Fibroblast responses to cyclic mechanical stretching depend on cell orientation to the stretching direction

Fibroblasts in intact tendons align with stretching direction, but they tend to orient randomly in healing tendons. Therefore, a question arises: Do fibroblast responses to mechanical stretching depend on their orientation? To address this question, human patellar tendon fibroblasts were grown in custom-made silicone dishes that possess microgrooved culture surfaces. The direction of the microgrooves was either parallel or normal to the direction of cyclic uniaxial stretching. Fibroblasts grown in these microgrooves had a polar morphology and oriented along the direction of the microgrooves regardless of the stretching conditions. Tendon fibroblasts expressed higher levels of alpha-smooth muscle actin when they were oriented parallel to the stretching direction than when they were oriented normal to the stretching direction. Also, cyclic stretching of the fibroblasts perpendicular to their orientation induced a higher activity level of secretory phospholipase A(2) compared with stretching of the cells parallel to their orientation. Thus, these results show that fibroblast responses to mechanical stretching depend on cell orientation to the stretching direction.     

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.     

LLC-PK1 cysts: a model for the study of epithelial polarity

In the present work, we have taken advantage of the properties of two recently isolated clonal subpopulations of the pig kidney-derived LLC-PK1 cell line to study aspects of the establishment of epithelial polarity. When grown in suspension, LLC-PK1/D + Sc cells reaggregated within a few hours and, during the following days of culture, formed free-floating, hollow spheres or cysts, lined by a monolayer of polarized cells. In contrast, LLC-PK1/D- cells were unable to develop such polarized structures even upon prolonged culture in suspension. The polarity of the LLC-PK1/D + Sc cells lining the cysts was inverted compared to that in intact renal tubules, the microvilli-rich "apical" pole being oriented toward the external medium. However, upon embedding these preformed cysts in collagen gels, a reversal of polarity was observed within hours, the microvilli-rich pole now facing the cyst cavity. Thus, in the same clonally derived cell population, cell-to-cell contact and interaction with the extracellular matrix differentially affect the orientation of cellular polarity. The LLC-PK1/D + Sc cysts provide a suitable in vitro model system for further study of the sequential events by which extracellular matrix components induce an appropriately oriented polarization. In addition, the comparison between LLC-PK1/D + Sc and D- cells, which differ in their ability to polarize in response to cell-to-cell contact, should help define some of the cellular determinants involved in epithelial organization.     

Extracellular matrix deposition by fibroblasts is necessary to promote capillary-like tube formation in vitro

The contribution of the cellular and fibrillar microenvironment to angiogenesis still remains unclear. Our purpose was to evaluate the effect of the extracellular matrix deposited by fibroblasts on the capacity of human endothelial cells to form capillaries in vitro. We have drastically decreased the amount of extracellular matrix surrounding fibroblasts in our model of endothelialized-reconstructed connective tissue (ERCT) by culturing it without ascorbate. Under these conditions, the number of capillary-like tubes (CLT) formed by endothelial cells was reduced by up to 10-fold after 31 days of culture compared to controls. This decrease was due neither to a variation of MMP-2 and MMP-9 secretion, nor to a reduction in the number of fibroblasts and/or endothelial cells, or a diminution of fibroblast growth factor 2 (FGF2) synthesis. The secretion of vascular endothelial growth factor (VEGF) by fibroblasts accounted for 25-70% of the capillary-like tube formation when tissues were cultured in the presence or absence of ascorbate, as demonstrated by VEGF-blocking studies. The culture of endothelial cells on a similar extracellular matrix but in the absence of living fibroblasts did not promote the formation of CLT, even when tissues were fed with fibroblast-conditioned medium. Thus, the deposition of a rich extracellular matrix by living fibroblasts appeared necessary, but not sufficient to promote capillary-like formation. Fibroblasts seem to induce endothelial cells to spontaneously form CLT by secreting and organizing an abundant extracellular matrix, which creates a microenvironment around cells that could in turn trap growth factors produced by fibroblasts and promote three-dimensional cell organization.     

