Osteoclast formation is related to bone matrix age

Summary Little is known about the relationship between the age of the skeleton and the development of multinucleated bone-resorbing cells, osteoclasts. It has been shown that mineralized bone implanted onto the chick chorioallantoic membrane (CAM) is effective in the recruitment and differentiation of osteoclast precursors. In studies reported here we used the CAM system to examine the influence of bone matrix age on osteoclast formation. Devitalized mineralized bone particles (75–250 μm) were prepared from rats of various ages (2, 4, 9, 12, and 16 months). The particles were implanted onto the chick chorioallantoic membrane and 8 days later implants were harvested and processed for morphometric or immunohistochemical analysis. Osteoclast number, cell area, nucleocytoplasmic ratio, and the presence of a distinctive osteoclast antigen, defined by the 121F monoclonal antibody, were determined. Bone particles of each age group resulted in the formation of osteoclast-like giant cells. Compared with multinucleated cells that formed in response to bone particles obtained from 2-month-old rats, matrix from the oldest age group (16 months) elicited significantly fewer and smaller cells which contained a smaller number of nuclei. These data suggest that with aging, bone undergoes qualitative and/or quantitative changes that affect the recruitment and differentiation of osteoclast precursor cells.     

An application of bioinformatics and text mining to the discovery of novel genes related to bone biology

The treatment and management of complex genetic diseases such as osteoporosis can greatly benefit from the integration of relevant research across many different disciplines. We created a text mining tool that analyzes the PubMed literature database and integrates the available genomic information to provide a detailed mapping of the genes and their interrelationships within a particular network such as osteoporosis. The results obtained from our text mining program show that existing genomic data within the PubMed database can effectively be used to predict potentially novel target genes for osteoporosis research that have not previously been reported in the literature. To filter the most significant findings, we developed a ranking system to rate our predicted novel genes. Some of our predicted genes ranked higher than those currently studied, suggesting that they may be of particular interest from a therapeutic standpoint. A preliminary analysis of the current biomedical literature in our research area using our tool suggests that S100A12, as well as a group of SMAD genes previously unstudied in relation to osteoporosis, may be highly relevant to the mechanism of action of bisphosphonates, that the function of osteocytes may be influenced by a family of important interleukins and interleukin-related molecules, and that the FYN oncogene may play an important role in regulating the apoptosis of bone cells in the context of degenerative bone diseases. An evaluation of our tool's predictive ability with an analysis of PubMed literature published before the year 2000 in the area of osteoporosis research shows that many of its top-rated novel target genes from that analysis were later studied and shown to be relevant to osteoporosis in the period between 2000 and 2006. We believe that our tool will be beneficial to researchers in the field of orthopaedics seeking to identify novel target genes in their research area, and it will allow them to delve deeper into the complex interplay between genes, biological systems and diseases.     

Sensitivity analysis of a novel mathematical model identifies factors determining bone resorption rates

The development of pharmaceutical treatments for bone disease can be enhanced by computational models that predict their effects on resorption and rates of remodeling. Therefore, a simple mathematical model was formulated to simulate erosion depth and duration of resorption, using Michaelis-Menten (M-M) equations to describe changing rates of cellular activity during the two phases of bone resorption. The model was based on histomorphometric data and cellular interactions that occur in the bone microenvironment cited from the literature. Availability of bone substrate for osteoclastic activity during Phase I was assumed to be limited by the ratio of RANKL (ligand for receptor activator for nuclear factor kappaB) to osteoprotegerin (OPG) ('effective RANKL'). The required presence of marrow stromal cell produced macrophage-colony stimulating factor (M-CSF) for osteoclast action was represented as a factor equal to 1 for healthy bone. Growth factors released from the matrix during Phase I were assumed to cause two negative feedback effects: (1) the inhibitory effect of transforming growth factor-beta1 (TGFbeta1)-induced production of OPG by marrow osteoblast stromal cells, reducing effective RANKL; (2) the apoptosis of osteoclast nuclei assumed to occur at high concentrations of TGFbeta. This signaled the end of Phase I. During Phase II, cellular activity to remove the collagen fibrils left behind by osteoclasts was also simulated by Michaelis-Menten kinetic equations. Results of sensitivity analysis revealed variation in resorption depth and duration to fluctuate within 6% and 7% of the baseline value for changes in most input parameters. However, resorption depth was reduced and the duration of resorption lengthened by both a decrease in matrix TGFbeta and an increase the apoptotic threshold. Furthermore, the duration of resorption, but not erosion depth, was sensitive to changes in the maximum rate of cellular activity during removal of collagen fibrils. This mathematical model, which simulates the changing rates of cellular activity, has identified factors that reduce the duration and depth of resorption. It also suggests new targets for modeling therapeutic intervention to slow the rate of bone remodeling.     

