Chick Limb Bud Mesenchymal Cells Research Articles

Homocysteine as a Pathological Biomarker for Bone Disease.

In the last few decades, perturbation in methyl-group and homocysteine (Hcy) balance have emerged as independent risk factors in a number of pathological conditions including neurodegenerative disease, cardiovascular dysfunction, cancer development, autoimmune disease, and kidney disease. Recent studies report Hcy to be a newly recognized risk factor for osteoporosis. Elevated Hcy levels are known to modulate osteoclastgenesis by causing detrimental effects on bone via oxidative stress induced metalloproteinase-mediated extracellular matrix degradation and decrease in bone blood flow. Evidence from previous studies also suggests that the decreased chondrocytes mediated bone mineralization in chick limb-bud mesenchymal cells and during the gestational period of ossification in rat model. However, Hcy imbalance and its role in bone loss, regression in vascular invasion, and osteoporosis, are not clearly understood. More investigations are required to explore the complex interplay between Hcy imbalance and onset of bone disease progression. This article reviews the current body of knowledge on regulation of Hcy mediated oxidative stress and its role in bone remodeling, vascular blood flow and progression of bone disease. J. Cell. Physiol. 232: 2704-2709, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA–ROCK–myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.

Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system

Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.

Alterations in the temporal expression and function of cadherin‐7 inhibit cell migration and condensation during chondrogenesis of chick limb mesenchymal cells in vitro

Endochondral bone formation requires a complex interplay among immature mesenchymal progenitor cells to form the cartilaginous anlagen, which involves migration, aggregation and condensation. Even though condensation of chondrogenic progenitors is an essential step in this process, its mechanism(s) has not been well studied. Here, we show that cadherin-7 plays a central role in cellular condensation by modulating cell motility and migration. In this study, many mesenchymal cells failed to migrate, and precartilage condensation was inhibited, after knockdown of endogenous cadherin-7 levels. Exposure of mesenchymal cells to SB203580 (a specific inhibitor of p38MAPK), LiCl (an inhibitor of GSK-3beta) or overexpression of beta-catenin resulted in inhibition of cadherin-7 levels and, subsequently, suppression of cell migration. Collectively, our results suggest that cadherin-7 controls cell migration in chick limb bud mesenchymal cells, and that p38MAPK and GSK signals are responsible for regulating cadherin-7-mediated cell migration.

Integrin signaling and cell spreading alterations by rottlerin treatment of chick limb bud mesenchymal cells

Endochondral skeletal development begins with the formation of a cartilaginous template where mesenchymal cells aggregate and increase in density prior to their overt differentiation into chondrocytes. Prechondrogenic condensation, in which mesenchymal cells aggregate, requires cell migration and proliferation. However, the molecular mechanisms promoting this aggregation remain to be elucidated. Here, we report that rottlerin suppresses migration and cell surface expression of integrin β1 in chondrogenic progenitors. Perturbation of integrin β1 function using an anti-integrin β1 blocking antibody suppressed the migration of wing bud mesenchymal cells. Furthermore, phosphorylation levels of Src and focal adhesion kinase (FAK) were decreased by rottlerin treatment. Cell treatment with PP2, an inhibitor of Src family kinase, or electroporation of FAK specific siRNA, suppressed cell migration in a wound-healing assay. Cells treated with rottlerin showed decreased phosphorylation of Akt, independent of PKCδ inhibition. In addition, an Akt inhibitor suppressed the migration of chick limb bud mesenchymal cells. Taken together, our results point to the novel finding that rottlerin may act as a negative regulator for cell migration, an essential step for prechondrogenic condensation, by regulating integrin β1 signaling at focal adhesion complexes via modulation of Akt activity.

