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Testosterone productivity and histostructural changes of autotransplanted rat Leydig cells.

To investigate the possibility of in vivo transplantation of Leydig cells as a new biologic androgen replacement therapy, the Leydig cells procured from 6 week-old male Sprague-Dawley rats were autotransplanted, and the level of testosterone secretion and histostructural changes were observed. The renal subcapsular and intraperitoneal transplant showed higher levels of testosterone compared to subcutaneous or scrotal counterparts, and the number of transplanted cells was correlated with the level of measured testosterone. Furthermore, if the Leydig cells were transplanted intraperitoneally after the uptake on synthetic collagen, testosterone levels were higher than the ones simply transplanted without synthetic collagen uptake, resulting in 27 fold increase at 3 months. The activity of 125I-hCG decreased 20 to 40% at each month after transplantation compared to the normal levels, but no statistical significance was noted among different periods. The histologic examination revealed neovascularized capillaries and well demarcated sheet-like group of eosinophilic Leydig cells were observed at 4 weeks. But the evidence of destructive changes such as a focal inflammation with central dystropic ossification could be noted after 3 month. On electron microscopy, the marked indentation of nucleus and presence of lipochrome pigment were seen, and the number and size of smooth endoplasmic reticulum and mitochondria were reduced after 3 month. In conclusion, testosterone output could be increased to the physiologic range by increasing the number of transplant cells or utilizing collagen uptake but further effort is necessary on delaying or preventing the structural and functional decrement of Leydig cells.

Animals↗

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro.

Connective tissue growth factor/hypertrophic chondrocyte-specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130-140; Shimo et al., 1999, J Biochem 126:137-145; Nakanishi et al., 2000, Endocrinology 141:264-273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using (125)I-labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos-2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross-linking study revealed the formation of (125)I-rCTGF/Hcs24-receptor complex with an apparent molecular weight of 280 kDa. The intensity of (125)I-rCTGF/Hcs24-receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet-derived growth factor-BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos-2 cells and a mouse osteoblast cell line MC3T3-E1 in a dose- and time-dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos-2 cells and MC3T3-E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3-E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3-E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein-2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re-defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called "ecogenin: endochondral ossification genetic factor."

3T3 Cells↗

Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum.

Maintenance of chick embryos in long-term culture without their calcareous egg-shell is a useful method for studying the relationship between calcium homeostasis and cell differentiation during skeletogenesis. Previously, we have shown that in shell-less (SL) embryos, calcium deficiency induces a cartilage-like phenotype in osteogenic tissues, such as calvaria (Jacenko and Tuan [1986] Dev. Biol. 115:215). In this investigation, we have studied the relationship between cartilage calcification and hypertrophy, and the expression of type X collagen, a specific product of hypertrophic chondrocytes. For this study, the cephalic (calcifying) and caudal (permanently cartilaginous) regions of sterna from day 18 and day 20 normal (NL) and SL embryos were metabolically labeled with [14C]-proline. Analysis of the biosynthetic products revealed significant differences in type X collagen expression in the cephalic region of sternal cartilage. In NL tissues, type X collagen production increased from 13.1% of total collagen at day 18 to 43.7% at day 20. In contrast, in SL embryos, type X collagen was not detectable until day 20, when it represented only 1% of total collagen. Comparison of the NL and SL embryos with respect to their serum calcium level and sternal calcium content and histology revealed a direct relationship between low systemic calcium and limited cartilage hypertrophy, undermineralization, and decreased type X collagen production in the sternal cephalic cartilage. Supplementation of CaCO3 to SL embryos increased their serum and sternal calcium, and restored cartilage hypertrophy, mineralization, and type X collagen synthesis in the cephalic portion of the sterna. These findings confirm that a critical relationship exists between calcium homeostasis, chondrocyte hypertrophy, mineralization, and type X collagen synthesis in the cephalic region of sternal cartilage. These results further demonstrate the importance of calcium in the morphogenetic events of endochondral ossification, in particular the transition from hyaline cartilage to hypertrophic cartilage, and eventually to bone.

