Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein.

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.

The role of glutamate in the regulation of bone mass and architecture.

Communication between the cells in bone underlies the way that the tissue functions physiologically, and in nearly all pathologies, the pathogenesis of skeletal diseases. The number of molecules involved in intercellular signalling in bone grows constantly and it is perhaps unsurprising that the list includes many with functions in other tissues. In recent years, evidence has accumulated to show that molecules involved in neurotransmission have paracrine roles in the skeleton. The focus of this review is the excitatory amino acid glutamate and its role in regulating bone formation and resorption. Specifically, this article will concentrate on the functional role of the system, and the reasons why mechanisms like synaptic transmission are relevant to what might appear to be a slow responding tissue, as the sites of expression of glutamate signalling components in bone have been reviewed already. While there is strong evidence for a regulatory role for glutamate in osteoblast and osteoclast differentiation and function in vitro, in vivo data is less advanced. Preliminary data from in vivo systems does however suggest that glutamate has a physiological function in the skeleton.

Compound heterozygous variants in NBAS as a cause of atypical osteogenesis imperfecta.

BACKGROUND: Osteogenesis imperfecta (OI), the commonest inherited bone fragility disorder, affects 1 in 15,000 live births resulting in frequent fractures and reduced mobility, with significant impact on quality of life. Early diagnosis is important, as therapeutic advances can lead to improved clinical outcome and patient benefit. REPORT: Whole exome sequencing in patients with OI identified, in two patients with a multi-system phenotype, compound heterozygous variants in NBAS (neuroblastoma amplified sequence). Patient 1: NBAS c.5741G>A p.(Arg1914His); c.3010C>T p.(Arg1004*) in a 10-year old boy with significant short stature, bone fragility requiring treatment with bisphosphonates, developmental delay and immunodeficiency. Patient 2: NBAS c.5741G>A p.(Arg1914His); c.2032C>T p.(Gln678*) in a 5-year old boy with similar presenting features, bone fragility, mild developmental delay, abnormal liver function tests and immunodeficiency. DISCUSSION: Homozygous missense NBAS variants cause SOPH syndrome (short stature; optic atrophy; Pelger-Huet anomaly), the same missense variant was found in our patients on one allele and a nonsense variant in the other allele. Recent literature suggests a multi-system phenotype. In this study, patient fibroblasts have shown reduced collagen expression, compared to control cells and RNAseq studies, in bone cells show that NBAS is expressed in osteoblasts and osteocytes of rodents and primates. These findings provide proof-of-concept that NBAS mutations have mechanistic effects in bone, and that NBAS variants are a novel cause of bone fragility, which is distinguishable from 'Classical' OI. CONCLUSIONS: Here we report on variants in NBAS, as a cause of bone fragility in humans, and expand the phenotypic spectrum associated with NBAS. We explore the mechanism underlying NBAS and the striking skeletal phenotype in our patients.

Microarray analysis of healing rat Achilles tendon: evidence for glutamate signaling mechanisms and embryonic gene expression in healing tendon tissue.

Tendon healing is a complex process consisting of a large number of intricate pathways roughly divided into the phases of inflammation, proliferation, and remodeling. Although these processes have been extensively studied at a variety of levels in recent years, there is still much that remains unknown. This study used microarray analyses to investigate the process at a genetic level in healing rat Achilles tendon at 1, 7, and 21 days postinjury, roughly representing the inflammation, proliferation, and remodeling phases. An interesting temporal expression profile was demonstrated, identifying both known and novel genes and pathways involved in the progression of tendon healing. Both inflammatory response and pro-proliferative genes were shown to be significantly upregulated from 24 h postinjury through to 21 days. Day 7 showed the largest increase in genetic activity, particularly with the expression of collagens and other extracellular matrix genes. Interestingly, there was also evidence of central nervous system-like glutamate-based signaling machinery present in tendon cells, as has recently been shown in bone. This type of signaling mechanism has not previously been shown to exist in tendon. Another novel finding from these analyses is that there appears to be several genes upregulated during healing which have exclusively or primarily been characterized as key modulators of proliferation and patterning during embryonic development. This may suggest that similar pathways are employed in wound healing as in the tightly regulated progression of growth and development in the embryo. These results could be of use in designing novel gene-based therapies to increase the efficacy and efficiency of tendon healing.

