Page 390 - Advances in Biomechanics and Tissue Regeneration

19.5 FUTURE DIRECTIONS 387

signaling through integrins [134]. Chondrogenesis is known to be modulated by calcium signaling cascades of specific
temporal sensitivity [135, 136]. Calcium influx via membrane-associated cation channels is a key event in initiating
chondrogenesis, potentially mediated by either the transient receptor potential (TRP) channels or voltage-gated cal-
cium channels (VGCC) [136]. As a ubiquitous second messenger, mechanically induced calcium signaling has been
shown to act as a regulator of mechanotransduction in multiple signaling pathways, including nuclear factor of acti-
vated T lymphocytes (NFAT), protein kinase C, nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB),
c-Jun N-terminal kinase 1 (JNK1), and cyclic adenosine monophosphate (cAMP) response element-binding protein
2+
(CREB) [137]. In addition, Sox9, the master transcription factor of chondrogenesis, is subject to Ca -calmodulin reg-
ulation [138]. Calcium signaling, mediated by transient receptor potential vanilloid 4 (TRPV4), an osmo-
mechanosensitive ion channel highly expressed in articular chondrocytes [139], has been shown to play a primary role
in promoting chondrogenesis [140] and transducing the mechanical signals that support cartilage extracellular matrix
maintenance and joint health [141–143]. Intracellular calcium signaling is also involved in the shear stress–driven
enhancement of PRG4 expression in superficial zone chondrocytes in which purinergic ATP/P2X7 and PKA/CREB
signaling pathways were also implicated [126]. Other studies have further indicated TGF-β/Smad, Erk1/2, p38, and
ciliary signaling [144, 145], as well as integrin/FAK signaling [110, 144], in mediating the responses of chondrocytes to
loading. Dynamic compression study with elastomeric polymeric scaffolds suggested that compression-driven hyper-
trophic development involves cross talk between TGFβ/SMAD2/3 signaling and integrin-ECM interactions, regulat-
ing the suppression of the BMP/GDP and integrin/FAK/ERK signaling [110].
Mechanical stress can directly induce nuclear conformational changes, exerting transcriptional and posttransla-
tional modification [33, 34]. Apart from the response of YAP expression and activity to passive mechanical force
[22], the role of YAP in transducing extrinsic mechanical force stimuli, in relation to chondrocytes and MSC chondro-
genesis, is being established. Using an integrated microfluidic perfusion device that subjects cells to controlled fluid
shear stresses (FSS), Zhong et al. showed that the levels of YAP nuclear distribution in both chondrocyte and MSC
increase, accompanying formation of fibroblastic morphology, with the increased strength of FSS [146]. Chondrocytes
under FSS undergo phenotypic conversion with the loss of Col II, while Col I expression increases. Treatment with
cytochalasin D reverted the FSS-induced morphological change, YAP distribution, and phenotypic conversion, reveal-
ing the connection between YAP and MSC/chondrocyte fates in a fluid flow–induced mechanical microenvironment
[146]. The combined effect of external mechanical treatment such as FSS [147] or tensile stress [34, 86] on chondrocytes
and MSCs already subjected to passive mechanical signal, directed by attachment to aligned fibers, was investigated.
Chondrocytes and MSCs on aligned fibrous meshes elicited further alterations in cell and nuclear morphology, depen-
dent on the direction of force application in relation to the fiber direction [86, 147], which resulted in generation of
fibrochondrocyte when force is parallel to the fiber direction, while yielding a more hyaline phenotype when force
is perpendicular to the fiber direction. The effect of applied tension is mediated by filamentous actin cytoskeleton
and can be curtailed with ROCK inhibitor and knockdown of YAP/TAZ [34, 147]. These studies suggest that the direc-
tion between applied force and the orientation of cells on the substrate have profound effects on intracellular mechan-
otransduction and play a significant role in regulating chondrogenesis. Of similar note, when comparing the response
of MSCs seeded in either agarose (spherical MSC morphology) or fibrin (spread MSC morphology with clear stress
fiber formation) hydrogels to the application of HP, it was demonstrated that while agarose provided a stronger pro-
chondrogenic environment, a more robust response to the application of HP was observed in fibrin hydrogels [114],
again demonstrating that cells with differing cytoskeleton contractility will alter their response to external mechanical
stimulation.

19.5 FUTURE DIRECTIONS

The impact of both mechanical environment and extrinsic mechanical force in directing chondrocyte phenotype and
MSC chondrogenic differentiation has been well recognized. Recent studies have been performed to decipher the intra-
cellular mechanotransduction mechanism in transmitting the various mechanical signals. Nonetheless, many ques-
tions remain, including the specific microenvironment cues best suited for imparting desirable mechanical
signaling for chondrocytes or MSCs, given that the differences in the developmental status of MSCs and the terminally
differentiated mature chondrocytes have been shown to prefer different microenvironment cues [42, 148–150]. The
majority of studies exploring the mechanobiology of chondrocytes and MSCs have been performed using in vitro sys-
tems, which have helped to improve our understanding on how cells respond to specific stimuli. Increasing studies
have started exploring the effect of multiple cues, including biophysical and biochemical factors, in combination with
externally applied force, to mimic the interplay between intrinsic and extrinsic mechanical cues in the in vivo

II. MECHANOBIOLOGY AND TISSUE REGENERATION