Application Of Mechanical Stimulation Research Articles

Effect of Uniaxial Compression Frequency on Osteogenic Cell Responses in Dynamic 3D Cultures.

The application of mechanical stimulation on bone tissue engineering constructs aims to mimic the native dynamic nature of bone. Although many attempts have been made to evaluate the effect of applied mechanical stimuli on osteogenic differentiation, the conditions that govern this process have not yet been fully explored. In this study, pre-osteoblastic cells were seeded on PLLA/PCL/PHBV (90/5/5 wt.%) polymeric blend scaffolds. The constructs were subjected every day to cyclic uniaxial compression for 40 min at a displacement of 400 μm, using three frequency values, 0.5, 1, and 1.5 Hz, for up to 21 days, and their osteogenic response was compared to that of static cultures. Finite element simulation was performed to validate the scaffold design and the loading direction, and to assure that cells inside the scaffolds would be subjected to significant levels of strain during stimulation. None of the applied loading conditions negatively affected the cell viability. The alkaline phosphatase activity data indicated significantly higher values at all dynamic conditions compared to the static ones at day 7, with the highest response being observed at 0.5 Hz. Collagen and calcium production were significantly increased compared to static controls. These results indicate that all of the examined frequencies substantially promoted the osteogenic capacity.

Mechano-bioengineering of the knee meniscus.

The meniscus is a fibrocartilaginous structure of the knee joint that serves a crucial role in joint health and biomechanics. Degeneration or removal of the meniscus is known to lead to a chronic and debilitating disease known as knee osteoarthritis, whose prevalence is expected to increase in the next few decades. Meniscus bioengineering has been developed as a potential alternative to current treatment methods, wherein meniscus-like tissues are engineered using cells, materials, and biomechanical stimuli. The application of mechanical stimulation in meniscus bioengineering has presented varied results but, for the most part, it has been shown to enhance meniscus-like tissue formation. In this review, we summarized literature over the last 10 years of various mechanical stimuli applied in bioengineering meniscus tissues. The role of individual loading types is examined, and the effects on engineered meniscus are evaluated on both molecular and tissue levels. In addition, simulated microgravity is highlighted as a new area of interest in meniscus engineering, and its potential use as a disease-driving platform is discussed. Taken together, with the increased understanding of the effects of mechanical stimulation on bioengineered meniscus tissues, the most suitable loading regime could be developed for meniscus tissue engineering and osteoarthritis modeling.

Application of subject-specific adaptive mechanical loading for bone healing in a mouse tail vertebral defect

Methods to repair bone defects arising from trauma, resection, or disease, continue to be sought after. Cyclic mechanical loading is well established to influence bone (re)modelling activity, in which bone formation and resorption are correlated to micro-scale strain. Based on this, the application of mechanical stimulation across a bone defect could improve healing. However, if ignoring the mechanical integrity of defected bone, loading regimes have a high potential to either cause damage or be ineffective. This study explores real-time finite element (rtFE) methods that use three-dimensional structural analyses from micro-computed tomography images to estimate effective peak cyclic loads in a subject-specific and time-dependent manner. It demonstrates the concept in a cyclically loaded mouse caudal vertebral bone defect model. Using rtFE analysis combined with adaptive mechanical loading, mouse bone healing was significantly improved over non-loaded controls, with no incidence of vertebral fractures. Such rtFE-driven adaptive loading regimes demonstrated here could be relevant to clinical bone defect healing scenarios, where mechanical loading can become patient-specific and more efficacious. This is achieved by accounting for initial bone defect conditions and spatio-temporal healing, both being factors that are always unique to the patient.

Flexible Light Sources.

Various light sources have been developed for the application of optical stimulation in the optogenetics field. Light transmission inside living tissue is limited to a distance of a few millimeters; hence, it is necessary to insert the light source near the nerve tissue to be stimulated. If a device is rigid, it causes mechanical stimulation to act on the nerve tissue. The application of mechanical stimulation may induce inflammation, obstructing neural activity. Fabricating such a device out of a soft material can prevent mechanical stimulation of cells and mitigate biological reactions such as inflammation or encapsulation. Minimizing the sizes of LED and other light sources as much as possible and mounting them on a flexible substrate can provide the entire device with flexibility. Micro-LEDs can be reduced to a size almost comparable to that of a cell and it has even been reported that some have been mounted on the tip of needle-shaped devices inserted into living tissue. A device using organic semiconductors is sufficiently soft to be bent, which is a characteristic not observed in inorganic semiconductors. Using organic LEDs can realize wide-area flexible light-emitting surfaces and they are widely anticipated to be the next generation of light sources. This chapter introduces technologies used to manufacture these soft light sources and examples of optical stimulation devices that incorporate them.

