Diffusion model to describe osteogenesis within a porous titanium scaffold.

In this study, we develop a two-dimensional finite element model, which is derived from an animal experiment and allows simulating osteogenesis within a porous titanium scaffold implanted in ewe's hemi-mandible during 12 weeks. The cell activity is described through diffusion equations and regulated by the stress state of the structure. We compare our model to (i) histological observations and (ii) experimental data obtained from a mechanical test done on sacrificed animal. We show that our mechano-biological approach provides consistent numerical results and constitutes a useful tool to predict osteogenesis pattern.

Skeletal unloading induces osteoblast apoptosis and targets alpha5beta1-PI3K-Bcl-2 signaling in rat bone.

The mechanisms underlying the altered osteoblastogenesis and bone loss in response to disuse are incompletely understood. Using the rat tail suspension model, we studied the effect of skeletal unloading on osteoblast and osteocyte apoptosis. Tail suspension for 2 to 7 days decreased tibial bone mass and induced early apoptotic loss of osteoblasts and delayed apoptotic loss of osteocytes. Surrenal gland weight and plasma corticosterone levels did not differ in loaded and unloaded rats at any time point, indicating that osteoblast/osteocyte apoptosis occurred independently of endogenous glucocorticoids. The mechanistic basis for the disuse-induced osteoblast/osteocyte apoptosis was examined. We found that alpha5beta1 integrin and phosphorylated phosphatidyl-inositol-3 kinase (p-PI3K) protein levels were transiently decreased in unloaded metaphyseal long bone compared to loaded bones. In contrast, p-FAK and p-ERK p42/44 levels were not significantly altered. Interestingly, the reduced p-PI3K levels in unloaded long bone was associated with decreased levels of the survival protein Bcl-2 with unaltered Bax levels, causing increased Bax/Bcl-2 levels. The results indicate that skeletal unloading in rats induces a glucocorticoid-independent, immediate increase in osteoblast apoptosis associated with decreased alpha5beta1-PI3K-Bcl-2 survival pathway in rat bone, which may contribute to the altered osteoblastogenesis and osteopenia induced by unloading.

Transforming growth factor-beta inhibits CCAAT/enhancer-binding protein expression and PPARgamma activity in unloaded bone marrow stromal cells.

The molecular mechanisms regulating the adipogenic differentiation of bone marrow stromal cells in vivo remain largely unknown. In this study, we investigated the regulatory effects of transforming growth factor beta-2 (TGF-beta2) on transcription factors involved in adipogenic differentiation induced by hind limb suspension in rat bone marrow stromal cells in vivo. Time course real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis of gene expression showed that skeletal unloading progressively increases the expression of CCAAT/enhancer-binding protein (C/EBP)alpha and C/EBPbeta alpha at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor gamma (PPARgamma2) transcripts at 7 days. TGF-beta2 administration in unloaded rats corrected the rise in C/EBPalpha and C/EBPbeta transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPARgamma2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBPalpha and C/EBPbeta expression by TGF-beta2 was associated with increased PPARgamma serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPARgamma transactivating activity. The sequential inhibitory effect of TGF-beta2 on C/EBPalpha, C/EBPbeta, and PPARgamma2 resulted in reduced LPL expression and abolition of bone marrow stromal cell adipogenic differentiation, which contributed to prevent bone loss induced by skeletal unloading. We conclude that TGF-beta2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBPalpha, C/EBPbeta, and PPARgamma expression and activity, which provides a sequential mechanism by which TGF-beta2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

Protracted systemic changes in bone biology after segmented unloading in the rat.

