Page 386 - Advances in Biomechanics and Tissue Regeneration

19.3 INFLUENCE OF EXTRACELLULAR CUES 383

19.3.2 Cell Shape and Dynamic Morphological Changes
While primary chondrocytes are typically round shaped, they undergo a dedifferentiation process upon cultivation
on 2-D substrates when cells adopt an elongated, fibroblastic morphology (with the formation of stress actin fibers),
which is accompanied by a drastic loss in chondrogenic phenotype with the downregulation of chondrogenic tran-
scription factor, SOX9, and Col II and concomitant upregulation of type I collagen I (Col I) [62–64]. Dedifferentiation
is reversible with fibroblastic chondrocytes reexpressing the chondrogenic phenotype upon regaining a rounded cell
shape by cultivation in 3-D hydrogel [64]. Treatment of fibroblastic chondrocytes with pharmacological agents (e.g.,
dihydrocytochalasin B, cytochalasin D, and staurosporine) that interfere with the integrity of the actin cytoskeleton,
resulting in rounding of dedifferentiated chondrocytes, can induce the reexpression of the chondrogenic phenotype
[65–68]. Inhibition of myosin/actin contractility by blebbistatin further enhanced staurosporine-induced redifferentia-
tion that involved PI3K, PKC, and mitogen-activated protein kinase (MAPK)–signaling pathways [67]. These studies
indicate that traction force generated from actin polymerization and myosin/actin contractility associated with the
development of fibroblastic morphology account for loss of chondrocyte phenotype during 2-D culture.
The role of cell shape on MSC chondrogenic differentiation was explored by subjecting transforming growth factor
(TGF)-β3 stimulated MSCs to fibronectin-coated micropatterned island that allowed cells to either flatten and spread
on large islands or maintain a rounded cell morphology on small islands. MSCs kept rounded were committed to a
chondrogenic lineage, while MSCs that were allowed to spread proceeded down a myogenic lineage [69]. Inhibition of
Rac1, a member of the Rho GTPase family associated with cytoskeleton tension, resulted in the inhibition of myogen-
esis while upregulating Sox9, indicating that structural changes to the cytoskeleton play key roles in determining MSC
lineage commitment. Although hydrogels maintain the encapsulated cells in spherical morphology and enhanced
MSC chondrogenesis has been observed with the provision of biochemical cues to improve cell-matrix interactions,
the inherent limitation with cells in hydrogel is that they are subjected to a “locked” morphology within the gel
and have limited direct cell-cell interactions, which are essential for early mesenchymal condensation process and ini-
tiation of MSC chondrogenesis [28]. ECM deposited by differentiated MSCs in poly(ethylene glycol) or HA hydrogel
was found to be restricted to the pericellular domain, even after prolonged culture period [70–72]. Robust chondro-
genesis of MSCs requires dynamic morphological changes, initiated through integrin engagement that induces the
association of their cytoplasmic domains with the actin cytoskeleton, adopting a fibroblastic morphology. Such
integrin-driven changes in cell morphology facilitate enhanced cell-cell interactions, through N-cadherin expression
during condensation events [25, 73]. Condensation is followed by a shift in cell morphology, involving cytoskeletal
rearrangement to form cortical actin, with transient expression of NCAD and N-CAM molecules [29, 58], a process
regulated by Rho kinase (ROCK)–driven actomyosin contractions and myosin II-generated differential cell cortex ten-
sion [35]. The importance of providing an orderly sequence of cell/matrix-induced cell-cell interaction in promoting
robust MSC chondrogenic differentiation has been recognized in several studies [32, 56]. In the study employing ori-
ented Col I ligand in interfacial polyelectrolyte complexation (IPC)–based hydrogels, Raghothaman et al. demon-
strated that proximal integrin-mediated cell-matrix contacts initiated early morphological dynamics and the onset
of N-cadherin/β-catenin-mediated chondrogenic induction resulted in superior chondrogenesis and the generation
of mature hyaline neocartilage in comparison with scaffold-free pellet culture (in which initial matrix-cell interaction
is not provided), and MSC in the conventional collagen hydrogel (in which cell-cell interaction is lacking) [32].

19.3.3 Substrate Topography

Cells can be forced to adopt specific morphology and cytoskeletal orientation by interaction with substrate topogra-
phy, especiallywithengineered surfacepatterns ofnanoscale featuresthat mimic nativeECM.MSCs on nonaligned nano-
fibers are well spread, with actin-rich processes extending isotropically. In contrast, cells on aligned nanofibers are
fibroblastic, extending along the fiber direction. Hyaline cartilage matrix production was preferentially upregulated
on randomly oriented nanofibers, whereas structurally anisotropic, aligned nanofibrous scaffold promotes fibrochondro-
genesis [74–76]. The effect of specific nanotopography in directing MSC chondrogenic differentiation was further dem-
onstrated using thermal imprinted nanotopographical patterns [77]. MSCs on nanograting topography formed actin
stress fiber organization and were induced into a fibrocartilaginous or superficial zone–like neocartilage formation, while
MSCs on nanopillars formed round morphology with their F-actin organized at the cell cortex, readily underwent cell
aggregation reminiscent of condensation, and were induced to form hyaline-like neocartilage tissue [77].Byvarying
the stiffness of substratum nanotopography, Wu et al. further explored the combined effect of substrate topography
and stiffness on MSC chondrogenesis, demonstrating that stiffer patterns inhibit the generation of the hyaline and fibro-
cartilage phenotype on the pillar and grating topography, respectively [78]. Formation of polygonal morphology on the

II. MECHANOBIOLOGY AND TISSUE REGENERATION