One mechanostat or many? Modifications of the site-specific response of bone to mechanical loading by nature and nurture.

The concept of the mechanostat was not new in 1983, when Harold Frost coined the term to describe a mechanism by which bone responded to habitual exercise and changes in loading with structurally appropriate alterations in bone architecture. However, the word "mechanostat" has a meaning that is immediately apparent, and its adoption has led to a much wider appreciation of the process of functional adaptation by other scientists than those whose primary research focus is in the biology of adaptation. One problem exists though: it is widely thought that in a single individual, there is a setting for the mechanostat, just as a single thermostat might set the temperature for a whole house, and this is reflected in the idea that bones throughout the skeleton require a specific strain magnitude for maintenance. Increases in loading above that threshold are expected to induce bone formation and a stiffer structure that then experiences again the habitual strain magnitude. Reductions in strain magnitude supposedly induce resorption to reduce tissue mass and architectural properties so that the lower loading restores habitual strain magnitude. That widely held belief of a single unifying number of strain is fundamentally flawed. The purpose of this article is to explain the real basis of the mechanostat; that the skeleton responds to a complex strain stimulus, made up of numerous different parameters, of which peak magnitude is only one, and that the strain stimulus is different in different parts of the skeleton, so there is no universal number to describe a tissue strain magnitude that underlies the mechanostat's setting. Furthermore, males and females have different responses to loading, and those responses change in response to many factors including genetic constitution, age, concomitant disease, nutrient availability, and exposure to drugs or biochemicals. In summary then, there is not a single mechanostat controlling the skeleton of each of us. At a fundamental tissue level, small functional units of bone each have their own multifactorial threshold target strain stimuli for a given set of dynamic modifying influences. Understanding the biology behind the way that each of these mechanostats functions independently is likely to have pervasive consequences on our ability to control bone mass by manipulation of loading, either directly through different exercise regimens, or in a targeted manner using tailored site and individual specific pharmaceuticals.

Glutamate signalling in non-neuronal tissues.

Since the discovery of its role in the CNS, glutamate, together with its involvement in signalling at synapses, has been the subject of a vast amount of research. More recently, it has become clear that glutamate signalling is also functional in non-neuronal tissues and occurs in sites as diverse as bone, pancreas and skin. These findings raise the possibility that glutamate acts as a more widespread 'cytokine' and is able to influence cellular activity in a range of tissue types. The impact of these discoveries is significant because they offer a rapid way to advance the development of therapeutics. Agents developed for use in neuroscience applications might be beneficial in the modulation of pathology peripherally, impacting on conditions such as osteoporosis, diabetes and wound healing.

PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats.

Using in situ hybridization, we correlated the expression of mRNA for the parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor with bone formation and resorption in undecalcified serial sections of bones from growing rats. In addition we investigated the presence of biologically active receptors in the same locations using an in vivo autoradiographic technique. In the ulnae of growing rats, there are well defined zones of cortical bone formation and resorption. These contribute to the modeling drifts by which the bone achieves its adult shape. Forming surfaces incorporate fluorochrome labels, are lined with osteoid, and have a layer of cuboidal osteoblasts that have a high alkaline phosphatase activity. Resorbing surfaces have no fluorochrome incorporation, no osteoid, and are lined with resorbing cells with high tartrate-resistant acid phosphatase (TRAP) activity. PTH/PTHrP receptor mRNA was expressed predominantly on forming but not on resorbing bone surfaces and colocalized with sites of binding of radiolabeled PTH after intravenous injection. PTH/PTHrP mRNA expression on osteocytes was inconclusive but radiolabeled PTH bound to a proportion of osteocytes in all regions of the cortex although binding was not specifically related to areas of bone formation or resorption. These results suggest that in growing animals the actions of PTH or PTHrP are connected more with bone formation than resorption. Such a role may be linked to the ability of PTH to induce bone formation in adults but does not explain the actions of the hormone in regulating resorption. Binding of PTH to osteocytes increases the evidence for a physiological role for these cells.

