Normal long bone growth and development in type X collagen-null mice.

To investigate the role of type X collagen in skeletal development, we have generated type X collagen-null mice. Surprisingly, mice without type X collagen were viable and fertile and had no gross abnormalities in long bone growth or development. No differences were detected between the type X collagen-null mice and controls when growth plates of both newborn and 3-week old mice were examined by histology and by immunostaining for extracellular matrix components of bone including osteopontin, osteocalcin and type II collagen. Our results suggest that type X collagen is not required for long bone development. However, mice and humans with dominant acting type X collagen mutations have bone abnormalities, suggesting that only the presence of abnormal type X collagen can modify bone growth and development.

Localization of types I, II, and III collagen mRNAs in developing human skeletal tissues by in situ hybridization.

Paraffin sections of human skeletal tissues were studied in order to identify cells responsible for production of types I, II, and III collagens by in situ hybridization. Northern hybridization and sequence information were used to select restriction fragments of cDNA clones for the corresponding mRNAs to obtain probes with a minimum of cross-hybridization. The specificity of the probes was proven in hybridizations to sections of developing fingers: osteoblasts and chondrocytes, known to produce only one type of fibrillar collagen each (I and II, respectively) were only recognized by the corresponding cDNA probes. Smooth connective tissues exhibited variable hybridization intensities with types I and III collagen cDNA probes. The technique was used to localize the activity of type II collagen production in the different zones of cartilage during the growth of long bones. Visual inspection and grain counting revealed the highest levels of pro alpha 1(II) collagen mRNAs in chondrocytes of the lower proliferative and upper hypertrophic zones of the growth plate cartilage. This finding was confirmed by Northern blotting of RNAs isolated from epiphyseal (resting) cartilage and from growth zone cartilage. Analysis of the osseochondral junction revealed virtually no overlap between hybridization patterns obtained with probes specific for type I and type II collagen mRNAs. Only a fraction of the chondrocytes in the degenerative zone were recognized by the pro alpha 1(II) collagen cDNA probe, and none by the type I collagen cDNA probe. In the mineralizing zone virtually all cells were recognized by the type I collagen cDNA probe, but only very few scattered cells appeared to contain type II collagen mRNA. These data indicate that in situ hybridization is a valuable tool for identification of connective tissue cells which are actively producing different types of collagens at the various stages of development, differentiation, and growth.

Expression of mRNAs coding for the alpha 1 chain of type XIII collagen in human fetal tissues: comparison with expression of mRNAs for collagen types I, II, and III.

This paper describes the topographic distribution of the multiple mRNAs coding for a novel human short-chain collagen, the alpha 1 chain of type XIII collagen. To identify the tissues and cells expressing these mRNAs, human fetal tissues of 15-19 gestational wk were studied by Northern and in situ hybridizations. The distribution pattern of the type XIII collagen mRNAs was compared with that of fibrillar collagen types I, II, and III using specific human cDNA probes for each collagen type. Northern hybridization showed the bone, cartilage, intestine, skin, and striated muscle to contain mRNAs for type XIII collagen. An intense in situ hybridization signal was obtained with the type XIII collagen cDNAs in the epidermis, hair follicles, and nail root cells of the skin, whereas the fibrillar collagen mRNAs were detected in the dermis. Cells in the intestinal mucosal layer also appeared to contain high levels of alpha 1(XIII) collagen mRNAs, but contained none of the fibrillar collagen mRNAs. In the bone and striated muscle, alpha 1(XIII) collagen mRNAs were detected in the mesenchymal cells forming the reticulin fibers of the bone marrow and endomycium. The hybridization signal obtained with the alpha 1(XIII) collagen cDNA probe in cartilaginous areas of the growth plates was similar, but less intense, to that obtained with the type II collagen probe. A clear hybridization signal was also detected at the (pre)articular surfaces and at the margins of the epiphyses, whereas it was weaker in the resting chondrocytes in the middle of the epiphyses. The brain, heart, kidney, liver, lung, placenta, spleen, testis, tendon, and thymus did not appear to contain alpha 1(XIII) collagen mRNAs.

Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

The N-myc proto-oncogene and IGF-II growth factor mRNAs are expressed by distinct cells in human fetal kidney and brain.

