Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration

Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.     

Chondrogenic differentiation in chick embryo osteoblast cultures.

Expression of specific differentiation markers was investigated by histochemistry, immunofluorescence, and biosynthetic studies in osteoblasts outgrown from chips derived from tibia diaphyses of 18-day-old chick embryos. The starting osteoblast population expressed type I collagen and alkaline phosphatase in addition to other bone and cartilage markers as the lipocalin Ch21; the extracellular matrix deposited by these cells was not stainable for cartilage proteoglycans, and mineralization was observed when the culture was maintained in the presence of ascorbic acid, calcium and beta-glycerophosphate. During culture, clones of cells presenting a polygonal chondrocyte morphology and surrounded by an Alcian-positive matrix appeared in the cell population. Type II collagen and type X collagen were synthesized in these areas of chondrogenesis. In addition, chondrocytes isolated from these cultures expressed Ch21 and alkaline phosphatase. Chondrocytes were generated also from homogeneous osteoblast populations derived from a single cloned cell. The coexistence of chondrocytes and osteoblasts was observed during amplification of primary clones as well as in subclones. The data show the existence, within embryonic bone, of cells capable in vitro of both osteogenic and chondrogenic differentiation.     

Growth plate chondrocytes store latent transforming growth factor (TGF)-β1 in their matrix through latent TGF-β1 binding protein-1

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-β) as well as a 290 kDa form containing latent TGF-β binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-β in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-β. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-β1 were found in these digests by TGF-β1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-β, in addition to LTBP1 that is associated with the 100 kDa latent TGF-β complex. These studies show for the first time that latent TGF-β is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-β, even at sites distant from the cell, in a relatively avascular environment. J. Cell. Physiol. 177:343–354, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes

Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na⁺ being 250–350 mM and extracellular osmolality 350–450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250–280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolity was altered over the range 250–450 mOsm by Na⁺ or sucrose addition. ³⁵S-sulphate and ³H-proline incorporation rates were at a maximum when the extracellular osmolality was 350–400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30–50% in DMEM to which NaCl or sucrose had been added to the increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of surcrose addition on incorporation rates were similar but not identical to those of Na⁺ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter-counter) increased 30–40% when the cells are removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition. © 1993 Wiley-Liss, Inc.     

Differential induction of stromelysin mRNA by bovine articular chondrocytes treated with interferon-γ and interleukin-1α

Articular chondrocytes from rheumatoid joints have been shown to express class II major histocompatibility (MHC) antigens that were correlated with the presence of interferon-gamma (IFN-γ) in the inflamed joint. Chondrocytes expressing MHC antigens function as antigens function as antigen presenting cells and thus stimulate lymphocyte proliferation. These responses suggest a powerful role for the IFN-γ stimulation of chondrocytes. The present studies were designed to examine the functional role of chondrocytes exposed to IFN-γ during cartilage degradation that occurs in synovial disease. Destruction of cartilage in arthritis is partially attributable to metalloproteinases released by the chondrocytes in response to interleukin-1 (IL-1). Bovine articular chondrocytes treated with interleukin-1 alpha (IL-1α) produced enhanced levels of stromelysin mRNA, however, Northern blots could not determine the percentage of cells responding. Exposure of bovine articular chondrocytes to IFN-γ induced the expression of bovine HLA-DR (boHLA-DR) antigen in 50% of the cells. Using a modified cell sorting technique, chondrocytes that expressed class II MHC antigens produced two fold greater stromelysin mRNA than chondrocytes that did not express this antigen. In contrast, collagen type II mRNA levels were similar in chondrocytes, regardless of the expression of class II MHC antigens. In situ hybridization studies showed that less than half of all cartilage chondrocytes were induced to synthesize stromelysin mRNA. These observations suggest that IFN-γ stimulates specific subpopulations of chondrocytes to be functionally active in inflammation-induced metalloprotease secretion. © 1993 Wiley-Liss, Inc.     