Ablation of Cathepsin K Activity in the Young Mouse Causes Hypermineralization of Long Bone and Growth Plates

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Activation of CREB by PKA promotes the chondrogeneic differentiation of chick limb bud mesenchymal cells

Cyclic AMP‐mediated signaling pathways regulate a number of cellular functions. In this study, we examined the regulatory role of cAMP signaling pathways in chondrogenesis of chick limb bud mesenchymal cells in vitro. Forskolin, which increases cellular cAMP levels by the activation of adenylate cyclase, enhanced chondrogenic differentiation. Inhibition of PKA with specific inhibitors (H89 or KT5720) blocked pre‐cartilage condensation stage, indicating that chondrogenesis is regulated by the increase in cellular cAMP level and subsequent activation of PKA. Downstream signaling pathway of PKA leading to gene expression was investigated by examination of several nuclear transcription factors. Forskolin treatment increased transcription level for a cartilage‐specific marker gene Sox9. However, inhibition of PKA with H89 led to restore expression of Sox9, indicating PKA activity was required to regulate the expression of Sox9 in chondrogenesis. In addition, CREB was highly phosphorylated at early stage of mesenchyme culture, and followed by progressive dephosphorylation. CBP and ATF, another CRE related proteins were transiently expressed at the early stage of chondrogenesis with a pattern similar to CREB phosphorylation. Electrophoretic mobility shift assays confirmed that the binding activity of CREB to the CRE is closely correlated to the phosphorylation pattern of CREB. Therefore, cAMP‐mediated signal transduction to nuclear events for the induction of genes appeared to be required at the early stage of chick limb bud chondrogenesis.

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells

Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

Integrity of the cortical actin ring is required for activation of the PI3K/Akt and p38 MAPK signaling pathways in redifferentiation of chondrocytes on chitosan

Cell shape alterations and accompanying cytoskeletal changes have diverse effects on cell function. We have already shown that dedifferentiated chondrocytes have a round cell morphology and undergo redifferentiation when cultured on chitosan membrane. In the present study, we investigate the role of the cytoskeleton in chondrocyte redifferentiation. Chondrocytes obtained from a micromass culture of chick limb bud mesenchymal cells were subcultured four times. Immunofluorescence analysis of F-actin showed cortical distribution of the actin cytoskeleton upon subculture of dedifferentiated chondrocytes on chitosan membrane. Treatment with cytochalasin D disrupted the cortical actin ring formed during cultivation of chondrocytes on the chitosan membrane, and inhibited chondrocyte redifferentiation. Moreover, cytochalasin D inhibited the phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK), induced during redifferentiation on chitosan membrane. LY294002, an inhibitor of phosphatidylinositol-3-OH-kinase (PI3K), suppressed chondrocyte redifferentiation. These findings suggest that integrity of the actin cytoskeleton is a crucial requirement for PI3K/Akt and p38 MAPK in chondrocyte redifferentiation.

Modulation of extracellular matrix protein phosphorylation alters mineralization in differentiating chick limb-bud mesenchymal cell micromass cultures

Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb-bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4 mM inorganic phosphate and no organic-phosphate esters; control cultures had 1 mM inorganic phosphate. Mineralization was monitored based on 45Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis. Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin-LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating that the phosphatases were, in part, providing a source of inorganic phosphate. To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin's previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.

Oxidative stress-induced poly(ADP-ribosyl)ation in chick limb bud-derived chondrocytes

Oxidative stress has been implicated in the pathogenesis of various diseases affecting chondrogenesis or the function of articular cartilage. DNA damage caused by oxidative stress may trigger the activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) which may contribute to tissue injury. We aimed at investigating the effects of peroxynitrite (100-600 microM) and hydrogen peroxide (0.1-4 mM) on PARP activation and extracellular matrix production of high density micromass cultures (HDC) prepared from chick limb bud mesenchymal cells. We found that both oxidative species strongly inhibited matrix formation of HDCs treated on day 2 but not on day 5. The PARP inhibitor 3-aminobenzamide (3-AB) stimulated matrix production in non-stressed cells and prevented suppressed matrix production in oxidatively stressed cells. Both hydrogen peroxide and peroxynitrite induced PARP activation and poly(ADP-ribose) accumulation. Decreased proliferation, viability and NAD+ content were not or only slightly improved by 3-AB, indicating that 3-AB directly affects matrix formation. In conclusion, oxidative stress stimulates poly(ADP-ribose) metabolism and inhibits extracellular matrix production of HDCs in a PARP-dependent manner. Our findings may have implications for potential therapeutic approaches aimed at restoring the matrix production capacity of chondrogenic cells.