Inhibition of bone resorption and stimulation of formation by mechanical loading of the modeling rat ulna in vivo.

During normal growth of the rat ulna, bone is resorbed from the medial periosteal surface. This occurs as part of the modeling process by which the bone achieves its adult shape. By attaching strain gauges to the ulnae of rats in vivo, we measured the strains imposed on that surface of the bone during normal locomotion. We then applied mechanical loads to the ulnae of other rats in vivo for 6 consecutive days, inducing strains approximately double those we had measured. Fluorochromes were given on the 1st and 5th days. The histology of the medial ulnar periosteal surface was correlated with the amount of fluorochrome incorporation and tartrate resistant acid phosphatase (TRAP) activity in serial sections. In the nonloaded ulnae, the surfaces were lined with bone resorbing cells. Corresponding areas of the loaded bones were lined with osteoid and osteoblasts. There was insignificant label incorporation in the nonloaded bones but almost continuous label incorporation in the corresponding regions of the loaded bones, which was significantly different from the nonloaded bones. TRAP activity of the periosteal cells in the loaded bones was significantly less than in the nonloaded limbs. It is widely acknowledged that loading induces bone formation, and this implies that it also has the ability to inhibit resorption. However, to date there has been little direct evidence for the inhibition of resorption in vivo by mechanical loading. The changes we have observed are similar to the sequence of cellular events that occur during the reversal phase of bone remodeling, in which osteoclastic resorption ceases and osteoblasts are recruited and begin formation. This model may help increase understanding of that process.

Direct transformation from quiescence to bone formation in the adult periosteum following a single brief period of bone loading.

The concept of resorption preceding formation in a coupled response is well established as the normal sequence of remodeling in adult bone. So prevalent is this concept, however, that the idea of the direct activation of osteogenic modeling in normal adult bone is often ignored. This experiment documents the direct transformation of the normal, quiescent, adult periosteum to active bone formation. The osteogenic stimulus was provided by a single short period of dynamic loading. Periosteal activation and the production of new bone within 5 days of loading was unaccompanied by resorption or the presence of osteoclasts. We therefore conclude that an adult resting periosteum can become directly converted to formation as a physiologic response to an appropriate osteogenic stimulus without the need for resorption. To distinguish this process from remodeling we suggest it be called renewed modeling. It is notable that a single short exposure to an "osteogenic" loading regime can influence the full cascade of cellular events between quiescence and active bone formation.

Early strain-related changes in enzyme activity in osteocytes following bone loading in vivo.

The skeleton's architecture is matched to the changing loads to which it is subjected because mechanical loading directly or indirectly influences the activity of cell populations to deposit, maintain, or remove bone tissue as appropriate. This responsiveness to load bearing presupposes that certain cells are sensitive to load itself or to its consequences within the tissue. The nature of this effect and the cells concerned have not yet been determined. In this series of experiments, bones were exposed in vivo to a single short period of dynamic loading, which if repeated daily had been shown to result in increased new bone formation. There was an increase in the activity of glucose 6-phosphate dehydrogenase (G6PD) in the periosteal cells adjacent to the bone surface 6 min after this single period of loading. This increase was proportional to the strain magnitude in the bone tissue in the immediate vicinity of the cells. In osteocytes, although the G6PD activity in each individual cell was unchanged by loading, the number of cells displaying activity was increased. This increase was also directly proportional to the applied strain in that area of the cortex (52% compared with 26% active osteocytes at a strain of 0.002). Activation of G6PD was unaccompanied by any equivalent changes in the activities of either glyceraldehyde 3-phosphate dehydrogenase (GA3PD) or lactate dehydrogenase (LDH). This finding is consistent with loading increasing the activity of the oxidative part of the pentose monophosphate shunt pathway. It is also consistent with stimulation of a synthetic process, such as the production of RNA from ribose 5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive in vivo mRNA expression by osteocytes of beta-actin, osteocalcin, connexin-43, IGF-I, c-fos and c-jun, but not TNF-alpha nor tartrate-resistant acid phosphatase.

