Effect Of Matrix Stiffness Research Articles

Effect of matrix stiffness on the proliferation and differentiation of umbilical cord mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a compatible cellular alternative for regenerative medicine and tissue engineering because of their powerful multipotency. Matrix stiffness plays a profound role on stem cell behavior. Nevertheless, the effect of matrix stiffness on umbilical cordmesenchymal stem cells (UC-MSCs) remains unexplored. To conduct an in-depth exploration, we cultured UC-MSCs on different stiffness (Young's modulus: 13–16, 35–38, 48–53, and 62–68 kPa) polyacrylamide gels coated with fibronectin. We found that the proliferation and adhesion of UC-MSCs varied when cultured on the different matrices, and the spreading capacity was stronger as the stiffness increased (*P<0.05). Real-time quantitative PCR results showed that the soft matrix promoted adipogenic differentiation, with higher expression levels of adipocytic markers like PPARγ and C/EBPα (*P<0.05). In contrast, cells tended to differentiate into muscle when cultured on the 48–53 kPa matrix, which was validated by increased expression of myogenic makers like desminand MOYG (*P<0.05). Moreover, increased expression of osteoblastic makers (*P<0.05), such as ALP, collagen type I, osteocalcin, and Runx2, confirmed that cells differentiated into bone on the high-stiffness matrix.

Effects of matrix stiffness on epithelial to mesenchymal transition-like processes of endometrial epithelial cells: Implications for the pathogenesis of endometriosis

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites. Our previous study revealed an epithelial to mesenchymal transition (EMT)-like process in red peritoneal endometriosis, whereas membrane localization of E-cadherin was well maintained in epithelial cells of deep infiltrating endometriosis (DIE). Here we show that endometrial epithelial cells (EEE) grown on polyacrylamide gel substrates (PGS) of 2 kilopascal (kPa), a soft matrix, initiate a partial EMT-like process with transforming growth factor-β1 (TGF-β1) stimulation. Increasing matrix stiffness with TGF-β1 stimulation reduced the number of cell-cell contacts. Cells that retained cell-cell contacts showed decreased expression of E-cadherin and zonula occludens 1 (ZO-1) to cell-cell junctions. Few deep endometriotic epithelial cells (DEE) grown on 30-kPa PGS, which may mimic in vivo tissue compliance of DIE, retained localization of E-cadherin to cell-cell junctions with TGF-β1 treatment. Immunohistochemical analysis showed no phosphorylated Smad 2/3 nuclear localization in E-cadherin+ epithelial cells of DIE. We hypothesize that EEE may undergo an EMT-like process after attachment of endometrium to peritoneum in a TGF-β1–rich microenvironment. However, TGF-β1 signaling may be absent in DIE, resulting in a more epithelial cell-like phenotype in a rigid microenvironment.

Effect of matrix stiffness on osteoblast functionalization.

Stiffness of bone tissue differs response to its physiological or pathological status, such as osteoporosis or osteosclerosis. Consequently, the function of cells residing in bone tissue including osteoblasts (OBs), osteoclasts and osteocytes will be affected. However, to the best of our knowledge, the detailed mechanism of how extracellular matrix stiffness affects OB function remains unclear. We conducted a study that exposed rat primary OBs to polydimethylsiloxane substrates with varied stiffness to investigate the alterations of cell morphology, osteoblastic differentiation and its potential mechanism in mechanotransduction. Distinctive differences of cell shapes and vinculin expression in rat osteoblasts were detected on different PDMS substrates. As representatives for OB function, expression of alkaline phosphatase, Runx2 and osteocalcin were identified and showed a decrease trend as substrates become soft, which is associated with the Rho/ROCK signalling pathway. Our results indicated substrate elasticity as a potent regulator in OBs functionalization, which may pave a way for further understanding of bone diseases as well as a potential therapeutic alternative in tissue regeneration.

The effects of matrix stiffness on mesenchymal stem cell chondrogenesis in 3d is dependent on crosslinking mechanisms and biochemical cues

The effects of matrix stiffness on mesenchymal stem cell chondrogenesis in 3d is dependent on crosslinking mechanisms and biochemical cues

Designing Visible Light-Cured Thiol-Acrylate Hydrogels for Studying the HIPPO Pathway Activation in Hepatocellular Carcinoma Cells.