Inhibition of chondrogenic differentiation in chick limb-bud mesenchyme microcultures treated with cyclosporine

Objectives: To explore the effects of cyclosporine (CsA) on skeletal development (chondrogenesis). Materials and Methods: Mesenchymal cells obtained from stage-23 to stage-24 chick-embryo limb buds were grown in 96-well plates using chemically defined tissue-culture medium. Cultures were treated with CsA (0.01-5.0 µg/ml) and incubated (37oC, 5% CO2) with daily medium changes for 4 days. After incubation of the cells in multiwell plate, cartilage differentiation (chondrogenesis) was assessed by selectively staining sulfated glycosaminoglycans (GAGs) in the cartilage matrix with Alcian blue, extracting the GAGs with 4 M guanidinium HCl, and spectrophotometric analysis of the extracts. Results: CsA treatment had concentration-dependent effects on chick limb-bud mesenchymal cell cultures. At 5 µg/ml, CsA caused cell loss, as judged microscopically by the paucity of cells remaining at the end of the culture period. CsA concentrations between 0.1 and 1 µg/ml caused a marked, dose-dependent decrease in chondrogenesis. At 0.01 µg/ml, CsA had no significant effect on chondrogenesis. At concentrations above 0.01 µg/ml, normalized data showed significant chondrogenic inhibition at 0.5 and 1.0 µg/ml CsA. Conclusions: The findings suggest a possible biological basis for CsA-associated effects on mesenchyme-derived tissues and provide a model system for further studies.

Review:Mesenchymal Stem Cells: Cell–Based Reconstructive Therapy in Orthopedics

Adult stem cells provide replacement and repair descendants for normal turnover or injured tissues. These cells have been isolated and expanded in culture, and their use for therapeutic strategies requires technologies not yet perfected. In the 1970s, the embryonic chick limb bud mesenchymal cell culture system provided data on the differentiation of cartilage, bone, and muscle. In the 1980s, we used this limb bud cell system as an assay for the purification of inductive factors in bone. In the 1990s, we used the expertise gained with embryonic mesenchymal progenitor cells in culture to develop the technology for isolating, expanding, and preserving the stem cell capacity of adult bone marrow-derived mesenchymal stem cells (MSCs). The 1990s brought us into the new field of tissue engineering, where we used MSCs with site-specific delivery vehicles to repair cartilage, bone, tendon, marrow stroma, muscle, and other connective tissues. In the beginning of the 21st century, we have made substantial advances: the most important is the development of a cell-coating technology, called painting, that allows us to introduce informational proteins to the outer surface of cells. These paints can serve as targeting addresses to specifically dock MSCs or other reparative cells to unique tissue addresses. The scientific and clinical challenge remains: to perfect cell-based tissue-engineering protocols to utilize the body's own rejuvenation capabilities by managing surgical implantations of scaffolds, bioactive factors, and reparative cells to regenerate damaged or diseased skeletal tissues.

Functional analysis of fibronectin isoforms in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces spreading and promotes condensation and chondrogenesis of limb mesenchymal cells

Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B +A + in precartilage mesenchyme to B +A − in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B +A +) FN isoform. Full-length cDNAs corresponding to four splice variants (B +A +, B +A −, B −A +, B −A −) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40% less on mesenchymal (B +A +) FN than on cartilage (B +A −) FN, (B −A +) FN, or plasma (B −A −) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B +A + FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.

BMP‐6 accelerates both chondrogenesis and mineral maturation in differentiating chick limb‐bud mesenchymal cell cultures

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation.

BMP-6 accelerates both chondrogenesis and mineral maturation in differentiating chick limb-bud mesenchymal cell cultures

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the “hypertrophic” chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 × 106cells/micromass; 4 mM Pi, 1.3 mM Ca2+, 10% FCS, and antibiotics) mineralization does not commence until day 14–16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of 45Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation. J. Cell. Biochem. 84: 509–519, 2002. © 2001 Wiley-Liss, Inc.

Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading.