To investigate whether the decreased bone formation observed in most experimental situations of disuse was caused by an increased inhibition by the bone microenvironment of osteoblast (OB) proliferation, we studied the inhibiting power on ROS 17/2.8 proliferation of the bone marrow extracellular fluid (IPEF) in loaded and unloaded bones of rats submitted to two situations of partial disuse: tail suspension (TS) for 3 days to 2 weeks and around the knee tenectomy (KT) for 2-10 weeks. Histomorphometric parameters and osteoblast precursors dynamics were studied in parallel. Bone volume was lost in the unloaded bones, but not in loaded bones, in both experimental situations. Bone formation was low at early times (7-14 days) in TS rats. However, in KT at later times (4-10 weeks), the osteoblastic index of the unloaded tibia was increased. IPEF was not increased in the unloaded bones 3-7 days after TS. It was decreased later in the course of unloading (after 2 weeks of TS and 2-10 weeks after KT). This decrease was observed in the loaded bones as well. Unexpectedly, we also found that the number of FCFUs was decreased in both loaded and unloaded limbs in TS and KT, and that the yield of cells obtained in primary culture from tibial metaphysis was decreased in both tibiae from KT animals. These data show that an increased IPEF does not play a role in the early inhibition of bone formation responsible for the loss of bone after unloading in the TS model. Its later decrease could be permissive for the increased osteoblastic index observed in the KT model. They also show that, contrary to the usual assumptions, bone biology is changed all over the skeleton after partial unloading, even if the changes result in bone loss in the unloaded bones only. Thus, as yet, unidentified systemic factors probably superimpose on the local factors that control bone volume.

Bone mass increases in less than 4 wk of voluntary exercising in growing rats.

INTRODUCTION: Physical exercise is known to augment bone mass, but the specific mechanisms by which physical activity influences skeletal metabolism is still not thoroughly understood. So far, time related events of bone tissue adaptation to physical training have not been investigated. We, therefore, studied the time-course effects of daily spontaneous exercise training on bone metabolism in the rat by biochemical and tissue analyses. METHODS: Forty-five 4-wk-old female Dark Agouti rats were used, randomly assigned to voluntary exercising groups of five animals for 1, 2, 3, and 4 wk, or to age-matched sedentary controls. At sacrifice, blood was sampled for determination of parameters of bone and calcium metabolism (osteocalcin, alkaline phosphatases, total and ionized calcium, phosphorus, parathyroid hormone). Right and left tibiae were removed for bone mass (dry and ash mass, mineral content) and histomorphometric analyses. RESULTS: We found that the animals performed large amounts of exercise (reaching 50 +/- 8 km x wk(-1)), and that this voluntary physical training induced significant changes in bone metabolism. An increase (approximately 32%) in serum alkaline phosphatase activity was already measurable at the end of the first week of exercising and persisted throughout the experiment, accompanied by a concomitant increase in osteoblastic bone formation (approximately 27%), as evaluated in bone tissue. These changes were associated with consecutive increases in tibial bone mass (approximately 7.6%), mineral content (approximately 7.4%) and metaphyseal bone volume (approximately 17%) measurable 4 wk after the onset of exercise, with interesting positive correlations between tibial bone mass parameters and soleus muscle mass. Conversely, no change was evidenced in biochemical parameters of calcium metabolism, except for an increase in phosphorus serum levels in trained animals. CONCLUSION: Daily spontaneous exercise training induced an increase in bone mass and bone volume in less than 4 wk. Further studies are needed to fully investigate these changes during the very first days of exercising.

Spaceflight inhibits bone formation independent of corticosteroid status in growing rats.

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (-12%; p < 0.05) associated with approximately 50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (-42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.

Skeletal unloading induces biphasic changes in insulin-like growth factor-I mRNA levels and osteoblast activity.

To determine the local mechanisms involved in the effects of skeletal unloading on bone formation, we studied the temporal pattern of mRNA levels for insulin-like growth factor-I (IGF-I), IGF-I receptor type I (IGF-IR), and transforming growth factor beta receptor type II (TGF-betaRII) in relation to osteoblast phenotypic markers and osteoblast activity in hindlimb suspended rats. Skeletal unloading decreased bone volume and the mineralizing and osteoblastic surfaces at 4, 7, and 14 days in the tibial metaphysis, whereas the mineral appositional rate returned to normal at 14 days of suspension. RT-PCR analysis showed that skeletal unloading decreased type 1 collagen (Col 1) and osteocalcin (OC) mRNA levels in metaphyseal bone at days 4 and 7, and the levels returned to normal at 14 days of suspension. Unloading also decreased mRNA levels for IGF-I, IGF-IR, and TGF-betaRII at 4-7 days in the metaphyseal bone. However, IGF-I and IGF-IR levels rose above normal at 14 days of suspension. The biphasic changes in IGF-I mRNA levels were strongly correlated with Col 1 and OC mRNA levels. The associated biphasic pattern of IGF-I/IGF-IR expression, osteoblast markers, and osteoblast activity strongly suggests an important role for IGF-I signaling in the local effect of skeletal unloading on metaphyseal bone formation.