We studied the expression of the N-myc proto-oncogene and the insulin-like growth factor-II (IGF-II) gene in human fetuses of 16-19 gestational wk. Both genes have specific roles in the growth and differentiation of embryonic tissues, such as the kidney and neural tissue. Since continued expression of N-myc and IGF-II mRNAs is also a characteristic feature of Wilms' tumor, a childhood neoplasm of probable fetal kidney origin, we were particularly interested in the possibility that their expression might be linked or coordinately regulated in the developing kidney. Expression of N-myc mRNA was observed in the brain and in the kidney by Northern hybridization analysis. In in situ hybridization of the kidney, N-myc autoradiographic grains were primarily located over epithelially differentiating mesenchyme while most of the mesenchymal stromal cells showed only a background signal with the N-myc probe. N-myc mRNA was detectable throughout the developing brain with a slight accentuation in the intermediate zone cells in between the subependymal and cortical layers. Thus, even postmitotic neuroepithelial cells of the fetal cerebrum expressed N-myc mRNA. In Northern hybridization, IGF-II mRNA signal was abundant in the kidney but much weaker, though definite, in the brain. The regional distribution of IGF-II mRNA in the kidney was largely complementary to that of N-myc. IGF-II autoradiographic grains were located predominantly over the stromal and blastemal cells with a relative lack of hybridization over the epithelial structures. In the brain, IGF-II mRNA was about two- to threefold more abundant in the subependymal and intermediate layers than in the cortical plate and ependymal zone, respectively. The fetal expression patterns of the N-myc and IGF-II mRNAs are reflected by the types of tumors known to express the corresponding genes during postnatal life such as Wilms' tumor. However, the apparent coexpression of the IGF-II and N-myc genes in immature kidneys occurs largely in distinct cell types.

Expression of the myc proto-oncogenes in developing human fetal brain.

We have analysed c-myc, N-myc and L-myc gene expression in developing human fetal brain by Northern hybridization, RNAase protection and in situ hybridization. The unique zonal organization of the developing fetal brain allows a particularly good assessment of the coupling of myc gene expression to cell proliferation and differentiation in vivo. By Northern and in situ hybridization, L-myc as well as c-myc and N-myc transcripts in the brain were found in the post-mitotic cortical and intermediate layers, as well as in the mitotically active layers containing the neuroepithelial precursor cells. Consistent results were also obtained for L-myc using RNAase protection analysis. Both the 3.6 and 3.8kb forms of the L-myc mRNA, resulting from alternative splicing of intron I, were detected in layers of neuroectodermal origin, but not in the meninges or choroid plexus. We also extended L-myc expression and splicing analyses to other developing human fetal tissues. L-myc mRNA was expressed in several other fetal tissues, particularly in fetal skin. Predominantly intron I containing L-myc mRNA was observed in fetal striated and cardiac muscle. Thus, L-myc is expressed in a wider spectrum of developing tissues than previously known. Our findings also, show that L-myc as well as N-myc and c-myc expression is uncoupled from cell division in developing brain.

Comparison on collagen gene expression in the developing chick embryo tendon and heart. Tissue and development time-dependent action of dexamethasone.

Glucocorticoids modulate various cellular functions such as proliferation, energy metabolism and the synthesis of proteins. In the present study, the response of collagen genes to dexamethasone in different stages of chick embryo development was studied in tendon and heart using Northern blot analysis and specific cDNA probes. The changes in collagen gene expression were compared to alterations in two reference mRNAs: actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of specific mRNAs measured per ribosomal RNA in tendon and heart varied markedly during normal development. In tendon the relative levels of alpha 1(I), alpha 2(I) and alpha 1(III) collagen mRNAs were highest between days 14-16 when also the synthesis of matrix proteins is most active. In heart the levels of these mRNAs peaked at day 12. In addition, qualitative differences were observed in the expression of actin genes between tendon and heart. Dexamethasone in high dose decreased collagen mRNA levels in tendons, while in heart a stimulatory effect was noted. Dexamethasone also decreased GAPDH mRNA levels in tendons. The alterations in gene expression after dexamethasone treatment in tendon and heart did not correlate with the level of specific glucocorticoid receptors, which varied markedly during the development of chick embryos. The cDNA for pro alpha 1(I) collagen hybridized to two transcripts corresponding to 6.2 and 5.1 kb in tendon and heart. During normal development of chick embryos the ratio of 6.2/5.1 kb mRNAs decreased markedly in heart, but no such change was observed in tendons. Dexamethasone, however, decreased the ratio of 6.2/5.1 kb transcripts in tendons. There was a significant correlation between the ratio 6.2/5.1 kb transcripts and total alpha 1(I) mRNA both in tendon and heart, suggesting that the 6.2 kb transcript may be associated with the rate of synthesis of type I collagen.

