Skeletal responses to space flight and the bed rest analog: a review

The potential for loss of bone mineral mass due to space flight was recognized by space scientists even before man's first venture into micro-gravity. Early life science studies in both the U.S. and Russian space programs attempted to measure the effects of reduced gravity on skeletal homeostasis, and these measurements have become more sophisticated with time. Bone-related measurements have typically included: bone mineral density measured by X-ray absorptiometry and more recently CT scanning; bonerelated hormones and other biochemical markers of bone turnover; and calcium excretion and balance. These measurements, conducted over the last 4 decades, have shed light on the nature of disuse bone loss and have provided preliminary information regarding bone recovery. Ground-based analog (bed rest) studies have provided information complementary to the space flight data and have allowed the testing of various countermeasures to bone loss. In spite of the wealth of knowledge obtained thus far, many questions remain regarding bone loss, bone recovery, and the factors affecting these skeletal processes. This paper will summarize the skeletal data obtained to date by the U.S. and Russian space programs and in ground-based disuse studies. In addition, related body composition data will be briefly discussed, as will possible countermeasures to space flight-induced bone loss.     

In vitro modeling of human tibial strains during exercise in micro-gravity

Prolonged exposure to micro-gravity causes substantial bone loss (Leblanc et al., Journal of Bone Mineral Research 11 (1996) S323) and treadmill exercise under gravity replacement loads (GRLs) has been advocated as a countermeasure. To date, the magnitudes of GRLs employed for locomotion in space have been substantially less than the loads imposed in the earthbound 1G environment, which may account for the poor performance of locomotion as an intervention. The success of future treadmill interventions will likely require GRLs of greater magnitude. It is widely held that mechanical tissue strain is an important intermediary signal in the transduction pathway linking the external loading environment to bone maintenance and functional adaptation; yet, to our knowledge, no data exist linking alterations in external skeletal loading to alterations in bone strain. In this preliminary study, we used unique cadaver simulations of micro-gravity locomotion to determine relationships between localized tibial bone strains and external loading as a means to better predict the efficacy of future exercise interventions proposed for bone maintenance on orbit. Bone strain magnitudes in the distal tibia were found to be linearly related to ground reaction force magnitude (R(2)>0.7). Strain distributions indicated that the primary mode of tibial loading was in bending, with little variation in the neutral axis over the stance phase of gait. The greatest strains, as well as the greatest strain sensitivity to altered external loading, occurred within the anterior crest and posterior aspect of the tibia, the sites furthest removed from the neutral axis of bending. We established a technique for estimating local strain magnitudes from external loads, and equations for predicting strain during simulated micro-gravity walking are presented.     

Forearm bone histology of the small theropod Daliansaurus liaoningensis (Paraves: Troodontidae) from the Yixian Formation,Liaoning, China

We present the first histological analysis of forelimb bones in a troodontid dinosaur, Daliansaurus liaoningensis, from the Early Cretaceous of China using osteohistological thin-sectioning and high-resolution synchrotron-based imaging. The thin wall compacta consists of primary bone, and three lines of arrested growth (LAG) in the radius (R) and two in the ulna (U) divide these into successive zones. Results show that the new fossil has four distinct bone depositional rates: (1) fastest deposition in inner zones R1 and U1 (fibro-lamellar bone with a plexiform-like vasculature); (2) slowed deposition in outer zones R1 and U1 + U2 (loss of vascular density and plexiform component); (3) fluctuating rates of deposition in zones R2 + R3 and in the inner zone U3 (alternating bands of circumferentially organised primary osteons and avascular bone); and (4) slowest deposition in zone R4 and the outer zone U3 (lamellar bone constituting the external fundamental system). Collectively, these growth characteristics suggest that the fossil is an individual that passed the exponential growth phase by the first year, and perished three years later. We conclude that the histology is consistent with an interpretation of this specimen as a late maturing individual that had not yet attained maximum somatic size.     