Contact guidance on oriented collagen gels

We have altered the shape of aligned hydrated collagen gels, without substantially altering their orientation, by air-drying them on coverslips. The original wet gels had a three-dimensional shape and elicited a strong contact guidance response when used as a substratum for heart fibroblasts or nerve axons, whereas the air-dried gels were totally flattened onto the plane support and were much less effective in guiding the cells. Treatment of the dried gels with dilute acetic acid slightly restored their three-dimensional shape and slightly restored their original contact guiding property. We interpret these results as indicating that contact guidance on such oriented fibrillar matrices is a direct cellular response to the shape of the substratum.     

Translating cell polarity into tissue elongation

Planar cell polarity, the orientation of single-cell asymmetries within the plane of a multicellular tissue, is essential to generating the shape and dimensions of organs and organisms. Planar polarity systems align cell behavior with the body axes and orient the cellular processes that lead to tissue elongation. Using Drosophila as a model system, significant progress has been made toward understanding how planar polarity is generated by biochemical and mechanical signals. Recent studies using time-lapse imaging reveal that cells engage in a number of active behaviors whose orientation and dynamics translate planar cell polarity into tissue elongation. Here we review recent progress in understanding the cellular mechanisms that link planar polarity to large-scale changes in tissue structure.     

The effect of a native collagen gel substratum on the synthesis of collagen by bovine brain capillary endothelial cells

Cultured capillary endothelial cells, derived from bovine brain, and maintained on a plastic substratum synthesized predominantly interstitial collagens of which approximately 75 per cent were secreted into the medium. When grown on a native hydrated collagen type I gel, although no marked alteration in the 'collagen synthetic pattern' was observed, the overall level of collagen synthesis was increased by approximately 100 per cent. More dramatic, however, was the alteration in the distribution of these molecules between medium and cell layer. Interstitial collagens produced by cells grown on collagen gels were almost exclusively associated with the cell layer or collagenous gel. These studies, thus, demonstrate that an extracellular matrix may exert a considerable influence on the cellular synthetic activities and possibly cellular polarity of capillary endothelial cells.     

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis

Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis have been studied. Throughout the larval period to stage 60, the connective tissue consists of a few immature fibroblasts surrounded by a sparse extracellular matrix: few collagen fibrils are visible except close to the thin basal lamina. At the beginning of the transition from larval to adult epithelial form around stage 60, extensive changes are observed in connective tissue. The cells become more numerous and different types appear as the collagen fibrils increase in number and density. Through gaps in the thickened and extensively folded basal lamina, frequent contacts between epithelial and connective tissue cells are established. Thereafter, with the progression of fold formation, the connective tissue cells become oriented according to their position relative to the fold structure. The basal lamina beneath the adult epithelium becomes thin after stage 62, while that beneath the larval epithelium remains thick. Upon the completion of metamorphosis, the connective tissue consists mainly of typical fibroblasts with definite orientation and numerous collagen fibrils. These observations indicate that developmental changes in the connective tissue, especially in the region close to the epithelium, are closely related spatiotemporarily to the transition from the larval to the adult epithelial form. This suggests that tissue interactions between the connective tissue and the epithelium play important roles in controlling the epithelial degeneration, proliferation, and differentiation during metamorphic climax.     

Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells

We have investigated the influence of culture substrata upon glycosaminoglycans produced in primary cultures of mouse mammary epithelial cells isolated from the glands of late pregnant mice. Three substrata have been used for experiments: tissue culture plastic, collagen (type I) gels attached to culture dishes, and collagen (type I) gels that have been floated in the culture medium after cell attachment. These latter gels contract significantly. Cells cultured on all three substrata produce hyaluronic acid, heparan sulfate, chondroitin sulfates and dermatan sulfate but the relative quantities accumulated and their distribution among cellular and extracellular compartments differ according to the nature of the culture substratum. Notably most of the glycosaminoglycans accumulated by cells on plastic are secreted into the culture medium, while cells on floating gels incorporate almost all their glycosaminoglycans into an extracellular matrix fraction. Cells on attached collagen gels secrete approx. 30% of their glycosaminoglycans and assemble most of the remainder into an extracellular matrix. Hyaluronic acid is produced in significant quantities by cells on plastic and attached gels but in relatively reduced quantity by cells on floating gels. In contrast, iduronyl-rich dermatan sulfate is accumulated by cells on floating gels, where it is primarily associated with the extracellular matrix fraction, but is proportionally reduced in cells on plastic and attached gels. The results are discussed in terms of polarized assembly of a morphologically distinct basal lamina, a process that occurs primarily when cells are on floating gels. In addition, as these cultures secrete certain milk proteins only when cultured on floating gels, we discuss the possibility that cell synthesized glycosaminoglycans and proteoglycans may play a role in the maintenance of a differentiated phenotype.     

