Do not overwork: cellular communication network factor 3 for life in cartilage.

Cellular communication network factor (CCN) 3, which is one of the founding members of the CCN family, displays diverse functions. However, this protein generally represses the proliferation of a variety of cells. Along with skeletal development, CCN3 is produced in cartilaginous anlagen, growth plate cartilage and epiphysial cartilage. Interestingly, CCN3 is drastically induced in the growth plates of mice lacking CCN2, which promotes endochondral ossification. Notably, chondrocytes in these mutant mice with elevated CCN3 production also suffer from impaired glycolysis and energy metabolism, suggesting a critical role of CCN3 in cartilage metabolism. Recently, CCN3 was found to be strongly induced by impaired glycolysis, and in our study, we located an enhancer that mediated CCN3 regulation via starvation. Subsequent investigations specified regulatory factor binding to the X-box 1 (RFX1) as a transcription factor mediating this CCN3 regulation. Impaired glycolysis is a serious problem, resulting in an energy shortage in cartilage without vasculature. CCN3 produced under such starved conditions restricts energy consumption by repressing cell proliferation, leading chondrocytes to quiescence and survival. This CCN3 regulatory system is indicated to play an important role in articular cartilage maintenance, as well as in skeletal development. Furthermore, CCN3 continues to regulate cartilage metabolism even during the aging process, probably utilizing this regulatory system. Altogether, CCN3 seems to prevent "overwork" by chondrocytes to ensure their sustainable life in cartilage by sensing energy metabolism. Similar roles are suspected to exist in relation to systemic metabolism, since CCN3 is found in the bloodstream.

Elevated Expression of CCN3 in Articular Cartilage Induces Osteoarthritis in Hip Joints Irrespective of Age and Weight Bearing.

Osteoarthritis (OA) occurs not only in the knee but also in peripheral joints throughout the whole body. Previously, we have shown that the expression of cellular communication network factor 3 (CCN3), a matricellular protein, increases with age in knee articular cartilage, and the misexpression of CCN3 in cartilage induces senescence-associated secretory phenotype (SASP) factors, indicating that CCN3 promotes cartilage senescence. Here, we investigated the correlation between CCN3 expression and OA degenerative changes, principally in human femoral head cartilage. Human femoral heads obtained from patients who received total hip arthroplasty were categorized into OA and femoral neck fracture (normal) groups without significant age differences. Gene expression analysis of RNA obtained from femoral head cartilage revealed that CCN3 and MMP-13 expression in the non-weight-bearing part was significantly higher in the OA group than in the normal group, whereas the weight-bearing OA parts and normal cartilage showed no significant differences in the expression of these genes. The expression of COL10A1, however, was significantly higher in weight-bearing OA parts compared with normal weight-bearing parts, and was also higher in weight-bearing parts compared with non-weight-bearing parts in the OA group. In contrast, OA primary chondrocytes from weight-bearing parts showed higher expression of CCN3, p16, ADAMTS4, and IL-1ß than chondrocytes from the corresponding normal group, and higher ADAMTS4 and IL-1ß in the non-weight-bearing part compared with the corresponding normal group. Acan expression was significantly lower in the non-weight-bearing group in OA primary chondrocytes than in the corresponding normal chondrocytes. The expression level of CCN3 did not show significant differences between the weight-bearing part and non-weight-bearing part in both OA and normal primary chondrocytes. Immunohistochemical analysis showed accumulated CCN3 and aggrecan neoepitope staining in both the weight-bearing part and non-weight-bearing part in the OA group compared with the normal group. The CCN3 expression level in cartilage had a positive correlation with the Mankin score. X-ray analysis of cartilage-specific CCN3 overexpression mice (Tg) revealed deformation of the femoral and humeral head in the early stage, and immunohistochemical analysis showed accumulated aggrecan neoepitope staining as well as CCN3 staining and the roughening of the joint surface in Tg femoral and humeral heads. Primary chondrocytes from the Tg femoral head showed enhanced expression of Ccn3, Adamts5, p16, Il-6, and Tnfα, and decreased expression of Col2a1 and -an. These findings indicate a correlation between OA degenerative changes and the expression of CCN3, irrespective of age and mechanical loading. Furthermore, the Mankin score indicates that the expression level of Ccn3 correlates with the progression of OA.

Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption.

Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1Δ105). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1.

Molecular and Genetic Interactions between CCN2 and CCN3 behind Their Yin-Yang Collaboration.

