Matrix Stiffness Method

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow-derived stem cell motility. We adopted an "inverse opal" processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells-including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within.

Tendinopathy is characterised by pathological changes in tendon matrix composition, architecture, and stiffness, alterations in tendon resident cell characteristics, and fibrosis, with inflammation also emerging as an important factor in tendinopathy progression. The sequence of pathological changes in tendinopathy and the cellular effects of the deteriorating matrix are largely unknown. This study investigated the effects of substrate stiffness on tendon-derived cells (TDCs) and THP-1 macrophages using PDMS substrates representing physiological tendon stiffness (1.88 MPa), a stiff gel (3.17 MPa) and a soft gel (0.61 MPa). Human TDCs were cultured on the different gel substrates and on tissue culture plastic. Cell growth was determined by alamarBlue™ assay, cell morphology was analysed in f-actin labelled cells, and phenotypic markers were analysed by real-time PCR. We found that in comparison to TDCs growing on gels with physiological stiffness, cell growth increased on soft gels at 48 h (23%, p = 0.003). Cell morphology was similar on all three gels. SCX expression was slightly reduced on the soft gels (1.4-fold lower, p = 0.026) and COL1A1 expression increased on the stiff gels (2.2-fold, p = 0.041). Culturing THP-1 macrophages on soft gels induced increased expression of IL1B (2-fold, p = 0.018), and IL8 expression was inhibited on the stiffer gels (1.9-fold, p = 0.012). We also found that culturing TDCs on plastic increased cell growth, altered cell morphology, and inhibited the expression of SCX, SOX9, MMP3, and COL3. We conclude that TDCs and macrophages respond to changes in matrix stiffness. The magnitude of responses measured in TDCs were minor on the range of substrate stiffness tested by the gels. Changes in THP-1 macrophages suggested a more inflammatory phenotype on substrates with non-physiological stiffness. Although cell response to subtle variations in matrix stiffness was moderate, it is possible that these alterations may contribute to the onset and progression of tendinopathy.

The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

Objective To investigate the mechanism of matrix stiffness promoting epithelial-mesenchymal transformation (EMT) and vasculogenic mimicry (VM) of glioma cells by yes-related proteins (YAP). Methods Human glioma U87 cell lines were cultured under different matrix stiffness conditions. Matrix stiffness 0.2, 16.0 and 64.0 kPa represented normal brain, glioma and extreme stiffness, respectively. CCK-assay was used to examine the proliferation of U87 cell line.The localization of YAP cells was detected by immunofluorescence staining. The expression of YAP targeting genes including CTGF and CRY61 were detected by real-time quantitative PCR. The expression and phosphorylation expression of YAP, proliferation signal including Akt and ERK1/2 were detected by Western blot. Immunofluorescence and Western blot were used to detect the expression of EMT-related proteins including Vimentin, E-cadherin and Twist. VM formation was detected in soft and stiff substrates under 3D culture. U87 cells were transfected with shYAP lentivirus, and then the expression of EMT proteins and VM formation were examined. Results With the increase of matrix stiffness, the proliferation activity of glioma cells and phosphorylation expression of Akt and ERK1/2 were increased (all P<0.05), the expression of phosphorylated Yap was decreased (P<0.05), while the expression levels of CTGF and CRY61 were increased (both P<0.05) as the nuclear translocation of YAP was increased. The results of immunofluorescence staining and Western blot showed that with the increase of matrix stiffness, the EMT component expression levels of Vimentin and Twist were increased, the epithelial marker E-cadherin was decreased (all P<0.05). Compared with that under soft matrix 3D culture, the formation of VM tubular structure in U87 cells was increased in stiff matrix (P<0.05). Interference with Yap expression inhibited the enhancement of the proliferation of glioma cells, the expression of Vimentin and Twist, and VM formation induced by matrix stiffness (all P<0.05). Conclusions Matrix stiffness promotes the proliferation of glioma cells, EMT and VM formation through YAP. Targeted matrix stiffness and YAP can be used as new strategies for glioma treatment. Key words: Glioma; Matrix stiffness; Yes-associated protein; Epithelial-mesenchymal transition; Vasculogenic mimicry