Decreased serum levels of 1,25-(OH)2 vitamin D during 1 year of sunlight deprivation in the Antarctic.

French Antarctic territories harbor bases that are devoted to scientific and technical work. Living and working conditions during 1-year sojourns in such an environment are quite acceptable, but the confinement and the drop in ultraviolet B radiation exposure during winter months raise the problem of preservation of normal vitamin D status. Seasonal variations in 25-hydroxyvitamin D [25(OH)D] levels have been well documented, but the effect of sunshine deprivation on 1,25 dihydroxyvitamin D [1,25(OH)2D] levels is quite controversial. The aim of this study was to address this question under the exceptional conditions of lack of sunshine exposure. Fifteen male Caucasian subjects participating in a 1-year mission in Antarctica were investigated. They were subjected to seven blood samplings, one before and six during their sojourn. Serum levels of 25(OH)D, 1,25(OH)2D, osteocalcin, and ICTP were measured. We found that levels of 25(OH)D and 1,25(OH)2D significantly decreased in these subjects during the mission, minimum levels being observed 10 months after their departure from France. ICTP concentrations did not change throughout this study, but osteocalcin levels were found to be higher at the end of the sojourn than before departure, which could argue for the existence of bone remodeling changes. Further studies are now needed to fully investigate bone metabolism changes and to address the question of vitamin D supplementation during this kind of sojourn.

Calcium-regulating hormones, bone mineral content, breaking load and trabecular remodeling are altered in growing pigs fed calcium-deficient diets.

Studies on calcium nutrition in appropriate large animal models can be directly relevant to humans. We have examined the effect of dietary Ca deficiency on various bone and bone-related variables, including plasma markers, histomorphometry, mineral content and breaking strength in pigs. Three groups of eight 38-d-old female pigs were fed adequate (0.9%; control), low (0.4%; LCa) or very low (0.1%; VLCa) Ca diets for 32 d. Plasma Ca significantly decreased over time only in the VLCa-deficient pigs. The concentrations of the parathyroid hormones (PTH) and calcitriol increased as Ca deficiency developed, and the plasma PTH and calcitriol levels varied inversely with dietary Ca. The total bone ash contents, bending moments, trabecular bone volume and the mineral apposition rate all decreased as the calcium intake decreased. The osteoclast surface areas were greater than those of controls in both Ca-deficient groups, whereas the osteoblast surface areas were greater only in the VLCa group. The plasma osteoblast-related markers (alkaline phosphatase, carboxy-terminal propeptide of type I procollagen and osteocalcin) were either greater or unaffected in the Ca-deficient pigs. The results indicate that deficient bone mineralization combined with an increased bone resorption led to bone loss and fragility. The differences in the changes in bone cells (number and activity) between LCa and VLCa groups might be due to differences (time and extent) of circulating PTH and calcitriol. The defective mineralization in both Ca-depleted groups resulted mainly from the lack of Ca because their osteoblast activity was either maintained or stimulated. The results also underline the progressive sensitivity of pigs to Ca supply and the usefulness of this model.

Effects of BMP-2 on osteoblastic cells and on skeletal growth and bone formation in unloaded rats.

A previous study showed that skeletal unloading induced by hindlimb suspension for 14 days in rats reduces osteoblastic cell proliferation, inhibits skeletal growth and bone formation and induces metaphyseal bone loss. This study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in this model. In vitro analysis showed that rhBMP-2 (25-100 ng/ml, 48-96 h) increased alkaline phosphatase activity, an early marker of osteoblast differentiation, in rat neonatal calvaria cells and adult marrow stromal cells, showing that rhBMP-2 induced the differentiation of osteoblast precursor cells in vitro. In contrast, rhBMP-2 did not increase rat calvaria or marrow stromal cell proliferation. Biochemical and histomorphometric analysis showed that systemic infusion with rhBMP-2 (2 microg/kg/day) in unloaded rats had no significant effect on serum osteocalcin levels and on histomorphometric indices of bone formation. Accordingly, rhBMP-2 infusion did not prevent the decreased skeletal growth, trabecular bone bone volume and bone mineral content induced by unloading. The present data indicate that, although rhBMP-2 stimulates osteoblastic cell differentiation, rhBMP-2 infusion is not effective in increasing bone formation and in preventing trabecular bone loss induced by unloading in rats.