Interleukin-1 increases collagen production and mRNA levels in cultured skin fibroblasts.

In the present study we show that highly purified human interleukin-1 increases collagen production nearly 2-fold and mRNA levels of type I and III collagen over 2.5-fold in cultured normal human dermal fibroblasts. To minimize the effects of transient prostaglanding E2 production in fibroblasts treated with interleukin-1, the cell cultures were preincubated for 24 h before these measurements were made. The effects of interleukin-1 were also tested on scleroderma fibroblasts exhibiting increased collagen production. Although collagen synthesis was stimulated by interleukin-1 to some degree, the cells grown from both affected and unaffected skin areas were found to be relatively unresponsive to the effects of interleukin-1, suggesting a role for this monokine in the earlier stages of the disease process. The results also suggest that interleukin-1 has a role in stimulation of collagen synthesis under certain normal and pathological conditions in addition to stimulating fibroblast proliferation.

Analysis of aggrecan and tenascin gene expression in mouse skeletal tissues by northern and in situ hybridization using species specific cDNA probes.

Cartilage matrix is an interacting multicomponent system of collagen fibrils, fibril-associated small proteoglycans, and large proteoglycans and glycoproteins entrapped within the fibrillar network. In order to better understand the relationships between these different components we have constructed short cDNA clones for detection of mRNAs for two major noncollagenous macromolecules of cartilage matrix, aggrecan and tenascin. We subsequently determined their corresponding mRNA levels by Northern analysis in a panel of total RNAs isolated from several newborn mouse tissues. The expression of aggrecan was strictly restricted to cartilages while tenascin mRNA was present at variable levels in most of the tissues studied. The cDNA clones were also used to identify the cells responsible for aggrecan and tenascin production in newborn mouse tissues by in situ hybridization. With this technique aggrecan mRNA was detected in chondrocytes throughout the developing skeleton in a pattern very similar but not identical to those of type II and IX collagen mRNAs. In the newborn mouse skeleton tenascin and aggrecan mRNAs were expressed essentially in a mutually exclusive manner, tenascin transcripts being present in osteoblasts, periosteal and perichondrial cells, and in cells at articular surfaces. None of these cells expressed the cartilage specific collagen or aggrecan genes. The results further suggest different patterns of gene expression in chondrocytes based on their location in the different cartilages.

Growth-dependent modulation of type I collagen production and mRNA levels in cultured human skin fibroblasts.

Five human skin fibroblast lines were studied for type I collagen production and type I procollagen mRNA levels through the different growth phases. The cells were plated at low density and followed for 11 days at daily intervals through the stages of rapid growth and visual confluency until the cultures reached stationary growth phase. Each day one culture flask was labeled with [3H]proline for 24 h, and analyzed for production of radiolabeled type I collagen into culture medium. The cell layers were counted and subjected to isolation of cytoplasmic RNA and determination of type I procollagen mRNA levels. The results revealed an approx. 2-fold increase in procollagen production and mRNA levels when the cells reached visual confluency. Thereafter the synthesis rates and mRNA levels remained relatively constant, although a decreasing tendency of both parameters was observed upon further culturing. The results confirm that determination of cell density is important when cell cultures are used for measurement of collagen synthesis or mRNA levels. For determination of pro alpha 2(I) collagen mRNA an 1193 bp cDNA clone was constructed using RNA extracted from human fetal calvaria. Sequencing of the clone revealed some nucleotide and amino acid differences between the previously published sequences. This suggests the presence of more individual variation in procollagen coding sequences than expected.

Cloning of cDNA for rat pro alpha 1(III) collagen mRNA. Different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue.