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast-like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures. Isolated mesenchymal cells from 12-day-old mouse limb buds were grown in alginate for up to 4 weeks. A sub-population of the cells differentiated to chondrocytes and exhibited a stable phenotype until the end of the culture period. After 3 to 4 days a cartilage-specific matrix started to develop. Fibroblast-like cells from this mixed culture did not survive; they became necrotic. When alginate was later on dissolved by chelating agents, only chondrocytes were isolated. During dissolution of alginate and centrifugation, chondrocytes did not lose their contact with their new matrix present on their surfaces. Cultivation of these chondrocytes or chondrones in mass culture yields a pure chondrocyte population. Immunoelectron microscopic investigations revealed collagen type II, fibronectin, decorin and chondroitin sulfate-proteoglycans in the chondrocyte capsules and in mass culture.     

In vitro biosynthesis by articular chondrocytes of a specific low molecular size proteoglycan pool

Proteoglycans synthesized by articular and epiphyseal chondrocytes in culture were compared. Proteoglycans extruded by the two types of cells into the culture medium are of identical Mr. On the other hand, the proteoglycans of cells or pericellular matrix synthesized by the articular chondrocytes are characterized by an heterogeneous fraction of low-Mr which is not present in the material derived from epiphyseal chondrocytes. There are at least two components in this fraction: the first seems to be a precursor of aggregated proteoglycans, the other may represent a component of cell coat. Stimulation of the cell cultures with vitamin D metabolites and somatomedin enhances proteoglycan biosynthesis but no modification is observed in the proteoglycan Mr.     

Emergence of chondrogenic progenitor stem cells in transplantation biology-prospects and drawbacks

Avascular tissues such as a cartilage contains a unique type of cell called as the chondrocyte. We, however, have not understood the origin of the chondrocyte population and how this population is maintained in the normal tissue. In spite of being considered to be a simple tissue, scientist had always faced difficulties to engineer this tissue. This is because different structural regions of the articular cartilage were never understood. In addition to this, the limited self-repair potential of cartilage tissue and lack of effective therapeutic options for the treatment of damaged cartilage has remained an unsolved problem. Mesenchymal stem cell based therapy may provide a solution for cartilage regeneration. This is due to their ability to differentiate into chondrogenic lineage when appropriate conditions are provided. An ideal cell source, a three-dimensional cell culture, a suitable scaffold material that accomplishes all the necessary properties and bioactive factors in specific amounts are required to induce chondrocyte differentiation and proliferation. Cartilage tissue engineering is a promising and rapidly expanding area of research that assures cartilage regeneration. However, many unsolved questions concerning the mechanism of engraftment of chondrocytes following transplantation in vivo, biological safety after transplantation and the retention of these cells for lifetime remain to be addressed that is possible only through years of extensive research. Further studies are therefore required to estimate the long-term sustainability of these cells in the native tissue, to identify well suited delivery materials and to have a thorough understanding of the mechanism of interaction between the chondrocytes and extracellular matrix and time is not far when this cell based therapy will provide a comprehensive cure to cartilage disease.     

Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A

Mesenchyme cells derived from limb buds of day 10 mouse embryos were plated out at confluent and sub-confluent cell densities. Cells in confluent cultures multiplied and differentiated into chondrocytes. The addition of vitamin A to the culture medium inhibited both cell proliferation and chondrogenesis. However, cytosine arabinoside, which also inhibited growth, did not block chondrogenesis. This indicates that the inhibition of growth in the vitamin A-treated cultures did not necessarily contribute to the inhibition of chondrogenesis. Cells in sub-confluent cultures multiplied but did not differentiate into chondrocytes. In contrast to confluent cultures, vitamin A did not inhibit growth in sub-confluent cultures. This observation suggests that vitamin A may inhibit growth by causing contact inhibition.     