Mechanical stretch induced calcium efflux from bone matrix stimulates osteoblasts

The mechanisms by which bone cells sense critically loaded regions of bone are still a matter of ongoing debate. Animal models to investigate response to microdamage involve post mortem immunohistological analysis and do not allow real-time monitoring of cellular response during the emergence of the damage in bone. Most in vitro mechanical stimulation studies are conducted on non-bone substrates, neglecting the damage-related alterations in the pericellular niche and their potential effects on bone cells. The current study reports spontaneous efflux of calcium ions (Ca(2+)) (1.924±0.742 pmol cm(-2)s(-1)) from regions of devitalized bone matrix undergoing post-yield strains, induced by a stress concentrator. When these samples are seeded with MC3T3-E1 osteoblasts, the strain-induced Ca(2+) efflux from bone elicits cell response at the stress concentration site as manifested by activation of intracellular calcium signaling (increase in fluorescence by 52%±27%). This activity is associated with extracellular calcium because the intracellular calcium signaling in response to mechanical loading subsides when experiments are repeated using demineralized bone substrates (increase in fluorescence by 6%±10%). These results imply a novel perspective where bone matrix acts as an intermediary mechanochemical transducer by converting mechanical strain into a chemical signal (pericellular calcium) to which cells respond. Such a mechanism may be responsible for triggering repair at locations of bone matrix undergoing critical deformation levels.     

Comprehensive genomic analysis identifies MDM2 and AURKA as novel amplified genes in juvenile angiofibromas

BACKGROUND: Frequent beta-catenin mutations have been detected in juvenile angiofibromas, but the tumor pathogenesis remains unknown. METHODS: Metaphase-comparative genomic hybridization (CGH) was used to identify chromosomal aberrations in 29 tumor specimens. Two tumors were investigated using genome DNA microarrays. RESULTS: Three hundred eleven chromosomal gains and losses were detected by metaphase-CGH. Frequent chromosomal gains were detected at 4q, 6, 12, and X, while frequent chromosomal losses affected regions of chromosomes 8, 16, 17, 22, and Y. Genome DNA microarray analysis in 2 tumors of the series confirmed chromosomal aberrations, detected by metaphase-CGH, and indicated genes such as AURKA (20q13.2) not being recognized by metaphase-CGH. CONCLUSION: Metaphase-CGH results confirmed numerous chromosomal aberrations in juvenile angiofibromas. The most frequent aberrations affected sex chromosomes. Further consensus regions of chromosomal aberrations were detected at 4q, 6, 8, 12, 16, 17, and 22. AURKA and MDM2 were identified as interesting novel amplified genes in juvenile angiofibromas.     

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts, osteoid, and bone matrix

Glycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicer's high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiéry's periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1-0.5 mumol in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.     

Proteomic analysis of a matrix stone: a case report

Matrix stones are radiolucent bodies that present as soft muco-proteinaceous material within the renal collecting system. Following wide-angle X-ray diffraction (XRD) and scanning electron microscopy (SEM), we homogenized a surgically removed matrix stone, extracted and purified protein, and analyzed samples using tandem mass spectrometry for proteomic composition. Resulting spectra were searched using ProteinPilot 2.0, and identified proteins were reported with >95% confidence. Primary XRD mineral analysis was a biological apatite, and SEM revealed fibrous, net-like laminations containing bacterial, cellular, and crystalline material. Of the 33 unique proteins identified, 90% have not been previously reported within matrix stones and over 70% may be considered inflammatory or defensive in nature. Characterization of other matrix stone proteomes, in particular from non-infectious populations, may yield insights into the pathogenesis of this rare stone as well as the mineralogical process that occurs within crystalline calculi.     

