Unstrained Cells Research Articles

Theoretical studies on intrinsic electron traps in strained amorphous silica

Charge trapping in amorphous silica (a-SiO2) dielectric layer can degrade electrical performances of semiconductor devices. Earlier studies demonstrate that a SiO4 tetrahedra unit with a wide O-Si-O angle (≥132.0∘) can act as an electron trapping site in unstrained a-SiO2 cells. With first-principle calculations, in conjunction with molecular dynamic calculations, this work investigates the impacts of uniaxial tensile strain on one-electron trapping behaviors of a-SiO2, including atomic configurations and electronic properties. Stable states of strained a-SiO2 cells with an extra electron indicate that a SiO4 with a wide O-Si-O angle (≥132.0∘) and a long Si-O bond (≥1.660Å) making up the angle can act as a novel intrinsic electron tap. An electron trapping on this trap can generate a stable defect complex with two components: a negatively charged non-bridging oxygen hole center and a neutral oxygen vacancy with an unpaired electron.

Mechanical Strain-Mediated Tenogenic Differentiation of Mesenchymal Stromal Cells Is Regulated through Epithelial Sodium Channels.

It has been suggested that mechanical strain may elicit cell differentiation in adult somatic cells through activation of epithelial sodium channels (ENaC). However, such phenomenon has not been previously demonstrated in mesenchymal stromal cells (MSCs). The present study was thus conducted to investigate the role of ENaC in human bone marrow-derived MSCs (hMSCs) tenogenic differentiation during uniaxial tensile loading. Passaged-2 hMSCs were seeded onto silicone chambers coated with collagen I and subjected to stretching at 1 Hz frequency and 8% strain for 6, 24, 48, and 72 hours. Analyses at these time points included cell morphology and alignment observation, immunocytochemistry and immunofluorescence staining (collagen I, collagen III, fibronectin, and N-cadherin), and gene expression (ENaC subunits, and tenogenic markers). Unstrained cells at similar time points served as the control group. To demonstrate the involvement of ENaC in the differentiation process, an ENaC blocker (benzamil) was used and the results were compared to the noninhibited hMSCs. ENaC subunits' (α, β, γ, and δ) expression was observed in hMSCs, although only α subunit was significantly increased during stretching. An increase in tenogenic genes' (collagen1, collagen3, decorin, tenascin-c, scleraxis, and tenomodulin) and proteins' (collagen I, collagen III, fibronectin, and N-cadherin) expression suggests that hMSCs underwent tenogenic differentiation when subjected to uniaxial loading. Inhibition of ENaC function resulted in decreased expression of these markers, thereby suggesting that ENaC plays a vital role in tenogenic differentiation of hMSCs during mechanical loading.

Abstract 1177: Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment

Abstract Introduction: Cells within the breast tumor mass are met with a variety of biophysical or mechanical signals that is associated with elevated compression or solid stress within the tumor interior, tension at the tumor periphery, and altered interstitial fluid pressure. Understanding how the mechanical stress within the tumor microenvironment (TME) regulates cancer cell phenotype is of interest, yet the response of breast cancer (BCa) cells to these forces is largely unknown. Our study aims to identify the impact of mechanical stress on BCa cell phenotype by mimicking the tension at the tumor periphery. Materials and Methods: BCa cells (MCF-7 or 4T1.2) were cultured to confluence on collagen coated FlexCell culture plates. These plates were then subjected to 10% uniaxial cyclic/oscillatory strain at 0.3 Hz, or 10% constant strain, or no strain for 48 hours. Strained or control cells were isolated for analysis of proliferation (MTT assay), and migration (8.0 μm pore transwell). Exosomes from conditioned media were isolated via differential centrifugation and purified exosomes were characterized by ImageStream. 5x105 4T1.2 cells or PKH-labeled strained or control cells were injected into the mammary fat pad of BALB/c mice. Tumor volume was measured over 14 days. Tumor-infiltrating immune cells and the uptake of exosomes were analyzed by flow cytometry on day-14 post implantation. Results: We determined significant increases in proliferation and migration of 4T1.2 and MCF-7 cells in vitro following exposure to oscillatory forces. The populations of CD63+, CD63+CD24+ and CD63+PD-L1+ exosomes were increased when 4T1.2 cells were exposed to oscillatory strain compared to unstrained control cells. Further, we investigated how oscillatory forces affect tumor growth and tumor-immune cell interactions in vivo by using a syngeneic, orthotopic mouse model of BCa. Mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces showed a significant increase in primary tumor growth at 8 and 11 days post tumor challenge. The percentages of tumor-infiltrating monocytic myeloid-derived suppressor cells (M-MDSC) and recruited macrophages were increased in the TME of mice implanted with 4T1.2 cells that were pre-exposed to oscillatory forces, while the granulocytic MDSC subset was not significantly different between the two groups. A marginal decrease in the percentage of CD8+ T cells was noted in the TME of mice implanted with strained 4T1.2 when compared to controls, suggesting immune suppression in the TME. Furthermore, exosome uptakes by M-MDSC and recruited macrophages were increased in the TME of mice implanted with PKH-labeled 4T1.2 cells, exposed to oscillatory strain. Conclusion: Together, these data indicate that exposure to mechanical stress changes BCa cell phenotype to an invasive and protumorigenic phenotype that promotes immunosuppressive effects in the TME. Citation Format: Yong Wang, Paige E. Severino, Kayla Goliwas, Kenneth Hough, Derek Van Vessem, Hong Wang, Andra R. Frost, Selvarangan Ponnazhagen, Joel L. Berry, Jessy S. Deshane. Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1177.