An improved method for estimating original mineral contents in excavated bone using sulfur

Trace element analysis in excavated bones is complicated by the lack of a reliable index for estimating the original amount of bone material. In this study, we subjected modern human bones to alkali treatment to simulate aging. Alkali treatment of vertebrae with attached muscle did not affect sulfur (S) content; it increased the magnesium (Mg), phosphorus (P), and zinc (Zn) contents, and tended to decrease iron (Fe) content of the bones. When vertebrae cleaned of muscle were used, alkali treatment did not affect S and Fe contents but increased Mg, P, Ca, and Zn contents Ca and S contents were higher in excavated bones (200–1300 yr old) than in their surrounding soils. In contrast, S, Mg, and Ca contents per dry weight did not differ between the excavated bones and the alkali-treated modern bones. These results indicate that S can provide a more accurate index of excavated bones than the often-used Ca content or dry wt measures, especially for bones excavated from calcium-rich soils.     

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.     

Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation

Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+ -ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+ -activated RBCs, suggesting a possible role for this GTPase in membrane shedding.     

CIB1, a ubiquitously expressed Ca2+-binding protein ligand of the InsP3 receptor Ca2+ release channel

A family of Ca(2+)-binding proteins (CaBPs) was shown to bind to the inositol 1,4,5-trisphosphate receptor (InsP(3)R) Ca(2+) release channel and gate it in the absence of InsP(3), establishing them as protein ligands (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). However, the neuronally restricted expression of CaBP and its inhibition of InsP(3)R-mediated Ca(2+) signaling when overexpressed (Kasri, N. N., Holmes, A. M., Bultynck, G., Parys, J. B., Bootman, M. D., Rietdorf, K., Missiaen, L., McDonald, F., De Smedt, H., Conway, S. J., Holmes, A. B., Berridge, M. J., and Roderick, H. L. (2004) EMBO J. 23, 312-321; Haynes, L. P., Tepikin, A. V., and Burgoyne, R. D. (2004) J. Biol. Chem. 279, 547-555) have raised questions regarding the functional implications of this regulation. We have discovered the Ca(2+)-binding protein CIB1 (calmyrin) as a ubiquitously expressed ligand of the InsP(3)R. CIB1 binds to all mammalian InsP(3)R isoforms in a Ca(2+)-sensitive manner dependent on its two functional EF-hands and activates InsP(3)R channel gating in the absence of InsP(3). In contrast, overexpression of CIB1 or CaBP1 attenuated InsP(3)R-dependent Ca(2+) signaling, and in vitro pre-exposure to CIB1 reduced the number of channels available for subsequent stimulation by InsP(3). These results establish CIB1 as a ubiquitously expressed activating and inhibiting protein ligand of the InsP(3)R.     

Direct effects of platelet-activating factor on isolated rat osteoclasts. Rapid elevation of intracellular free calcium and transient retraction of pseudopods

Platelet-activating factor (PAF, 1-O-alkyl-(2R)-acetylglycero-3-phosphocholine) is a potent inflammatory mediator whose actions on bone cells have not been investigated previously. In this study, we examined effects of PAF on osteoclast morphology and intracellular free calcium. Osteoclasts, the large multinucleated cells responsible for bone resorption, were isolated from neonatal rat long bones, and the cytosolic free calcium concentration ([Ca2+]i) of individual fura-2-loaded cells was monitored by microspectrofluorimetry. In one series of experiments, PAF was applied focally to single, isolated osteoclasts (1 nM to 1 microM racemic mixture, in an application micropipette). Within 10 s of PAF application, [Ca2+]i increased from basal levels of 74 +/- 6 nM to peak levels of 209 +/- 28 nM (mean +/- S.E. of 24 cells responding). These results indicate that PAF acted directly on osteoclasts. In more than 75% of cells tested, PAF, at concentrations greater than or equal to 10 pM (final concentration, in the bath), induced biphasic elevation of [Ca2+]i. This response was highly specific for PAF, in that vehicle, lyso-PAF (the biologically inactive precursor/metabolite of PAF), and (S)-PAF (the inactive enantiomer of PAF) all failed to change [Ca2+]i. Moreover, [Ca2+]i elevation was blocked by the specific PAF antagonist CV-3988. To determine the source of Ca2+, cells were bathed in Ca(2+)-free medium, where PAF still caused an increase in [Ca2+]i, establishing that the response to PAF arose, at least in part, by release of Ca2+ from internal stores. In addition to changes in [Ca2+]i, PAF caused retraction followed by respreading of peripheral pseudopods. These findings indicate that rat osteoclasts respond to PAF by release of internal calcium and alterations in cell morphology and suggest that PAF may regulate resorption in inflammatory bone diseases.     