A cDNA clone for rat pro alpha 1(III) collagen mRNA was isolated from a cDNA library constructed for poly(A)+ RNA from 15-day experimental granulation tissue. Two clones, pRGR1 and pRGR5, were characterized by restriction mapping and sequencing. Comparison with human type III collagen sequences revealed 92% identity at the level of translated amino acids, and 88% identity at nucleotide level in the coding region. In the 3'-untranslated sequence the identity was even higher (90%). The clones were used together with cDNA clones for type I collagen chains, fibronectin and gamma-actin to study the expression of the corresponding mRNAs during the development of experimental sponge-induced granulation tissue in rats. These studies revealed a marked activation of type I and type III collagen genes during the second week of granuloma development followed by a transient reduction in their levels during the third week. The mRNA levels for both collagen types remained relatively unchanged from day 25. The molar ratio of pro alpha 1(III) and pro alpha 1(I) collagen mRNAs was at a maximum on day 6, and then decreased to reach a plateau by the end of the third week. Fibronectin mRNA levels were found to increase slower; the maximum value was reached during the fifth week of granuloma development. The mRNA levels of gamma-actin increased continuously up to the end of the fourth week, thus following the cellular maturation of the tissue.

Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs.

We have constructed DNA probes for the specific detection of mouse pro alpha 1(I), pro alpha 1(II), pro alpha 1(III) and alpha 1(IX) collagen transcripts. To avoid cross-hybridization the probes for fibrillar collagens cover mainly sequences in the 3' untranslated region of the gene. Sequencing and Northern analysis confirmed that the clones share minimal sequence similarity and detect only the specific mRNAs under normal hybridization and washing conditions. The clone for mouse alpha 1(IX) collagen covers coding sequences but is sufficiently divergent from other collagen transcripts to allow specific detection of the corresponding mRNA.

Interferon-alpha and interferon-gamma reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture.

The effects of interferon-alpha and interferon-gamma on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-gamma treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-gamma reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-alpha on procollagen mRNA levels was somewhat weaker than that of interferon-gamma. The results suggest potential use of interferon-gamma in treatment and prevention of human fibrotic conditions.

Cathepsin expression during skeletal development.

Cysteine proteinases, cathepsins B, H, K, L and S, have been implicated in several proteolytic processes during development, growth, remodeling and aging, as well as in a variety of pathological processes. For systematic analysis of cathepsin gene expression we have produced cDNA clones for mouse and human cysteine cathepsins. Northern analysis of a panel of total RNAs isolated from 16-19 different human and mouse tissues revealed the presence of mRNAs for cathepsin B, H, K, L and S in most tissues, but each with a distinct profile. Of the different cathepsin mRNAs, those for cathepsin K were clearly the highest in bone and cartilage. However, relatively high mRNA levels for the other cathepsins were also present in these tissues. To better understand the roles of different cathepsins during endochondral ossification in mouse long bones, cathepsin mRNAs were localized by in situ hybridization. Cathepsin K mRNAs were predominantly seen in multinucleated chondroclastic and osteoclastic cells at the osteochondral junction and on the surface of bone spicules. The other cathepsin mRNAs were also seen in osteoclasts, and in hypertrophic and proliferating chondrocytes. These observations were confirmed by immunohistochemistry and suggest that all cysteine cathepsins are involved in matrix degradation during endochondral ossification.

Increased type I collagen mRNA levels in cultured scleroderma fibroblasts.

Pro alpha 1(I)collagen mRNA levels in fibroblasts cultured from affected and non-affected skin areas of two scleroderma patients were measured by hybridization of RNA blots with a specific cDNA clone. Collagen synthesis was estimated with an inhibition ELISA for type I collagen and with densitometric scans of fluorograms of [3H]proline-labelled medium proteins. Affected scleroderma fibroblasts exhibited 2-7-fold higher levels of pro alpha 1(I)collagen mRNAs to account for the increased synthesis of collagen by the same cells. This suggests that the control of collagen synthesis in scleroderma is altered at transcriptional level.

Specific hybridization probes for mouse alpha 2(IX) and alpha 1(X) collagen mRNAs.

We have used polymerase chain reaction (PCR) technology and available cross-species sequence information to construct cDNA probes for mouse alpha 2(IX) and alpha 1(X) collagen transcripts. Sequencing confirmed the identification of the clones. Northern analysis proved sufficient divergence of the cloned sequences from other collagen transcripts: specific detection of the mouse 2.9 kb alpha 2(IX) and 3.3 kb alpha 1(X) collagen mRNAs was seen under normal hybridization and washing conditions.

Bone defect repair in immobilization-induced osteopenia: a pQCT, biomechanical, and molecular biologic study in the mouse femur.

