The elasticity of the hydrogel biomaterials were tuned to mimic certain tissue types;
adipose at 2 kPa, muscle at 6 kPa and osteoid (demineralised bone tissue) at 38 kPa (Engler et al., 2006) with the hypothesis that MSCs will interpret these properties and differentiate accordingly. As such, cells were stained for formation of cell types of typical mesenchyme lineage. In addition, cells were also checked for neuronal development as development along this lineage by MSCs has been observed on rigidity-tuned hydrogels (Kopen et al., 1999, Mareschi et al., 2006, Woodbury et al., 2002).
Immunofluorescent-labelled biomarkers showed that differential behaviour of cells on the F2/S hydrogels were not as clear-cut as suggested by the aforementioned hypothesis.
Cells showed a heterogeneous mix of lineages on the substrates staining positively for more than one cell lineage.
Cells cultured on the soft (2 kPa) F2/S hydrogel surface stained positively for the early neurological biomarker nestin, adipogenic marker PPAR-γ and the chondrogenic marker SOX-9. There was no observed production of the osteogenic biomarker RUNX-2 (Figure 2-7).
Figure 2-6 Analysis of morphological properties of MSCs cultured on 2 kPa, 6 kPa and 38 kPa F2/S substrates. Cells were analysed for degree of cell spreading (cell lengths measured from distal tips) on each substrate (A) and cytoskeletal properties (F-actin, shown in red and cell nucleus shown in blue) were also investigated (B-D). Cells cultured on 2 kPa F2/S hydrogels (B) showed the presence of filopodia like out growths emanating from the cell body. Shorter processes were observed on the 6 kPa substrate (C) while cells on the 38 kPa substrate were lacking in the outgrowths showing a diffused actin composition (D). Error bars in (A) denote standard error; *** indicates statistical significance where p < 0.001 as calculated using one way ANOVA; n > 40; Scale bar – 50 μm.
Cells cultured on the stiff (6 kPa) F2/S hydrogel surface stained positively for nestin, PPAR-γ, SOX-9 and RUNX-2 with the highest population observed for PPAR-γ and SOX-9 (Figure 2-8). Cells cultured on the rigid (38 kPa) F2/S hydrogel surface stained positively for nestin, PPAR-γ, SOX-9 and RUNX-2 with the highest population observed for SOX-9 and RUNX-2 (Figure 2-9).
Cells stained for myoblast formation (MyoD), as was expected for the stiff (6 kPa) hydrogel, did not give any positive results. As such it was concluded that the hydrogels did not support cell differentiation along the myogenic lineage.
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Figure 2-7 Immunofluorescence microscopy images to ascertain phenotypical development of MSCs cultured on 2 kPa F2/S hydrogel surfaces. Cells were cultured on 2 kPa F2/S surfaces and maintained for 1 week. Fluorescence staining was carried out for detection of the differentiation biomarkers (shown in green) nestin, for neurogenesis (A), PPAR-γ for adipogenesis (B), MyoD for myogenesis (C), SOX-9 for chondrogenesis (D) and RUNX-2 for osteogenesis (E). Cells had stained positively for neurogenic, adipogenic and chondrogenic development but were negative for myogenesis and osteogenesis. Cell nuclei are shown in blue (DAPI). Scale bar – 50 μm.
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Figure 2-8 Immunofluorescence microscopy images to ascertain phenotypical development of MSCs cultured on 6 kPa F2/S hydrogel surfaces. Cells were cultured on 6 kPa F2/S surfaces and maintained for 1 week. Fluorescence staining was carried out for detection of the differentiation biomarkers (shown in green) nestin, for neurogenesis (A), PPAR-γ for adipogenesis (B), MyoD for myogenesis (C), SOX-9 for chondrogenesis (D) and RUNX-2 for osteogenesis (E). Cells had stained positively for all tested lineages with the exception of myogenesis. Cell nuclei are shown in blue (DAPI). Scale bar – 50 μm.
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Figure 2-9 Immunofluorescence microscopy images to ascertain phenotypical development of MSCs cultured on 38 kPa F2/S hydrogel surfaces. Cells were cultured on 38 kPa F2/S surfaces and maintained for 1 week. Fluorescence staining was carried out for detection of the differentiation biomarkers (shown in green) nestin, for neurogenesis (A), PPAR-γ for adipogenesis (B), MyoD for myogenesis (C), SOX-9 for chondrogenesis (D) and RUNX-2 for osteogenesis (E). Cells had stained positively for all tested lineages with the exception of myogenesis. Cell nuclei are shown in blue (DAPI). Scale bar – 50 μm.