Osteocytes have been proposed to be the cells primarily responsible for sensing the effects of mechanical loading in bone. Osteocytes respond to loading in vivo, and have been shown to express osteotropic agents and their receptors, and cell/matrix adhesion molecules in vitro, but the functional significance of such findings is not clear. One obstacle to increased understanding of the role of osteocytes in the regulation of bone mass is that the cells are not easily accessible for study. In situ studies are difficult, and although it is possible to extract and culture osteocytes from neonatal bones, the responses of such cells might be very different from those in older bones in situ. We have developed a technique to investigate osteocyte gene expression in vivo, using the reverse transcriptase linked polymerase chain reaction (PCR), and have shown that they express mRNA for beta-actin (beta-ACT), osteocalcin (OC), connexin-43 (Cx43), insulin-like growth factor I (IGF-I), c-fos and c-jun, but not tumor necrosis factor alpha (TNF-alpha) or tartrate-resistant acid phosphatase (TRAP). The principle behind the method is that after removal of the periosteum, tangential cryostat sections of a tubular bone contain RNA only from osteocytes and a very small number of endothelial cells as long as the marrow cavity is not broached. Using this method, we have investigated gene expression in cells from rat ulnar cortical bone under forming and resorbing bone surfaces. In addition, we have investigated the effect on gene expression of mechanical loading which, if repeated daily, initiates new bone formation on quiescent or resorbing surfaces. Although the expression of the genes we have studied in osteocytes is different from those expressed by the periosteal surfaces overlying the cortex, we have not detected loading-related changes in osteocyte gene expression in any cortical bones. This may be because of the extreme sensitivity of the PCR technique which can only resolve large differences in expression. The use of quantitative methods in the future may allow demonstration of regulated gene expression in osteocytes.

Evidence for a novel glutamate-mediated signaling pathway in keratinocytes.

Phenotypic alterations in keratinocyte behavior are essential for maintaining epidermal integrity during growth and wound repair and rely on co-ordinated cell signaling events. Numerous growth factors and cytokines have been shown to be instrumental in guiding such changes in keratinocyte activity; here we provide evidence which proposes a novel epidermal signaling pathway mediated by the excitatory amino acid glutamate. Glutamate is the major excitatory neurotransmitter at synaptic junctions within the central nervous system; however, we have identified expression in vivo of several regulatory molecules associated with glutamate signaling in keratinocytes. In resting rat skin epidermis, different classes of glutamate receptors, transporters, and a recently described clustering protein were shown to display distinct distribution patterns, supportive of a multifunctional cellular communication pathway. Immunoreactive N-methyl-D-aspartate-type, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type, and metabotropic-type glutamate receptors were colocalized with the specific glutamate transporter EAAC1 in basal layer keratinocytes, and GLT-1, a related transporter, was expressed suprabasally. In full-thickness rat skin wounds, marked modifications in the distribution of N-methyl-D-aspartate receptors and EAAC1 were observed during re-epithelialization, and alterations in N-methyl-D-aspartate receptor expression accompanied embryonic epidermal development, implicating glutamate signaling in these important biologic events. Furthermore, we provide evidence that these receptors are functional in vitro. These data provide strong evidence to support a role for glutamate in the control of epidermal renewal, and therefore suggest potentially novel therapeutic targets for the treatment of skin disease and enhancement of wound healing.