Effect of endurance training on postexercise parathyroid hormone levels in elderly men.

The present study was designed to evaluate the effects of 6-wk endurance training on serum parathyroid hormone (PTH) levels and on other parameters at rest and after a maximal exercise test (MET) in 24 55- to 73-yr-old men. Before training, MET was found to induce a significant increase in PTH levels as compared with resting values. This MET-induced rise in PTH was accompanied by enhanced total calcium, phosphate, alkaline phosphatase (ALP), osteocalcin, and albumin levels. After the training period (75-80% maximal heart rate, 1 h.d-1, 4 d.wk-1), the changes induced by MET in calcium, phosphate, ALP, and albumin levels followed the same pattern as before training. Conversely, the MET-induced increase in PTH levels was found markedly more pronounced after training than in untrained conditions (+21.9% vs +11.1%, respectively, P < 0.05). Furthermore, lower values of osteocalcin were found after training as compared with pretraining values, both at rest and after maximal exercise. These findings indicate that 6 wk of endurance training enhanced exercise-related release of PTH and reduced osteocalcin levels in elderly men. This might be of importance regarding bone status in the elderly, as exercise is proposed as a preventive measure against osteopenia.

TGF-beta2 prevents the impaired chondrocyte proliferation induced by unloading in growth plates of young rats.

Growth plate width and cartilage organization are altered during skeletal unloading in growing rats. Immunohistochemical studies have identified TGF-beta in calcified cartilage, and TGF-beta is known to induce mitogenic effects on chondrocytes in vitro. On the other hand, IGF-1 was shown to be expressed in the proximal tibial growth plate and to mediate GH-induced longitudinal bone growth in rats. We therefore investigated the effect of recombinant human (rh) IGF-1 and rhTGF-beta2 infusion on the changes induced by unloading in the cellular organization of the growth plate in growing rats. Hindlimb unloading for 14 days induced a 13% reduction in growth cartilage height in the proximal tibia. This effect was mostly related to a 17% and 14% decrease in the proliferative zone height and chondrocyte number, respectively. In unloaded rats treated with a systemic infusion of rhTGF-beta2 (2microg/kg/day) the number of chondrocytes in the proliferative zone was not different from those of normal loaded animals. In contrast, rhIGF-1 treatment at a 2mg/kg/day dose was not effective in counteracting the effects of unloading on growth plate height and chondrocyte number. These results show that systemic administration of rhTGF-beta2 prevents in large part the reduced growth of chondrocytes in the proliferative zone and the reduced epiphyseal growth plate growth induced by unloading in rats.

Effects of spaceflight and recovery on rat humeri and vertebrae: histological and cell culture studies.

Skeletal changes associated with spaceflight in the rat have been well documented, but few data are available on bone tissue and bone cell metabolism after subsequent on-Earth recovery. We therefore investigated the effects of microgravity and subsequent recovery on trabecular bone morphology and cellular activities in rat humeri and thoracic vertebrae and compared histomorphometric parameters in caudal vertebrae with the behavior of vertebral osteoblastic cells in culture. We report here that humeral weight showed normal growth during the experiment but was unaffected by spaceflight or recovery from spaceflight. However, the 14-day spaceflight resulted in inhibition of static indexes of bone formation in humeral proximal metaphyses and thoracic vertebral bodies. This was associated with a decrease in bone volume in humeral metaphyses. After 14 days of on-Earth recovery, osteoblastic and osteoid surfaces returned toward normal and bone volume was normalized in humeri, whereas the static bone formation parameters were not restored in thoracic vertebrae. In addition, histological indexes of bone formation and osteoblastic cell growth in vitro were not affected by spaceflight in caudal vertebrae. This study shows that rat humeri and thoracic and caudal vertebrae exhibit different patterns of response to spaceflight and subsequent on-Earth recovery, which could be due, at least in part, to the different loading pattern of these bones, and also to differences in bone turnover rate.

