3-D cell polymer constructs are apparently composed of dissimilarly differentiated cells (see above). However, only mature adipocytes exhibit full functionality. In order to assess cellular functionality within distinct construct areas, the extent of lipolytic cell response was analyzed. The lipolysis rates of the respective construct parts were determined by analyzing the concentration of glycerol released into the incubation buffer. Under control conditions, cells from all groups exhibited basal lipolysis rates. However, only entire constructs and external parts responded significantly (p<0.01) to addition of a lipolysis stimulating factor (10 µM isoproterenol) (Fig. 7). Both groups released 3.49-fold more glycerol as compared to control conditions. In contrast, cell response of internal areas did not change significantly (1.41-fold increase) (Fig. 7). Accordingly, significant inhibition of lipolysis, facilitated by supplementing 100 µM propranolol, could only be triggered in adipocytes from complete constructs (0.47-fold decrease) and outer rings (0.51-fold decrease). Again, cells from inner discs did not change glycerol release significantly (0.64-fold decrease) (Fig. 7). Comparison of absolute values (control conditions: 1.90 ± 0.03 µg (entire), 1.10 ± 0.02 µg (external), and
0.60 ± 0.03 µg (internal)) furthermore demonstrated that externally situated cells contributed to a major extent to the overall lipolytic cell response of entire constructs.
0 1 2 3 4 5
entire external internal Released Glycerol (Relative to Control)
control
+ isoproterenol + propranolol
Fig. 7:
Functionality of 3T3-L1 adipocytes within differential sites of 3-D cell-polymer constructs as assessed by analysis of released glycerol subsequent to treatment with a lipolysis stimulating (10 µM isoproterenol) and a lipolysis inhibiting (100 µM propranolol) agent. Three independent experiments were performed. Data are expressed as mean ± SD (n=3) of one representative experiment. Statistically significant differences to control conditions (p < 0.01) are denoted by «. Absolute values under control conditions were 1.90 ± 0.03 µg (entire), 1.10 ± 0.02 µg (external), and 0.60 ± 0.03 µg (internal).
Discussion
The focus of the presented study was to thoroughly characterize engineered adipose tissue constructs after differentiation over 9 days. In particular, it was aimed at comprehensively evaluating the fat-like properties in differential areas of the constructs and to compare the gained results with those determined from conventional 2-D cell culture.
It is commonly acknowledged that in vitro engineered tissues may be subject to insufficient nutrient and oxygen supply and may therefore suffer from the limitation to consist of differential areas [16]. However, to the best of our knowledge, no study has been conducted so far addressing this heterogeneity more in detail. Adipose tissue constructs were generated from PGA polymer meshes and 3T3-L1 preadipocytes as previously described [6].
As preliminary analysis indicated a heterogeneous composition, we intended to take this potential restriction particularly into consideration and examined not only entire constructs but also different construct parts divided by die-punching with a stainless-steel dermal punch.
Thereby, potential differences could be assessed in detail allowing to better figure out the usefulness of the adipose equivalents for basic research.
At first, histological investigations of entire constructs were performed in order to determine preliminarily detected differences of the generated constructs more in detail. By means of light and scanning electron microscopy, a cellular gradient was observed, which revealed that only outer construct areas consisted of well-differentiated, lipid-filled adipocytes, whereas interior regions mainly contained fibroblastic, less-differentiated cells (Fig. 1, 3). Accordingly, laminin, a component of the basement membrane secreted by adipocytes, could only be detected in outer regions of the constructs (Fig. 2). With the objective of comprehensively evaluating the microscopically detected gradient, entire constructs as well as the separated parts were analyzed with regard to triglyceride accumulation, expression of typical fat cell genes, and functionality of the differently located cells. All applied methodologies revealed that the outer rings featured appropriate properties with respect to fat-like characteristics, whereas the inner parts displayed inferior quality (Fig. 4-7). By analyzing entire constructs, a mixture of those distinct areas was recorded and, thus, yielded impaired overall results compared to the findings measured from external parts alone. In detail, the determination of GPDH activity and intracellular triglyceride content showed that outwardly located adipocytes exhibited enhanced lipogenesis relative to the cells harvested from either entire constructs or inner parts (Fig. 4, 5). In terms of adipocyte gene expression, cells situated in interior areas expressed diminished Glut-4, laminin-b1, angiotensinogen, and beta3-AR mRNA levels as compared to the cells derived from complete constructs and the corresponding external rings (Fig. 6). In contrast, gene expression of adipocytes derived from outer regions was either equivalent to that of entire constructs (PPARg, Glut-4, laminin-b1, and angiotensinogen) or was enhanced (beta3-AR). Analysis of functionality further demonstrated that both entire constructs and outer parts yielded basal lipolysis rates under control conditions and appropriate cell response subsequent to supplementation of stimulating or inhibiting factors (Fig. 7). In contrast, internal ones did not exhibit a significant cell response to either stimulating or inhibiting conditions.
