Imranul Alam

Mechanical Stimulation of Bone in Vivo Reduces Osteocyte Expression of Sost/Sclerostin

Sclerostin, the protein product of the Sost gene, is a potent inhibitor of bone formation. Among bone cells, sclerostin is found nearly exclusively in the osteocytes, the cell type that historically has been implicated in sensing and initiating mechanical signaling. The recent discovery of the antagonistic effects of sclerostin on Lrp5 receptor signaling, a crucial mediator of skeletal mechanotransduction, provides a potential mechanism for the osteocytes to control mechanotransduction, by adjusting their sclerostin (Wnt inhibitory) signal output to modulate Wnt signaling in the effector cell population. We investigated the mechanoregulation of Sost and sclerostin under enhanced (ulnar loading) and reduced (hindlimb unloading) loading conditions. Sost transcripts and sclerostin protein levels were dramatically reduced by ulnar loading. Portions of the ulnar cortex receiving a greater strain stimulus were associated with a greater reduction in Sost staining intensity and sclerostin-positive osteocytes (revealed via in situ hybridization and immunohistochemistry, respectively) than were lower strain portions of the tissue. Hindlimb unloading yielded a significant increase in Sost expression in the tibia. Modulation of sclerostin levels appears to be a finely tuned mechanism by which osteocytes coordinate regional and local osteogenesis in response to increased mechanical stimulation, perhaps via releasing the local inhibition of Wnt/Lrp5 signaling.

Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2

We have investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta tricalcium phosphate (beta-TCP). The purpose of this study was to evaluate these BCP ceramics as carriers for rhBMP-2. BCP ceramics impregnated with the different doses of recombinant human bone morphogenetic protein 2 (rhBMP-2) (1, 5 and 10g) were used for the experimental purpose and the ceramics without rhBMP-2 were used as control. The pellets were placed into subcutaneous pockets on the dorsum of 4-week-old male Wistar rats. The animals were sacrificed 2 and 4 weeks after implantation. Bone induction was estimated by alkaline phosphatase (ALP) activity measured at 2 weeks after implantation. Pellets were also examined radiologically, histologically and histomorphometrically. The results showed that all experimental pellets exhibited new bone formation whereas the control pellets produced only fibrous connective tissue. Here, 100% HAP ceramic showed most amount of bone formation, whereas 25% HAP to 75% TCP ceramic produced the bone least in amount among different BCP ceramics at the end of 4 weeks. This study indicates that formation of new bone depends on the ceramic content with high HAP-TCP ratio and high dose of rhBMP-2.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Stochastic resonance in osteogenic response to mechanical loading

Stochastic resonance, in which noise enhances the response of a nonlinear system to a weak signal, has been observed in various biological sensory systems. We speculated that bone formation in response to mechanical loading could be enhanced by adding noise (vibration) to a standard exercise regimen. To test this hypothesis, three different loading regimens were applied to the ulnae of mice: (1) high amplitude, low frequency sinusoidal loading at 2 Hz with an amplitude of 3 N to simulate exercise; (2) low amplitude, broad frequency vibration with frequency components 0‐50 Hz and 0.3 N of mean amplitude; (3) the sinusoidal wave combined with vibration (S+V) to invoke stochastic resonance. The simulated exercise regimen induced new bone formation on the periosteal surface of the ulna, however the addition of vibration noise with exercise enhanced the osteogenic response by almost 4‐fold. Vibration by itself had no effect on bone formation. It was concluded that adding low magnitude vibration greatly enhanced bone formation in response to loading, suggesting a contribution of stochastic resonance in the osteogenic response.

Low-amplitude, broad-frequency vibration effects on cortical bone formation in mice

