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Chapter 7 Adipose TE based on MSCs and bFGF Scanning electron microscopy (SEM)
Tissue development and cellular distribution were monitored on the surface and in the interior of cell-polymer constructs using SEM. In the presence of bFGF, the scaffold surface was partially covered by cells and structured sheets considered to be extracellular matrix (ECM) material after just two weeks and was completely covered after four weeks (Fig. 4a). Thus, discrete adipocytes could not be observed at the surface, but rather only in the interior of the
Fig. 4a SEM of the surface of cell-polymer constructs (100-fold magnification): Constructs cultivated over 1, 2, and 4 weeks in absence and presence of bFGF revealed a clear tissue development in the course of time, more pronounced after treatment of MSCs with bFGF compared to control group. Figures show single cells or groups of cells attached to the polymeric scaffold and, at later points of time, figures show cell-extracellular matrix areas
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Chapter 7 Adipose TE based on MSCs and bFGF
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Thus, discrete adipocytes could not be observed at the surface, but rather only in the interior of the scaffold. In contrast, in the absence of bFGF, the structure of the scaffold (compare Fig.
2) was still clearly identifiable after one and two weeks; after four weeks, the scaffold was only partially covered by cells and sheets (Fig. 4a).
Fig. 4b SEM of the interior of cell-polymer constructs (500-fold magnification): Constructs cultivated in absence and presence of bFGF are shown after 1 and 4 weeks. Undifferentiated MSCs are designated by the black arrow, differentiated adipocytes by the white arrows. An increase in cell size and changes in morphology with bulged cell membranes due to fat storage were observed. An augmentation of intracellular lipid droplets was observable in the course of time and in presence of bFGF compared to control group. Scale bars: 20 µm.
In the interior of the constructs, differentiated adipocytes exhibited a multivacuolar phenotype (Fig. 4b). The size of the adipocytes was increased in bFGF-treated constructs as compared to the control group (Fig. 4b). In the presence of bFGF, groups of adipocytes were observed even after one week, recognizable by the bulged cell membranes, which are caused by lipid droplets. After four weeks, cells were additionally embedded in structures regarded as extracellular matrix. In the absence of bFGF, most cells were undifferentiated MSCs and only a few differentiated adipocytes were observed.
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Chapter 7 Adipose TE based on MSCs and bFGF Glycerol-3-phosphate dehydrogenase (GPDH) activity
The degree of adipogenesis of MSCs in the cell-polymer constructs was reflected in measurements of the GPDH activity, which was determined three days after each induction.
GPDH is a late marker of adipogenic differentiation, since it is a key enzyme in the biosynthesis of triglycerides. GPDH activity was significantly elevated after supplementation of bFGF compared to the control group after one (6.0-fold), two (3.8-fold), and three (1.8-fold) weeks (Fig. 5). In contrast, values of GPDH activity were equal after four weeks. The kinetics of GPDH activity was different for the two experimental groups. In the absence of bFGF, GPDH activity increased the first three weeks; after that no further increase was observed. In contrast, in the presence of bFGF, GPDH activity reached a maximum after two weeks followed by a steady decrease.
Fig. 5 GPDH activity of cell-polymer constructs: GPDH, a key enzyme involved in triacylglycerol synthesis, was determined at day 3 after each induction of adipogenesis and standardized per mg protein. White bars represent control group without bFGF, gray bars represent bFGF-treated cell-polymer constructs. Values are expressed as mean ± SD (n=3).
Statistically significant differences of bFGF treated cell compared to control group are denoted by ∗ (p < 0.01) and ∗∗ (p<0.05).
Chapter 7 Adipose TE based on MSCs and bFGF
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RT-PCR was performed three days after each induction in order to assess adipocytic gene expression of peroxisome proliferator-activated receptor γ2 (PPARγ2), a key transcription factor of adipogenesis, and of glucose transporter 4 (GLUT4), a late marker of adipogenesis.
The housekeeping gene 18S served as control gene. Basic FGF-treated cells yielded a clearly higher PPARγ2 expression compared to control without bFGF in the complete time course (Fig. 6). The differential expression was most pronounced after one and two weeks and attenuated after four weeks. In cell-polymer constructs cultivated in the absence of bFGF, PPARγ2 expression steadily increased with time. Basic FGF-treated constructs showed an initially high level of PPARγ2 expression, which was only slightly increased thereafter.
GLUT4 expression was elevated in the bFGF group after one and two weeks and similar after four weeks compared to the control group. In the absence of bFGF, similarly to the expression profile of PPARγ2, GLUT4 expression steadily increased with time. In contrast, GLUT4 levels in the presence of bFGF were initally high and were maintained at later time points.
Fig. 6 RT-PCR analysis of cell-polymer constructs: adipocyte-specific gene expression of PPARγ, a key transcription factor in adipogenesis, and glucose transporter 4 (GLUT4) were evaluated at day 3 after induction of adipogenesis. Gene expression of cells cultured in absence (- bFGF) and presence (+ bFGF) of bFGF were compared in the course of time. The housekeeping gene 18S served as internal standard.