Consequently, only external areas of the constructs displayed a coherent adipose tissue consisting of mature adipocytes featuring lipid accumulation, secretion of a laminin containing basement membrane, appropriate gene expression, and functionality. Therefore, to demonstrate the potential of the 3-D culture approach for basic research, these results were used for comparison with conventional 2-D cell culture. Encouraging data were obtained by measurement of both GPDH activity and intracellular triglyceride content revealing increased
lipid accumulation of 3-D cultivated cells relative to that determined from 2-D cultured ones.
Investigation of gene expression by means of RT-PCR showed that laminin-1b mRNA levels were increased in 2-D cultures as compared to 3-D. This observation may be attributed to the fact that the ECM secreted by 3-D cultivated cells is largely kept incorporated within the constructs, which in turn may lead to down-regulation of laminin-1b. In contrast, ECM synthesized in 2-D cell culture is more likely to be aspirated with every medium change, which can be supported by the finding that preadipocyte differentiation in 2-D cell culture results in enhanced medium viscosity [17]. As a consequence, the cells possibly up-regulate laminin-b1 mRNA to maintain the levels required for a physiological environment.
The heterogeneous composition of the in vitro engineered adipose tissue constructs may be ascribed to both the insufficient nutrient and oxygen supply to the internally situated cells and the inadequate removal of catabolites away from them [16,18]. Furthermore, tissue-inducing factors are required for appropriate fat formation. In detail, preadipocytes are reported to only undergo adipogenesis when stimulated with an hormonal cocktail containing either pharmacological concentrations of insulin or IGF-1, a glucocorticoid, and a cAMP-enhancing agent [14,19,20]. In vivo, the supply of substances would be regulated by diffusion processes and simultaneous onset of vascularization [18,21], whereas in vitro-cultures are dependent upon diffusion processes alone. Hence, the quality of the interior areas of the engineered cell-polymer constructs is generally limited to the maximum distance over which factors can effectively diffuse to the cells (app. 100-200 µm [22]). Accordingly, it seems likely that restricted local concentrations of nutrients, oxygen, and tissue-inducing substances, necessary to trigger appropriate adipocyte differentiation and structural reorganization, contributed to the underdevelopment of interior areas. Although dynamic culture conditions (as used in our approach) are known to considerably improve mass transfer of essential biochemical factors as compared to static culture [23-25], they appear to not be adequate enough to allow for homogeneous tissue quality. Hence, efforts have to be undertaken to engineer adipose tissue equivalents homogeneously composed of coherent fat-like structures throughout the whole construct. This challenge is most likely to be met by enhancing the transfer of nutrients, oxygen, and adipogenesis stimulating factors into the constructs, e.g.
through cultivation in perfusion reactors or by development of cell carriers with modified pore size and pore structure [25,26]. Furthermore, the engineering of smaller constructs may prove beneficial. By exhibiting a larger surface to volume ratio, more cells are in direct
contact with the culture medium and, thus, are likely to develop into larger coherent tissue structures relative to the overall construct volume.
In summary, engineered fat-like constructs were shown to exhibit typical adipose tissue characteristics, which were comparable to those of conventional 2-D cell culture.
Nevertheless, die-punching of the constructs allowed for a more precise analysis of the results. Thereby, it could be assessed that the constructs consisted of well- and less-differentiated areas. By comparing adipocytes derived from high quality outer parts with fat-cells differentiated under 2-D conditions, a slight advantage could be revealed with respect to lipid accumulation and laminin-b1 expression. However, only the 3-D culture approach allowed for formation of a coherent tissue-like context. Until meeting the challenge of engineering homogenous fat equivalents, it is suggested that future experiments aimed at elucidating intrinsic adipose tissue properties will be conducted with separated outer parts, rather than entire constructs. Taking into consideration the above mentioned findings, significance for basic research was clearly demonstrated. Nevertheless, it must be noted that the histological cross-sections did not reveal unilocular signet-ring cells. As this lack may be attributed to insufficient cultivation time, future studies over prolonged periods of differentiation will be required and eventually contribute to improving the exhibition of fat-like characteristics.
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