Glutamate signalling in bone.

The identification of novel signalling pathways in a tissue provides new avenues for pharmacological manipulation of tissue function. Where the pathway concerned is one that has been the subject of extensive research in another body system, progress towards new therapies can be rapid. The discovery that glutamate has functions in bone that share striking similarities with its role in synaptic neurotransmission opens the way to manipulate skeletal pathophysiology using modulators of glutamate release, uptake or receptor function. The purpose of this review is to describe the way that a role for glutamate as a signalling molecule in bone was discovered, to summarise the evidence for this role. In addition, it will identify points that are unresolved, to highlight areas where new research could provide significant advances. Furthermore, it will indicate how studies already performed but analysed without consideration of the non-neuronal functions of modulators of glutamate signalling, could contain information of significant value for the advance of therapeutic approaches to bone diseases.

Immobilisation induced bone loss in the sheep is not modulated by calcitonin treatment.

The effect of calcitonin on immobilisation-induced loss of cortical and trabecular bone was studied in adult female sheep. The left calcaneus of 24 adult female Welsh mountain sheep was protected from normal loading by placing an external fixator across the hock joint, from the tibia to the metatarsus. In vivo strain gauge recordings from similar animals showed that this procedure resulted in a 50% reduction in principal strain magnitude in the shaft of the calcaneus during walking. All animals received intramuscular injections three times weekly. Half of the sheep received 100 i.u. of salmon calcitonin while the remainder received vehicle alone. The experiment was conducted blind. Over the 12 week period of the experiment, dual photon absorptiometry was performed at monthly intervals to measure the bone mineral content (BMC) of the calcanei. In all the animals, the loss of bone associated with the functional protection afforded by the fixator was highly significant (p < 0.0001). There was however, no significant difference in either the amount or rate of bone loss between animals which had received calcitonin and those which had not. Post mortem measurement of the cortical thickness of the shaft of the calcaneus revealed a specific pattern of loss which was also not different in the two groups. In this model, calcitonin treatment was ineffective in preventing or retarding loss of bone associated with reduced functional load-bearing over a 12 week period.

Interruption of disuse by short duration walking exercise does not prevent bone loss in the sheep calcaneus.

The effect on calcaneal bone loss of interrupting immobilization by daily periods of normal walking was investigated in adult female sheep. The left calcaneus of 28 sheep was protected from normal loading by placing an external fixator across the hock joint, from the tibia to the metatarsus. In vivo strain gauge recordings from 3 animals showed that, at a test location on the calcaneus, this resulted in a significant reduction in principal strain magnitude during walking at 1.5 m s-1 from a peak of -228 x 10(-6) before application of the fixator, to 71 x 10(-6) with the fixator in place. In addition to reducing peak strains during normal activity, the fixator also abolished the high magnitude transient strains (peak -1147 x 10(-6)) normally experienced when the sheep made sudden movements. Thirteen animals (group 1) had fixators applied, and were given no further treatment. In a further 6 animals (group 2), the fixator was removed for 20 min per day, during which they walked on a motorized treadmill at 1.5 m s-1. In the remaining 6 sheep (group 3), incomplete fixator bars were applied to allow uninterrupted joint movement. Over the 12-week period of the experiment, dual photon absorptiometric scans showed that the bone mineral content (BMC) of the calcanei of group 1 fell by 22%. In group 2, where immobilization was interrupted by daily walking, the BMC fell by 21%. In group 3, with incomplete bars, there was no significant reduction in BMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of syndecan expression by osteosarcoma cell lines in response to cytokines but not osteotropic hormones.