Cellular communication network factor (CCN) 2 and 3 are the members of the CCN family that conduct the harmonized development of a variety of tissues and organs under interaction with multiple biomolecules in the microenvironment. Despite their striking structural similarities, these two members show contrastive molecular functions as well as temporospatial emergence in living tissues. Typically, CCN2 promotes cell growth, whereas CCN3 restrains it. Where CCN2 is produced, CCN3 disappears. Nevertheless, these two proteins collaborate together to execute their mission in a yin-yang fashion. The apparent functional counteractions of CCN2 and CCN3 can be ascribed to their direct molecular interaction and interference over the cofactors that are shared by the two. Recent studies have revealed the mutual negative regulation systems between CCN2 and CCN3. Moreover, the simultaneous and bidirectional regulatory system of CCN2 and CCN3 is also being clarified. It is of particular note that these regulations were found to be closely associated with glycolysis, a fundamental procedure of energy metabolism. Here, the molecular interplay and metabolic gene regulation that enable the yin-yang collaboration of CCN2 and CCN3 typically found in cartilage development/regeneration and fibrosis are described.

Maternal Gut Microbiome Decelerates Fetal Endochondral Bone Formation by Inducing Inflammatory Reaction.

To investigate the effect of the maternal gut microbiome on fetal endochondral bone formation, fetuses at embryonic day 18 were obtained from germ-free (GF) and specific-pathogen-free (SPF) pregnant mothers. Skeletal preparation of the fetuses' whole bodies did not show significant morphological alterations; however, micro-CT analysis of the tibiae showed a lower bone volume fraction in the SPF tibia. Primary cultured chondrocytes from fetal SPF rib cages showed a lower cell proliferation and lower accumulation of the extracellular matrix. RNA-sequencing analysis showed the induction of inflammation-associated genes such as the interleukin (IL) 17 receptor, IL 6, and immune-response genes in SPF chondrocytes. These data indicate that the maternal gut microbiome in SPF mice affects fetal embryonic endochondral ossification, possibly by changing the expression of genes related to inflammation and the immune response in fetal cartilage. The gut microbiome may modify endochondral ossification in the fetal chondrocytes passing through the placenta.

Cellular communication network factor 3 in cartilage development and maintenance.

Cellular communication network factor (CCN) 3 is one of the classical members of the CCN family, which are characterized by common molecular structures and multiple functionalities. Although this protein was discovered as a gene product overexpressed in a truncated form in nephroblastoma, recent studies have revealed its physiological roles in the development and homeostasis of mammalian species, in addition to its pathological association with a number of diseases. Cartilage is a tissue that creates most of the bony parts and cartilaginous tissues that constitute the human skeleton, in which CCN3 is also differentially produced to exert its molecular missions therein. In this review article, after the summary of the molecular structure and function of CCN3, recent findings on the regulation of ccn3 expression and the roles of CCN3 in endochondral ossification, cartilage development, maintenance and disorders are introduced with an emphasis on the metabolic regulation and function of this matricellular multifunctional molecule.

UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions.

The uncoupling protein 1 (UCP1) gene is known to be highly expressed in brown adipose tissue (BAT) that functions in thermogenesis. It has been shown that UCP1 mRNA is localized to the mouse adrenal gland, but its significance remains elusive. To explore how UCP1 expression in the adrenal gland is regulated, we generated a reporter knock-in mouse in which the GFP gene was inserted into the UCP1 locus using CRISPR-Cas9 system. Firstly, we confirmed by Western blot analysis UCP1-driven GFP protein expression in interscapular BAT of the knock-in mice kept at 4 °C. Immunohistochemistry showed that GFP protein was detected in the adrenal gland of the knock-in mice. More intense GFP expression was observed in the adrenal medulla than in the cortex of the reporter mice irrespectively of cold exposure. Immunohistochemistry using anti-UCP1 antibody, as well as Western blot analysis verified UCP1 protein expression in the wild-type adrenal medulla. These results suggest that the mouse adrenal gland is a novel organ expressing UCP1 protein and its expression is not upregulated by cold exposure.

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1.

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of Ccn3 mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with p21 and p53 mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 in vitro, either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by H2O2 caused a dose-dependent increase in Ccn3 gene and CCN3 protein expression, along with enhanced expression of p21 and p53 mRNA and proteins, as well as SA-ß gal activity. Overexpression of CCN3 also enhanced p21 promoter activity via p53. Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of p21 and p53 mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction.

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf-/- ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf-/- embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

Burden of asthma exacerbations and health care utilization in pediatric patients with asthma in the US and England.

BACKGROUND: Data on asthma burden in pediatric patients are limited; this real-world study investigated exacerbation frequency and health care resource utilization (HCRU) in pediatric asthma patients from the US and England. METHODS: Data from pediatric patients (aged 6-17 years) in the Optum claims database (US) or Clinical Practice Research Datalink with linkage to Hospital Episode Statistics (England) were analyzed. Patients were categorized into four hierarchical groups: treated asthma (patients with ≥1 baseline asthma medication), severe asthma (plus Global Initiative for Asthma Step 4/5), severe refractory asthma ([SRA] plus ≥2 baseline severe asthma exacerbations), and eosinophilic SRA (SRA plus blood eosinophil count ≥150 cells/µL). Exacerbation frequency and HCRU during the 12 months postindex were described. RESULTS: Of 151 549 treated asthma patients in the US, 18 086 had severe asthma, 2099 SRA, and 109 eosinophilic SRA. There were 32 893 treated asthma patients in England, of whom 2711 had severe asthma, 265 SRA, and 8 eosinophilic SRA. In the 12 months postindex, ≥1 exacerbation occurred in 12.4% and 10.8% of patients with severe asthma, and 32.6% and 42.6% with SRA in the US and England, respectively. The proportions of patients with ≥1 asthma hospitalization in the 30 days after the first asthma exacerbation were 2.7% and 4.4% (treated), 3.5% and 8.2% (severe asthma), and 6.0% and 16.8% (SRA) in the US and England, respectively. CONCLUSION: This study provides insights into current asthma management practices in the US and England and indicates that some patients with severe disease have an unmet need for effective management.