In vitro Models of Bone Remodelling and Associated Disorders.

Disruption of bone remodelling by diseases such as osteoporosis results in an imbalance between bone formation by osteoblasts and resorption by osteoclasts. Research into these metabolic bone disorders is primarily performed in vivo; however, in the last decade there has been increased interest in generating in vitro models that can reduce or replace our reliance on animal testing. With recent advances in biomaterials and tissue engineering the feasibility of laboratory-based alternatives is growing; however, to date there are no established in vitro models of bone remodelling. In vivo, remodelling is performed by organised packets of osteoblasts and osteoclasts called bone multicellular units (BMUs). The key determinant of whether osteoclasts form and remodelling occurs is the ratio between RANKL, a cytokine which stimulates osteoclastogenesis, and OPG, its inhibitor. This review initially details the different circumstances, conditions, and factors which have been found to modulate the RANKL:OPG ratio, and fundamental factors to be considered if a robust in vitro model is to be developed. Following this, an examination of what has been achieved thus far in replicating remodelling in vitro using three-dimensional co-cultures is performed, before overviewing how such systems are already being utilised in the study of associated diseases, such as metastatic cancer and dental disorders. Finally, a discussion of the most important considerations to be incorporated going forward is presented. This details the need for the use of cells capable of endogenously producing the required cytokines, application of mechanical stimulation, and the presence of appropriate hormones in order to produce a robust model of bone remodelling.

Influence of hydrodynamic pressure on chondrogenic differentiation of human bone marrow mesenchymal stem cells cultured in perfusion system

The natural conditions of chondrocytes in native cartilage including mechanical forces and surface topology could be simulated to enhance chondrogenesis. A perfusion system recapitulating the hydrodynamic pressure of cartilage tissue is designed. Mesenchymal stem cells (MSCs) are isolated and seeded on aligned nanofibrous PCL/PLGA scaffolds that mimic the structure of superficial zone of articular cartilage. The cell-seeded scaffolds are placed into the perfusion bioreactor and exposed to chondrogenic differentiating medium. The chondrogenesis is then investigated by histological analysis and real time PCR for cartilage-specific genes. The highest expression levels of aggrecan and type II collagen are observed in the cells cultured in the presence of differentiating medium and mechanical stimulation. The expression level of type II collagen is higher than aggrecan in presence of differentiating medium and absence of mechanical stimulation. On the contrary, the expression ratio of aggrecan is higher than type II collagen in presence of mechanical stimulation and absence of differentiating medium. These results show the dominant role of mechanical stimulation and differentiating medium on upregulated expression of aggrecan and type II collagen, respectively. The application of mechanical stimulation upon cells-seeded scaffolds could mimic superficial zone of articular cartilage tissue and increase derivation of chondrocytes from MSCs.

Thigmomorphogenesis – Control of plant growth by mechanical stimulation

Abstract Controlled regulation of plant growth is a general prerequisite for the production of marketable ornamental plants. Consumers as well as retailers prefer stronger, more compact plants with greener leaves as these not only better meet a certain desired visual quality but also allow for a maximization of production per unit area as well as facilitation of packaging and transport. The same applies for the production of young vegetable plants. Special attention is paid to solid, compact and resilient plants that survive transport and planting without any problems. During the last decades plant growth control has mainly been achieved through the application of chemical plant growth regulators that generally interfere with the function of growth regulating hormones. However, there is an increasing demand to replace chemical treatments by other means such as the modulation of growth conditions, including temperature, light and fertilization. Alternatively, the application of mechanical stimulation has been shown to induce plant responses that yield some of the commercially relevant phenotypes including increased compactness, higher girth, darker leaves and a delay in flowering. The ability of plants to sense and respond to mechanical stimuli is an adaptive trait associated with increased fitness in many environmental settings. Mechanical stimulation in nature occurs e.g. through wind, rain, neighboring plants or predatory animals and induces a range of morphogenic responses that have been summarized under the term thigmomorphogenesis. We are only just about to begin to understand the molecular mechanisms underlying mechanosensing and the associated morphogenic changes in plants. However, a number of examples suggest that mechanical stimulation applied in a greenhouse setting can be used to alter plant growth in order to produce marketable plants. In this review will briefly summarize the current knowledge concerning the biological principles of thigmomorphogenesis and discuss the potential of mechanical growth regulation in commercial plant production especially with respect to organic horticulture.

