation of coherent 3T3-L1-polymer constructs exhibiting tissue-like interactions proves helpful and finally allows for reorganization into fat pads. For this purpose, the constructs were implanted and, subsequent to their excision, histologically investigated regarding adipose tissue formation.