The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK −/− mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK −/− mice also exhibited a marked deficiency of B cells in the spleen. RANK −/− mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

Although the skeleton's adaptability to load-bearing has been recognized for over a century, the specific mechanical components responsible for strengthening it have not been identified. Here we show that after mechanically stimulating the hindlimbs of adult sheep on a daily basis for a year with 20-minute bursts of very-low-magnitude, high-frequency vibration, the density of the spongy (trabecular) bone in the proximal femur is significantly increased (by 34.2%) compared to controls. As the strain levels generated by this treatment are three orders of magnitude below those that damage bone tissue, this anabolic, non-invasive stimulus may have potential for treating skeletal conditions such as osteoporosis.

Thrombospondin (TSP) 2, and its close relative TSP1, are extracellular proteins whose functions are complex, poorly understood, and controversial. In an attempt to determine the function of TSP2, we disrupted the Thbs2 gene by homologous recombination in embryonic stem cells, and generated TSP2-null mice by blastocyst injection and appropriate breeding of mutant animals. Thbs2−/− mice were produced with the expected Mendelian frequency, appeared overtly normal, and were fertile. However, on closer examination, these mice displayed a wide variety of abnormalities. Collagen fiber patterns in skin were disordered, and abnormally large fibrils with irregular contours were observed by electron microscopy in both skin and tendon. As a functional correlate of these findings, the skin was fragile and had reduced tensile strength, and the tail was unusually flexible. Mutant skin fibroblasts were defective in attachment to a substratum. An increase in total density and in cortical thickness of long bones was documented by histology and quantitative computer tomography. Mutant mice also manifested an abnormal bleeding time, and histologic surveys of mouse tissues, stained with an antibody to von Willebrand factor, showed a significant increase in blood vessels. The basis for the unusual phenotype of the TSP2-null mouse could derive from the structural role that TSP2 might play in collagen fibrillogenesis in skin and tendon. However, it seems likely that some of the diverse manifestations of this genetic disorder result from the ability of TSP2 to modulate the cell surface properties of mesenchymal cells, and thus, to affect cell functions such as adhesion and migration.

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The means by which muscle function modulates bone homeostasis is poorly understood. To begin to address this issue, we have developed a novel murine model of unilateral transient hindlimb muscle paralysis using botulinum toxin A (Botox). Female C57BL/6 mice (16 weeks) received IM injections of either saline or Botox (n = 10 each) in both the quadriceps and calf muscles of the right hindleg. Gait dysfunction was assessed by multi-observer inventory, muscle alterations were determined by wet mass, and bone alterations were assessed by micro-CT imaging at the distal femur, proximal tibia, and tibia mid-diaphysis. Profound degradation of both muscle and bone was observed within 21 days despite significant restoration of weight bearing function by 14 days. The muscle mass of the injected quadriceps and calf muscles was diminished −47.3% and −59.7%, respectively, vs. saline mice (both P < 0.001). The ratio of bone volume to tissue volume (BV/TV) within the distal femoral epiphysis and proximal tibial metaphysis of Botox injected limbs was reduced −43.2% and −54.3%, respectively, while tibia cortical bone volume was reduced −14.6% (all P < 0.001). Comparison of the contralateral non-injected limbs indicated the presence of moderate systemic effects in the model that were most probably associated with diminished activity following muscle paralysis. Taken as a whole, the micro-CT data implied that trabecular and cortical bone loss was primarily achieved by bone resorption. These data confirm the decisive role of neuromuscular function in mediating bone homeostasis and establish a model with unique potential to explore the mechanisms underlying this relation. Given the rapidly expanding use of neuromuscular inhibitors for indications such as pain reduction, these data also raise the critical need to monitor bone loss in these patients.

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low-magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10-s rest interval between each load cycle transformed a locomotion-like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (؉SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 ؎ 1.5%) was doubled after 3 consecutive days of low-magnitude loading (3.8 ؎ 1.5%; p ‫؍‬ 0.03). However

The ability of physical stimuli demonstrated as potently osteogenic in the young adult skeleton were evaluated for their capacity to stimulate new bone formation in the aging skeleton. Using the externally loadable, functionally isolated turkey ulna preparation, the ulnae of 1-year-old (n = 5), and 3-year-old (n = 3) turkeys were subjected to 300 cycles per day of a load regimen generating a high but physiologic level of normal strain (3,000 microstrain). Following 8 weeks of loading, areal properties and histomorphometry were performed on both the experimental and intact control ulnae. Bone cross-sectional areas in the 1-year-old animal increased by 30.2% (+/- 7.8%) as compared with the intact contralateral control ulnae, whereas the areal properties of the older skeleton remained essentially unchanged (-3.3 +/- 7.5%). Renewed bone formation in the experimental ulnae of the 1-year-old animals was characterized by the activation of periosteal bone apposition (4.0 +/- 0.4 microns/day). In comparison, periosteal bone formation in the 3-year-old males was activated in only 1 animal, and this at a significantly attenuated level (less than 0.8 micron/day). The histomorphometric evaluation of intracortical bone remodeling revealed no significant differences between the control and experimental ulnae in either age group. However, osteon mean wall thickness and bone formation sigma were significantly increased in the 3-year-old males (P less than 0.05). In conclusion, these data suggest that a physical signal that is clearly osteogenic in the young adult skeleton is hardly acknowledged in older bone tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

In the mouse, ovariectomy (OVX) leads to signi®cant reductions in cancellous bone volume while estrogen (17b-estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2-dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) a and b expression of primary culture MSCs isolated from OVX and sham-operated mice. MSCs, treated in vitro with 10 À7 M E2, displayed a signi®cant increase in ERa mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERb mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up-regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF-b1, BMP-2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERa was the predominant form expressed in MSCs obtained from both OVX and sham-operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERa expression, osteogenic gene expression and, inhibits apoptosis and ERb expression in MSCs obtained from OVX and sham-operated mice. Coexpression of ERa, but not ERb, and osteogenic differentiation markers might indicate that ERa function as an activator and ERb function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERa activation. J. Cell. Biochem. Suppl. 36:144±155, 2001. ß 2001 Wiley-Liss, Inc.Key words: estrogen; estrogen receptor; mesenchymal stem cells (MSCs); osteogenesis; apoptosis; osteoporosis Estrogen deprivation results in postmenopausal osteoporosis; long-term treatment with estrogen in appropriate doses reduces the risk of hip fractures by 50 to 60% and the risk of vertebral deformation by 90% [Notelovitz, 1997]. Bone loss in ovariectomized (OVX) animals is similar to that in postmenopausal women [Frost and Jee, 1992]. Estrogen maintains bone mass by preserving the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption [Turner et al., 1994]. The protective effect of estrogen on the skeleton of postmenopausal women appears to be mediated by suppression of bone resorption, with little evidence to suggest that conventional doses of estrogen also stimulate osteoblast activity. However, recent studies demonstrate that prolonged exposure of postmenopausal women to relatively high doses of estrogen results in sustained stimulation of osteoblast function [Tobias and Compston, 1999]. Systemically administered 17b-estradiol (E2) enhanced bone formation in animals [Takano-Yamamoto and Rodan, 1990;