Mechanical loading of the skeleton is necessary to maintain bone structure and strength. Large amplitude strains associated with vigorous activity typically result in the greatest osteogenic response; however, data suggest that low-amplitude, broad-frequency vibration results in new bone formation and may enhance adaptation through a stochastic resonance (SR) phenomenon. That is, random noise may maximally enhance bone formation to a known osteogenic stimulus. The aims of this study were to (1) assess the ability of different vibration signals to enhance cortical bone formation during short- and long-term loading and (2) determine whether vibration could effect SR in bone. Two studies were completed wherein several osteogenic loading waveforms, with or without an additive low-amplitude, broad-frequency (0-50 Hz) vibration signal, were applied to the mouse ulna in axial compression. In study 1, mice were loaded short-term (30 s/day, 2 days) with either a carrier signal alone (1 or 2 N sine waveform), vibration signal alone [0.1 N or 0.3 N root mean square (RMS)] or combined carrier and vibration signal. In study 2, mice were loaded long-term (30 s/day, 3 days/week, 4 weeks) with a carrier signal alone (static or sine waveform), vibration signal alone (0.02 N, 0.04 N, 0.08 N or 0.25 N RMS) or combined carrier and vibration signal. Sequential calcein bone labels were administered at 2 and 4 days and at 4 and 29 days after the first day of loading in study 1 and 2, respectively; bone formation parameters and changes in geometry were measured. Combined application of the carrier and vibration signals in study 1 resulted in significantly greater bone formation than with either signal alone (P < 0.001); however, this increase was independently explained by increased strain levels associated with additive vibration. When load and strain levels were similar across loading groups in study 2, cortical bone formation and changes in geometry were not significantly altered by vibration. Vibration alone did not result in any new bone formation. Our data suggest that low-amplitude, broad-frequency vibration superimposed onto an osteogenic waveform or vibration alone does not enhance cortical bone adaptation at the frequencies, amplitudes and loading periods tested.

Mechanotransduction in bone does not require a functional cyclooxygenase-2 (COX-2) gene

COX‐2 is a key enzyme involved in the response of bone to loading. However, using mice with a null mutation of the COX‐2 gene, we found that a functional COX‐2 gene is not required for mechanotransduction. This paradoxical finding may have resulted, in part, from mechanically induced COX‐1 activity.

Whole-genome scan for linkage to bone strength and structure in inbred Fischer 344 and Lewis rats.

A genome‐wide genetic linkage analysis identified several chromosomal regions influencing bone strength and structure in F2 progeny of Fischer 344 x Lewis inbred rats.

Genome screen for bone mineral density phenotypes in Fisher 344 and Lewis rat strains

In humans, peak bone mineral density (BMD) is the primary determinant of osteoporotic fracture risk among older individuals, with high peak BMD levels providing protection against osteoporosis in the almost certain event of bone loss later in life. A genome screen to identify quantitative trait loci (QTLs) contributing to areal BMD (aBMD) and volumetric BMD (vBMD) measurements at the lumbar spine and femoral neck was completed in 595 female F2 rats produced from reciprocal crosses of inbred Fischer 344 and Lewis rats. Significant evidence of linkage was detected to rat Chromosomes 1, 2, 8, and 10, with LOD scores above 8.0. The region on rat Chromosome 8 is syntenic to human Chromosome 15, where linkage to spine and femur BMD has been previously reported and confirmed in a sample of premenopausal women.

Generation of the first autosomal dominant osteopetrosis type II (ADO2) disease models

Autosomal dominant osteopetrosis type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl(-)/1H(+) antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene.

Heterogeneous stock rat: A unique animal model for mapping genes influencing bone fragility

Previously, we demonstrated that skeletal mass, structure and biomechanical properties vary considerably among 11 different inbred rat strains. Subsequently, we performed quantitative trait loci (QTL) analysis in four inbred rat strains (F344, LEW, COP and DA) for different bone phenotypes and identified several candidate genes influencing various bone traits. The standard approach to narrowing QTL intervals down to a few candidate genes typically employs the generation of congenic lines, which is time consuming and often not successful. A potential alternative approach is to use a highly genetically informative animal model resource capable of delivering very high resolution gene mapping such as Heterogeneous stock (HS) rat. HS rat was derived from eight inbred progenitors: ACI/N, BN/SsN, BUF/N, F344/N, M520/N, MR/N, WKY/N and WN/N. The genetic recombination pattern generated across 50 generations in these rats has been shown to deliver ultra-high even gene-level resolution for complex genetic studies. The purpose of this study is to investigate the usefulness of the HS rat model for fine mapping and identification of genes underlying bone fragility phenotypes. We compared bone geometry, density and strength phenotypes at multiple skeletal sites in HS rats with those obtained from five of the eight progenitor inbred strains. In addition, we estimated the heritability for different bone phenotypes in these rats and employed principal component analysis to explore relationships among bone phenotypes in the HS rats. Our study demonstrates that significant variability exists for different skeletal phenotypes in HS rats compared with their inbred progenitors. In addition, we estimated high heritability for several bone phenotypes and biologically interpretable factors explaining significant overall variability, suggesting that the HS rat model could be a unique genetic resource for rapid and efficient discovery of the genetic determinants of bone fragility.