Animals↗

Insulin enhancement of in vitro wound healing in fetal rat parietal bones.

PURPOSE: This study evaluated the effect of insulin on fracture in fetal rat intramembranous bone using an organ culture system in which bone mineralizes in vitro. MATERIALS AND METHODS: Twenty-day-old fetal rat parietal bones were fractured parallel to the frontal plane and cultured in a serum-free medium with or without insulin for 4 to 12 days. They were then processed for light microscopy. RESULTS: In the specimens not treated with insulin, bony bridges were observed in less than 10% on day 4 and 8, and only 45% on day 12. In bones treated with insulin at 10(-6) mol/L, bony bridges were observed in 73% on day 4 and 100% on day 8 and 12. No callus or cartilage formation was observed during the wound repair process, indicating that intramembranous ossification occurred. CONCLUSION: These findings suggest that insulin has a direct effect on bone cells, which enhances the healing of fractures in intramembranous bone.

Animals↗

Early development of the primitive cranial vault in the chick embryo.

The calcified tissues involved in the early morphogenesis of the cranial vault were studied by microradiographic analysis and histological techniques in 12 chick embryos on the 9th, 12th, and 14th days of incubation. On the 9th day, the frontal, parietal, and squamosal bones are comprised of a thin lamina of chondroid tissue deposited at a short distance from the fibers of the dura mater. Woven bone formation takes place in the calvarial mesenchyme only after the 12th day of incubation and occurs mainly on the external side of the chondroid primordium. The present data obviously indicate that the primitive desmocranium of the chick embryo, which is usually known to be formed by intramembranous ossification, consists first of chondroid tissue. This tissue represents thus the initial modality of skeletogenic differentiation within the cephalic mesenchyme of the cranial vault.

Animals↗

Adaptive bone formation in acellular vertebrae of sea bass (Dicentrarchus labrax L.).

Mammalian bone is an active tissue in which osteoblasts and osteoclasts balance bone mass. This process of adaptive modelling and remodelling is probably regulated by strain-sensing osteocytes. Bone of advanced teleosts is acellular yet, despite the lack of osteocytes, it is capable of an adaptive response to physical stimuli. Strenuous exercise is known to induce lordosis. Lordosis is a ventrad curvature of the vertebral column, and the affected vertebrae show an increase in bone formation. The effects of lordosis on the strain distribution in sea bass (Dicentrarchus labrax L.) vertebrae are assessed using finite element modelling. The response of the local tissue is analyzed spatially and ontogenetically in terms of bone volume. Lordotic vertebrae show a significantly increased strain energy due to the increased load compared with normal vertebrae when loaded in compression. High strain regions are found in the vertebral centrum and parasagittal ridges. The increase in strain energy is attenuated by a change in architecture due to the increased bone formation. The increased bone formation is seen mainly at the articular surfaces of the vertebrae, although some extra bone is formed in the vertebral centrum. Regions in which the highest strains are found do not spatially correlate with regions in which the most extensive bone apposition occurs in lordotic vertebrae of sea bass. Mammalian-like strain-regulated bone modelling is probably not the guiding mechanism in adaptive bone modelling of acellular sea bass vertebrae. Chondroidal ossification is found at the articular surfaces where it mediates a rapid adaptive response, potentially attenuating high stresses on the dorsal zygapophyses.

Adaptation, Physiological↗

Effects of spaceflight and simulated weightlessness on longitudinal bone growth.