Random Loss and Selective Fusion of Bones Originate Morphological Complexity Trends in Tetrapod Skull Networks

The tetrapod skull has undergone a reduction in number of bones in all major lineages since the origin of vertebrates, an evolutionary trend known as Williston’s Law. Using connectivity relations between bones as a proxy for morphological complexity we showed that this reduction in number of bones generated an evolutionary trend toward more complex skulls. This would imply that connectivity patterns among bones impose structural constraints on bone loss and fusion that increase bone burden due to the formation of new functional and developmental dependencies; thus, the higher the number of connections, the higher the burden. Here, we test this hypothesis by exploring plausible evolutionary scenarios based on selective versus random processes of bone loss and fusion. To do this, we have built a computational model that reduces iteratively the number of bones by loss and fusion, starting from hypothetical ancestral skulls represented as Gabriel networks in which bones are nodes and suture connections are links. Simulation results indicate that losses and fusions of bones affect skull structure differently whether they target bones at random or selectively depending on the number of bone connections. Our findings support a mixed scenario for Williston’s Law: the random loss of poorly connected bones and the selective fusion of the most connected ones. This evolutionary scenario offers a new explanation for the increase of morphological complexity in the tetrapod skull by reduction of bones during development.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of clinostat-microgravity on bone and calcium metabolism in rats

Decreases in bone minerals and tissue volume after space flight have been observed in humans and animals, with a variety of results. Such data obtained from space flight experiments have given unsatisfactory results due to short periods of space flight and differences in age, body weights, and strain of animals used. Therefore, ground-based animal models have been developed in order to elucidate changes in bone affected by space flight. For example, a tail-suspended rat model has been established to study the effects of microgravity on bones by producing hind limb unloading. However, problems with this model due to the remaining forelimb loading and the unusual changes in blood current require the development of a new model simulating the physiological conditions of space flight. So we developed a three-dimension clinostat as an apparatus to produce a simulated microgravity similar to space flight by rotating rats equally in all directions. The purpose of the present study is to examine the effects of clinostat-microgravity on bone metabolism in rats.     

Ca/P concentration ratio at different sites of normal and osteoporotic rabbit bones evaluated by Auger and energy dispersive X-ray spectroscopy

Osteoporosis is a systemic skeletal disorder associated with reduced bone mineral density and the consequent high risk of bone fractures. Current practice relates osteoporosis largely with absolute mass loss. The assessment of variations in chemical composition in terms of the main elements comprising the bone mineral and its effect on the bone’s quality is usually neglected. In this study, we evaluate the ratio of the main elements of bone mineral, calcium (Ca), and phosphorus (P), as a suitable in vitro biomarker for induced osteoporosis. The Ca/P concentration ratio was measured at different sites of normal and osteoporotic rabbit bones using two spectroscopic techniques: Auger electron spectroscopy (AES) and energy-dispersive X-ray spectroscopy (EDX). Results showed that there is no significant difference between samples from different genders or among cortical bone sites. On the contrary, we found that the Ca/P ratio of trabecular bone sections is comparable to cortical sections with induced osteoporosis. Ca/P ratio values are positively related to induced bone loss; furthermore, a different degree of correlation between Ca and P in cortical and trabecular bone is evident. This study also discusses the applicability of AES and EDX to the semiquantitative measurements of bone mineral’s main elements along with the critical experimental parameters.     