Various polymerization mechanisms have been developed to prepare peptide-immobilized poly(ethylene glycol) (PEG) hydrogels, a class of biomaterials suitable for studying cell biology in vitro. Here, a visible light mediated thiol-acrylate photopolymerization scheme is reported to synthesize dually degradable PEG-peptide hydrogels with controllable crosslinking and degradability. The influence of immobilized monothiol pendant peptide is systematically evaluated on the crosslinking of these hydrogels. Further, methods are proposed to modulate hydrogel crosslinking, including adjusting concentration of comonomer or altering the design of multifunctional peptide crosslinker. Due to the formation of thioether ester bonds, these hydrogels are hydrolytically degradable. If the dithiol peptide linkers used are susceptible to protease cleavage, these thiol-acrylate hydrogels can be designed to undergo partial proteolysis. The differences between linear and multiarm PEG-acrylate (i.e., PEGDA vs PEG4A) are also evaluated. Finally, the use of the mixed-mode thiol-acrylate PEG4A-peptide hydrogels is explored for in situ encapsulation of hepatocellular carcinoma cells (Huh7). The effects of matrix stiffness and integrin binding motif (e.g., RGDS) on Huh7 cell growth and HIPPO pathway activation are studied using PEG4A-peptide hydrogels. This visible light poly-merized thiol-acrylate hydrogel system represents an alternative to existing light-cured hydrogel platforms and shall be useful in many biomedical applications.

Three-dimensional matrix stiffness and adhesive ligands affect cancer cell response to toxins.

There is an immediate need to develop highly predictive in vitro cell-based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial-based three-dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate-based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell-matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin-based cell-matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell-matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening.

3D Scaffolds with Different Stiffness but the Same Microstructure for Bone Tissue Engineering.

A growing body of evidence has shown that extracellular matrix (ECM) stiffness can modulate stem cell adhesion, proliferation, migration, differentiation, and signaling. Stem cells can feel and respond sensitively to the mechanical microenvironment of the ECM. However, most studies have focused on classical two-dimensional (2D) or quasi-three-dimensional environments, which cannot represent the real situation in vivo. Furthermore, most of the current methods used to generate different mechanical properties invariably change the fundamental structural properties of the scaffolds (such as morphology, porosity, pore size, and pore interconnectivity). In this study, we have developed novel three-dimensional (3D) scaffolds with different degrees of stiffness but the same 3D microstructure that was maintained by using decellularized cancellous bone. Mixtures of collagen and hydroxyapatite [HA: Ca10(PO4)6(OH)2] with different proportions were coated on decellularized cancellous bone to vary the stiffness (local stiffness, 13.00 ± 5.55 kPa, 13.87 ± 1.51 kPa, and 37.7 ± 19.6 kPa; bulk stiffness, 6.74 ± 1.16 kPa, 8.82 ± 2.12 kPa, and 23.61 ± 8.06 kPa). Microcomputed tomography (μ-CT) assay proved that there was no statistically significant difference in the architecture of the scaffolds before or after coating. Cell viability, osteogenic differentiation, cell recruitment, and angiogenesis were determined to characterize the scaffolds and evaluate their biological responses in vitro and in vivo. The in vitro results indicate that the scaffolds developed in this study could sustain adhesion and growth of rat mesenchymal stem cells (MSCs) and promote their osteogenic differentiation. The in vivo results further demonstrated that these scaffolds could help to recruit MSCs from subcutaneous tissue, induce them to differentiate into osteoblasts, and provide the 3D environment for angiogenesis. These findings showed that the method we developed can build scaffolds with tunable mechanical properties almost without variation in 3D microstructure. These preparations not only can provide a cell-free scaffold with optimal matrix stiffness to enhance osteogenic differentiation, cell recruitment, and angiogenesis in bone tissue engineering but also have significant implications for studies on the effects of matrix stiffness on stem cell differentiation in 3D environments.