The effects of acoustic vibration on fibroblast cell migration

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0–1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration.

Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a potential alterative cell source for cartilage tissue engineering applications.

Differentiation of Nucleus Pulposus Cells Alters Their Responsivity to Mechanics and Glucose Reduction

IntroductionNutritional supply of cells within the avascular intervertebral disk occurs mainly by diffusion from the blood vessels ending at the endplate of the vertebral body. With proceeding disk degeneration and calcification of the endplate the main route of transport of nutrients and metabolites is impaired. The consequential reduced nutrient supply is supposed to be a key contributor to disk degeneration. In short-term approaches using undifferentiated nucleus pulposus (NP) cells, gene expression of matrix proteins was decreased after glucose reduction but mechanosensitivity was not altered. Aim of this study was to investigate effects of long-term differentiation of NP cells regarding their responsivity to glucose reduction and mechanical stimulation.Materials and MethodsNP cells isolated from bovine caudal discs (n = 7) were cultured in alginate beads for 3 weeks for differentiation in high glucose (25 mM) chondrogenic medium supplemented with 10 ng/mL TGF-β1. After differentiation, part of the samples was exposed to reduced glucose concentration (5 mM) for 3 days. Afterwards glucose concentration was further reduced to either 0 mM, 0.5 mM, or 5 mM for 24 hours. Subsequently, half of all samples were mechanically loaded by hydrostatic pressure for 30 minutes at 0.1 Hz and 2.5 MPa or maintained at unloaded conditions. Gene expression of aggrecan, collagen type II and type I, MMP2, MMP3, MMP13, and aggrecanase was analyzed by real-time RT-PCR. Statistical significance was tested by Wilcoxon test (p < 0.05).ResultsTotal 3 weeks of differentiation in high-glucose (25 mM) chondrogenic medium caused a slight upregulation of aggrecan (2.7×), collagen type II (1.9×), and collagen type I (2.6×) expression in NP cells compared to day 0. While MMP2 (3.2×) expression increased significantly, a significant decrease of MMP3 (0.06×), MMP13 (0.006×), and aggrecanase (0.5×) expression was determined. Subsequent reduction of glucose concentration from high-glucose medium to medium containing 5 mM glucose decreased significantly expression of collagen type II (0.01×) and collagen type I (0.003×) to gene expression levels below that of day 0. Further reduction of glucose concentration revealed minor influences on gene expression.Effects of mechanical stimulation depended strongly on the medium in which NP cells were cultured. In NP cells, which were maintained in high-glucose chondrogenic medium without glucose reduction, mechanically induced effects were strongest. Within this group, mechanical loading increased significantly expression of c-FOS (3.3×) and collagen type I (4.6×), whereas expression of collagen type II (1.4×) increased only moderately. Furthermore, a decrease of gene expression of MMP2 (0.6×), MMP3 (0.02×), MMP13 (0.0005×) and aggrecanase (0.003×) after mechanical stimulation was observed. In cells exposed to glucose reduction, the mechanically induced changes in gene expression were minimal and no distinct correlation with glucose concentration could be pointed out.ConclusionAfter successful in vitro differentiation of NP cells, subsequent glucose deprivation revealed a strong downregulating impact on gene expression of matrix molecules and increased expression of matrix-degrading enzymes. Application of mechanical stimulation could not change these effects of glucose reduction. However, in high glucose chondrogenic medium supplemented with TGF-β1, hydrostatic pressure increased expression of matrix molecules and decreased expression of matrix-degrading enzymes. These findings indicate that mechanical sensitivity of NP cells depends on environmental conditions, as NP cells were less mechanosensitive under glucose deprivation. These interactions of glucose supply, presence of TGF-β1, and mechanical loading in the regulation of disk matrix turnover are investigated in further studies.Acknowledgement Funded by FP7 EU project GENODISK (HEALTH-F2-2008-201626).I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Differentiation of Nucleus Pulposus Cells Alters Their Responsivity to Mechanics and Glucose Reduction