A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment.

Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

MicroRNA-218, microRNA-191*, microRNA-3070a and microRNA-33 are responsive to mechanical strain exerted on osteoblastic cells.

MicroRNA (miRNA) is an important regulator of cell differentiation and function. Mechanical strain is important in the growth and differentiation of osteoblasts. Therefore, mechanresponsive miRNA may be important in the response of osteoblasts to mechanical strain. The purpose of the present study was to select and identify the mechanoresponsive miRNAs of osteoblasts. Mouse osteoblastic MC3T3-E1 cells were cultured in cell culture dishes and stimulated with a mechanical tensile strain of 2,50 με at 0.5 Hz, and the activity of alkaline phosphatase (ALP), mRNA levels of ALP, osteocalcin (OCN), and collagen type I (Col I), and protein levels of bone morphogenetic proteins (BMPs) in the cell culture medium were assayed. Following miRNA microarray and reverse transcription-quantitative polymerase chain reaction analyses, differentially expressed miRNAs in the mechanically strained cells and unstrained cells were selected and identified. Using bioinformatics analysis, the target genes of the miRNAs were then predicted. The results revealed that the mechanical strain of 2,500 με increased the activity of ALP, the mRNA levels of ALP, OCN and Col I, and the protein levels of bone morphogenetic protein(BMP)-2 and BMP-4 Continuous mechanical stimulation for 8 h had the most marked stimulant effects. miR-218, miR-191*, miR-3070a and miR-33 were identified as differentially expressed miRNAs in the mechanically strained MC3T3-E1 cells. Certain target genes of these four miRNAs were involved in osteoblastic differentiation. These findings indicated that a mechanical strain of 2,500 με, particularly for a period of 8 h, promoted osteoblastic differentiation, and the four mechanoresponsive miRNAs identified may be a potential regulator of osteoblastic differentiation and their response to mechanical strain.

Diffusion of Si impurities in Ni under stress: A first-principles study

We perform a first-principles study of the effect of strain on the migration of Si atoms in Ni. For that purpose, migration barriers are computed using the nudged elastic band method and attempt frequencies are computed using the direct force method. Good agreement is found with tracer diffusion experiments. We used the elastic dipole model to calculate effects of strain on migration barriers by performing calculations on unstrained cells, therefore reducing significantly the computing time. We validate this approach by comparing results with migration barriers calculated on strained cells and obtain an excellent agreement up to a strain of 1%. Computing all the jump frequencies in the neighborhood of Si solutes, the effect of strain is found to be nearly independent of the relative position of the solute atom. A simple elastic analysis models the changes in the vacancy jump with strain; this correlates with the changes in geometry for the ``cage'' of atoms surrounding the hopping atom at the saddle point.