Bone cells are regulated by interactions with both growth factors and components of the extracellular matrix (ECM). Syndecans are cell-surface heparan sulfate proteoglycans known to play a role in cell adhesion and migration, and binding of growth factors. This study was performed to investigate the expression of syndecans by osteoblasts. Reverse transcription-linked polymerase chain reaction (RT-PCR) and Northern analysis detected syndecan transcripts in the human osteosarcoma cell lines MG-63, TE-85, SaOS-2, and U2OS; human osteoblast-like cells; rat calvarial osteoblasts; and in human bone. Western blot analysis of proteoglycans from MG-63 and TE-85 cells detected multiple heparan sulfate proteoglycan core proteins consistent with syndecan expression. Regulation of syndecan-1, -2, and -4 expression was investigated in TE-85, MG-63, and SaOS-2 cells, in response to interleukin (IL)-1beta, and IL-6, parathyroid hormone [PTH(1-34)], and 1,25(OH)2-vitamin D3. Northern analysis demonstrated that in the osteosarcoma cell lines there was no regulation of syndecan transcript levels in response to PTH(1-34) or 1,25(OH)2-vitamin D3 for 24 or 48 h. In contrast, when MG-63 and SaOS-2 cells were incubated with IL-1beta (0.01-10 ng/mL) and IL-6 (0.1-50 ng/mL) there was a dose-dependent decrease in mRNA levels for syndecan-1 and -2 at 24 and 48 h, but in response to IL-1beta upregulation in the levels of syndecan-4 transcripts. In addition, Northern analysis was performed on RNA isolated from neonatal rat calvarial osteoblasts cultured under conditions that promote osteogenesis for 0, 5, 13, 21, and 35 days. Syndecan-1 expression was observed to decrease during the culture period, syndecan-2 transcript levels increased, and there appeared to be no overall change in syndecan-4 levels. Controlled expression of syndecans by cells of the osteoblast lineage may be important in the regulation of osteoblastic proliferation and differentiation.

Osteoblast-derived acetylcholinesterase: a novel mediator of cell-matrix interactions in bone?

The adhesive interactions that occur between bone cells and the developing matrix during bone formation help guide coupled remodeling and the maintenance of bone mass. Here, we provide evidence that acetylcholinesterase (AChE) is a novel osteoblast-derived mediator of cell-matrix interactions in bone. These findings complement an increasing body of evidence which suggests that AChE, in addition to its role in terminating cholinergic signaling, may be instrumental in regulating cellular differentiation and adhesion. We have shown, using RT-PCR, that osteosarcoma cell lines and primary cultures of osteoblasts express AChE mRNA. Expression appeared to be differentiation-dependent, and restricted to AChE splice variants containing the T subunit (exon 6). Immunofluorescent localization demonstrated that these osteoblastic cells expressed protein for AChE with an intracellular vesicular distribution. Immunohistochemistry on tissue sections confirmed AChE expression by osteoblasts in vivo, and revealed the presence of AChE along cement lines, also identified by enzyme histochemistry. In vitro functional studies indicated that osteoblast-like cells adhered specifically to and spread on AChE substrates, but did not interact with butyrylcholinesterase, a closely related protein. Our evidence strongly implicates AChE as a novel bone matrix protein, capable of mediating cell-matrix interactions, and as such may be a principal participant in organized bone formation and the regulation of remodeling.

Evidence for targeted vesicular glutamate exocytosis in osteoblasts.