[The suspension device for hindlimb unloading in the rat--results of a video monitoring study]. / Le dispositif de suspension et de privation d'appui chez le rat--resultat d'une surveillance video.

Several physiological modifications reported in rats after spaceflight have been shown to be mimicked by an experimental procedure which permits skeletal unloading of the animal. Therefore, the model of hindlimb suspension is used by many authors interested in spaceflight and in hypokinesia studies. The aim of our work was to describe this model and propose a new device for headdown suspension and hindlimb unloading purposes. Using a video monitoring system, we observed that animals adapt to their new situation. Indeed, we have verified that the apparatus allows most of animals' functions. The growth curve which was not significantly modified, confirmed the present observations showing that rat feeding activity seems to not be altered. The rat is also able to move in the cage, to rest and sleep, to groom. Moreover, most of problems encountered during previous experiments have been understood and a new device with the same principles, but protected against disfunction, is proposed.

Electrical stimulation of leg muscles increases tibial trabecular bone formation in unloaded rats.

Rat head-down hindlimb suspension (HS) has been shown to induce hindlimb cancellous bone loss. As HS is known to associate unloading with progressive disappearance of hindlimb muscle contractions, we investigated whether persisting muscle motion could modify suspension-induced bone disorders or even prevent them. Chronic electrical stimulation (ES) was applied to leg muscles of rats during 3-wk hindlimb suspension, the lack of support for hindlimbs maintaining a hypodynamic situation. The histomorphometric characteristics of the proximal tibial metaphysis were analyzed. At the end of this protocol of combined suspension and stimulation, trabecular bone loss remained similar to that of nonstimulated HS animals. However, trabecular bone cell activity parameters showed greater bone formation after muscle stimulation in unloaded animals, with significantly increased osteoblastic, osteoid, and mineralizing surfaces. In addition, periosteal mineral apposition rate and cancellous bone formation rate, markedly decreased by suspension, were not significantly different in suspended stimulated compared with normal loaded animals. This enhanced formation activity could be related to persistence of muscle activity, as shown by partial preservation of muscle mass. However, direct electrical effects on bone cannot be excluded. Thus, despite muscle stimulation, with enhanced bone formation, isolated suppression of hypokinesia has not been able to counteract bone effects of unloading. This finding supports the hypothesis of the importance of mechanical loading to maintain bone architecture.

Systemic administration of transforming growth factor-beta 2 prevents the impaired bone formation and osteopenia induced by unloading in rats.

We investigated the effect of recombinant human transforming growth factor beta 2 (rhTGF-beta 2) administration on trabecular bone loss induced by unloading in rats. Hind limb suspension for 14 d inhibited bone formation and induced osteopenia as shown by decreased bone volume, calcium and protein contents in long bone metaphysis. Systemic infusion of rhTFG-beta 2 (2 micrograms/kg per day) maintained normal bone formation rate, and prevented the decrease in bone volume, bone mineral content, trabecular thickness and number induced by unloading. In vitro analysis of tibial marrow stromal cells showed that rhTGF-beta 2 infusion in unloaded rats increased the proliferation of osteoblast precursor cells, but did not affect alkaline phosphatase activity or osteocalcin production. Northern blot analysis of RNA extracted from the femoral metaphysis showed that rhTGF-beta 2 infusion in unloaded rats increased steady-state levels of type I collagen mRNA but not alkaline phosphatase mRNA levels. rhTGF-beta 2 infusion at the dose used had no effect on metaphyseal bone volume and formation, osteoblast proliferation or collagen expression in control rats. The results show that systemic administration of rhTGF-beta 2 enhances osteoblast precursor cell proliferation and type I collagen expression by osteoblasts, and prevents the impaired bone formation and osteopenia induced by unloading.

Changes in rat atrial ANF granules induced by hindlimb suspension.