Indirect measurements have suggested that spaceflight impairs bone elongation in rats. To test this possibility, our laboratory measured, by the fluorochrome labeling technique, bone elongation that occurred during a spaceflight experiment. The longitudinal growth rate (LGR) in the tibia of rats in spaceflight experiments (Physiological Space Experiments 1, 3, and 4 and Physiological-Anatomical Rodent Experiment 3) and in two models of skeletal unloading (hind-limb elevation and unilateral sciatic neurotomy) were calculated. The effects of an 11 day spaceflight on gene expression of cartilage matrix proteins in rat growth plates were also determined by northern analysis and are reported for the first time in this study. Measurements of longitudinal growth indicate that skeletal unloading generally did not affect LGR, regardless of age, strain, gender, duration of unloading, or method of unloading. There was, however, one exception with 34% suppression in LGR detected in slow-growing, ovariectomized rats skeletally unloaded for 8 days by hind-limb elevation. This detection of reduced LGR by hind-limb elevation is consistent with changes in steady-state mRNA levels for type II collagen (-33%) and for aggrecan (-53%) that were detected in rats unloaded by an 11 day spaceflight. The changes detected in gene expression raise concern that spaceflight may result in changes in the composition of extracellular matrix, which could have a negative impact on conversion of growth-plate cartilage into normal cancellous bone by endochondral ossification.

Animals↗

Ultrastructural investigation of calcification and ossification in experimental fracture healing with special reference to osteogenic role of fibroblasts.

OBJECTIVE: To investigate ultrastructurally the process of calcification and ossification in experimental fracture healing. METHODS: A standardized fracture of the radius was made in rabbits. Undecalcified callus tissues from different fracture regions were subjected to ultrastructural observation under transmission electron microscope. RESULTS: In the mitochondria and membrane-bound vesicles of the fibroblasts, osteoblasts and chondrocytes, high electron density calcium granules were seen. These granules provided raw materials for the deposition of calcium salt crystals. Around these cells, round, oval or irregularly shaped matrix vesicles could also be discerned, and they became calcified and formed flocculent calcospherules. Then in the Type I collagen fibrils around the fibroblasts and osteoblasts, and in the Type II collagen fibrils around the chondrocytes, calcium salt crystals also appeared. Innumerable flocculent calcospherules and calcified collagen fibrils coalesced together, expanded and finally turned into pieces of bone tissues. The fibroblasts also possessed the indispensable prerequisites for calcification and ossification in fracture healing. They either transformed into osteocytes or degenerated, perished and eventually were replaced by bone tissues. CONCLUSIONS: Sequence of events taking place in calcification and ossification in experimental fracture healing was depicted. Calcification of matrix vesicles and collagen fibrils triggered these processes. Evidence of osteogenic role played by fibroblasts was provided.

Animals↗

[Comparative pathology of vertebral hyperostosis].

The changes seen in vertebral hyperostosis are due to a slow ossification of the perivertebral connective tissue causing osteophytic spurs and/or bridging of intervertebral ankylosis. The same changes seen in man are also seen in all land and aquatic mammals. This confrontation of comparative pathology emphasizes certain essential points in common. There is a relation between the vertebral hyperostosis and local anatomic and physiologic conditions linked to a ligament insertions. It occurs in bipodisis or tetrapodisis as well as in an aquatic environment. It must be distinguished from an osteoarthritic intervertebral remodeling and the evolution of its bone tissue is dissociated from that of the adjacent vertebral bone tissue. It is frequently observed in relation to general conditions (aging -- diabetes in man -- dietary imbalance in the animal). This example emphasizes the interest that comparative pathology can have for the rheumatologist.

Animals↗

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Animals↗

Bone within a bone.

The "bone within a bone" appearance is a well-recognized radiological term with a variety of causes. It is important to recognize this appearance and also to be aware of the differential diagnosis. A number of common conditions infrequently cause this appearance. Other causes are rare and some remain primarily of historical interest, as they are no longer encountered in clinical practice. In this review we illustrate some of the conditions that can give the bone within a bone appearance and discuss the physiological and pathological aetiology of each where known.

Anemia, Sickle Cell↗

Dominance of SOX9 function over RUNX2 during skeletogenesis.