Immunoelectron microscopy of osteonectin and type I collagen in osteoblasts and bone matrix

Summary The pathway of production, secretion, and extracellular deposition of type I collagen and osteonectin was studied by immunoelectron microscopy using respective polyclonal antibodies. Protein A gold and immunogold methods yielded to similar results in human callus tissue used as a model of bone formation. The intracellular distribution of osteonectin in active osteoblasts is found as a faint immunolabeling of vesicular Golgi fields and some lamellae of rough endoplasmic reticulum. A more intensive labeling occurs in opaque cytoplasmic vesicles pointing to the process of secretion as some of the vesicles are connected with the basal cell membrane. Our type I collagen antibody did not label the respective intracellular compartments. Extracellulary, the type I collagen antibody showed a continuous labeling from the immature subcellular osteoid to the mineralized bone. Osteonectin antibodies were bound first to the deeper layer of osteoid maturation with intensity increasing below the mineralization front. Osteonectin is thought to be associated with mineralization of bone matrix.     

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts,osteoid, and bone matrix

Summary Glycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicer's high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiéry's periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1–0.5μm in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.     

Meta analysis identifies a novel susceptibility locus associated with heel bone strength in the Korean population

IntroductionCalcaneal quantitative ultrasound has been recognized as a non-invasive method for evaluation of bone strength and prediction of osteoporotic fracture.MethodsTo extend a thorough genetic catalog for osteoporotic bone properties, we performed a genome-wide association study (rural cohort I, n = 1895) of speed of sound (SOS) using the 1000 genome-based imputation in the discovery stage and then carried out in silico lookups (rural cohort II and III, n = 2,967) and de novo genotyping (rural cohort IV, n = 4,296) in the replication stage.ResultsIn the combined meta-analysis (n = 9,158), we identified a novel variant associated with SOS (rs2445771 in the GLDN gene, P = 2.27 × 10^− 9) reaching genome-wide significance in the Korean population. We further demonstrated that allele-specific regulatory modifications found to be associated with functional enrichments by ENCODE annotations. Conclusion: Our findings could provide additional insights into understanding of genetic and epigenetic regulations on bone metabolism.     

Gene expression analysis in human osteoblasts exposed to dexamethasone identifies altered developmental pathways as putative drivers of osteoporosis

Background  

Osteoporosis, a disease of decreased bone mineral density represents a significant and growing burden in the western world. Aging population structure and therapeutic use of glucocorticoids have contributed in no small way to the increase in the incidence of this disease. Despite substantial investigative efforts over the last number of years the exact molecular mechanism underpinning the initiation and progression of osteoporosis remain to be elucidated. This has meant that no significant advances in therapeutic strategies have emerged, with joint replacement surgery being the mainstay of treatment.     

Effects of acute and chronic PTH stimulation on osteoblasts and the under-lying bone matrix

Calcified Tissue International -     

Leptin increases extracellular matrix mineralization of human osteoblasts from heterotopic ossification and normal bone

Heterotopic ossification (HO) is the pathologic formation of bone in soft tissue. The exact pathomechanism is unknown but probably involves a disturbed osteoblast differentiation. Leptin, known as the obesity gene, may regulate normal osteoblast function in vitro. The aim of the present in vitro study was to further analyze the pathomechanisms of HO, including a possible role of leptin in ectopic bone formation. Human osteoblasts were cultivated either from normal bone or from resected HO. Both groups were incubated with increasing doses of leptin. Phenotype expression and mineralization of extracellular matrix were measured after 7, 14, and 21 days. In both groups, leptin increased both the formation of bone nodules and Ca-45 incorporation. This is the first study to analyze the effect of leptin on bone cells from ectopic ossification. Similar to the in vitro behavior of normal osteoblasts, cells from HO respond to leptin exposure with an increased mineralization of the extracellular matrix. This mechanism may be involved in the pathogenesis of ectopic bone formation in vivo.     

Exome sequencing identifies novel genes and variants in patients with Hirschsprung disease

BackgroundHirschsprung disease (HSCR) is a complex genetic disease characterized by the absence of ganglion cells in the intestines, leading to a functional obstruction in infants. At least 24 genes have been identified for the pathogenesis of HSCR. They contributed to approximately 72% of HSCR cases. We aimed to elucidate further the genetic basis of HSCR in Indonesia using the whole-exome sequencing (WES) approach.MethodsWES was performed in 39 sporadic non-syndromic HSCR patients and 16 non-HSCR subjects as controls. Variants presented in controls were excluded, followed by in silico prediction tools and population allele frequency databases to select rare variants. We determined the minor allele frequency (MAF) using gnomAD (MAF <0.1%).ResultsWe involved 24 (61.5%) males and 15 (38.5%) females. Most patients (62%) had short-segment aganglionosis and underwent the Duhamel procedure (41%). We identified several candidate novel variants in HSCR-related genes, including UBR4, GDNF, and ECE1. Moreover, we also identified some novel candidate genes, including a possible compound heterozygous variant in the MUTYH gene: the first variant, a known protein-truncating variant associated with colorectal cancer (CRC), p.Glu452Ter and the second variant is novel, p.Ala39Val. Moreover, the type of variants was not associated with the aganglionosis type.ConclusionsWe identified several novel genes and variants, including the variant associated with CRC, that might contribute to the pathogenesis of HSCR. No genotype–phenotype associations were noted. Our study further confirms the complex network involved in enteric nervous system development and HSCR pathogenesis.Level of evidenceLevel III.     

采用基因表达谱芯片技术筛选新基因AngRem104的功能相关基因

目的 采用基因芯片技术来筛选基因AngRem104的功能相关基因，为进一步的基因功能研究提供线索。方法 建立新基因AngRem104的正义和反义表达的系膜细胞模型，应用含有4000个人类基因的cDNA表达谱芯片，对过量表达AngRem104基因和抑制表达AngRem104基因的人肾小球系膜细胞RNA表达进行检测。部分基因的差异表达由RT－PCR方法来验证。结果 筛选出的差异表达基因共96个，其中94个基因上调表达，2个基因下调表达。差异表达的基因涉及到细胞外基质和受体蛋白、细胞信号传导相关基因、DNA结合及转录相关蛋白、免疫相关蛋白、细胞骨架和动力蛋白和细胞合成及代谢相关蛋白等。特别是纤连蛋白（FN）及整合素β1(integrin β1）表达明显上调。RT－PCR的方法也证实了AngRem104与FN表达的相关性。结论 应用作为研究基因功能有效手段的基因表达谱芯片技术来筛选新基因AngRem104的功能相关基因，发现AngRem104与FN的表达有关，为其功能研究提供了重要线索。     

基于生物信息学分析骨峰值及骨质疏松症相关基因

目的:基于生物信息学探讨与女性骨峰值(peak bone mass,PBM)及骨质疏松症(osteoporosis,OP)相关的基因并进行验证。方法:通过基因表达数据库(gene expression omnibus database,GEO),利用DNA微阵列技术对高PBM和低PBM成年女性的单核细胞进行全基因组范围内的基因差异表达研究,通过聚类分析、GO富集和京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)分析差异基因,并进一步分析差异表达基因间的相互作用网络。建立OP大鼠模型,进行股骨颈组织染色,进一步验证差异基因的表达。结果:差异基因筛选共得到283个基因,与高PBM样本相比,低PBM样本中有135个基因表达上调,148个基因表达下调,总共有7个通路与12个差异基因被富集,涉及矿物质吸收与转运、细胞免疫等方面的多个基因表达存在差异。其中,CACNA1D基因编码的L型钙离子通道蛋白1.3(voltage-gated Ca²⁺ channel 1.3,CaV1.3)在OP大鼠模型股骨颈组织中表...