It is well known that the heart releases a factor called ANF (atrial natriuretic factor) or ANP (atrial natriuretic peptide) capable of inducing rapid diuretic and natriuretic actions. This factor is stored in secretory granules mainly located in myocytes in both atria. The main secretory stimulus is the distention of the atrial cavity resulting, for example, from enhanced venous return. However, the cellular events which occur after the stimulation remain to be clarified. The aim of this investigation was to study the intra-cellular events preceding the ANF release, using the rat hindlimb suspension as model of stimulation. In this model, Wistar rats were placed in a 30 degrees anti-orthostatic position and a blood shift towards the heart was obtained. Different durations (1/4 h, 1/2 h, 3/4 h, 1 h, 2 h and 6 h) were studied. The ANF plasma level was investigated by Radio Immuno Assay and granule immunoreactivity was measured by counting gold particles on micrographs. The ANF plasma level was significantly increased (+60%) after 1 h of suspension. The response was transient and then decreased to basal values. Morphological criteria established at the beginning of this study, and measured throughout the experiment, were found transiently modified after suspension. The surface of the perinuclear area was transitory enlarged by 36% 30 min after suspension. Moreover, in the same time immunoreactivity of the secretory granules was enhanced without changes in granule size. These results suggest an increase in the ANF synthesis and storage in the granules during the stimulation. However, the cellular regulatory mechanism of the ANF synthesis which could explain the transitory aspect of these events, requires further investigation.

Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats.

We previously found that the inhibition of bone formation and trabecular osteopenia induced by skeletal unloading in rats are associated with reduced proliferation of osteoblastic cells lining the bone surface. In this study, we examined the effects of insulin-like growth factor-I (IGF-I) on trabecular bone formation, bone mineral density, and proliferation of marrow-derived osteoblastic cells in unloaded rats. Skeletal unloading of hind limbs was induced by tail suspension, and recombinant human IGF-I was administered at two different doses (1.3 or 2.0 mg/kg.day) in control and unloaded rats by continuous infusion for 14 days. Treatment with IGF-I had no effect on plasma glucose levels, body weight, or longitudinal bone growth. The double calcein-labeled surface, bone formation rate, and trabecular number measured at the tibial metaphysis were lower in unloaded rats compared to controls and were increased after IGF-I treatment. The increased number of bone-forming sites induced by IGF-I was associated with partial prevention of trabecular bone loss in unloaded rats. In contrast to the beneficial effects of IGF-I on bone formation and bone mineral content in unloaded rats, IGF-I had no effect in control rats. To evaluate the cellular mechanisms of action of IGF-I, marrow stromal cells were derived from the tibia of unloaded and control rats and studied in vitro. Unloading was associated with a decreased proliferation of alkaline phosphatase-positive (ALP+) marrow stromal cells. Treatment with IGF-I increased the number of ALP+ cells in unloaded rats, but not in control rats. IGF-I treatment increased ALP activity and osteocalcin production by marrow-derived cells in suspended and control rats, suggesting that IGF-I stimulated the proliferation and differentiation of osteoblast precursor cells. These results indicate that IGF-I infusion enhanced the recruitment of osteoblastic cells, increased trabecular bone formation, and partially prevented trabecular bone loss in unloaded rats, which supports the hypothesis that IGF-I may mediate in part the effects of loading on bone formation.

Skeletal unloading in rat decreases proliferation of rat bone and marrow-derived osteoblastic cells.

The effects of skeletal unloading on osteoblastic cells were evaluated in tail-suspended rats. Hindlimb elevation for 14 days induced osteopenia, decreased histomorphometric indexes of bone formation in tibial metaphysis, and reduced plasma osteocalcin and alkaline phosphatase (ALP) levels compared with controls. The in vitro proliferation of osteoblastic cells isolated from the endosteal bone surface of suspended tibias was decreased by 42 and 31% at 2 and 4 days of culture, respectively, compared with controls, as shown by [3H]thymidine labeling and cell number. The proliferation of ALP-positive marrow stromal cells was also decreased by 20-24% at 1 and 2 days of culture. However, ALP activity in bone-derived cells and marrow stromal cells was not different in unloaded and control rats, and the number of bone cells synthesizing osteocalcin, osteonectin, and type I or type III collagen was identical in the two groups. The results indicate that the inhibition of bone formation induced by skeletal unloading is related to a decreased proliferation of putative osteoblast precursor cells present along the endosteal bone surface and in the marrow stroma.