Chapter 5. Mechanotopography-driven design of dispersible nanofiber-laden hydrogel as a 3D
5.3. Results and Discussion
5.3.4. Myofibroblast differentiation
It has been well established that TGF-β plays a central role in mediating fibrosis, inducing differentiation into myofibroblasts and increased collagen deposition [8, 42-44]. Therefore, treating the fibroblasts encapsulated in the nanofiber-laden hydrogels with TGF-β was expected to elicit fibrotic phenotypes dependent on the hydrogel conditions. First, the effect of TGF-β on the proliferation was evaluated (Fig. 6). As expected, there was a substantial decrease in proliferation rate at all conditions, indicative of transition into differentiation state [45]. Interestingly, the decrease in proliferation was more noticeable for those with higher nanofiber concentration and increasing presence of GO or rGO.
This coincided with the significant change in fibroblast morphology, in which there were more cells undergoing spreading, which has been widely implicated in differentiation [46, 47]. This observation indicated that under the influence of TGF-β, the cells within nanofiber-laden hydrogels with higher mechanical properties and surface roughness, especially those with GO-Gel NF and rGO-Gel NF, were possibly undergoing significant myofibroblast differentiation, since it has been well documented that fibroblasts undergo significant morphological changes during wound healing, where increased matrix rigidity promotes fibroblasts to acquire myofibroblast phenotypes, including cell spreading, in order to interact with increased collagen fibrils and further remodel the tissue via matrix contraction [46, 47].
Taken together, the presence of TGF-β resulted in diminishing the proliferative capacity of fibroblasts and pushed them towards the path of differentiation within hydrogels. This phenomenon was more frequently observed in nanofiber-laden hydrogels than MGel hydrogels with increasing MGel concentration, again, due to the limited spatial availability.
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Figure 5.6. (a, b) Fluorescent images of fibroblasts encapsulated in MGel hydrogels in the presence of TGF- at various MGel concentrations, Gel NF hydrogels, GO-Gel NF hydrogels, and rGO-Gel NF hydrogels at various nanofiber concentrations (scale: 100μm). (c–f) (c-f) Relative proliferation of fibroblast over time were quantified by MTT assay. (g, h) The proliferation rates (kP) defined by fitting the trend line in (a) and (b) with Eq. (1) (*p < 0.05).
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To further confirm and analyze in detail the myofibroblast differentiation of fibroblasts within nanofiber-laden hydrogels, the expression level of α-SMA, a characteristic marker for myofibroblast differentiation, and cytoskeletal actin organization were identified via immunocytochemistry. In particular, the effects of TGF-β and the mechanotopographical features of nanofiber-laden hydrogels were evaluated. First, the fibroblasts in nanofiber-laden hydrogels without TGF-β treatment were evaluated (Fig. 7b) For MGel hydrogels, the elongated cell morphology was shown only at lower hydrogel rigidity, while both cell aspect ratio and cell area decreased substantially at higher hydrogel rigidity, as expected (Fig. a, c & d). In contrast, the fibroblasts in Gel NF hydrogels showed much greater cell elongation, with the cell aspect ratio of 8 or higher, up to 5 wt% nanofiber (Fig. a, c & d), a further indication that nanofiber-laden hydrogels possessed greater inner space for the cells to extend and migrate at similar rigidity. In addition, the gelatin-based nanofibers also provided additional adhesion sites for the cells. However, the cell aspect ratio did continue to decrease with nanofiber concentration, albeit in lesser extent, which was likely caused by the significantly increased presence of nanofibers surrounding cells acting as a barrier. In both hydrogels with and without nanofibers, however, α-SMA was not noticeably present, indicating that the microenvironmental factors were not sufficient for inducing differentiation.
Incorporating GO or rGO into nanofibers resulted in increased cell area and reduced cell aspect ratio, showing the cells became more evenly spread than elongated. This behavior was more pronounced for rGO-Gel NF hydrogels. With increased adhesive properties of graphene-laden nanofibers providing more sites for adhesion, cells became more spread to allow a greater number of adhesions [48]. More importantly, however, the fibroblasts in GO-Gel NF hydrogels and rGO-Gel NF hydrogels showed meaningful amount of α-SMA expression, but mostly around the nuclei and not throughout the cytoskeletal area which is expected for mature myofibroblasts. This surprising result pointed to the fibroblasts under the early differentiation stage and highlighted that modulating both mechanical and topographical properties of hydrogels, imparted by adopting graphene-laden nanofibers, alone could guide the myofibroblast differentiation, even without providing inflammatory factors [49].
Quantitative real-time PCR (qRT-PCR) was employed to measure the amounts of mRNA for α- SMA (ACTA2) produced by the fibroblasts in hydrogels. Regardless of hydrogel mechanics and nanofiber incorporation, ACTA2 expression remained critically low, which was in line with the low presence of α-SMA in the actin cytoskeleton. This result also suggested that the effects of hydrogel mechanics and surface topography were not significant enough to induce more extensive myofibroblast differentiation without any soluble factors.
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Figure 5.7. (a, b) Immunocytochemistry images of -smooth muscle actin (red) and actin (green) of fibroblasts in MGel hydrogels at various MGel concentrations, Gel NF hydrogels, GO-Gel NF hydrogels, and rGO-Gel NF hydrogels at various nanofiber concentrations. (c, d) Cell phenotype such as aspect ratios and (e, f) cell areas of fibroblasts were measured and quantified from (a) and (b) (*p<0.05, n=30).
