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Standing is a common modality used in the management of children with developmental disabilities. The purpose of this article is to examine the scientific basis for standing programs, with specific emphasis on the known effects of weight bearing on bone development. Guidelines for the use of standing programs are presented, and the supporting rationale is discussed.
An extensive literature on bone morphogenetic protein (BMP) induced generation and regeneration shows general agreement about one observation. The incidence and quantity of bone were greatest when BMP was delivered with a carrier of various biologic and non biologic polymers. In the present research, neutral lipids either endogenous in demineralized bone matrix (DBM) or exogenous in orign were employed as a delivery system for induced heterotopic bone formation in the rectus muscle in rats. Total neutral lipids including cholesterol were measured by correlated gravimetric and Sudan Black B dye binding methods. The heterotopic bone was measured by computer assisted radiomorphometric and histologic methods. Bone formation was measured by total calcium, DNA-P, and alkaline phosphatase activity. Composites of BMP and neutral lipids, separable from phospholipids by extraction with absolute acetone, were consistently osteoinductive. A significant quantity of the total bone lipid was closely associated with and extractable from the bone matrix non-collagenous protein fraction which had high levels of BMP activity. Lathyritic matrix was very low both in dye binding and osteoinductive activity. These observations suggest the possibility that lipids may serve as a BMP carrier in the process of induced bone development.
A major challenge in developmental biology is to correlate genome-wide gene expression modulations with developmental processes in vivo. In this study, we analyzed the role of Runx2 during intramembranous and endochondral bone development, by comparing gene expression profiles in 14.5 dpc wild-type and Runx2 (-/-) mice. A total of 1277, 606 and 492 transcripts were found to be significantly modulated by Runx2 in calvaria, forelimbs and hindlimbs, respectively. Bioinformatics analysis indicated that Runx2 not only controls the processes of osteoblast differentiation and chondrocyte maturation, but may also play a role in axon formation and hematopoietic cell commitment during bone development. A total of 41 genes are affected by the Runx2 deletion in both intramembranous and endochondral bone, indicating common pathways between these two developmental modes of bone formation. In addition, we identified genes that are specifically involved in endochondral ossification. In conclusion, our data show that a comparative genome-wide expression analysis of wild-type and mutant mouse models allows the examination of mutant phenotypes in complex tissues.
Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzon's syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzon's syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.
We examined the immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14-16 (E14-16) in Meckel's cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckel's cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckel's cartilage, tartrate-resistant acid phosphatase (TRAP)-positive cells appeared on the lateral side of the cartilage. Furthermore, the dental follicle showed moderate immunoreactivity for RANKL and OPG, whereas osteoblasts derived from perichondral cells were immunonegative for RANKL and OPG in that area. On E16, cartilage resorption by TRAP-positive cells had progressed at the differential position, and intensely immunoreactive products of RANKL were overlapped on and found to exist next to TRAP-positive cells in the resorption area. In developing metatarsal tissue, OPG immunoreactivity was intense in periosteal osteoblasts, whereas RANKL was only faintly seen in some of the periosteal cells. In epiphyseal chondrocytes of the developing femur, RANKL immunoreactivity was moderate, and OPG scarcely detected. These results indicate a peculiarity of RANKL and OPG immunolocalization in resorption of Meckel's cartilage. Growth of the incisor teeth may be involved in the time- and position-specific resorption of Meckel's cartilage through local regulation of the RANKL/OPG system in dental follicular cells and periosteal osteoblasts, whereas RANKL and OPG in chondrocytes seem to contribute to resorption through regulation of the chondroclast function.