Introduction Nutritional supply of cells within the avascular intervertebral disk occurs mainly by diffusion from the blood vessels ending at the endplate of the vertebral body. With proceeding disk degeneration and calcification of the endplate the main route of transport of nutrients and metabolites is impaired. The consequential reduced nutrient supply is supposed to be a key contributor to disk degeneration. In short-term approaches using undifferentiated nucleus pulposus (NP) cells, gene expression of matrix proteins was decreased after glucose reduction but mechanosensitivity was not altered. Aim of this study was to investigate effects of long-term differentiation of NP cells regarding their responsivity to glucose reduction and mechanical stimulation. Materials and Methods NP cells isolated from bovine caudal discs ( n = 7) were cultured in alginate beads for 3 weeks for differentiation in high glucose (25 mM) chondrogenic medium supplemented with 10 ng/mL TGF-β1. After differentiation, part of the samples was exposed to reduced glucose concentration (5 mM) for 3 days. Afterwards glucose concentration was further reduced to either 0 mM, 0.5 mM, or 5 mM for 24 hours. Subsequently, half of all samples were mechanically loaded by hydrostatic pressure for 30 minutes at 0.1 Hz and 2.5 MPa or maintained at unloaded conditions. Gene expression of aggrecan, collagen type II and type I, MMP2, MMP3, MMP13, and aggrecanase was analyzed by real-time RT-PCR. Statistical significance was tested by Wilcoxon test ( p &lt; 0.05). Results Total 3 weeks of differentiation in high-glucose (25 mM) chondrogenic medium caused a slight upregulation of aggrecan (2.7×), collagen type II (1.9×), and collagen type I (2.6×) expression in NP cells compared to day 0. While MMP2 (3.2×) expression increased significantly, a significant decrease of MMP3 (0.06×), MMP13 (0.006×), and aggrecanase (0.5×) expression was determined. Subsequent reduction of glucose concentration from high-glucose medium to medium containing 5 mM glucose decreased significantly expression of collagen type II (0.01×) and collagen type I (0.003×) to gene expression levels below that of day 0. Further reduction of glucose concentration revealed minor influences on gene expression. Effects of mechanical stimulation depended strongly on the medium in which NP cells were cultured. In NP cells, which were maintained in high-glucose chondrogenic medium without glucose reduction, mechanically induced effects were strongest. Within this group, mechanical loading increased significantly expression of c-FOS (3.3×) and collagen type I (4.6×), whereas expression of collagen type II (1.4×) increased only moderately. Furthermore, a decrease of gene expression of MMP2 (0.6×), MMP3 (0.02×), MMP13 (0.0005×) and aggrecanase (0.003×) after mechanical stimulation was observed. In cells exposed to glucose reduction, the mechanically induced changes in gene expression were minimal and no distinct correlation with glucose concentration could be pointed out. Conclusion After successful in vitro differentiation of NP cells, subsequent glucose deprivation revealed a strong downregulating impact on gene expression of matrix molecules and increased expression of matrix-degrading enzymes. Application of mechanical stimulation could not change these effects of glucose reduction. However, in high glucose chondrogenic medium supplemented with TGF-β1, hydrostatic pressure increased expression of matrix molecules and decreased expression of matrix-degrading enzymes. These findings indicate that mechanical sensitivity of NP cells depends on environmental conditions, as NP cells were less mechanosensitive under glucose deprivation. These interactions of glucose supply, presence of TGF-β1, and mechanical loading in the regulation of disk matrix turnover are investigated in further studies. Acknowledgement Funded by FP7 EU project GENODISK (HEALTH-F2-2008-201626). I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

8H-01 磁気駆動式マイクロピラーデバイスを用いた細胞焦点接着部位への力学刺激負荷(OS-11(1) MEMS技術を用いた細胞解析・制御(1))

8H-01 磁気駆動式マイクロピラーデバイスを用いた細胞焦点接着部位への力学刺激負荷(OS-11(1) MEMS技術を用いた細胞解析・制御(1))

Controlled induction of distributed microdeformation in wounded tissue via a microchamber array dressing