Fibroblast cytoskeletal remodeling contributes to connective tissue tension

The visco-elastic behavior of connective tissue is generally attributed to the material properties of the extracellular matrix rather than cellular activity. We have previously shown that fibroblasts within areolar connective tissue exhibit dynamic cytoskeletal remodeling within minutes in response to tissue stretch ex vivo and in vivo. Here, we tested the hypothesis that fibroblasts, through this cytoskeletal remodeling, actively contribute to the visco-elastic behavior of the whole tissue. We measured significantly increased tissue tension when cellular function was broadly inhibited by sodium azide and when cytoskeletal dynamics were compromised by disrupting microtubules (with colchicine) or actomyosin contractility (via Rho kinase inhibition). These treatments led to a decrease in cell body cross-sectional area and cell field perimeter (obtained by joining the end of all of a fibroblast's processes). Suppressing lamellipodia formation by inhibiting Rac-1 decreased cell body cross-sectional area but did not affect cell field perimeter or tissue tension. Thus, by changing shape, fibroblasts can dynamically modulate the visco-elastic behavior of areolar connective tissue through Rho-dependent cytoskeletal mechanisms. These results have broad implications for our understanding of the dynamic interplay of forces between fibroblasts and their surrounding matrix, as well as for the neural, vascular, and immune cell populations residing within connective tissue.     

The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

Background  

During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules.     

Patterns of cell polarity in the developing mouse limb

Most cells have a morphological polarity with the centrioles and Golgi apparatus occupying one pole of the cell and the nucleus the other. This structural polarity often correlates with functional polarity as in secretory epithelia where the Golgi apparatus moves to the pole of the cell from which secretory materials are exreted. In limb development an interaction of unknown mechanism occurs between the epithelium and mesenchyme. We have evaluated the pattern of cell polarity using silver impregnation of the Golgi apparatus in limb epithelium and mesenchyme of mouse embryos from day 9.5, when limbs are first visible, to day 15, when cartilage formation is complete. Cells in the epithelium almost always have the Golgi apparatus in the apex of the cell, i.e., oriented away from the basement membrane. The layer of mesenchyme cells just beneath the basement membrane initially has only 16 to 25% of the cells oriented toward the basement membrane. A marked shift in orientation occurs between days 12 and 13 so that from days 13 to 15 up to 53% of the mesenchyme cells are oriented toward the basement membrane. This shift in orientation occurs more slowly in the mesenchyme at a depth of four cells below the basement membrane. This changing pattern of mesenchymal cell polarity occurs at a time when there is an apparent increase in the amount of extracellular matrix, especially in the region just below the basement membrane.     

Differential proliferation rates generate patterns of mechanical tension that orient tissue growth

Orientation of cell divisions is a key mechanism of tissue morphogenesis. In the growing Drosophila wing imaginal disc epithelium, most of the cell divisions in the central wing pouch are oriented along the proximal–distal (P–D) axis by the Dachsous‐Fat‐Dachs planar polarity pathway. However, cells at the periphery of the wing pouch instead tend to orient their divisions perpendicular to the P–D axis despite strong Dachs polarization. Here, we show that these circumferential divisions are oriented by circumferential mechanical forces that influence cell shapes and thus orient the mitotic spindle. We propose that this circumferential pattern of force is not generated locally by polarized constriction of individual epithelial cells. Instead, these forces emerge as a global tension pattern that appears to originate from differential rates of cell proliferation within the wing pouch. Accordingly, we show that localized overgrowth is sufficient to induce neighbouring cell stretching and reorientation of cell division. Our results suggest that patterned rates of cell proliferation can influence tissue mechanics and thus determine the orientation of cell divisions and tissue shape.     