As part of a program of research aimed at determining the role of mechanical forces in connective tissue differentiation, we have developed a model for investigating the effects of dynamic compressive loading on chondrocyte differentiation in vitro. In the current study, we examined the influence of cyclic compressive loading of chick limb bud mesenchymal cells to a constant peak stress of 9.25 kPa during each of the first 3 days in culture. Cells embedded in agarose gel were subjected to uniaxial, cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h. In addition, load durations of 12, 54, or 120 min were evaluated while holding frequency constant at 0.33 Hz. For a 2 h duration, there was no response to loading at 0.03 Hz. A significant increase in chondrocyte differentiation was associated with loading at 0.15 Hz, and an even greater increase with loading at 0.33 Hz. Holding frequency constant at 0.33 Hz, a loading duration of 12 min elicited no response, whereas chondrocyte differentiation was enhanced by loading for either 54 or 120 min. Although not statistically significant from the 120 min response, average cartilage nodule density and glycosaminoglycan synthesis rate were highest in the 54 min duration group. This result suggests that cells may be sensitive to the level of cumulative (nonrecoverable) compressive strain, as well as to the dynamic strain history.

Homocysteine decreases chondrocyte-mediated matrix mineralization in differentiating chick limb-bud mesenchymal cell micro-mass cultures

The differentiating chick limb-bud mesenchymal cell micro-mass culture system has been used as a model for monitoring the effects of matrix modification on cell-mediated calcification. In this study, we show that treating these micro-mass cultures with homocysteine (Hcys) impairs cartilage calcification. Cultures were treated from day 2 to day 7 with two nonphysiological concentrations of Hcys equivalent to 100× and 1000× avian serum levels (0.36 and 3.6 mmol/L), and from days 9–13 with one tenth the concentration. Mineralization assays were done at days 16, 19, and 21, and matrix and cell properties were examined between days 5 and 21. Mineral accretion, based on differential 45Ca uptake (mineralizing minus control cultures), was significantly reduced in the high-Hcys-concentration group, and slightly reduced in the low-Hcys-concentration group. Electron microscopy at culture day 21 showed that the collagen matrix was less abundant and its banding pattern less obvious in the Hcys-treated groups than in the untreated cultures. Pyridinoline (Pyr) and deoxypyridinoline (d-Pyr) contents were not detectable in day 21 cultures with either 0.36 or 3.6 mmol/L homocysteine, whereas values in mineralizing and nonmineralizing controls ranged from 0.06 to 0.08 and 0.03 to 0.06 (moles/mole collagen) for Pyr and d-Pyr, respectively. Fourier transform infrared (FTIR) imaging also indicated a decreased content of pyridinoline cross-links. Hcys caused other matrix changes as well. Whereas at culture day 5 there was no significant difference in the number of chondrocyte nodules formed, by day 11 the proteoglycan content (measured by Alcian blue dye binding at 595 nm) was significantly reduced in both mineralizing and control cultures in the high- and low-Hcys groups. In contrast, there were no detectable differences in type X collagen and alkaline phosphatase staining in the mineralizing cultures with or without Hcys supplements. Because vital dye stains and electron microscopy studies indicated that cells in the control and experimental groups did not differ in terms of viability, the observed differences cannot be attributed to toxicity. Thus, Hcys treatment, which causes matrix disorganization, decreases the ability of the matrix to support mineralization.

Vitamin C-sulfate inhibits mineralization in chondrocyte cultures: a caveat.

Differentiating chick limb-bud mesenchymal cell micro-mass cultures routinely mineralize in the presence of 10% fetal calf serum, antibiotics, 4 mM inorganic phosphate (or 2.5 mM beta-glycerophosphate), 0.3 mg/ml glutamine and either 25 microg/ml vitamin C or 5-12 microg/ml vitamin C-sulfate. The failure of these cultures to produce a mineralized matrix (assessed by electron microscopy, 45Ca uptake and Fourier transform infrared microscopy) led to the evaluation of each of these additives. We report here that the "stable" vitamin C-sulfate (ascorbic acid-2-sulfate) causes increased sulfate incorporation into the cartilage matrix. Furthermore, the release of sulfate from the vitamin C derivative appears to be responsible for the inhibition of mineral deposition, as demonstrated in cultures with equimolar amounts of vitamin C and sodium sulfate.