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Next, the effect of TGF-β on the fibroblasts in nanofiber-laden hydrogels was explored (Fig. 8). First, the α-SMA expression increased dramatically and found throughout the cytoskeleton in all nanofiber- laden hydrogels, which highlighted the critical role of TGF-β in myofibroblast differentiation (Fig. 8a
& b) However, the α-SMA expression was the highest in rGO-Gel NF hydrogels, followed by GO-Gel NF hydrogels and then Gel NF hydrogels. For MGel hydrogels, the α-SMA expression was much lower and mostly localized around the nuclei. Greater α-SMA expression in nanofiber-infused hydrogels highlight the importance of localized crosslinking, which results in both softer area for ECM deformability and stiff area for spreading and contractile force, leading to myofibroblast transition. In rGO-Gel NF hydrogel, it was more evident due to increased surface roughness.
Generally, fibroblasts in conventional hydrogels having high rigidity are not capable of generating long-range cellular force transmission due to spatial limitation, leading to negligible matrix remodeling, spreading, and proliferation. Recent study has shown that 3D soft matrix induced much more enhancement of proliferation, cell spreading, and myofibroblast transition than the stiff matrix due to enough spatial availability and easier deformation of soft matrix caused by cell traction force and matrix metalloproteinase [50, 51] In contrast, increased matrix rigidity promotes greater cellular force generation by forming aligned actin filaments, which is crucial evidence for fibrotic phenotype [52].
With the nanofiber-laden hydrogel system, it became possible to provide not only spatial availability and softer matrix for cellular spreading but also the locally stiff matrix for promoting actomyosin- mediated cellular tension at the same time. Furthermore, rGO-Gel NF, and GO-Gel NF to a lesser extent, with greater surface roughness promoted more extensive cellular adhesions, leading to sufficient actomyosin-based contractility, formation of stable focal adhesion [53, 54]. This ultimately helped facilitate α-SMA incorporation onto stress fibers and induce more mature myofibroblast differentiation.
This explanation was further corroborated by a detailed examination of cell adhesion and degree of spreading via SEM imaging (Fig. 9). It was clearly evident that the cell spreading became more extensive with the presence of nanofibers and incorporation of GO or rGO into the nanofibers (Gel NF
< GO-Gel NF < rGO-Gel NF). Furthermore, especially with rGO-Gel NF, there were far greater number of filopodial projections along the leading edge of the fibroblast membranes, anchored on the nanofibers.
This visual evidence clearly demonstrated that the nanofibers with higher surface roughness and hydrophobicity, imparted by the presence of GO and rGO, allowed greater cell adhesion and spreading, and ultimately led to more mature myofiber differentiation.
ACTA2 mRNA expression by the fibroblasts in nanofiber-laden hydrogels was closely in line with α- SMA expression, in which the expression level increased with GO or rGO incorporation into nanofibers (Gel NF < GO-Gel NF < rGO-Gel NF). This result clearly demonstrated that the mechanical and surface topographical properties of nanofibers within hydrogels influenced the gene expression of fibroblasts through altered mechanotransduction. It is also noteworthy that ACTA2 mRNA expressions of fibroblasts in MGel hydrogels with varying MGel concentrations were also significantly increased by
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TGF-β. However, there was a lack of α-SMA presence within the actin cytoskeleton of fibroblasts, especially within MGel hydrogels at higher MGel concentration where there was less cell spreading due to limited space. This result suggested that significant extent of actin cyctoskeletal development during cell spreading is required for proper expression and inclusion of α-SMA within the actin cytoskeleton, leading to more mature myofibroblast differentiation.
The cell area in Gel NF hydrogels became lower than those in MGel hydrogels, coupled with higher cell aspect ratios and α-SMA expression, demonstrating greater extent of myofibroblast differentiation (Fig. 8c & e). Interestingly, however, the difference in cell aspect ratios and cell area between Gel NF, GO-Gel NF, and rGO-Gel-NF hydrogels decreased, especially at higher nanofiber concentrations where α-SMA expression was the highest (Fig. 8d & f). This interesting change in cellular morphology suggested that the cells formed more adhesions to adhesive nanofibers with greater extent of spreading.
Even though a certain degree of elongation is a hallmark of differentiation, there was an optimal level of mechanotopographical properties that led to the cell morphology with maximal adhesions for myofibroblast differentiation. Overall, the nanofiber-laden hydrogels with controllable mechanotopographical properties could be employed as a highly effective 3D cell culture platform for in-depth investigation of tissue fibrosis.
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Figure 5.8. (a, b) Immunocytochemistry images of -smooth muscle actin (red) and actin (green) of fibroblasts in MGel hydrogels in the presence of TGF- at various MGel concentrations, Gel NF hydrogels, GO-Gel NF hydrogels, and rGO-Gel NF hydrogels at various nanofiber concentrations. (c, d) Cell phenotype such as aspect ratios and (e, f) cell areas of fibroblasts were measured and quantified from (a) and (b) (*p<0.05, n=30).
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Figure 5.9. SEM images of fibroblast encapsulated within MGel (11%) hydrogel, Gel NF hydrogel, GO-Gel NF hydrogel, and rGO-Gel NF hydrogel (2% nanofiber) (scale bar: 10μm).
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