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As the cells cultured on each of the F2/S substrates had stained positively for a number of cell lineages using immunocytochemistry techniques, the degree to which the observed population heterogeneity had occurred was then ascertained by quantifying biomarker gene expression on each of the substrate types using qRT-PCR.
Cells were analysed for expression of early and subsequently expressed biomarkers particular to neuronal, adipogenic, chondrogenic and osteogenic development after 1 week in culture.
While also reflecting the heterogeneity observed using immunofluorescence, quantitative results obtained by qRT-PCR was able to give a more defined picture of the adopted differentiation lineages of cell populations cultured on the hydrogels. From this, primary differentiation lineages on each F2/S were determined.
Cells on the 2 kPa substrate showed high expression of nestin compared to the other two hydrogels suggesting initial differentiation along the neuronal route. Elevated levels of β3-tubulin were also observed for cells cultured on this substrate albeit not statistically significant (Figure 2-10). A statistically significant increase in GLUT-4 expression was also noted on this hydrogel illustrating adipogenic development. Levels however, were far less compared to that observed for nestin and β3-tubulin (approximately 900 and 8 times respectively). On the 6 kPa substrate, type II collagen was observed to have the highest expression of all tested genes (Figure 2-11). In addition, SOX-9 levels were also observed to be highest on the 6 kPa hydrogels compared to the 2 kPa and 38 kPa substrates – expression levels of SOX-9 were approximately 17 times greater on 6 kPa than on 38 kPa F2/S and 194 times higher than measured on 2 kPa F2/S.
Cells cultured on the 38 kPa substrate showed highest expression of osteopontin (Figure 2-12). The earlier expressed RUNX-2 was also observed as showing higher expression on the 38 kPa hydrogel compared to the remaining two substrates – expression levels of RUNX-2 were approximately 5 times greater on 38 kPa than on 6 kPa F2/S and 14 times higher than measured on 2 kPa F2/S.
Although there is some underlying overlap, these results suggest that the stiff (6 kPa) and rigid (38 kPa) substrates are best adept at influencing chondrogenic and osteogenic development respectively.
Relative to the control, expression of adipogenic markers PPAR-γ and GLUT-4 were found to be elevated on all three substrates but did not show any preference to any one substrate in particular.
Figure 2-10 Gene expression analysis of MSCs undergoing phenotypical development on 2 kPa F2/S hydrogel surfaces. Cells were cultured for 1 week on 2 kPa F2/S hydrogels and samples subsequently analysed for expression of early and latterly expressed differentiation specific biomarkers. Cells were analysed for production of nestin & β3-tubulin for neurogenesis, PPAR-γ & GLUT-4 for adipogenesis, SOX-9 & COL2A1 for chondrogenesis and RUNX-2 & OPN for osteogenesis. Gene expression was measured as fold change compared to cells maintained on plain surfaces as a negative control (held nominally at 1). Error bars denote standard error from the mean; n = 4 replicates; * notes statistical significance compared to the control where p < 0.05 and *** where p < 0.001 as calculated using one way ANOVA followed by Bonferroni post hoc tests.
Figure 2-11 Gene expression analysis of MSCs undergoing phenotypical development on 6 kPa F2/S hydrogel surfaces. Cells were cultured for 1 week on 6 kPa F2/S hydrogels and samples subsequently analysed for expression of early and latterly expressed differentiation specific biomarkers. Cells were analysed for production of nestin & β3-tubulin for neurogenesis, PPAR-γ & GLUT-4 for adipogenesis, SOX-9 & COL2A1 for chondrogenesis and RUNX-2 & OPN for osteogenesis. Gene expression was measured as fold change compared to cells maintained on plain surfaces as a negative control (held nominally at 1). Error bars denote standard error from the mean; n = 4 replicates; *** notes statistical significance compared to the control where p < 0.001 as calculated using one way ANOVA followed by Bonferroni post hoc tests.
Figure 2-12 Gene expression analysis of MSCs undergoing phenotypical development on 38 kPa F2/S hydrogel surfaces. Cells were cultured for 1 week on 38 kPa F2/S hydrogels and samples subsequently analysed for expression of early and latterly expressed differentiation specific biomarkers. Cells were analysed for production of nestin & β3-tubulin for neurogenesis, PPAR-γ & GLUT-4 for adipogenesis, SOX-9 & COL2A1 for chondrogenesis and RUNX-2 & OPN for osteogenesis. Gene expression was measured as fold change compared to cells maintained on plain surfaces as a negative control (held nominally at 1). Error bars denote standard error from the mean; n = 4 replicates; ** notes statistical significance compared to the control where p < 0.01 as calculated using one way ANOVA followed by Bonferroni post hoc tests.