Regulated intercellular signaling is essential for the maintenance of bone mass. In recent work we described how osteoblasts and osteoclasts express functional receptors for the excitatory amino acid, glutamate, indicating that a signaling pathway analogous to synaptic neurotransmission exists in bone. Here, we show that osteoblasts also express the essential molecular framework for regulated glutamate exocytosis to occur as is present in presynaptic neurons. A combination of reverse transcription-polymerase chain reaction (RT-PCR) and northern and western blotting is used to show expression of the target membrane-SNARE (soluble NSF attachment protein receptor), proteins SNAP-25 and syntaxin 4 and the vesicular-SNARE protein VAMP (synaptobrevin), the minimum molecular requirements for core exocytotic complex formation. Immunofluorescent localizations reveal peripheral SNAP-25 expression on osteoblastic cells, particularly at intercellular contact sites, colocalizing with immunoreactive glutamate and the synaptic vesicle-specific protein, synapsin I. We also identify multiple accessory proteins associated with vesicle trafficking, including munc18, rSec8, DOC2, syntaxin 6, and synaptophysin, which have varied roles in regulated glutamate exocytosis. mRNA for the putative Ca(2+)-dependent regulators of vesicle recycling activity, synaptotagmin I (specialized for fast Ca(2+)-dependent exocytosis as seen in synaptic neurotransmission), and the GTP-binding protein Rab3A are also identified by northern blot analysis. Finally, we demonstrate that osteoblastic cells actively release glutamate in a differentiation-dependent manner. These data provide compelling evidence that osteoblasts are able to direct glutamate release by regulated vesicular exocytosis, mimicking presynaptic glutamatergic neurons, showing that a process with striking similarity to synaptic neurotransmission occurs in bone.

Tetracyclines induce apoptosis in osteoclasts.

Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption through inhibition of matrix metalloproteinases. Here we report that some tetracyclines also induce apoptosis in rabbit osteoclasts and inhibit differentiation and activity of osteoclasts in murine osteoblast/marrow cocultures. Apoptosis of mature rabbit osteoclasts increased from 5.5 +/- 1.4% (mean +/- SD) in control cultures to 44.9 +/- 6.3% (p < 0.001) and 18.9 +/- 4.0% (p < 0.005) with CMT-3 and doxycycline (10 microg/mL), respectively. CMT-2 or CMT-5 did not alter osteoclast viability even at 25 microg/mL. In murine osteoblast/marrow cocultures over 11 days, CMT-3 and doxycycline (5 microg/mL) reduced the formation of mature osteoclasts and inhibited resorption to 21 +/- 9% (p < 0.01) and 49 +/- 4% (p < 0.01) of untreated cultures. Induction of osteoclast apoptosis is an additional property of tetracyclines that may contribute to their ability to inhibit bone resorption.

Intracortical remodeling in adult rat long bones after fatigue loading.

Intracortical remodeling in the adult skeleton removes and replaces areas of compact bone that have sustained microdamage. Although studies have been performed in animal species in which there is an existing baseline of remodeling activity, laboratory rodents have been considered to have limited suitability as models for cortical bone turnover processes because of a lack of haversian remodeling activity. Supraphysiological cyclic axial loading of the ulna in vivo was used to induce bending with consequent fatigue and microdamage. Right ulnae of adult Sprague-Dawley rats were fatigue-loaded to a prefailure stopping point of 30% decrease in ulnae whole bone stiffness. Ten days after the first loading, left ulnae were fatigued in the same way. Ulnae were harvested immediately to allow comparison of the immediate response of the left ulna to the fatigue loads, and the biological response of the right leg to the fatigue challenge. Histomorphometry and confocal microscopy of basic fuchsin-stained bone sections were used to assess intracortical remodeling activity, microdamage, and osteocyte integrity. Bone microdamage (linear microcracks, as well as patches of diffuse basic fuchsin staining within the cortex) occurred in fatigue-loaded ulnar diaphyses. Ten days after fatigue loading, intracortical resorption was activated in ulnar cortices. Intracortical resorption occurred in preferential association with linear-type microcracks, with microcrack number density reduced almost 40% by 10 days after fatigue. Resorption spaces were also consistently observed within areas of the cortex in which no bone matrix damage could be detected. Confocal microscopy studies showed alterations of osteocyte and canalicular integrity around these resorption spaces. These studies reveal that: (1) rat bone undergoes intracortical remodeling in response to high levels of cyclic strain, which induce microdamage in the cortex; and (2) intracortical resorption is associated both with bone microdamage and with regions of altered osteocyte integrity. From these studies, we conclude that rats can initiate haversian remodeling in long bones in response to fatigue, and that osteocyte death or damage may provide one of the stimuli for this process.