Effects of electric fields on fibroblast contractility and cytoskeleton

We used silicone rubber substrata and fluorescent staining of cytoskeletal components to study the mechanisms by which electrical voltage gradients cause reorientation of embryonic chick fibroblasts in tissue culture. No evidence was found for a direct stimulation of cell contractility, either parallel or perpendicular to the voltage gradient. Instead, there was a gradual weakening in cell contractility in the axis parallel to this gradient, accompanied by progressive retraction of lamellae oriented along this axis, apparently due to selective weakening of cell-substratum adhesions. The cells then elongated perpendicular to the electric field, and strengthened their contractility in that axis. Fluorescence microscopy showed that cytoplasmic actin stress fibers and microtubules oriented perpendicular to the imposed voltage gradient. Many more cases were observed in which cell morphology had reoriented, but the actin fibers had not, as compared to the converse (cytoskeleton oriented, but no morphology). This disparity further supports the interpretation that the redirection of cell contractility is a consequence of morphological reorientation, rather than its cause. We also studied the effects of reversing the polarity of the electric fields at constant intervals (of as long as 1 minute). Fibroblasts failed to orient in response to such alternating fields, even after long exposure, but these same cells did reorient in response to pulsed currents in a consistent direction separated by "rest periods" (with no current). This combination of results is more consistent with an electrophoretic mechanism than with one depending on voltage-induced changes in membrane permeabilities.     

Metabolism of glycoproteins and fibronectin in skin fibroblasts of patients with rheumatoid arthritis

The distribution in the cellular monolayer of the de novo synthetized pre-labeled glycoproteins and fibronectin upon culturing of fibroblasts in the medium with low serum content was analyzed. It was found that in rheumatoid arthritis (RA) the amount of total glycoproteins on the surface and within fibroblasts is higher and in the extracellular matrix is lower than in skin fibroblasts of healthy donors (HD). However, the amount of pre-labeled fibronectin on the surface of skin fibroblasts from patients with RA was considerably lower than in those from HD This finding as well as a rapid decrease in the amount of pre-labeled fibronectin in the extracellular matrix of RA fibroblasts is indicative of a more rapid metabolism of this protein in RA. In the skin fibroblasts from HD there was a practically uniform decrease in the amount of pre-labeled fibronectin in the cellular monolayer. The presence of caseinolytic activity in the culture medium even upon the first day of cell culturing in the serum-free medium, as well as the effect of various proteinase inhibitors on glycoprotein content in the cellular monolayer provide evidence that the rate of glycoprotein and fibronectin metabolism, especially in connective tissue cells of patients with RA, might possibly be determined not only by the level of their synthesis but also by the level of proteolytic activity in the connective tissue cells.     

Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell–FN interactions.     

The cellular matrix: a feature of tensile bearing dense soft connective tissues

The term connective tissue encompasses a diverse group of tissues that reside in different environments and must support a spectrum of mechanical functions. Although the extracellular matrix of these tissues is well described, the cellular architecture of these tissues and its relationship to tissue function has only recently become the focus of study. It now appears that tensile-bearing dense connective tissues may be a specific class of connective tissues that display a common cellular organization characterized by fusiform cells with cytoplasmic projections and gap junctions. These cells with their cellular projections are organised into a complex 3-dimensional network leading to a physically, chemically and electrically connected cellular matrix. The cellular matrix may play essential roles in extracellular matrix formation, maintenance and remodelling, mechanotransduction and during injury and healing. Thus, it is likely that it is the interaction of both the extracellular matrix and cellular matrix that provides the basis for tissue function. Restoration of both these matrices, as well as their interaction must be the goal of strategies to repair these connective tissues damaged by either injury or disease.     