The Mechanical Behaviour of Chondrocytes Predicted with a Micro-structural Model of Articular Cartilage

The integrity of articular cartilage depends on the proper functioning and mechanical stimulation of chondrocytes, the cells that synthesize extracellular matrix and maintain tissue health. The biosynthetic activity of chondrocytes is influenced by genetic factors, environmental influences, extracellular matrix composition, and mechanical factors. The mechanical environment of chondrocytes is believed to be an important determinant for joint health, and chondrocyte deformation in response to mechanical loading is speculated to be an important regulator of metabolic activity. In previous studies of chondrocyte deformation, articular cartilage was described as a biphasic material consisting of a homogeneous, isotropic, linearly elastic solid phase, and an inviscid fluid phase. However, articular cartilage is known to be anisotropic and inhomogeneous across its depth. Therefore, isotropic and homogeneous models cannot make appropriate predictions for tissue and cell stresses and strains. Here, we modelled articular cartilage as a transversely isotropic, inhomogeneous (TI) material in which the anisotropy and inhomogeneity arose naturally from the microstructure of the depth-dependent collagen fibril orientation and volumetric fraction, as well as the chondrocyte shape and volumetric fraction. The purpose of this study was to analyse the deformation behaviour of chondrocytes using the TI model of articular cartilage. In order to evaluate our model against experimental results, we simulated indentation and unconfined compression tests for nominal compressions of 15%. Chondrocyte deformations were analysed as a function of location within the tissue. The TI model predicted a non-uniform behaviour across tissue depth: in indentation testing, cell height decreased by 43% in the superficial zone and between 11 and 29% in the deep zone. In unconfined compression testing, cell height decreased by 32% in the superficial zone, 25% in the middle, and 18% in the deep zones. This predicted non-uniformity is in agreement with experimental studies. The novelty of this study is the use of a cartilage material model accounting for the intrinsic inhomogeneity and anisotropy of cartilage caused by its microstructure.     

Studies on Cartilage: Electron Microscope Observations on Normal Rabbit Ear Cartilage

Normal rabbit ear cartilage studied with the light and electron microscope shows chondrocytes in which large lipide spherules, and an abundance of glycogen, a few small mitochondria, and relatively few elements of the endoplasmic reticulum can be identified. The chondrocytes contain, in addition, a material which stains strongly with acid fuchsin and appears in the electron microscope as a relatively dense felt-work. In electron micrographs, the matrix of normal rabbit ear cartilage consists of two components: a uniformly distributed moderately dense substance which appears as a fine meshwork without any particular pattern extending from cartilage cell border to cartilage cell border; and a three-dimensional anastomotic network of more dense material, which is best described as "felt-like" lying between the cells. The similarity between the felt-like material of the matrix and the elastic fibers described in previous electron microscope observations is discussed.     

Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling

The majority of bones comprising the adult vertebrate skeleton are generated from hyaline cartilage templates that form during embryonic development. A process known as endochondral ossification is responsible for the conversion of these transient cartilage anlagen into mature, calcified bone. Endochondral ossification is a highly regulated, multistep cell specification program involving the initial differentiation of prechondrogenic mesenchymal cells into hyaline chondrocytes, terminal differentiation of hyaline chondrocytes into hypertrophic chondrocytes, and finally, apoptosis of hypertrophic chondrocytes followed by bone matrix deposition. Recently, extensive research has been carried out describing roles for the three major mitogen-activated protein kinase (MAPK) signaling pathways, the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminal kinase (JNK) pathways, in the successive stages of chondrogenic differentiation. In this review, we survey this research examining the involvement of ERK1/2, p38, and JNK pathway signaling in all aspects of the chondrogenic differentiation program from embryonic through postnatal stages of development. In addition, we summarize evidence from in vitro studies examining MAPK function in immortalized chondrogenic cell lines and adult mesenchymal stem cells. We also provide suggestions for future studies that may help ameliorate existing confusion concerning the specific roles of MAPK signaling at different stages of chondrogenesis.     