Comparison of methods to determine variations in unstrained unit cell parameter across welds

Many alloys undergo complex changes in local chemistry in the vicinity of weldments due to the thermal excursion during welding. The resulting changes in solute concentrations can lead to significant local variations in the unstrained unit cell parameter which, if not accounted for, can lead to serious error when determining residual stress by diffraction methods. Age-hardening aluminium alloys are particularly susceptible to such effects. The present paper compares three methods (plane stress assumption, sin2ψ method, and comb correction method) for evaluating the stress-free unit cell parameter variation for friction stir welds in AA7449-W51 plates of two different thicknesses. All three methods gave comparable results for thin (5 mm) sheet, but for the thicker (12.2 mm) plate the results calculated on the basis of the plane stress assumption diverged from the other two, largely because in this case the other methods indicate there to be a significant triaxiality of stress. In the example cases, hardness and unstrained unit cell parameter variations were found to be strongly correlated across the welds. The advantages and disadvantages of the three methods are compared.

Chronic oscillatory strain induces MLCK associated rapid recovery from acute stretch in airway smooth muscle cells

A deep inspiration (DI) temporarily relaxes agonist-constricted airways in normal subjects, but in asthma airways are refractory and may rapidly renarrow, possibly due to changes in the structure and function of airway smooth muscle (ASM). Chronic largely uniaxial cyclic strain of ASM cells in culture causes several structural and functional changes in ASM similar to that in asthma, including increases in contractility, MLCK content, shortening velocity, and shortening capacity. However, changes in recovery from acute stretch similar to a DI have not been measured. We have therefore measured the response and recovery to large stretches of cells modified by chronic stretching and investigated the role of MLCK. Chronic, 10% uniaxial cyclic stretch, with or without a strain gradient, was administered for up to 11 days to cultured cells grown on Silastic membranes. Single cells were then removed from the membrane and subjected to 1 Hz oscillatory stretches up to 10% of the in situ cell length. These oscillations reduced stiffness by 66% in all groups (P < 0.05). Chronically strained cells recovered stiffness three times more rapidly than unstrained cells, while the strain gradient had no effect. The stiffness recovery in unstrained cells was completely inhibited by the MLCK inhibitor ML-7, but recovery in strained cells exhibiting increased MLCK was slightly inhibited. These data suggest that chronic strain leads to enhanced recovery from acute stretch, which may be attributable to the strain-induced increases in MLCK. This may also explain in part the more rapid renarrowing of activated airways following DI in asthma.

Numerical Analysis of a Solar Cell with Tensile-Strained Ge as a Novel Narrow-Band-Gap Absorber

A narrow-band-gap (0.6–0.7 eV) bottom cell absorber material is one of the most important and relatively undeveloped components for future five- or six-junction solar cells. Tensile-strained Ge is a promising material for a novel bottom cell absorber since it has a high absorption coefficient and an “adjustable” lattice constant. In this study, we numerically demonstrate the possibility of tensile-strained Ge as a bottom cell material for multijunction solar cells. The design examples of lattice-matched five-junction cells using tensile-strained Ge as bottom cells are also presented. It is shown that sub-µm tensile-strained Ge can produce same efficiency as that of a 100–300 µm bulk unstrained Ge cell. Using tensile-strained Ge as a bottom cell gives a higher efficiency and solves the lattice mismatch problem compared with traditional bottom cell materials such as a Ge substrate and a high In-composition InxGa1-xAs.

Numerical Analysis of a Solar Cell with Tensile-Strained Ge as a Novel Narrow-Band-Gap Absorber

A narrow-band-gap (0.6–0.7 eV) bottom cell absorber material is one of the most important and relatively undeveloped components for future five- or six-junction solar cells. Tensile-strained Ge is a promising material for a novel bottom cell absorber since it has a high absorption coefficient and an "adjustable" lattice constant. In this study, we numerically demonstrate the possibility of tensile-strained Ge as a bottom cell material for multijunction solar cells. The design examples of lattice-matched five-junction cells using tensile-strained Ge as bottom cells are also presented. It is shown that sub-µm tensile-strained Ge can produce same efficiency as that of a 100–300 µm bulk unstrained Ge cell. Using tensile-strained Ge as a bottom cell gives a higher efficiency and solves the lattice mismatch problem compared with traditional bottom cell materials such as a Ge substrate and a high In-composition InxGa1-xAs.

Role of Nitric Oxide in Activity Control of Mechanically Gated Ionic Channels in Cardiomyocytes: NO-Donor Study

Whole-cell ionic currents through mechanically gated channels (MGC) were recorded in isolated cardiomyocytes under voltage clamp conditions. In unstrained cells, NO donors SNAP and DEA-NO activated MGC and induced MG-like currents. In contrast, in stretched cells with activated MGC, these NO-donors inactivated and inhibited MGC.

