Hyaluronidase expression in cultured growth plate chondrocytes during differentiation

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.This research was supported by Grants-in-Aid (no. 11557166) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan     

The metabolism of hyaluronan in cultured rabbit growth plate chondrocytes during differentiation

Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes.     

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.     

Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes

Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn(2+). Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn(2+) with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn(2+) as the chondrocytes mature. Intracellular labile Zn(2+) was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged by multi-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short-term exposure to Zn(2+), both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn(2+). Treatment with the membrane-permeant Zn(2+) chelator TPEN rapidly reduced the levels of labile Zn(2+) and triggered apoptosis. Cytosolic Zn(2+) levels were significantly reduced following 24-h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn(2+) was accompanied by a significant reduction in the cytosolic metal-binding protein metallothionein. Examination of Zn(2+)-treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn(2+)-loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn(2+). A dose-dependent increase in zinquin fluorescence was observed in cells treated with a range of Zn(2+) concentrations. Short-term treatment of cultured chondrocytes with apoptosis-inducing chemicals resulted in transient increases in intracellular labile Zn(2+). These results indicate that Zn(2+) is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn(2+) provides a mechanism for its movement to matrix vesicles and the extracellular matrix.     

PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

In developing murine growth plates, chondrocytes near the articular surface (periarticular chondrocytes) proliferate, differentiate into flat column-forming proliferating cells (columnar chondrocytes), stop dividing and finally differentiate into hypertrophic cells. Indian hedgehog (Ihh), which is predominantly expressed in prehypertrophic cells, stimulates expression of parathyroid hormone (PTH)-related peptide (PTHrP) which negatively regulates terminal chondrocyte differentiation through the PTH/PTHrP receptor (PPR). However, the roles of PTHrP and Ihh in regulating earlier steps in chondrocyte differentiation are unclear. We present novel mouse models with PPR abnormalities that help clarify these roles. In mice with chondrocyte-specific PPR ablation and mice with reduced PPR expression, chondrocyte differentiation was accelerated not only at the terminal step but also at an earlier step: periarticular to columnar differentiation. In these models, upregulation of Ihh action in the periarticular region was also observed. In the third model in which the PPR was disrupted in about 30% of columnar chondrocytes, Ihh action in the periarticular chondrocytes was upregulated because of ectopically differentiated hypertrophic chondrocytes that had lost PPR. Acceleration of periarticular to columnar differentiation was also noted in this mouse, while most of periarticular chondrocytes retained PPR signaling. These data suggest that Ihh positively controls differentiation of periarticular chondrocytes independently of PTHrP. Thus, chondrocyte differentiation is controlled at multiple steps by PTHrP and Ihh through the mutual regulation of their activities.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.     

The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes

Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K_d of 145 nM. Near saturation, ∼103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K_d of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated ³H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.     

PDGF BB stimulates proliferation and differentiation in cultured chondrocytes from rat rib growth plate.

The role of two isoforms (PDGF AA and PDGF BB) of platelet derived growth factor either alone or in combination with insulin-like growth factor I, on the regulation of proliferation and differentiation of rat rib growth plate chondrocytes was analyzed. PDGF BB increased DNA-synthesis in a dose dependent manner with a half maximal effect at 1 ng/ml. When PDGF BB was combined with IGF-I, an additive effect on DNA-synthesis was observed. PDGF AA and BB alone or combined with IGF-I had no appreciable effects on proteoglycan synthesis. Both homodimers caused an increase in AP-activity, indicating stimulation of cell differentiation. Cultured chondrocytes bound 125I-PDGF AA and 125I-PDGF BB and after stimulation with PDGF expressed c-fos protein. Thus, both homodimers play an important role in chondrocyte differentiation and together with IGF-I interact in the regulation of longitudinal bone growth.     

The effect of hormones on bone growth is mediated through mechanical stress

Mechanical stresses play a key role in regulating cell growth and cell differentiation. Using mechanical and physiological data available in the literature, we are able to construct a growth curve of a child, which we compare to the standard curve. It appears likely that the impact of hormones on pubertal growth rate sprout followed by growth arrest can be solely explained by increased mechanical stresses. The uptake of hormones by the muscles results in increased mechanical stress on the chondrocyte before and at the puberty, resulting in a peak in growth followed by growth cessation.     

Development of a serum-free system to study the effect of growth hormone and insulinlike growth factor-I on cultured postembryonic growth plate chondrocytes

Summary We have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of chondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, chondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 μg/ml) produced a dose-dependant decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.     

Treatment of growth plate injury with microencapsulated chondrocytes

Background

Tissue-engineered cartilage has provided a promising method in the treatment of physeal growth arrest. This study was designed to investigate transplantation of microencapsulated allogeneic chondrocytes to treat the injured growth plate.