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes.

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed.

Circadian production of melatonin in cartilage modifies rhythmic gene expression.

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan, and Sox9, but inhibited Col10a1 expression in primary BALB/c mouse chondrocytes. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2, and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1, and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

The efficacy and safety of fluticasone/salmeterol compared to fluticasone in children younger than four years of age.

BACKGROUND: Fluticasone propionate 50 µg/salmeterol xinafoate 25 µg (FP/SAL) is widely used in adults and children with asthma, but there is sparse information on its use in very young children. METHODS: This was a randomized, double-blind, multicentre, controlled trial conducted in children aged 8 months to 4 years. During a 2-week run-in period, they all received FP twice daily. At randomization, they commenced FP/SAL or FP twice daily for 8 weeks. All were then given FP/SAL only, in a 16-week open-label study continuation. Medications were inhaled through an AeroChamber Plus with attached face mask. The primary end-point was mean change in total asthma symptom scores from baseline to the last 7 days of the double-blind period. Analyses were undertaken in all children randomized to treatment and who received at least one dose of study medication. RESULTS: Three hundred children were randomized 1:1 to receive FP/SAL or FP. Mean change from baseline in total asthma symptom scores was -3.97 for FP/SAL and -3.01 with FP. The between-group difference was not statistically significant (P = 0.21; 95% confidence interval: -2.47, 0.54). No new safety signals were seen with FP/SAL. CONCLUSION: This is the first randomized, double-blind study of this size to evaluate FP/SAL in very young children with asthma. FP/SAL did not show superior efficacy to FP; no clear add-on effect of SAL was demonstrated. No clinically significant differences in safety were noted with FP/SAL usage.

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.

Chondrocyte burst promotes space for mineral expansion.

Analysis of tissue development from multidisciplinary approaches can result in more integrative biological findings, and can eventually allow the development of more effective bioengineering methods. In this study, we analyzed the initial steps of mineral formation during secondary ossification of mouse femur based on biological and bioengineering approaches. We first found that some chondrocytes burst near the mineralized area. External factors that could trigger chondrocyte burst were then investigated. Chondrocyte burst was shown to be modulated by mechanical and osmotic pressure. A hypotonic solution, as well as mechanical stress, significantly induced chondrocyte burst. We further hypothesized that chondrocyte burst could be associated with space-making for mineral expansion. In fact, ex vivo culture of femur epiphysis in hypotonic conditions, or under mechanical pressure, enhanced mineral formation, compared to normal culture conditions. Additionally, the effect of mechanical pressure on bone formation in vivo was investigated by immobilization of mouse lower limbs to decrease the body pressure onto the joints. The results showed that limb immobilization suppressed bone formation. Together, these results suggest chondrocyte burst as a novel fate of chondrocytes, and that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering.

Bioinspired Mineralization Using Chondrocyte Membrane Nanofragments.

Biomineralization involves complex processes and interactions between organic and inorganic matters, which are controlled in part by the cells. The objectives of this study were, first, to perform a systematic and ultrastructural investigation of the initial mineral formation during secondary ossification center of mouse femur based on material science and biology viewpoint, and then develop novel biomaterials for mineralization based on the in vivo findings. First, we identified the very initial mineral deposition at postnatal day 5 (P5) at the medial side of femur epiphysis by nanocomputed tomography. Initial minerals were found in the surroundings of hypertrophic chondrocytes. Interestingly, histological and immunohistochemical analyses showed that initial mineralization until P6 was based on chondrocyte activity only, i.e., it occurred in the absence of osteoblasts. Moreover, electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane nanofragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane nanofragments were not mineralized material, and could be the nucleation site for the newly formed calcospherites. The phospholipids in the cell membrane nanofragments could be a source of phosphate for subsequent calcium phosphate formation, which initially was amorphous, and later transformed into apatite crystals. Finally, artificial cell nanofragments were synthesized from ATDC5 chondrogenic cells, and in vitro assays showed that these nanofragments could promote mineral formation. Taken together, these results indicated that cell membrane nanofragments were the nucleation site for mineral formation, and could potentially be used as material for manipulation of biomineralization.

Production of Recombinant CCN2 Protein in Escherichia coli.

Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.

Protocols for Screening for Binding Partners of CCN Proteins: Yeast Two-Hybrid System.

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.