The MGF expression of osteoblasts in response to mechanical overload

Cell proliferation and mRNA expression of insulin-like growth factor (IGF-I) and "mechanogrowth factor" (MGF) were studied in osteoblasts in response to overload. Static and cyclic-stretching were used to apply superphysiological strains to cells. Overload was found to increase cell growth. IGF-I and its splicing variant, MGF, were measured using reverse transcriptase-polymerase chain reaction method and were found to be regulated differentially by mechanical signals at the mRNA level. Cyclic-stretching had a more significant effect on cell proliferation and mRNA expression levels of IGF-I and MGF, while unstrained cells did not express MGF at the mRNA level. These results demonstrated that gene expression is regulated by mechanical stimulation. MGF expression in osteoblasts in response to strain may be related to an autocrine mechanism.

Cyclic Strain Inhibits Notch Receptor Signaling in Vascular Smooth Muscle Cells In Vitro

Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the Jkappa immunoglobulin gene-dependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-protein- and mitogen-activated protein kinase-dependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-x(L) ratio). Notch 3 receptor overexpression significantly reversed the strain-induced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, through regulation of Notch receptor and downstream target gene expression.

Mechanical strain enhances proteoglycan message in fibroblasts from asthmatic subjects.

Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation. We hypothesized that fibroblasts from asthmatic patients would respond to excessive mechanical strain with up-regulation of message for PGs. We obtained fibroblasts from asthmatic patients (AF) and normal volunteers (NF) using endobronchial biopsy. Cells were maintained in culture until the fifth passage and then grown on a flexible collagen-coated membrane. Using the Flexercell device, cells were then subjected to cyclic stretch at 30% amplitude at 1 Hz for 24 h. Control cells were unstrained. Total RNA was extracted from the cell layer and quantitative RT-PCR performed for decorin, lumican and versican mRNA. In unstrained cells, the expression of decorin mRNA was greater in AF than NF. With strain, NF showed increased expression of versican mRNA and AF showed increased expression of versican and decorin mRNA. The relative increase in versican mRNA expression with strain was greater in AF than NF. These data support the hypothesis that proteoglycan message is increased in asthmatic fibroblasts subject to mechanical strain. This finding has implications for the mechanisms governing airway wall remodelling in asthma.

Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells

Ectopic calcification of vascular tissue is associated with several cardiovascular pathologies and likely involves active regulation by vascular smooth muscle cells and osteoblast-like vascular cells. This process often occurs in sites with altered mechanical environments, suggesting a role for mechanical stimuli in calcification. In this study, we investigated the effect of mechanical stimulation on the proliferation, osteogenic differentiation, calcification, and mitogen-activated protein kinase (MAPK) signaling in calcifying vascular cells (CVCs), a subpopulation of aortic smooth muscle cells putatively involved in vascular calcification. Application of equibiaxial cyclic strain (7%, 0.25 Hz) to CVCs had no effect on cell proliferation, but accelerated alkaline phosphatase expression and significantly increased mineralization by 3.1-fold over unstrained cells. Fluid motion in the absence of strain also enhanced mineralization, but to a lesser degree. Because MAPK pathways mediate mechanically regulated osteoblast differentiation, we tested whether similar signaling was involved in mineralization by CVCs. In static cultures, pharmacological inhibition of the extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase pathways significantly attenuated mineral production by as much as −94%, compared with uninhibited CVCs. Strikingly, although mechanical stimulation activated each of the MAPK pathways, inhibition of these pathways had no effect on the mechanically induced enhancement of alkaline phosphatase activity or mineralization. These novel data indicate that mechanical signals regulate calcification by CVCs, and although MAPK signaling is critical to CVC osteogenic differentiation and mineralization, it is not involved directly in transduction of mechanical signals to regulate these processes under the conditions utilized in this study.