The present study was carried out to determine whether immobilization-induced (Im) osteopenic bone possesses the same reparative capacity as normal healthy bone. Furthermore, the effects of mechanical loading versus immobilization on bone defect healing were studied. Three-week cast-immobilization was used to induce local osteopenia in mice. A standardized metaphyseal bone defect of the distal femur was created unilaterally both in immobilization-induced (Im) osteopenic mice and in nonimmobilized (Mo) age-matched control animals. After creation of the bone defect, the animals in both groups were further divided into two groups: 3-week cast-immobilization (Im-Im and Mo-Im) groups, and unrestricted weight-bearing (Im-Mo and Mo-Mo) groups. The healing process was followed up to 3 weeks using RNA analysis, histomorphometry, biomechanical testing, and pQCT measurements. At 3 weeks of healing without immobilization, bone mineral density (BMD), as well as bone bending stiffness and strength were higher in normal (Mo-Mo) than in osteopenic (Im-Mo) bone. Although the levels of mRNAs characteristic to chondrocytes (Sox9 and type II collagen), hypertrophic chondrocytes (Type X collagen), osteoblasts (type I collagen and osteocalcin), and osteoclasts (cathepsin K) during the bone defect healing exhibited similarities in their expression profiles, mechanical loading conditions also caused characteristic differences. Mechanical loading during healing (Mo-Mo group) induced stronger expression of cartilage- and bone-specific genes and resulted in higher BMD than that seen in the cast-immobilized group (Mo-Im). In biomechanical analysis, increased bending stiffness and strength were also observed in animals that were allowed weight-bearing during healing. Thus, our study shows that bone healing follows the same molecular pathway both in osteopenic and normal bones and presents evidence for reduced or delayed regeneration of noncritical size defects in immobilization-induced osteopenic bone.

Combined effect of BMP-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation.

Adenovirus-mediated recombinant human BMP-2 (RAdBMP-2) gene transfer has been found to have significant osteoinductive properties. The hypothesis of the current study was that bioactive glass surface could provide favorable osteoconductive conditions for cellular action of osteoinductive RAdBMP-2 gene transfer. In the rat proximal tibia, a portion of the medullary cavity was evacuated and filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (BG/RAdBMP-2). Control defects filled with BG microspheres were injected with adenovirus carrying the LacZ reporter gene (BG/RAdLacZ) or saline (BG). Empty control defects were also used. Bone healing response was analyzed at 4 days, and at 2 and 8 weeks by radiography, peripheral quantitative computed tomography (pQCT), histomorphometry, and backscattered electron imaging of scanning electron microscopy (BEI-SEM) equipped with energy dispersive X-ray analysis (EDXA). In empty controls, the amount of intramedullary new bone peaked at 2 weeks, whereas defects filled with bioactive glass with and without RAdBMP-2 gene transfer showed a constant time-related increase of intramedullary new bone. At 8 weeks, there was significantly more new bone in defects treated with BG and RAdBMP-2 than in defects left to heal without filling (p < 0.001). Compared with the other controls (BG only or BG/RAdLacZ), the difference was not significant. In the current model, the osteopromotive effect of bioactive glass microspheres appears synergistic with the osteoinductive action of BMP-2 gene transfer, or one overshadows the other, as no additive effect was observed.

Expression profiles of mRNAs for osteoblast and osteoclast proteins as indicators of bone loss in mouse immobilization osteopenia model.

An experimental mouse model for disuse osteopenia was developed using unilateral cast immobilization. Analysis of the distal femurs and proximal tibias by quantitative histomorphometry revealed significant osteopenia within 10-21 days of immobilization. At 3 weeks, bone loss was also demonstrated with peripheral quantitative computed tomography as diminished bone mineral content and as concomitant reduction in the cross-sectional moment of inertia. These structural and geometrical alterations resulted in decreased strength of the distal femurs tested by cantilever bending. Analysis of the underlying cellular and molecular mechanisms of bone loss revealed a rapid increase in bone resorption within 3 days of immobilization. The mRNA levels for cathepsin K, matrix metalloproteinase-9, and tartrate resistant acid phosphatase were all significantly increased during the 21-day immobilization period, but with different expression profiles. These increases were paralleled by an increased number of osteoclasts as measured by histomorphometry. By day 6 of immobilization, the balance of bone turnover was further shifted toward net bone loss as the mRNA levels for major bone components (type I collagen and osteocalcin) were decreased. In histomorphometric analysis this was observed as reduced rates of mineral apposition and bone formation after 10 days of immobilization. The results of this study demonstrate that immobilization has a dual negative effect on bone turnover involving both depressed bone formation and enhanced bone resorption.