Mechanical stimuli are known to play an important role in determining the structure and function of living cells and tissues. Recent studies have highlighted the role of mechanical signals in mammalian dermal wound healing. However, the biological link between mechanical stimulation of wounded tissue and the subsequent cellular response has not been fully determined. The capacity for researchers to study this link is partially limited by the lack of instrumentation capable of applying controlled mechanical stimuli to wounded tissue. The studies outlined here tested the hypothesis that it was possible to control the magnitude of induced wound tissue deformation using a microfabricated dressing composed of an array of open-faced, hexagonally shaped microchambers rendered in a patch of silicone rubber. By connecting the dressing to a single vacuum source, the underlying wounded tissue was drawn up into each of the microchambers, thereby inducing tissue deformation. For these studies, the dressings were applied to full-thickness murine dermal wounds with 200 mmHg vacuum for 12 h. These studies demonstrated that the dressing was capable of inducing wound tissue deformation with values ranging from 11 to 29%. Through statistical analysis, the magnitude of the induced deformation was shown to be a function of both microchamber height and width. These results demonstrated that the dressing was capable of controlling the amount of deformation imparted in the underlying tissue. By allowing the application of mechanical stimulation with varying intensities, such a dressing will enable the performance of sophisticated mechanobiology studies in dermal wound healing.

Interactive effects of mechanical stretching and extracellular matrix proteins on initiating osteogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are characterized by their abilities to differentiate into different lineages, including osteoblasts. Besides soluble factors, mechanical strain and extracellular matrix (ECM) proteins play important roles in osteogenic differentiation of hMSCs. However, interactions between them are still not fully understood. The purpose of this study was to investigate the combined effects of insoluble chemical and mechanical factors (ECM proteins vs. cyclic stretching) in driving hMSCs into osteogenic differentiation. To avoid the influence from osteogenic supplements, hMSCs were cultured in regular medium and subjected to cyclic mechanical stretching using a Flexcell Tension system (3% elongation at 0.1 Hz) when they were grown on substrates coated with various ECM proteins (collagen I (Col I), vitronectin (VN), fibronectin (FN), and laminin (LN)). Using alkaline phosphatase (ALP) activity and mineralized matrix deposition as respective indicators of the early and late stages of osteogenesis, we report herein that all of the ECM proteins tested supported hMSC differentiation into osteogenic phenotypes in the absence of osteogenic supplements. Moreover, cyclic mechanical stretching activated the phosphorylation of focal adhesion kinase (FAK), upregulated the transcription and phosphorylation of core-binding factor alpha-1 (Cbfa1), and subsequently increased ALP activity and mineralized matrix deposition. Among the ECM proteins tested, FN and LN exhibited greater effects of supporting stretching-induced osteogenic differentiation than did Col I and VN. The ability of ECM proteins and mechanical stretching to regulate osteogenesis in hMSCs can be exploited in bone tissue engineering via approximate matrix design or application of mechanical stimulation.

Optimization of Dynamic Culture Conditions: Effects on Biosynthetic Activities of Chondrocytes Grown in Collagen Sponges

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.

Properties of engineered vascular constructs made from collagen, fibrin, and collagen–fibrin mixtures

Vascular constructs were formed by embedding rat aortic smooth muscle cells in three-dimensional matrices of Type I collagen, fibrin, or a mixture of collagen and fibrin in a 1:1 ratio, at total matrix protein concentrations of 2 and 4 mg/ml. Morphological and mechanical properties were evaluated after 6 days in culture, and the effect of cyclic mechanical strain on collagen–fibrin mixture constructs was also studied. Constructs made with the lower protein concentration compacted to the greatest degree, and fibrin was found to enhance gel compaction. Each matrix type exhibited a characteristic stress–strain profile. Pure collagen had the highest linear modulus and pure fibrin had the lowest. The ultimate tensile stress was strongly dependent on the degree of gel compaction, and collagen–fibrin mixtures at 2 mg/ml total protein content exhibited the highest values. Application of cyclic mechanical strain to collagen–fibrin mixture constructs caused a significant increase in gel compaction and a decrease in cell proliferation. The linear modulus, ultimate tensile stress and toughness of the constructs were all augmented by mechanical strain. These results demonstrate that the properties of engineered vascular tissues can be modulated by the combination of selected extracellular matrix components, and the application of mechanical stimulation.