Mesothelial cells cover the surface of the internal organs and the walls of body cavities, facilitating the movement between organs by secretion of a lubricating fluid. Upon injury, mesothelial cells undergo a mesothelial-mesenchymal transition (MMT) and give rise to myofibroblasts during organ fibrosis, including in the liver. Although transforming growth factor-β1 (TGF-β1) was shown to induce MMT, molecular and cellular mechanisms underlying MMT remain to be clarified. In the present study, we examined how the extracellular environment, soluble factors, and cell density control the phenotype of liver mesothelial cells by culturing them at different cell densities or on hydrogels of different stiffness. We found that TGF-β1 does not fully induce MMT in mesothelial cells cultured at high cell density or in the absence of fetal bovine serum. Extracellular lysophosphatidic acid (LPA) synergistically induced MMT in the presence of TGF-β1 in mesothelial cells. LPA induced nuclear localization of WW domain-containing transcription regulator1 (WWTR1/TAZ) and knockdown of Taz, which suppressed LPA-induced MMT. Mesothelial cells cultured on stiff hydrogels upregulated nuclear localization of TAZ and myofibroblastic differentiation. Knockdown of Taz suppressed MMT of mesothelial cells cultured on stiff hydrogels, but inhibition of TGF-β1 signaling failed to suppress MMT. Our data indicate that TAZ mediates MMT induced by TGF-β1, LPA, and a stiff matrix. The microenvironment of a stiff extracellular matrix is a strong inducer of MMT.

Inherent negative refraction on acoustic branch of two dimensional phononic crystals

Matrix dimensionality and stiffness cooperatively regulate osteogenesis of mesenchymal stromal cells

Fibrosis progression in systemic sclerosis (SSc) has been attributed to matrix stiffness. Despite extensive research on fibroblast heterogeneity and subset imbalances in fibrotic disorders, the interplay between biomechanical cues and fibroblast dynamics remains largely unexplored. Here, we investigate how matrix stiffness alters fibroblast transcriptional state and influences lineage specification in fibrotic skin. We employed a collagen I-based 3-dimensional culture system to expose fibroblasts to varying levels of matrix stiffness, followed by RNA sequencing to identify stiffness-responsive gene expression signature. We integrated single-cell RNA sequencing data from SSc and healthy skin samples to identify fibroblast subsets associated with this signature. Spatial transcriptomic analyses were performed to localise these fibroblasts and their associations with the fibrotic niche. Fibroblasts subjected to increased matrix stiffness exhibited a distinct transcriptional signature, amplified in SSc patients and enriched in PI16+ progenitor-like cells within the SFRP2+ fibrotic compartment. Further analysis indicated that PI16+ fibroblasts are predisposed to SFRP2+COMP+ PU.1+ myofibroblasts differentiation, whereas blocking mechanotransduction by focal adhesion kinase inhibition disrupts this process, suggesting that matrix stiffness is a key driver of this lineage transition. Spatial mapping revealed colocalisation of the PI16+ and COMP+ subsets in extracellular matrix-dense regions, highlighting the functional relevance of this relationship in fibrotic progression. Our findings suggest that increased matrix stiffness promotes fibroblast precursor differentiation into SFRP2+ COMP+ PU.1+ myofibroblasts, thereby sustaining the vicious cycle of persistent fibrosis in absence of inflammatory triggers. These insights reveal new aspects of fibrosis pathogenesis and highlight biomechanical signals as therapeutic targets in SSc.

Injury to the central nervous system (CNS) usually leads to the activation of astrocytes, followed by glial scar formation. The formation of glial scars from active astrocytes in vivo has been found to be dependent on the cell microenvironment. However, how astrocytes respond to different microenvironmental cues during scar formation, such as changes in matrix stiffness, remains elusive. In this work, we established an in vitro model to assess the responses of astrocytes to matrix stiffness changes that may be related to pathophysiology. The investigated hydrogel backbones are composed of collagen type I and alginate. The stiffness of these hybrid hydrogels can be dynamically changed by association or dissociation of alginate chains through adding crosslinkers of calcium chloride or a decrosslinker of sodium citrate, respectively. We found that astrocytes obtain different phenotypes when cultured in hydrogels of different stiffnesses. The obtained phenotypes can be switched in situ when changing matrix stiffness in the presence of cells. Specifically, matrix stiffening reverts astrogliosis, whereas matrix softening initiates astrocytic activation in 3D. Moreover, the effect of matrix stiffness on astrocytic activation is mediated by Yes-associated protein (YAP), where YAP inhibition enhances the upregulation of GFAP and contributes to astrogliosis. To investigate the underlying mechanism of matrix stiffness-dependent GFAP expression, we also developed a mathematical model to describe the time-dependent dynamics of biomolecules involved in the matrix stiffness mechanotransduction process of astrocytes. The modeling results further indicate that the effect of matrix stiffness on cell fate and behavior may be related to changes in the cytoskeleton and subsequent activity of YAP. The results from this study will guide researchers to re-examine the role of matrix stiffness in reactive astrogliosis in vivo and inspire the development of a novel therapeutic approach for controlling glial scar formation following injury, enabling axonal regrowth and improving functional recovery by exploiting the benefits of mechanobiology studies.