Application and comparison of two models to study effects of calcium sources in sheep

The aim of this research was to assess and compare two compartmental models by studying effects of different Ca sources on Ca and P metabolism of sheep. Brazilian male sheep (20) were fed a basal diet supplemented with different sources of Ca, being: limestone (L), alfalfa hay (AH), shell meal (OSM) and citrus pulp (CTP). After 21 days, each sheep was given, as a single dose via the right jugular vein, 7.4 MBq of radio-calcium (⁴⁵Ca) and 7.4 MBq of radio-phosphorus (³²P). Calcium and P metabolism were evaluated by comparing the Vitti–Dias (VD) and Fernández–Lopes (FL) models [Dias, R.S., Kebreab, E., Vitti, D.M.S.S., Roque, A.P., Bueno, I.C.S., France, J., 2006. A revised model for studying phosphorus and calcium kinetics in growing sheep. J. Anim. Sci. 84, 2787–2794; Lopes, J.B., Vitti, D.M.S.S., Abdalla, A.L., Haddad, M.L., Figueredo, A.V., Moraes, R.C.B., 2001. Modelo do fluxo biológico do fósforo de fontes de fosfato em suínos, usando o ³²P como marcador. Rev. Bras. Zoot. 30, 165–173], by contrasting flows between gut and plasma, plasma and bone, and plasma and tissue. There were no differences in Ca and P intakes for the treatments. Ca flows from tissue and bone to plasma and vice versa were similar among treatments, though net bone and net tissue Ca retentions were higher for treatments L and OSM and lower, as well as negative, for AH and CTP (P<0.05). Net bone Ca results were consistent between the VD and FL models, although net tissue Ca retention was slightly higher for the VD. The presence of pectin in CTP and oxalate in AH could have affected Ca balance on these treatments. Total Ca absorption was higher (P<0.05) for L with both models. The chemical form of Ca in the different sources affected its metabolism, but did not affect P metabolism. Both models had higher P resorption than P absorption in bone, suggesting that the sheep were mobilizing P. It could be inferred that impaired digestion induced P mobilization from bone to supply P for metabolic needs. Both the VD and FL models had the same pattern for the P flows, and for net bone and tissue P retentions. Both models can be used to assess Ca and P kinetics in ruminants, and both suggest that our sheep tended to be deficient in Ca and P as well as that the inorganic sources of Ca were better utilized.     

Effects of cholera toxin on cyclic AMP accumulation and bone resorption in cultured mouse calvaria

We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic differentiation of U.S.S.R. house mice: electrophoretic study of proteins

Protein electrophoresis at 24 loci was used to characterize house mice from 56 localities in the U.S.S.R., concentrating on samples from Moldavia to Primorye (extreme south-east of the U.S.S.R.). Mus -2A is the most widespread form, extending over the European part of the U.S.S.R., Middle Asia and Siberia as far east as the Pacific Ocean. In Moldavia the group is sympatric with Mus-iB . It is found with Mus -4A in Transcaucasus, where it may hybridize with Mus -1. In Primorye Mus -2A and M. raddei have a wide zone of hybridization with Mus -2C.     

Staphylococcus aureus protein A binds to osteoblasts and triggers signals that weaken bone in osteomyelitis

Osteomyelitis is a debilitating infectious disease of the bone. It is predominantly caused by S. aureus and is associated with significant morbidity and mortality. It is characterised by weakened bones associated with progressive bone loss. Currently the mechanism through which either bone loss or bone destruction occurs in osteomyelitis patients is poorly understood. We describe here for the first time that the major virulence factor of S. aureus, protein A (SpA) binds directly to osteoblasts. This interaction prevents proliferation, induces apoptosis and inhibits mineralisation of cultured osteoblasts. Infected osteoblasts also increase the expression of RANKL, a key protein involved in initiating bone resorption. None of these effects was seen in a mutant of S. aureus lacking SpA. Complementing the SpA-defective mutant with a plasmid expressing spa or using purified protein A resulted in attachment to osteoblasts, inhibited proliferation and induced apoptosis to a similar extent as wildtype S. aureus. These events demonstrate mechanisms through which loss of bone formation and bone weakening may occur in osteomyelitis patients. This new information may pave the way for the development of new and improved therapeutic agents to treat this disease.