Biphasic response of cell invasion to matrix stiffness in three-dimensional biopolymer networks

When cells come in contact with an adhesive matrix, they begin to spread and migrate with a speed that depends on the stiffness of the extracellular matrix. On a flat surface, migration speed decreases with matrix stiffness mainly due to an increased stability of focal adhesions. In a three-dimensional (3-D) environment, cell migration is thought to be additionally impaired by the steric hindrance imposed by the surrounding matrix. For porous 3-D biopolymer networks such as collagen gels, however, the effect of matrix stiffness on cell migration is difficult to separate from effects of matrix pore size and adhesive ligand density, and is therefore unknown. Here we used glutaraldehyde as a crosslinker to increase the stiffness of self-assembled collagen biopolymer networks independently of collagen concentration or pore size. Breast carcinoma cells were seeded onto the surface of 3-D collagen gels, and the invasion depth was measured after 3days of culture. Cell invasion in gels with pore sizes >5μm increased with higher gel stiffness, whereas invasion in gels with smaller pores decreased with higher gel stiffness. These data show that 3-D cell invasion is enhanced by higher matrix stiffness, opposite to cell behavior in two dimensions, as long as the pore size does not fall below a critical value where it causes excessive steric hindrance. These findings may be important for optimizing the recellularization of soft tissue implants or for the design of 3-D invasion models in cancer research.

Evaluation of the impact of matrix stiffness on encapsulated HepaRG spheroids

Background The drug development process is widely hampered by the lack of human models that recapitulate liver functionality and efficiently predict toxicity of new chemical compounds. Moreover, liver failure is a global medical problem, with transplantation being the only effective treatment currently available. The bipotent liver progenitor cell line HepaRG can be differentiated into cholangiocyte and hepatocyte-like cells that express major functions of mature hepatocytes, representing a valuable tool to model hepatic function [1]. Current two-dimensional (2D) protocols for the differentiation into mature hepatocyte-like cells fail to recapitulate the complex cell-cell interactions, which are crucial for maintaining polarity and inherent mature hepatic functionality. Herein, we present a three-dimensional (3D) strategy for the culture of HepaRG cells based on the encapsulation of aggregates. The effect of matrix stiffness on expansion and differentiation was evaluated through encapsulation with different concentrations of alginate (1.1% and 2%). Further characterization of the hepatic features will reveal the extent of the hepatic functionality of the generated spheroids. Materials and methods HepaRG cells were routinely propagated in static conditions as previously described [2]. Briefly, culture medium Williams E was supplemented with 1% (v/v) Glutamax, 1% (v/v) pen/strep, 5 μ g/ml insulin and 50 μ M hydrocortisone hemissuccinate and 10% (v/v) FBS and cultures were maintained at 37 ° C, 5% CO2 .S pinner vessels with ball impeller (Wheaton) were inoculated with inoculums ranging from 5 to 8 × 10 5 cell/mL and an agitation ranging from 35 to 45 rpm to attain the desired aggregation conditions. Aggregate size was determined by measuring Ferret’s diameter using the Image J software (NIH). After 3 days of aggregation, spheroids were encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate (UP MVG NovaMatrix, Pronova Biomedical) in NaCl 0.9% (w/v) solution. Encapsulation was performed in an electrostatically driven microencapsulation unit VarV1 (Nisco) and cultures were maintained for 14 days in stirred culture conditions. Viability was determined by the double stain viability test - alginate beads were collected from stirred cultures, incubated with fluorescein diacetate (10 μg/ mL) and TO-PRO3 ® (1 μM) and observed on a fluores

Abstract 251: Engineered matrix to study the effect of microenvironment on cancer stem cell maintenance.

Abstract Introduction: Tumors are highly heterogeneous. The heterogeneity of the tumor tissue is rooted in the existence of cancer stem cells (CSCs). Therefore, understanding the mechanism of CSC maintenance and its regulation by the microenvironment is critical to cancer prevention and treatment. Since cancer takes many years to grow, it is important to develop in vitro models to study the molecular basis of tumorigenesis and progression. 3D cell culture systems with biologic materials that support adhesion and growth of many cell types have emerged as another approach to cancer stem cell research. However, it is not possible to isolate individual factors in the microenvironment and the effect on cell response with naturally derived materials. In an effort to control the cell microenvironment, we have developed novel inert permissive gels with controlled physical, mechanical, and biological properties that support the maintenance of CSCs and tumorsphere formation. The objective of this work was to investigate the effect of matrix stiffness and a CD44 binding peptide, conjugated to the inert hydrogel matrix, on tumorsphere formation and CSC maintenance. Methods: Mouse 4T1 and human MCF7 breast cancer cells were encapsulated in the inert PEGDA gel with conjugation of CD44BP. Control groups included dissolved CD44BP and the gel conjugated with mutant CD44BP. Tumorsphere size and density, and expression of CSC markers were determined with incubation time in tumor CSC culture medium. Effect of CD44BP conjugation on breast CSC maintenance was compared with those gels conjugated with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined with time. Results: The gel stiffness had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers by the encapsulated cells peaked after 8 days of incubation. 4T1 and MCF7 cells encapsulated in the gel with 5-kPa stiffness formed the largest and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. Conjugation of CD44 binding peptide to the inert gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. Conclusion: The PEGDA hydrogel cell culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors. This model system can be used to understand the effect of individual factors in the microenvironment on epithelial to mesenchymal transition (EMT) and tumorigenesis. Citation Format: Xiaoming Yang, Samaneh Kamali, Esmaiel Jabbari. Engineered matrix to study the effect of microenvironment on cancer stem cell maintenance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 251. doi:10.1158/1538-7445.AM2013-251

Fabrication and Characterization of the Composites Reinforced with Multi-Walled Carbon Nanotubes

Carbon nanotubes with superior mechanical, electrical and thermal properties have received intensive attention in recent years. In this study, multi-walled carbon nanotubes (MWCNT) were infused into a liquid epoxy, and the solution was sonicated for three hours to separate the aggregation of the MWCNTs and achieve good dispersion. The trapped air was removed from the mixture using a high vacuum. To investigate the effect of matrix stiffness on the mechanical properties of the MWCNT nanocomposites, the mixture ratio between the epoxy and hardener was varied. Two different contents (1% wt. and 2% wt.) of the multi-walled carbon nanotubes were added into the epoxy matrix. Tensile tests were conducted to determine the Young's modulus, yielding stress and tensile strength of the nanocomposites. The natural frequency and damping ratio of the nanocomposites were evaluated using free vibration tests. Experimental results show that the Young's modulus and natural frequency of MWCNT/epoxy nanocomposites increase with increase of the addition of multi-walled carbon nanotubes. While the damping ratio of the nanocomposites decreases with increase of the multi-walled carbon nanotubes. The reinforcement role of the multi-walled carbon nanotubes is less significant in a hard matrix when compares with a soft matrix.

Interplay of cell adhesion matrix stiffness and cell type for non-viral gene delivery

Non-viral gene delivery has the potential to treat a wide array of diseases but has been hindered by limited expression in vivo, possibly due to complex cellular microenvironments at delivery sites. Previous studies have reported that extracellular matrix properties, including stiffness, influence non-viral gene transfection efficiencies. This study reports that the effect of matrix stiffness on non-viral gene delivery differs among cell types due to varying sensitivities to matrix rigidity. Plasmid DNA encoding bone morphogenetic protein (BMP)-2 was delivered to fibroblasts, bone marrow stromal cells, and myoblasts cultured on fibronectin-conjugated poly(ethylene glycol) diacrylate hydrogels with varied elastic moduli, and the cellular uptake and subsequent expression of plasmid DNA were examined. While exogenous BMP-2 expression increased with increasing matrix stiffness for all three cell types, the effects of matrix stiffness were most pronounced for fibroblasts. Mechanistic studies conducted in parallel indicate that matrix stiffness influenced the projected area and nuclear aspect ratio for fibroblasts but had minimal effects on the morphology of bone marrow stromal cells and myoblasts. Overall, we believe that the results of this study will be useful for developing advanced non-viral gene delivery strategies for improved therapeutic efficacy.

Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials

Co-cultures of endothelial cells (EC) and mesenchymal stem cells (MSC) in three-dimensional (3D) protein hydrogels can be used to recapitulate aspects of vasculogenesis in vitro. MSC provide paracrine signals that stimulate EC to form vessel-like structures, which mature as the MSC transition to the role of mural cells. In this study, vessel-like network formation was studied using 3D collagen/fibrin (COL/FIB) matrices seeded with embedded EC and MSC and cultured for 7days. The EC:MSC ratio was varied from 5:1, 3:2, 1:1, 2:3 and 1:5. The matrix composition was varied at COL/FIB compositions of 100/0 (pure COL), 60/40, 50/50, 40/60 and 0/100 (pure FIB). Vasculogenesis was markedly decreased in the highest EC:MSC ratio, relative to the other cell ratios. Network formation increased with increasing fibrin content in composite materials, although the 40/60 COL/FIB and pure fibrin materials exhibited the same degree of vasculogenesis. EC and MSC were co-localized in vessel-like structures after 7days and total cell number increased by approximately 70%. Mechanical property measurements showed an inverse correlation between matrix stiffness and network formation. The effect of matrix stiffness was further investigated using gels made with varying total protein content and by crosslinking the matrix using the dialdehyde glyoxal. This systematic series of studies demonstrates that matrix composition regulates vasculogenesis in 3D protein hydrogels, and further suggests that this effect may be caused by matrix mechanical properties. These findings have relevance to the study of neovessel formation and the development of strategies to promote vascularization in transplanted tissues.

Matrix Modulus Affects Invasion Rate of Tumor Cells through Synthetic Hydrogels

ABSTRACTUnderstanding factors affecting cell invasion influences the design of engineered constructs for tissue regeneration. The objective of this work was to investigate the effect of matrix stiffness on invasion of tumor cells through a synthetic hydrogel with well-defined properties. A novel star acrylate-functionalized polyethylene glycol-co-lactide (SPELA) macromer was synthesized to produce hydrogels with well-defined water content, elastic modulus, degree of crosslinking and hydrophilicity. The hydrogel was formed by photo-polymerization of the macromer with or without integrin-binding cell adhesive RGD peptide. Cell invasion experiments were carried out in a transwell with SPELA hydrogel as the invading matrix and 4T1 mouse breast cancer cells. The invading cells on the lower membrane side were counted with an inverted fluorescent microscope. The concentration of SPELA macromer ranged from 10-25 wt% and that of RGD ranged from 1x10-4 to 1x10-2 M. The shear modulus of the hydrogel varied from 200 Pa to 25 kPa as the SPELA concentration increased from 10 to 25 wt%. Cell invasion slightly increased with increasing RGD concentration. However, RGD concentration &gt;1% resulted in a significant decrease in cell migration. As the matrix stiffness increased from 0.15 to 0.4, 3, 5, 6, 14, and 25 kPa the invasion rate decreased from 18.0 to 5.5, 6, 5.7, 5.2, 1.5, and 1.0 cells/mm2/h, respectively. There was a sharp decrease in invasion rate for matrix stiffness greater than 10 kPa. Results demonstrate that matrix stiffness plays a major role in invasion of tumor cell through a gelatinous matrix.

Frequency-Dependent Focal Adhesion Instability and Cell Reorientation Under Cyclic Substrate Stretching

The adhesion-based cell mechanosensitivity plays central roles in many physiological and pathological processes. Recently, quantitative understanding of cell responses to external force has been intensively pursued. However, the frequency dependent cell responses to the substrate stretching have not yet been fully understood. Here we developed a multiscale modeling framework for studying cell reorientation behaviors under substrate stretching, in which the mechano-chemical coupling at molecular, subcellular, and cellular scales was considered. The effect of matrix stiffness was also considered in a FEM based mechano-chemical coupling simulation. We showed that the collapsing time of focal adhesion decreases with the increasing of the loading frequency, however, the cell reorientation time exhibits a biphasic frequency-dependent behavior. Our results suggested that this biphasic behavior might be caused by the competition between the frequency-dependent collapsing of focal adhesions and the less frequency-dependent formation of stress fibers aligning away from the loading direction. At the low loading frequency, the collapsing of focal adhesion dominates the reorientation process, however, at the high loading frequency the polymerization of stress fiber dominates the reorientation. Moreover, we showed that the compliance of matrix may help accelerate the cell reorientation because focal adhesion is prone to be instable on soft matrix.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α–smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β

