Molecular responses of human dermal fibroblasts to dual cues: Contact guidance and mechanical load

Abstract

Fibroblast contraction in wound healing involves the interaction of several cell types, cytokines, and extracellular matrix molecules. We have previously developed fibroblast alignment models using precise uniaxial mechanical loads in 3D culture and using contact guidance on fibronectin strands. Our aim here was to use contact guidance to place fibroblasts in their potentially most sensitive configuration, i.e., perpendicular to the axis of loading, to present cells with conflicting guidance cues. Gene expression at the mRNA level of cells recovered from different zones of the 3D collagen gel (with distinct orientation) was determined by quantitative RT-PCR for the matrix proteases MMP1, 2, and 3, and inhibitors TIMP1 and 2.Our results show a 2-, 4-, and 3-fold increase in MMP1, 2, and 3, respectively, in the non-aligned strain zone, relative to the aligned strain zone. These results suggest that cells unable to align to applied loads remodel their matrix far more rapidly than orientated cells. Where fibroblasts were held in an alignment perpendicular to the applied load by contact guidance, the fall in MMP mRNA expression was largely abolished, indicating that these cells remained in a mechano-activated state. The protease inhibitors TIMP1 and 2 were poorly mechano-responsive, further suggesting that changes in MMP expression result in functional matrix remodelling. These results indicate how mechanical loading in tissues may influence matrix remodelling, particularly under conflicting guidance cues.

Stretch-induced modulation of matrix metalloproteinases in mineralizing osteoblasts via extracellular signal-regulated kinase-1/2

“…This can lead to spatially directed turnover of the extracellular matrix by directing the osteoclast to a specific location, ultimately resulting in an accurate adaptation of matrix architecture and composition that enables the osteoblasts to better resist mechanical deformation as a result of the forces that act upon them. This theory is supported by findings by Mudera et al, 38 who showed that fibroblasts unable to align to applied loads remodel their matrix far more rapidly than orientated cells.…”

Remodeling of Engineered Tissue Anisotropy in Response to Altered Loading Conditions

“…The investigators did not examine the cytoskeleton of cells attached to the fibronectin cables, and hence it is unclear whether the cells were responding in any morphological way to the mechanical stimulus. However, the cells on fibronectin exhibited trends in matrix protease gene expression indicative of a mechano-active state 24 .…”

“…In another related study, Mudera and co-workers devised a composite matrix of large fibronectin cables embedded in a fibrous collagen gel and oriented perpendicular to the direction of uniaxial mechanical loading 24 . Locally, fibroblasts attached and spontaneously aligned along the fibronectin cables and remained thus during mechanical stimulation, even though neighboring cells in the collagen matrix realigned parallel to the mechanical stimulus.…”

“…In contrast, Barocas et al 1 have shown that smooth muscle cells suspended in gels with magnetically prealigned collagen fibrils can overcome the effects of contact guidance by reorganizing the matrix in response to mechanical constraints imposed during tissue culture, revealing that loading effects can predominate. More recently, Mudera et al demonstrated that on large fibronectin strands embedded within a fine collagen matrix, fibroblasts aligned with the fibronectin, whereas the alignment of neighboring cells in the surrounding collagen was governed by the applied mechanical loading 24 , indicating that substrate feature size may be a key factor. Based on this background, it remains unclear how contact guidance and mechanical stimulation independently impact structural alignment in living replacement load-bearing tissues.…”

Spatiotemporal dynamics of actin-rich adhesion microdomains: influence of substrate flexibility

“…Indeed, it has been shown that the extracellular matrix rigidity plays a crucial role in several cellular processes, ranging from locomotion (Lo et al, 2000;Pelham and Wang, 1997), to anchorage dependency of growth control (Burridge and ChrzanowskaWodnicka, 1996;Chen et al, 1997;Folkman and Moscona, 1978), phagocytosis (Beningo and Wang, 2002), differentiation (Cukierman et al, 2001;Deroanne et al, 2001) and ECM remodelling (Mudera et al, 2000;Tranqui and Tracqui, 2000;Urech et al, 2005). Since the rosettes of podosomes are adherent structures, we investigate the influence of the matrix rigidity on the dynamic properties of these structures in 3T3 fibroblasts transfected with GFP-actin.…”