Dynamic osmotic loading of chondrocytes using a novel microfluidic device

Many cells exhibit disparate responses to a mechanical stimulus depending on whether it is applied dynamically or statically. In this context, few studies have examined how cells respond to dynamic changes of the extracellular osmolality. In this study, we hypothesized that the cell size change response of cultured articular chondrocytes would be dependent on the frequency of applied osmotic loading. To test this hypothesis, we developed a novel microfluidic device, to apply hydrostatic pressure-driven dynamic osmotic loading by applying composition modulated flow, adapted from Tang and co-workers. This microfluidic device was used to study osmotic loads of +/-180 mOsm at a frequency up to 0.1 Hz with a constant minimal fluid-shear stress, and permit real-time monitoring of cell responses. Bovine articular chondrocytes were observed to exhibit increasing changes in cell volume with decreasing osmotic loading frequency. When the cell volume response was modeled by an exponential function, chondrocytes exhibited significantly different volume change responses to dynamic osmotic loading at 0.0125 Hz and static osmotic loading applied for a period of four minutes (Delta = +/-180 mOsm relative to the isotonic 360 mOsm). The intracellular calcium response at 0.0125 Hz was also monitored and compared with the response to static loading. Coupled with phenomenological or constitutive models, this novel approach could yield new information regarding cell material properties in response to dynamic loading that may contribute new insights into mechanisms of cellular homeostasis and mechanotransduction.     

Modelling of location- and time-dependent deformation of chondrocytes during cartilage loading.

Experimental evidence suggests that the biosynthetic activity of chondrocytes is regulated primarily by the mechanical environment. In order to study the mechanisms underlying remodeling, adaptation, and degeneration of articular cartilage in a joint subjected to changing loads, it is important to know the time-dependent fluid pressure and stress-strain state in chondrocytes. The purpose of the present study was to develop a theoretical model to simulate the mechanical behaviour of articular cartilage and to describe the time-dependent stress-strain state and fluid pressure distribution in chondrocytes during cartilage deformation. It was assumed that the volume occupied by the chondrocytes is small and that cartilage can be treated as a macroscopically homogenized material with effective material properties which depend on the material properties of the cells and matrix and the volumetric fraction of the cells. Model predictions on the time-dependent distribution of fluid pressure and stress and on the time-dependent cell deformation during confined and unconfined compression tests agree with previous theoretical predictions and experimental observations. The proposed model supplies the tools to study the mechanisms of degeneration, adaptation and remodelling of cartilage associated with cell loading and deformation.     

Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (phi = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose-coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (phi = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quiescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.     

Syndecan-3 is a selective regulator of chondrocyte proliferation

Chondrocyte proliferation is important for skeletal development and growth, but the mechanisms regulating it are not completely clear. Previously, we showed that syndecan-3, a cell surface heparan sulfate proteoglycan, is expressed by proliferating chondrocytes in vivo and that proliferation of cultured chondrocytes in vitro is sensitive to heparitinase treatment. To further establish the link between syndecan-3 and chondrocyte proliferation, additional studies were carried out in vivo and in vitro. We found that the topographical location of proliferating chondrocytes in developing chick long bones changes with increasing embryonic age and that syndecan-3 gene expression changes in a comparable manner. For in vitro analysis, mitotically quiescent chondrocytes were exposed to increasing amounts of fibroblast growth factor-2 (FGF-2). Proliferation was stimulated by as much as 8-10-fold within 24 h; strikingly, this stimulation was significantly prevented when the cells were treated with both fibroblast growth factor-2 (FGF-2) and antibodies against syndecan-3 core protein. This neutralizing effect was dose-dependent and elicited a maximum of 50-60% inhibition. To establish specificity of neutralizing effect, cultured chondrocytes were exposed to FGF-2, insulin-like growth factor-1, or parathyroid hormone, all known mitogens for chondrocytes. The syndecan-3 antibodies interfered only with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone. Protein cross-linking experiments indicated that syndecan-3 is present in monomeric, dimeric, and oligomeric forms on the chondrocyte surface. In addition, molecular modeling indicated that contiguous syndecan-3 molecules might form stable complexes by parallel pairing of beta-sheet segments within the ectodomain of the core protein. In conclusion, the results suggest that syndecan-3 is a direct and selective regulator of the mitotic behavior of chondrocytes and its role may involve formation of dimeric/oligomeric structures on their cell surface.     