Focal adhesion kinase is involved in mechanosensing during fibroblast migration.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase localized at focal adhesions and is believed to mediate adhesion-stimulated effects. Although ablation of FAK impairs cell movement, it is not clear whether FAK might be involved in the guidance of cell migration, a role consistent with its putative regulatory function. We have transfected FAK-null fibroblasts with FAK gene under the control of the tetracycline repression system. Cells were cultured on flexible polyacrylamide substrates for the detection of traction forces and the application of mechanical stimulation. Compared with control cells expressing wild-type FAK, FAK-null cells showed a decrease in migration speed and directional persistence. In addition, whereas FAK-expressing cells responded to exerted forces by reorienting their movements and forming prominent focal adhesions, FAK-null cells failed to show such responses. Furthermore, FAK-null cells showed impaired responses to decreases in substrate flexibility, which causes control cells to generate weaker traction forces and migrate away from soft substrates. Cells expressing Y397F FAK, which cannot be phosphorylated at a key tyrosine site, showed similar defects in migration pattern and force-induced reorientation as did FAK-null cells. However, other aspects of F397-FAK cells, including the responses to substrate flexibility and the amplification of focal adhesions upon mechanical stimulation, were similar to that of control cells. Our results suggest that FAK plays an important role in the response of migrating cells to mechanical input. In addition, phosphorylation at Tyr-397 is required for some, but not all, of the functions of FAK in cell migration.

The Sensitivity and Control of the Scallop Mantle Edge

ABSTRACT Application of mechanical stimulation or crude starfish extracts to the mantle edge of Aequipecten irradians elicited afferent impulse activity in the radial pallial nerves and local movements of the stimulated mantle edge. The evoked afferent spike activity was not recorded from primary receptor cells. The local mantle edge movements were controlled by peripherally located neurones and resembled jet formation on the velum of intact scallops. The central efferent neurones that supply the adductor muscle and much of the mantle edge are situated in the visceroparietal ganglion. Cobaltous chloride back-filling of the radial pallial nerves of the right side revealed the routes of the nerve fibres and the locations of the cell bodies in the visceroparietal ganglion. One group of motor neurones has fibres that are spatio-topically arranged across the visceroparietal ganglion and play a role in jet formation on corresponding portions of the mantle edge on both valves. It is apparent that axons from this group of mantle edge efferents traverse the ganglion without chemical synaptic connection. Two groups of mantle edge efferents that control concerted movements of the mantle edge on both shells appear to have cell bodies in the lateral margins of the dorso-central lobes. One group of motor neurones controls the raising of the velum curtain to an erect position around the shell margin. The output from the second group of efferents can be synchronized with the motor output to the adductor muscle to ensure that the velum folds into the mantle cavity, and thus is protected, as the shells are closed. Fibres in the radial pallial nerves have conduction velocities of up to 2·35 m/s at a temperature of 25 °C.

Mechanical and chemical sensitivity at the mantle edge of the file clamLima scabra(Born)

Crude starfish extracts or mechanical stimulation applied to the isolated mantle edge of Lima scabra evoked afferent impulse activity in the palliai nerves and local movements of the velum. The elicited spike activity was not a primary afferent response and the induced velum movements, which resembled jet formation on the mantle edge of intact clams, were apparently controlled by peripherally located neurons. Adductor muscle activity and efferent palliai nerve spikes were elicited by decreases in ambient illumination and by the application of mechanical stimulation or starfish extract to the mantle edge. Efferent impulse activity in the palliai nerves was correlated with protective movements of the mantle edge at the adduction. Similarities between the adductions evoked from L. scabra and scallop escape adductions are discussed.

Excitability and functional organization of cutaneous tactile units of the bullfrog (R. catesbeiana).

Frog skin touch receptors are discrete structures which appear as dome-shaped translucent elevations of the epidermis. These domes are particularly responsive to direct touch. Tactile stimulation of adjacent skin never caused impulse activity, except when the skin distortion disturbed the domes. On stimulation at threshold intensity, the response of all units studied always consisted of a single impulse whether the stimulus energy was electrical or mechanical. While most tactile units discharged one impulse upon application of mechanical stimulation, a few also discharged one impulse upon stimulus release (on-off response). No after discharge was seen at stimulus strengths of three or four times threshold or after repetitive stimulation at 1,000 pps. The range of distribution of active domes/receptive field was 1--14. Generally the most sensitive domes appeared to be in or near center field. Twice as many of the tactile units responded more readily to cold than to warmth. The impulse frequency of units responding to thermal stimulation ranged from 3-11/sec. The conduction velocity of tactile units measured was within a range of 9--25 m/sec, with a mean of 12 m/sec. The results suggest that the following: 1) the existence of discrete structures which respond to tactile stimuli; a phylogenetic relationship might be postulated; 2) cold/touch receptors which appear to be present in greater quantity in the frog's skin than are warm/touch receptors, 3) touch units which should be considered primarily as mechanoreceptors with a possible secondary function as thermoreceptors.