To accommodate developing tooth germ in the alveolar bone, active bone resorption and the recruitment of numerous osteoclasts are essential. Recently, the signaling of receptor activator of nuclear factor-KappaB (RANK) and its ligand (RANKL) was reported to play a pivotal role in osteoclast formation and activation. The aim of this study was to examine the expression of RANKL and the contribution of RANK-RANKL signaling to the process of tooth germ and alveolar bone development. In situ hybridization showed RANKL was expressed in dental follicle cells and osteoblasts on the alveolar bone surface surrounding developing tooth germs. To elucidate the function of RANKL, mouse mandibular explants on embryonic day 14 were subjected to organ culture with osteoprotegerin (OPG), an inhibitor of RANK-RANKL signaling as a decoy receptor of RANKL. Many tooth germs were compressed with the surrounding bone tissue in the OPG-treated explants, whereas these abnormalities were not seen in untreated explants. The numbers of tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cells aligning on the alveolar bone surface were significantly decreased in OPG-treated explants compared with untreated explants. Moreover, TRAP-positive osteoclastic cells were not observed along the alveolar bone surfaces depressing tooth germs. These observations suggest that osteoclastogenesis in the alveolar bone, which is essential for the accommodation of normal tooth development, is mediated by RANK-RANKL signaling.
The von Hippel Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that promotes proteolysis of the transcription factor hypoxia-inducible-factor 1alpha (HIF1alpha), the key molecule in the hypoxic response. We have used conditional inactivation of murine VHL (Vhlh) in all cartilaginous elements to investigate its role in endochondral bone development. Mice lacking Vhlh in cartilage are viable, but grow slower than control littermates and develop a severe dwarfism. Morphologically, Vhlh null growth plates display a significantly reduced chondrocyte proliferation rate, increased extracellular matrix, and presence of atypical large cells within the resting zone. Furthermore, stabilization of the transcription factor HIF1alpha leads to increased expression levels of HIF1alpha target genes in Vhlh null growth plates. Lastly, newborns lacking both Vhlh and Hif1a genes in growth plate chondrocytes display essentially the same phenotype as Hif1a null single mutant mice suggesting that the Vhlh null phenotype could result, at least in part, from increased activity of accumulated HIF1alpha. This is the first study reporting the novel and intriguing findings that pVHL has a crucial role in endochondral bone development and is necessary for normal chondrocyte proliferation in vivo.
Recent evidence suggests that sensory nerve fibers of the dental pulp secrete calcitonin gene related peptide alpha exactly where secondary or tertiary dentin is mineralized. In addition, calcitonin gene related peptide raises the level of cyclic adenosine monophosphate in osteoblasts, indicating a potential effect on secretory activity in bone. Because calcitonin and calcitonin gene related peptide are formed from the same gene, the authors tested whether calcitonin gene related peptide or calcitonin has an osteogenic potential in vitro. To this end, 0.01, 0.1, and 1 microg/ml of salmon calcitonin or rat calcitonin gene related peptide in Bigger, Gwatkin, Jackson b medium was added daily to 3 x 10(6) rat light density bone marrow leukocytes that were separated with the Ficoll-Paque density gradient separation method, then seeded on a previously prepared fibroblast layer in Petri dishes. After 7 days, the number and size of bone colonies formed in the calcitonin gene related peptide (0.1 or 1 microg/ml) added group was significantly greater than that of the control group. There was no statistically significant difference between the calcitonin added and control groups. Calcitonin gene related peptide has an osteogenic stimulating effect, either by stimulating stem cell mitosis or osteoprogenitor cell differentiation (or both), whereas salmon calcitonin has no effect.
Serum thymidine kinase (S-TK) was measured weekly in 16 randomly selected patients with AIDS or AIDS-related complex (ARC; Centers for Disease Control group IV A or group IV C-2) who participated in a controlled study of the efficacy of zidovudine therapy. S-TK increased significantly (P less than 0.01) in the zidovudine group, whereas it remained stable in the placebo (control) group. On the basis of this observation, the value of S-TK measurements as a predictor of bone marrow toxicity during zidovudine therapy was investigated in 42 patients with AIDS or ARC who received zidovudine as part of their usual treatment. There was a significant association between S-TK, haemoglobin and neutrophil counts measured after the first 4 weeks of therapy and the risk of developing bone marrow toxicity during the following 6 months. Combined, measurements of S-TK and neutrophil counts seem to be well suited for the identification of patients who have a high probability for developing bone marrow toxicity during zidovudine treatment.
Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-beta superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-beta 1 and porcine TGF-beta 1 and beta 2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35SO4 into glycosaminoglycans. Mineralization was monitored by 45Ca incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondrocytes, resulting in increased appositional growth. In contrast to OP-1, TGF-beta 1 and beta 2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-beta inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-beta exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-beta have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development.
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Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.
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Delayed skeletal maturation has been long accepted as a correlate of mental retardation. Three hundred mentally retarded children were examined for skeletal age and failed to show any overall delay of bone age as compared to chronological age. Further, birth weight was found to be unrelated to bone age maturity. Children with severe levels of retardation displayed significant delay in bone development only when profound physical disability was also present. In eight diagnostic categories of mental retardation, only those with metabolic syndromes showed any significant delay in skeletal system maturation. Conversely, mongoloid children showed a larger-than-chance percentage of advanced bone maturation. Skeletal measures therefore are perhaps more reflective of etiological states than of diagnostic classifications in mental retardation.
Both endogenous and exogenous glucocorticoid excess are well establish as causes of osteoporosis, however the reversibility of bone mass following the restauration of normal steroid levels is not well documented. In this longitudinal study, we mesured bone mineral density (expressed as Z-score) by dual-photon and X-ray absorptiometry of the lumbar spine (L2-L4) in 9 patients cured of Cushing's syndrome who were followed for the next 48 months (16-76). Initial Z-scores ranged from -2 to -1 standard-deviation (SD) in 6 patients consistent with osteopenia, and were below -3 SD in 2 patients consistent with osteoporosis. One patient developed lumbar spine fractures. There is no relationship between the severity of the Cushing's syndrome (assessed by the urinary free cortisol) and initial bone reduction (inital Z-score), nor between length of Cushing's symptoms and initial bone reduction. Our data show a marked variation (+74 +/- 9%) in bone mass in patients successfully treated for Cushing's syndrome. Seven patients completely recovered from steroid-induced osteoporosis, one patient partially recovered but remained osteopenic. One post-menopausal women presented several lumbar spine fractures despite successfull treatment of Cushing's syndrome. This longitudinal study confirms that if steroid-induced bone loss may improve substantially by cure of steroid excess even without other treatment, osteoporosis may worsen particularly in post-menopausal women. These results are important to take into account to properly manage patients with steroid-induced osteoporosis.
An analysis of function and marker expression during cytotoxic cell differentiation in vitro from T cell-depleted bone marrow precursors is described. Cytotoxic activity is not detectable during the first five days of culture, but rises abruptly soon after. Antibody plus complement depletion studies show that cytotoxic cells derive from Thy-1-negative precursors and undergo a continual increase in Thy-1 and Lyt-2 marker expression as the culture progresses. A reciprocal decrease in asialo-GM1 antigen expression on effector cells is seen. The J11d-negative precursor cells acquire J11d (an antigen known to mark cortical thymocytes) at an intermediate stage of culture, but revert to the J11d-negative phenotype prior to functional acquisition. At least some effectors are T cell receptor positive as illustrated by an anti-T cell receptor antibody-mediated killing assay. These patterns precisely correlate with those detected among developing T cells in vivo. Results may indicate that a programmed course of differentiation inherent to bone marrow cells may be triggered in the absence of a thymic environment.
The fine structure of early developing bone was examined using a modification of a new non-aqueous processing method in which anhydrous glutaraldehyde dissolved in dimethylsulphoxide (GLUT-DMSO) is used as a fixative. Preliminary results show that the basic morphological features of the cellular and extracellular compartments of developing bone were preserved. The cytoplasm of osteoblasts revealed numerous well-preserved mitochondria and other membranous organelles. Inside the mitochondria there were large and conspicuous electron-opaque granules of mineral which were more prominent and numerous than in specimens prepared by conventional aqueous procedures. The collagenous matrix also revealed electron-opaque deposits of mineral. These results support the evidence for the existence of mitochondrial calcium phosphate in osteoblasts in the form of granules.
Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.