Mesenchymal stem cell-derived osteochondroprogenitors express two master transcription factors, SOX9 and RUNX2, during condensation of the skeletal anlagen. They are essential for chondrogenesis and osteogenesis, respectively, and their haploinsufficiency causes human skeletal dysplasias. We show that SOX9 directly interacts with RUNX2 and represses its activity via their evolutionarily conserved high-mobility-group and runt domains. Ectopic expression of full-length SOX9 or its RUNX2-interacting domain in mouse osteoblasts results in an osteodysplasia characterized by severe osteopenia and down-regulation of osteoblast differentiation markers. Thus, SOX9 can inhibit RUNX2 function in vivo even in established osteoblastic lineage. Finally, we demonstrate that this dominant inhibitory function of SOX9 is physiologically relevant in human campomelic dysplasia. In campomelic dysplasia, haploinsufficiency of SOX9 results in up-regulation of the RUNX2 transcriptional target COL10A1 as well as all three members of RUNX gene family. In summary, SOX9 is dominant over RUNX2 function in mesenchymal precursors that are destined for a chondrogenic lineage during endochondral ossification.

Animals↗

Low-intensity pulsed ultrasound accelerates rat femoral fracture healing by acting on the various cellular reactions in the fracture callus.

Low-intensity pulsed ultrasound (LIPUS) has been shown to accelerate fracture healing in both animal models and clinical trials, but the mechanism of action remains unclear. In fracture healing, various consecutive cellular reactions occurred until repair. We investigated whether the advanced effects of LIPUS depended on the duration and timing of LIPUS treatment in a rat closed femoral fracture model to determine the target of LIPUS in the healing process. Sixty-nine Long-Evans male rats that have bilateral closed femoral fractures were used. The right femur was exposed to LIPUS (30 mW/cm2 spatial and temporal average [SATA], for 20 minutes/day), and the left femur was used as a control. Rats were divided into four groups according to timing and duration of treatment (Ph-1, days 1-8; Ph-2, days 9-16; Ph-3, days 17-24; throughout [T], days 1-24 after the fracture). Animals were killed on day 25. After radiographs and microfocus X-ray computed tomography (muCT) tomograms were taken, the hard callus area (HCA), bone mineral content (BMC) at the fracture site, and mechanical torsion properties were measured, and histological analysis was conducted. Interestingly, the maximum torque of the LIPUS-treated femur was significantly greater than that of the controls in all groups without any changes in HCA and BMC. The multiviewing of three-dimensional (3D) muCT reconstructions and histology supported our findings that the partial LIPUS treatment time was able to accelerate healing, but longer treatment was more effective. These results suggest that LIPUS acts on some cellular reactions involved in each phase of the healing process such as inflammatory reaction, angiogenesis, chondrogenesis, intramembranous ossification, endochondral ossification, and bone remodeling.

Animals↗

IGF-I, TGF-beta, and BMP-4 are expressed during distraction osteogenesis of the pig mandible.

The mechanisms that regulate bone formation during distraction osteogenesis (DO) are not completely understood. Our hypothesis is that local cytokines that stimulate osteoblast activity are potential regulators of this process. The purpose of this study was to determine gene expression of insulin-like growth factor I (IGF-I), transforming growth factor-beta (TGF-beta), and bone morphogenetic protein 4 (BMP-4) in distracted wounds. A semiburied, rigid distraction device was placed across an osteotomy at the right mandibular angle in 9 Yucatan minipigs. Distraction was begun immediately at a rate of 1 mm/day. The animals were sacrificed after 4 and 7 days of distraction, and after 7 days of distraction plus 4 days of neutral fixation. Excised wound tissues were processed for histologic and gene expression analyses. Competitive reverse-transcription polymerase chain reaction (RT-PCR) assays were developed and validated for porcine genes. Histologic analysis showed membranous ossification within the DO wound. Gene expression of IGF-I, TGF-beta and BMP-4 was detected during distraction and neutral fixation. These results show that gene expression analyses can be performed in a large animal model of mandibular DO. As the pig mandible closely resembles that of the human in morphology and physiology, this is an important step toward characterization of the early molecular events in the DO wound.

Animals↗

Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization.

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.

Adaptation, Physiological↗