The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices

Tumors have a unique niche system that plays an important role in their occurrence and development. At present, there is increasing interest in the biomechanical properties of niches. The increased stemness of cancer cells is closely related to bladder cancer progression and recurrence. However, how biomechanical properties in the niche regulate bladder cancer stemness remains unclear. Here, we show that as bladder cancer progresses, matrix stiffness increases, and tumor stemness increases. Mechanistically, high matrix stiffness mediates β-catenin nuclear translocation by increasing the nuclear pore size. On the other hand, it promotes the expression of the nuclear cytoskeletal protein Lamin A/C, inhibits the nuclear export of β-catenin, and finally, it upregulates the Wnt pathway to increase the stemness of cancer cells. These findings reveal a role for matrix stiffness in the regulation of stemness in bladder cancer cells and suggest that targeting matrix stiffness may be an effective strategy to delay bladder cancer progression.

Hepatocellular carcinoma (HCC) almost exclusively develops in the context of liver fibrosis or cirrhosis, both of which are characterized by increased matrix stiffness. Matrix stiffness promotes HCC invasion and metastasis via upregulation of stearoyl-CoA desaturase 1 (SCD1). However, the downstream effector of the SCD1-dependent mechanoresponsive pathway remains unclear. HCC cells were grown on mechanically tunable polyacrylamide gels in 2% Matrigel-supplemented culture media and subjected to the invitro invasion assay. The key molecule that responded to matrix stiffness was identified. The underlying mechanism by which the molecule participated in invitro matrix stiffness-induced invasion and invivo metastasis was explored. High matrix stiffness caused an increase in hyaluronan-mediated motility receptor (HMMR) expression, which enhanced the invasion of HCC cells invitro and augmented the response of HCC cells to the HMMR ligand hyaluronic acid. Stiffness-dependent HMMR upregulation occurred through integrin β1 and a positive feedback loop that included focal adhesion kinase (FAK)/SCD1/oleic acid (OA)/CCAAT-enhancer binding protein α (C/EBPα). HMMR knockdown significantly inhibited high matrix stiffness-induced HCC cell invasion invitro and suppressed HCC metastasis invivo. In human HCC samples, the combination of collagen content, a surrogate for increased matrix stiffness, and increased HMMR expression predicted poor survival of HCC patients. We define a new mechanotransduction axis of SCD1/HMMR that enhances HCC invasion and metastasis in response to increased matrix stiffness, providing a new therapeutic target against stiffness-driven HCC invasion and metastasis.

The migration of metastatic breast cancer cells is regulated by matrix stiffness via YAP signalling

Angiogenesis from endothelial progenitor cells is co‐regulated by vascular endothelial growth factor and matrix stiffness. Endothelial Progenitor Cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostatis and contribute to adult vascular regeneration. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate into endothelial cells, and line the lumen of the blood vessels. Evidently the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behavior. Using our controllable in vitro model of angiogenesis we show that vascular endothelial growth factor (VEGF) and matrix stiffness regulate angiogenesis from EPCs. When exposed to low levels of VEGF, EPCs proliferate independently of matrix stiffness. Exposure to high VEGF concentrations results in upregulation of MMPs, demonstrating EPCs migratory and invasive abilities. Under these conditions, the stiffness of the matrix affects the progression of angiogenesis. Immunofluorescence and TEM images confirm the formation of vacuoles, which fully develop into lumen within a chain elongated cell on the softest matrix. RNAi studies further suggest that EPCs sense matrix stiffness through signaling cascades downstream of integrin ligation leading to the activation of the RhoGTPase and Cdc42. These results suggest that dual regulation of VEGF and matrix compliances are required to modulate angiogenesis dynamics of EPCs.Grant Funding SourceAHA

Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior