Influence of Micropatterning on Human Periodontal Ligament Cells’ Behavior

The periodontal ligament (PDL) is highly ordered connective tissue located between the alveolar bone and cementum. An aligned and organized architecture is required for its physiological function. We applied micropatterning technology to arrange PDL cells in 10- or 20-μm-wide extracellular protein patterns. Cell and nuclear morphology, cytoskeleton, proliferation, differentiation, and matrix metalloproteinase system expression were investigated. Micropatterning clearly elongated PDL cells with a low cell-shape index and low spreading area. The nucleus was also elongated as nuclear height increased, but the nuclear volume remained intact. The cytoskeleton was rearranged to form prominent bundles at cells’ peripheral regions. Moreover, proliferation was promoted by 10- and 20-μm micropatterning. Osteogenesis and adipogenesis were each inhibited, but micropatterning increased PDL cells’ stem cell markers. β-catenin was expelled to cytoplasm. YAP/TAZ nuclear localization and activity both decreased, which might indicate their role in micropatterning-regulated differentiation. Collagen Ι expression increased in micropatterned groups. It might be due to the decreased expression of matrix metalloproteinase-1, 2 and the tissue inhibitor of metalloproteinase-1 gene expression elevation in micropatterned groups. The findings of this study provide insight into the effects of a micropatterned surface on PDL cell behavior and may be applicable in periodontal tissue regeneration.     

A “sweet-spot” for fluid-induced oscillations in the conditioning of stem cell-based engineered heart valve tissues

Fluid-induced shear stresses are involved in the development of cardiovascular tissues. In a tissue engineering framework, this stimulus has also been considered as a mechanical regulator of stem cell differentiation. We recently demonstrated that the fluid-oscillating effect in combination with a physiologically-relevant shear stress magnitude contributes to the formation of stem cell-derived de novo heart valve tissues. However, the range of oscillations necessary to induce favorable gene expression and engineered tissue formation is unknown. In this study, we took a computational approach to establish a range of oscillatory shear stresses that may optimize in vitro valvular tissue growth. Taking a biomimetic approach, three physiologically-relevant flow waveforms from the human: (i) aorta, (ii) pulmonary artery and (iii) superior vena cava were utilized to simulate pulsatile flow conditions within a bioreactor that housed 3 tissue specimens. Results were compared to non-physiological pulsatile flow (NPPF) and cyclic flexure-steady flow (Flex-Flow) conditions. The oscillatory shear index (OSI) was used to quantify the fluid-induced oscillations occurring on the specimen surfaces. The range of mean OSI under the physiological conditions investigated was found to be 0.18 ≤ OSI ≤ 0.23. On the other hand, NPPF and Flex-Flow environments yielded a mean OSI of 0.37 and 0.11 respectively, which were 46% higher and 45% lower than physiological conditions. Moreover, we subsequently conducted OSI-based human bone marrow stem cell (HBMSC) culture experiments which resulted in preferential valvular gene expression and phenotype (significant upregulation of BMP, KLF2A, CD31 and α-SMA using an OSI of 0.23 in comparison to a lower OSI of 0.10 or a higher OSI of 0.38; p < .05). These findings suggest that a distinct range or a “sweet-spot” for physiological OSI exists in the mechanical conditioning of tissue engineered heart valves grown from stem cell sources. We conclude that in vitro heart valve matrix development could be further enhanced by simultaneous exposure of the engineered tissues to physiologically-relevant magnitudes of both fluid-induced oscillations and shear stresses.     

Computational simulation of fracture healing: influence of interfragmentary movement on the callus growth

Bone fractures heal through a complex process involving several cellular events. This healing process can serve to study factors that control tissue growth and differentiation from mesenchymal stem cells. The mechanical environment at the fracture site is one of the factors influencing the healing process and controls size and differentiation patterns in the newly formed tissue. Mathematical models can be useful to unravel the complex relation between mechanical environment and tissue formation. In this study, we present a mathematical model that predicts tissue growth and differentiation patterns from local mechanical signals. Our aim was to investigate whether mechanical stimuli, through their influence on stem cell proliferation and chondrocyte hypertrophy, predict characteristic features of callus size and geometry. We found that the model predicted several geometric features of fracture calluses. For instance, callus size was predicted to increase with increasing movement. Also, increases in size were predicted to occur through increase in callus diameter but not callus length. These features agree with experimental observations. In addition, spatial and temporal tissue differentiation patterns were in qualitative agreement with well-known experimental results. We therefore conclude that local mechanical signals can probably explain the shape and size of fracture calluses.     

A prediction of cell differentiation and proliferation within a collagen–glycosaminoglycan scaffold subjected to mechanical strain and perfusive fluid flow

Mesenchymal stem cell (MSC) differentiation can be influenced by biophysical stimuli imparted by the host scaffold. Yet, causal relationships linking scaffold strain magnitudes and inlet fluid velocities to specific cell responses are thus far underdeveloped. This investigation attempted to simulate cell responses in a collagen–glycosaminoglycan (CG) scaffold within a bioreactor. CG scaffold deformation was simulated using μ-computed tomography (CT) and an in-house finite element solver (FEEBE/linear). Similarly, the internal fluid velocities were simulated using the afore-mentioned μCT dataset with a computational fluid dynamics solver (ANSYS/CFX). From the ensuing cell-level mechanics, albeit octahedral shear strain or fluid velocity, the proliferation and differentiation of the representative cells were predicted from deterministic functions. Cell proliferation patterns concurred with previous experiments. MSC differentiation was dependent on the level of CG scaffold strain and the inlet fluid velocity. Furthermore, MSC differentiation patterns indicated that specific combinations of scaffold strains and inlet fluid flows cause phenotype assemblies dominated by single cell types. Further to typical laboratory procedures, this predictive methodology demonstrated loading-specific differentiation lineages and proliferation patterns. It is hoped these results will enhance in-vitro tissue engineering procedures by providing a platform from which the scaffold loading applications can be tailored to suit the desired tissue.     

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-β, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.     

Dynamic patterns of mechanical stimulation co-localise with growth and cell proliferation during morphogenesis in the avian embryonic knee joint

Muscle contractions begin in early embryonic life, generating forces that regulate the correct formation of the skeleton. In this paper we test the hypothesis that the biophysical stimulation generated by muscle forces may be a causative factor for the changes in shape of the knee joint as it grows. We do this by predicting the spatial and temporal patterns of biophysical stimuli, where cell proliferation and rudiment shape changes occur within the emerging tissues of the joint over time. We used optical projection tomography (OPT) to create anatomically accurate finite element models of the embryonic knee at three time points (stages) of development. OPT was also used to locate muscle attachment sites and AFM was used to determine material properties. An association was found between the emergence of joint shape, cell proliferation and the pattern of biophysical stimuli generated by embryonic muscle contractions. Elevated rates of growth and cell proliferation in the medial condyle were found to co-localise with elevated patterns of biophysical stimuli including maximum principal stresses and fluid flow, throughout the time period studied, indicating that cartilage growth and chondrocyte proliferation in the epiphysis is potentially related to local patterns of biophysical stimuli. The development of the patella and articular cartilages, which is known to be affected by in ovo immobilisation, could be contributed to by specific patterns of fluid flow, pore pressure and stress in the joint interzone. This suggests that both cartilage growth and tissue differentiation in the embryonic joint is regulated by specific patterns of biophysical stimuli and that these stimuli are needed for the correct development of the joint.     

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction

Cells and tissues are exposed to multiple mechanical stresses during development, tissue homoeostasis and diseases. While we start to have an extensive understanding of the influence of mechanics on cell differentiation and proliferation, how excessive mechanical stresses can also lead to cell death and may be associated with pathologies has been much less explored so far. Recently, the development of new perturbative approaches allowing modulation of pressure and deformation of tissues has demonstrated that compaction (the reduction of tissue size or volume) can lead to cell elimination. Here, we discuss the relevant type of stress and the parameters that could be causal to cell death from single cell to multicellular systems. We then compare the pathways and mechanisms that have been proposed to influence cell survival upon compaction. We eventually describe the relevance of compaction‐induced death in vivo, and its functions in morphogenesis, tissue size regulation, tissue homoeostasis and cancer progression.     

Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing

A new quantitative tissue differentiation theory which relates the local tissue formation in a fracture gap to the local stress and strain is presented. Our hypothesis proposes that the amounts of strain and hydrostatic pressure along existing calcified surfaces in the fracture callus determine the differentiation of the callus tissue. The study compares the local strains and stresses in the callus as calculated from a finite element model with histological findings from an animal fracture model. The hypothesis predicts intramembranous bone formation for strains smaller approximately +/- 5% and hydrostatic pressures smaller than +/- 0.15 MPa. Endochondral ossification is associated with compressive pressures larger than about -0.15 MPa and strains smaller than +/- 15%. All other conditions seemed to lead to connective tissue or fibrous cartilage. The hypothesis enables a better understanding of the complex tissue differentiation seen in histological images and the mechanical conditions for healing delayed healing or nonunions.     

Informing tendon tissue engineering with embryonic development

Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.     

Cell cycling and cell enlargement in developing leaves of Arabidopsis.

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.     

Localized decrease of beta-catenin contributes to the differentiation of human embryonic stem cells

Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies.     

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates

Summary Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 μm² spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 μm² failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-μm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 μm². Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-μm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 μm²), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis—growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.     

A finite element model of cell-matrix interactions to study the differential effect of scaffold composition on chondrogenic response to mechanical stimulation

Mechanically induced cell deformations have been shown to influence chondrocyte response in 3D culture. However, the relationship between the mechanical stimulation and cell response is not yet fully understood. In this study a finite element model was developed to investigate cell-matrix interactions under unconfined compression conditions, using a tissue engineered encapsulating hydrogel seeded with chondrocytes. Model predictions of stress and strain distributions within the cell and on the cell boundary were shown to exhibit space-dependent responses that varied with scaffold mechanical properties, the presence of a pericellular matrix (PCM), and the cell size. The simulations predicted that when the cells were initially encapsulated into the hydrogel scaffolds, the cell size hardly affected the magnitude of the stresses and strains that were reaching the encapsulated cells. However, with the inclusion of a PCM layer, larger cells experienced enhanced stresses and strains resulting from the mechanical stimulation. It was also noted that the PCM had a stress shielding effect on the cells in that the peak stresses experienced within the cells during loading were significantly reduced. On the other hand, the PCM caused the stresses at the cell-matrix interface to increase. Based on the model predictions, the PCM modified the spatial stress distribution within and around the encapsulated cells by redirecting the maximum stresses from the periphery of the cells to the cell nucleus. In a tissue engineered cartilage exposed to mechanical loading, the formation of a neo-PCM by encapsulated chondrocytes appears to protect them from initially excessive mechanical loading. Predictive models can thus shed important insight into how chondrocytes remodel their local environment in order to redistribute mechanical signals in tissue engineered constructs.     

Micropatterned structural control suppresses mechanotaxis of endothelial cells

Vascular endothelial cell migration is critical in many physiological processes including wound healing and stent endothelialization. To determine how preexisting cell morphology influences cell migration under fluid shear stress, endothelial cells were preset in an elongated morphology on micropatterned substrates, and unidirectional shear stress was applied either parallel or perpendicular to the cell elongation axis. On micropatterned 20-μm lines, cells exhibited an elongated morphology with stress fibers and focal adhesion sites aligned parallel to the lines. On 115-μm lines, cell morphology varied as a function of distance from the line edge. Unidirectional shear stress caused unpatterned cells in a confluent monolayer to exhibit triphasic mechanotaxis behavior. During the first 3 h, cell migration speed increased in a direction antiparallel to the shear stress direction. Migration speed then slowed and direction became spatially heterogeneous. Starting 11-12 h after the onset of shear stress, the unpatterned cells migrated primarily in the downstream direction, and migration speed increased significantly. In contrast, mechanotaxis was suppressed after the onset of shear stress in cells on micropatterned lines during the same time period, for the cases of both parallel and perpendicular flow. The directional persistence time was much longer for cells on the micropatterned lines, and it decreased significantly after flow onset. Migration trajectories were highly correlated among micropatterned cells within a three-cell neighborhood, and shear stress disrupted this spatially correlated migration behavior. Thus, presetting structural morphology may interfere with mechanisms of sensing local physical cues, which are critical for establishing mechanotaxis in response to hemodynamic shear stress.     

Collagen Promotes Higher Adhesion,Survival and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.     

The effects of misalignment during in vivo loading of bone: Techniques to detect the proximity of objects in three-dimensional models

Theories of mechanical adaptation of bone suggest that mechanical loading causes bone formation at discrete locations within bone microstructure experiencing the greatest mechanical stress/strain. Experimental testing of such theories requires in vivo loading experiments and high-resolution finite element models to determine the distribution of mechanical stresses. Finite element models of in vivo loading experiments typically assume idealized boundary conditions with applied load perfectly oriented on the bone, however small misalignments in load orientation during an in vivo experiment are unavoidable, and potentially confound the ability of finite element models to predict locations of bone formation at the scale of micrometers. Here we demonstrate two different three-dimensional spatial correlation methods to determine the effects of misalignment in load orientation on the locations of high mechanical stress/strain in the rodent tail loading model. We find that, in cancellous bone, the locations of tissue with high stress are maintained under reasonable misalignments in load orientation (p<0.01). In cortical bone, however, angular misalignments in the dorsal direction can alter the locations of high mechanical stress, but the locations of tissue with high stress are maintained under other misalignments (p<0.01). We conclude that, when using finite element models of the rodent tail loading model, small misalignments in loading orientation do not affect the predicted locations of high mechanical stress within cancellous bone.     

Expression of keratinocyte biomarkers is governed by environmental biomechanics

In solid body tissues, environmental biomechanics is indispensable for tissue homeostasis. While characteristics of homeostasis include morphogenesis, proliferation and differentiation, the influences through biomechanics in corneal keratinocytes are poorly understood. Here we show for the first time that corneal keratinocytes, established in a defined biomechanical microenvironment of micropatterned soft pillars, exhibit favoritism of late and terminal differentiation at large pillar patterns of 11 μm with matched small 5 μm arrays. At 11 μm, epithelial cells expressed decreased levels of early differentiation marker cytokeratin 19 (KRT19), which was antagonized by an increase in biomarkers of late and terminal differentiation, i.e. cytokeratin 12 (KRT12), involucrin and filaggrin. Keratinocytes showed proper morphogenesis on 5 μm arrays, whereas 11 μm yielded in morphological disorders. While the propensity of keratinocyte proliferation appeared attenuated at large pillar patterns, stem cell marker ABCG2 was weak though homogeneous at 5 μm, but strong at 11 μm. Thus, corneal keratinocytes reveal interference of biomarker expression, morphogenesis and proliferation, which are at least in part characteristics of tissue homeostasis by mechanisms, depending on environmental biomechanics of micropattern-allocated cell adhesion points in vitro.     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

Variability observed in mechano-regulated in vivo tissue differentiation can be explained by variation in cell mechano-sensitivity

Computational simulations of tissue differentiation have been able to capture the main aspects of tissue formation/regeneration observed in animal experiments-except for the considerable degree of variability reported. Understanding and modelling the source of this variability is crucial if computational tools are to be developed for clinical applications. The objective of this study was to test the hypothesis that differences in cell mechano-sensitivity between individuals can explain the variability of tissue differentiation patterns observed experimentally. Simulations of an experiment of tissue differentiation in a mechanically loaded bone chamber were performed. Finite element analysis was used to determine the biophysical environment, and a lattice-modelling approach was used to simulate cell activity. Differences in cell mechano-sensitivity among individuals were modelled as differences in cell activity rates, with the activation of cell activities regulated by the mechanical environment. Predictions of the tissue distribution in the chambers produced the two different classes of results found experimentally: (i) chambers with a layer of bone across the chamber covered by a layer of cartilage on top and (ii) chambers with almost no bone, mainly fibrous tissue and small islands of cartilage. This indicates that the differing cellular response to the mechanical environment (i.e., subject-specific mechano-sensitivity) could be a reason for the different outcomes found when implants (or tissue engineered constructs) are used in a population.     

Vibration induced osteogenic commitment of mesenchymal stem cells is enhanced by cytoskeletal remodeling but not fluid shear

Consistent across studies in humans, animals and cells, the application of vibrations can be anabolic and/or anti-catabolic to bone. The physical mechanisms modulating the vibration-induced response have not been identified. Recently, we developed an in vitro model in which candidate parameters including acceleration magnitude and fluid shear can be controlled independently during vibrations. Here, we hypothesized that vibration induced fluid shear does not modulate mesenchymal stem cell (MSC) proliferation and mineralization and that cell's sensitivity to vibrations can be promoted via actin stress fiber formation. Adipose derived human MSCs were subjected to vibration frequencies and acceleration magnitudes that induced fluid shear stress ranging from 0.04 Pa to 5 Pa. Vibrations were applied at magnitudes of 0.15g, 1g, and 2g using frequencies of both 100 Hz and 30 Hz. After 14 d and under low fluid shear conditions associated with 100 Hz oscillations, mineralization was greater in all vibrated groups than in controls. Greater levels of fluid shear produced by 30 Hz vibrations enhanced mineralization only in the 2g group. Over 3 d, vibrations led to the greatest increase in total cell number with the frequency/acceleration combination that induced the smallest level of fluid shear. Acute experiments showed that actin remodeling was necessary for early mechanical up-regulation of RUNX-2 mRNA levels. During osteogenic differentiation, mechanically induced up-regulation of actin remodeling genes including Wiskott–Aldrich syndrome (WAS) protein, a critical regulator of Arp2/3 complex, was related to the magnitude of the applied acceleration but not to fluid shear. These data demonstrate that fluid shear does not regulate vibration induced proliferation and mineralization and that cytoskeletal remodeling activity may play a role in MSC mechanosensitivity.