Analysis of the mechanical behavior of chondrocytes in unconfined compression tests for cyclic loading

Experimental evidence indicates that the biosynthetic activity of chondrocytes is associated with the mechanical environment. For example, excessive, repetitive loading has been found to induce cell death, morphological and cellular damage, as seen in degenerative joint disease, while cyclic, physiological-like loading has been found to trigger a partial recovery of morphological and ultrastructural aspects in osteoarthritic human articular chondrocytes. Mechanical stimuli are believed to influence the biosynthetic activity via the deformation of cells. However, the in situ deformation of chondrocytes for cyclic loading conditions has not been investigated experimentally or theoretically. The purpose of the present study was to simulate the mechanical response of chondrocytes to cyclic loading in unconfined compression tests using a finite element model. The material properties of chondrocytes and extracellular matrix were considered to be biphasic. The time-histories of the shape and volume variations of chondrocytes at three locations (i.e., surface, center, and bottom) within the cartilage were predicted for static and cyclic loading conditions at two frequencies (0.02 and 0.1 Hz) and two amplitudes (0.1 and 0.2 MPa). Our results show that cells at different depths within the cartilage deform differently during cyclic loading, and that the depth dependence of cell deformation is influenced by the amplitude of the cyclic loading. Cell deformations under cyclic loading of 0.02 Hz were found to be similar to those at 0.1 Hz. We conclude from the simulation results that, in homogeneous cartilage layers, cell deformations are location-dependent, and further are affected by load magnitude. In physiological conditions, the mechanical environment of cells are even more complex due to the anisotropy, depth-dependent inhomogeneity, and tension-compression non-linearity of the cartilage matrix. Therefore, it is feasible to speculate that biosynthetic responses of chondrocytes to cyclic loading depend on cell location and load magnitude.     

An established rat cell line expressing chondrocyte properties

Chondrocytes express a well-characterized set of marker proteins making these cells useful for studies on differentiation and regulation of gene expression. Because of the inherent instability of primary rat chondrocytes in culture, and because several rat chondrocyte genes have been cloned and characterized (including the collagen II promoter and enhancer), a rat chondrocyte cell line would be especially useful. To obtain this line we infected primary fetal rat costal chondrocytes with a recombinant retrovirus (NIH/J-2) carrying the myc and raf oncogenes, which have been shown to have an "immortalizing" function. Following infection, a rapidly proliferating clonal line was isolated that maintained a stable phenotype through 45 passages (11/2 year in culture). This line, termed IRC, grows in suspension culture as multicellular aggregates and in monolayer culture as polygonal cells which accumulate an alcian blue-stainable matrix. IRC cells synthesize high levels of cartilage proteoglycan core protein, and link protein, but show reduced collagen II expression. In addition, the cells express virally derived myc mRNA and protein, but do not express v-raf. Retinoic acid, which is a known modulator of chondrocyte phenotype, down-regulates expression of chondrocyte marker proteins, while stimulating v-myc expression by IRC cells. These data suggest that v-myc expression by chondrocytes results in rapid cell division and maintenance of many aspects of the differentiated phenotype. These "immortalized" cells, however, remain responsive to agents such as retinoic acid which modulate cell phenotype. The potential exists for development of chondrocyte cell lines from diseased cartilage, as well as from human cartilage.