Bone marrow mesenchymal stem cells (MSCs) are a valuable cell source for tissue engineering and regenerative medicine. Transforming growth factor β (TGF-β) can promote MSC differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. Here we showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers and lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase (LPL). TGF-β increased SMC marker expression on stiff substrates. However, TGF-β increased chondrogenic marker expression and suppressed adipogenic marker expression on soft substrates, while adipogenic medium and soft substrates induced adipogenic differentiation effectively. Rho GTPase was involved in the expression of all aforementioned lineage markers, but did not account for the differential effects of substrate stiffness. In addition, soft substrates did not significantly affect Rho activity, but inhibited Rho-induced stress fiber formation and α-actin assembly. Further analysis showed that MSCs on soft substrates had weaker cell adhesion, and that the suppression of cell adhesion strength mimicked the effects of soft substrates on the lineage marker expression. These results provide insights of how substrate stiffness differentially regulates stem cell differentiation, and have significant implications for the design of biomaterials with appropriate mechanical property for tissue regeneration.

Influence of Multi-Walled Carbon Nanotubes on the Mechanical Properties of Nanocomposites

The effects of matrix stiffness and the content of multi-walled carbon nanotubes on the mechanical properties of the nanocomposites have been examined in this investigation. The matrix stiffness was controlled by changing the mixture ratio between the epoxy and hardener. Two different contents (1 wt.%. and 2 wt.%) of the multi-walled carbon nanotubes (MWCNT) were added to the epoxy matrix. Three-Point-Bending and Shore’s hardness tests were conducted to determine the Young’s modulus and hardness of the nanocomposites, respectively. Experimental results showed that the Young’s modulus of the nanocomposites was significantly increased with the increase of the addition of MWCNTs. However, the improvement of the hardness of the epoxy was insignificant with the addition of the MWCNTs. The reinforcement role of the multi-walled carbon nanotubes decreased while increasing the stiffness matrix.

Electrospun cross‐linked gelatin fibers with controlled diameter: The effect of matrix stiffness on proliferative and biosynthetic activity of chondrocytes cultured in vitro

Nanofibrous scaffolds were prepared from gelatin solutions and were further cross-linked with glutaraldehyde (GA). The fiber diameter was varied from 100 to 1000 nm by controlling the applied voltage (4-15 kV) and the concentration of the gelatin solution (4-15%). The tensile moduli and the tensile strength of the noncross-linked scaffolds varied from 20 to 120 MPa and 0.5 to 3.5 MPa, respectively. Cross-linking with GA led to an increase in both the tensile modulus and strength and correlated with cross-linker concentration. Gelatin-based matrices were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. High cellular viabilities and rounded morphology of chondrocytes was observed at the end of 7 days in culture with added matrix deposition and flattening of cells at 15 days. Matrix stiffness was noted to impact cell densities and the expression of chondrocytic markers, especially aggrecan. The ratios of collagen-II (C-II) to collagen-I (C-I) of 0.62 and 1.33 were noted on gelatin nanofibrous scaffolds cross-linked with 0.1% GA at the end of 7 and 15 days in culture, respectively. C-II/C-I ratios of 1.30 and 2.58 were noted on scaffolds cross-linked with 1.0% GA at the end of 7 and 15 days in culture, respectively.

The Effect of Matrix Stiffness on the Mechanical Properties of the Composite Reinforced with Multi-Wall Carbon Nanotube

Carbon nanotubes have shown the superior mechanical, electrical and thermal properties. These outstanding properties as well as a high aspect ratio and low density make carbon nanotubes an ideal reinforcement to develop superior nanocomposites. Mechanical tests indicated that the reinforcement role of carbon nanotubes is affected by the stiffness matrix. It is well known that the degree of cure of the epoxy has great influence on their mechanical properties. In this investigation, the matrix stiffness is controlled by changing the mixture ratio between the epoxy and hardener. Two different contents (1% wt. and 2% wt.) of the multi-wall carbon nanotubes in the epoxy are proposed in this work. Tensile tests are conducted to determine the mechanical properties of the nanocomposites, including the Young’s modulus, yield stress, tensile strength and fracture strain. Experimental results show that the mechanical properties are increasing with the increase of the addition of multi-wall carbon nanotubes. The reinforcement role of the multi-wall carbon nanotubes is decreasing while increasing the stiffness matrix.