The effects of mechanical strain on synovial fibroblasts

Purpose: Arthritic diseases of the temporomandibular joint, such as rheumatoid arthritis and osteoarthritis, suggest that inflammatory mediators and metalloproteinases may play a role in their pathogenesis. Recent clinical evidence from physical therapy and other modalities has shown a significant decrease in temporomandibular joint symptoms in patients with early disease. This project examines the effect of mechanical strain on synovial fibroblasts' production of inflammatory mediators including prostaglandin E2 (PGE2) and proteinases. Materials and Methods: An established synovial fibroblast cell line (HIG-82) was grown to confluency in modified Eagle's medium supplemented with 10% fetal calf serum. The monolayer of fibroblasts was then subjected to mechanical strain using the Flexercell Strain Unit (Flexcell International Corporation, McKeesport, PA) at 3 cycles per minute, with 10 seconds' elongation of up to 24% and 10 seconds of relaxation. Levels of PGE2 were determined by radioimmunoassay using commercially available product and measured in nanograms per milliliter of supernatant. Proteinases collagenase, gelatinase, and stromelysin were measured by H3 radioactive labeling of acidic anhydride to the specific substrate. Enzymatic proteolysis of the radiolabeled substrate was then measured in supernate as units per milliliter. Statistical analysis of all results was performed using Student's t test in triplicate. Results: PGE2 levels of mechanically activated cells was 18.1 ± 13.4 ng/mL, with control levels being 58.0 ± 9.2 ng/mL. This is a statistically significant decrease, between strained and unstrained cells with P <.05. In control cells, proteinase activity that degrades collagen, gelatin, or casein was 4.27 ± 1.5, 4.62 ± 0.11, or 0.11 ± 0.01 U/mL, respectively. Levels for mechanically strained cells were 3.99 ± 1.90, 4.02 ± 0.90, and 0.12 ± 0.01 U/mL, respectively. These results show that there is a significant decrease in PGE2 levels of synovial fibroblasts undergoing mechanical strain. Proteinases examined show no difference in levels between mechanically activated fibroblasts and their controls. Conclusion: This decrease in PGE2 production in synovial fibroblasts could help elucidate the mechanism by which physical therapy, and in particular continuous passive motion, may decrease inflammatory mediators of the temporomandibular joint. © 2003 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 61:707-712, 2003

Cyclic strain enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway

Physical stimuli play critical roles in the development, regeneration, and pathology of many mesenchymal tissues, most notably bone. While mature bone cells, such as osteoblasts and osteocytes, are clearly involved in these processes, the role of their progenitors in mechanically mediated tissue responses is unknown. In this study, we investigated the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Application of equibiaxial cyclic strain (3%, 0.25 Hz) to hMSCs cultured in osteogenic media inhibited proliferation and stimulated a 2.3-fold increase in matrix mineralization over unstrained cells. The strain stimulus activated the extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase pathways, but had no effect on c-Jun N-terminal kinase phosphorylation or activity. Strain-induced mineralization was largely mediated by ERK1/2 signaling, as inhibition of ERK1/2 attenuated calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype, suggesting an inhibitory role for p38 signaling in the modulation of strain-induced osteogenic differentiation. These results demonstrate that mechanical signals regulate hMSC function, suggesting a critical role for physical stimulation of this specific cell population in mesenchymal tissue formation.

Critical behavior of a strain percolation model for metals.

Extensive simulations of a strain percolation model for a deforming metal have been performed to examine its strain behavior. We find that the total strain exhibits critical power-law behavior that is well explained by two-dimensional percolation theory. Near the critical point, most of the strained cells organize themselves around a state having the minimum or at least marginally stable strain regardless of the initial conditions. A strain much greater than the minimum stable strain generally decays to a lower value when transmitted to an unstrained cell. The universal behavior of the total strain in the system is a consequence of the self-organizing character of the strain in the critical cluster. Although the probability distributions for the total strain and cluster size appear to exhibit nonuniversal behavior, this may merely represent a transient response before crossover to a true asymptotic, universal behavior occurs. Other critical aspects of the model are also discussed.

The stiffness of bone marrow cell–knit composites is increased during mechanical load

A novel device for mechanical stimulation of primary adult rat bone marrow cells cultured on three-dimensional knitted textiles has been prototyped. A method has been developed ensuring a well-defined, high-density, and reproducible cell seeding on the knitted fabric. After culturing for 18–52 days the cell–knit composites were subjected to uniaxial 2% stretching and relaxation. The frequency was altered between 0.1 Hz (196 min, loading phase) and 0.01 Hz (360 min, resting phase). Identically treated knits without cells exhibited a slight stiffness reduction, whereas the stiffness of knits with cells increased from cycle to cycle. The stiffness increase was found to depend on the duration of the culture period before mechanical loading. Our data suggest that the extracellular matrix deposited by the cells on the knit and intact microtubuli of living cells cause the observed stiffness increase. In comparison to the unstrained static cell–knit composites cell proliferation and bone cell differentiation were reduced by the mechanical load.

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose

Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.