Methods

Allogeneic chondrocytes were encapsulated within alginate-polylysinealginate semipermeable membranes. Microencapsulated chondrocytes co-cultured with Bone Mesenchymal Stem Cells (BMSCs) were evaluated whether it could promote chondrogenesis of BMSCs. An experiment model of an injured growth plate was made by resecting the lateral half of the right distal femur physis in rabbits. Microencapsulated allogeneic chondrocytes, allogeneic chondrocytes as well as empty microcapsules were grafted into growth plate defects of 6-week-old rabbits. Histological and radiographic examinations were examined after transplantation up to 16 weeks.

Results

The histological study showed that BMSCs co-cultured with microencapsulated chondrocytes could produce GAG and II collagen similarly with chondrocytes. Angular deformity and length discrepancy in the group with microencapsulated allogeneic chondrocytes were less than those in other groups (p < 0.001). The histological study confirmed the viability of microencapsulated chondrocytes at 16 weeks postoperatively. The neogenetic chondrocytes of columnar arrangement have been found in the growth plate defect to prevent early ossification and closure of the growth plate.

Conclusions

The histological study confirmed the viability of microencapsulated chondrocytes at 16 weeks postoperatively. The neogenetic chondrocytes of columnar arrangement have been found in the growth plate defect to prevent early ossification and closure of the growth plate.     

Effects of leptin to cultured growth plate chondrocytes

OBJECTIVE: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. METHODS: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. RESULTS: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. CONCLUSION: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.     

Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1

Cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes. Differentiated chondrocytes in articular cartilage undergo dedifferentiation and apoptosis during arthritis, in which NO production plays a critical role. Here, we investigated the roles and mechanisms of action of insulin-like growth factor-1 (IGF-1) in the chondrogenesis of mesenchymal cells and the maintenance and survival of differentiated articular chondrocytes. IGF-1 induced chondrogenesis of limb bud mesenchymal cells during micromass culture through the activation of phosphatidylinositol 3-kinase (PI3K) and Akt. PI3K activation is required for the activation of protein kinase C (PKC)-alpha and p38 kinase and inhibition of ERK1/2. These events are necessary for chondrogenesis. The growth factor additionally blocked NO-induced dedifferentiation and apoptosis of primary culture articular chondrocytes. NO production in chondrocytes induced down-regulation of PI3K and Akt activities, which was blocked by IGF-1 treatment. Stimulation of PI3K by IGF-1 resulted in blockage of NO-induced activation of p38 kinase and ERK1/2 and inhibition of PKCalpha and PKCzeta, which in turn suppressed dedifferentiation and apoptosis. Our results collectively indicate that IGF-1 regulates differentiation, maintenance of the differentiated phenotype, and apoptosis of articular chondrocytes via a PI3K pathway that modulates ERK, p38 kinase, and PKC signaling.     

The mechanical response of active human muscle during and after stretch

Five subjects contracted forearm supinator muscles which were stretched after development of maximal isometric torque. The ratio of torque at the end of stretch over isometric torque at that position was calculated as excess torque. Excess torque increased with stretch velocity and decreased with stretch amplitude, and it was not dependent upon final muscle length. The rate of decay of torque following stretch could not be shown to depend upon stretch variables. The absence of significant changes in myoelectric activity suggested that with high initial forces, reflex activity did not account for the observed changes. Time-constants of decay (0.15 s to 1.8 s) were much greater than time-constants of rise (approx. 0.07 s) of isometric torque at the same muscle length. This indicates that interaction of series elastic and contractile elements is not the sole cause of prolonged torque following stretch. It is concluded that stretch temporarily enhances the intrinsic contractile properties of a group of human muscles in a manner similar to, but quantitatively different from that seen in isolated muscle preparations.     

The effects of mechanical stress on the growth, differentiation, and paracrine factor production of cardiac stem cells

Stem cell therapies have been clinically employed to repair the injured heart, and cardiac stem cells are thought to be one of the most potent stem cell candidates. The beating heart is characterized by dynamic mechanical stresses, which may have a significant impact on stem cell therapy. The purpose of this study is to investigate how mechanical stress affects the growth and differentiation of cardiac stem cells and their release of paracrine factors. In this study, human cardiac stem cells were seeded in a silicon chamber and mechanical stress was then induced by cyclic stretch stimulation (60 cycles/min with 120% elongation). Cells grown in non-stretched silicon chambers were used as controls. Our result revealed that mechanical stretching significantly reduced the total number of surviving cells, decreased Ki-67-positive cells, and increased TUNEL-positive cells in the stretched group 24 hrs after stretching, as compared to the control group. Interestingly, mechanical stretching significantly increased the release of the inflammatory cytokines IL-6 and IL-1β as well as the angiogenic growth factors VEGF and bFGF from the cells in 12 hrs. Furthermore, mechanical stretching significantly reduced the percentage of c-kit-positive stem cells, but increased the expressions of cardiac troponin-I and smooth muscle actin in cells 3 days after stretching. Using a traditional stretching model, we demonstrated that mechanical stress suppressed the growth and proliferation of cardiac stem cells, enhanced their release of inflammatory cytokines and angiogenic factors, and improved their myogenic differentiation. The development of this in vitro approach may help elucidate the complex mechanisms of stem cell therapy for heart failure.