THE FIBROBLAST AND WOUND REPAIR

This review of connective tissue repair has attempted to place into historical perspective information obtained by newer approaches. The literature review is incomplete, as it was unfortunately necessary to leave many interesting studies out of the discussion. Emphasis has been placed upon what is known of the inflammatory response, the fine structure of the connective tissue cells in healing wounds and with correlated chemical findings in these tissues. An optimal inflammatory response appears to be an important, rapid, non-specific stimulus for fibroplasia. It is not clear how inflammation exerts this effect. The inflammatory cells and their enzymes markedly alter the extracellular matrix of injured tissue. The matrix of connective tissue may itself participate in the control of its own synthesis and degradation. It is possible that modification of this environment by injury and/or inflammation with ensuing matrix alteration may provide a stimulus for cell migration and protein synthesis. The converse may also be true, that is, a given level of matrix concentration may have an inhibitory effect upon the connective tissue cells. The inter-relationships between the connective tissue matrix and the cells, and the possibilities of feedback mechanisms playing a role in maintaining a balance between these two are important areas for future investigation. In this regard, additional questions may be asked concerning the role of the fibroblast in remodelling and degradation of connective tissue. It is not yet clear how important a balance between collagenolytic enzymes and the solubility states, or stability, of collagen are in each connective tissue. It will be interesting to determine which cells make collagenolytic and/or proteolytic enzymes upon appropriate stimulus. It is possible to distinguish between the fibroblast and the monocyte, or potential macrophage with the electron microscope. The rough endoplasmic reticulum with its large numbers of attached ribosomes is extensively developed in the fibroblast in contrast to the monocyte. The endoplasmic reticulum sequesters collagen precursors and other secretory proteins for transport either directly to the extracellular space, as appears to be the case for collagen, or to the Golgi complex as is the case for other exportable proteins. Collagen precursors are secreted into the environment and are not shed from within the cell surface. A number of cytoplasmic alterations have been described for fibroblasts and other cells during various pathological states. The significance of these alterations is not clear. It will be important to distinguish between specific and non-specific responses to injury, if these alterations are to aid us in understanding the various cellular responses. The source of the fibroblasts in granulation tissue appears to be mesenchymal cells from adjacent tissues rather than blood-borne precursors. Although contact inhibition can be demonstrated in vitro, it is not clear how important this phenomenon is in vivo, nor are the reasons for the ability of some tissues to heal by regeneration rather than by scar tissue formation understood. These and many other questions remain to be answered. The healing wound is multifaceted and presents the opportunity for systematic investigation into the problems of cell proliferation, cell and matrix interactions, and protein synthesis in vivo and it also can help to further our understanding of the ubiquitous fibroblast and its complex extracellular matrix.     

Dynamic Fibroblast Cultures

Mechanical forces play an important role in the organization, growth and function of tissues. Dynamic extracellular environment affects cellular behavior modifying their orientation and their cytoskeleton. In this work, human fibroblasts have been subjected for three hours to increasing substrate deformations (1-25%) applied as cyclic uniaxial stretching at different frequencies (from 0.25Hz to 3Hz). Our objective was to identify whether and in which ranges the different deformations magnitude and rate were the factors responsible of the cell alignment and if actin cytoskeleton modification was involved in these responses. After three hours of cyclically stretched substrate, results evidenced that fibroblasts aligned perpendicularly to the stretch direction at 1% substrate deformation and reached statistically higher orientation at 2% substrate deformation with unmodified values at 5-20%, while 25% substrate deformation induced cellular death. It was also shown that a percentage of cells oriented perpendicularly to the deformation were not influenced by increased frequency of cyclical three hours deformations (0.25-3Hz). Cyclic substrate deformation was shown also to involve actin fibers which orient perpendicularly to the stress direction as well. Thus, we argue that a substrate deformation induces a dynamic change in cytoskeleton able to modify the entire morphology of the cells.     

Reciprocal interactions between cells and extracellular matrix during remodeling of tissue constructs

Cells remodel extracellular matrix during tissue development and wound healing. Similar processes occur when cells compress and stiffen collagen gels. An important task for cell biologists, biophysicists, and tissue engineers is to guide these remodeling processes to produce tissue constructs that mimic the structure and mechanical properties of natural tissues. This requires an understanding of the mechanisms by which this remodeling occurs. Quantitative measurements of the contractile force developed by cells and the extent of compression and stiffening of the matrix describe the results of the remodeling processes. Not only do forces exerted by cells influence the structure of the matrix but also external forces exerted on the matrix can modulate the structure and orientation of the cells. The mechanisms of these processes remain largely unknown, but recent studies of the regulation of myosin-dependent contractile force and of cell protrusion driven by actin polymerization provide clues about the regulation of cellular functions during remodeling.