Luc Malaval

SERUM BONE GLA-PROTEIN: A SPECIFIC MARKER FOR BONE FORMATION IN POSTMENOPAUSAL OSTEOPOROSIS

Mean (+/- SD) serum bone Gla-protein (BGP or osteocalcin) was normal (7.0 +/- 3.3 ng/ml) in 26 patients with untreated postmenopausal osteoporosis ( PMO ). But 9 patients had values either above (4) or below (5) the normal values obtained in 35 age-matched control women (6.9 +/- 1.25 ng/ml). Serum BGP correlated positively with relative osteoid volume, relative osteoid surfaces, tetracycline labelled surfaces, and bone formation rate but not with resorption surfaces. Based on normal values for osteoid volume, patients were classified as having high (HF, 9 patients), normal (NF, 12 patients) and low osteoid formation (LF, 5 patients). Serum BGP (+/- SEM) was significantly lower in LF group (2.7 +/- 0.9 ng/ml) and significantly higher in HF group (9.7 +/- 0.8 ng/ml) than in the NF group (7.0 +/- 0.6 ng/ml). Serum alkaline phosphatase and urinary hydroxyproline did not discriminate between these three groups and did not correlate significantly with any of the measured histomorphometric indices in biopsy specimens in these patients. Serum BGP appears to be a specific marker for bone formation and can predict the histological profile in PMO . Serum BGP might be useful in investigating patients with PMO and should be valuable in assessing the effects of treatments that increase bone formation.

Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue‐specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9–10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase‐negative cells at the periphery of colonies, to postmitotic, osteocalcin‐producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late‐stage, BrdU‐negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase‐negative cells, and again later in both late preosteoblast (BrdU‐positive) and osteoblast (BrdU‐negative, osteocalcin‐positive) cells. In late‐stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule “tissue‐like” structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue‐specific marker expression during osteoblastic differentiation. J. Cell. Biochem. 74:616–627, 1999.

Effects of whole body vibration on the skeleton and other organ systems in man and animal models : What we know and what we need to know

Previous investigations reported enhanced osseous parameters subsequent to administration of whole body vibration (WBV). While the efficacy of WBV continues to be explored, scientific inquiries should consider several key factors. Bone remodeling patterns differ according to age and hormonal status. Therefore, WBV protocols should be designed specifically for the subject population investigated. Further, administration of WBV to individuals at greatest risk for osteoporosis may elicit secondary physiological benefits (e.g., improved balance and mobility). Secondly, there is a paucity of data in the literature regarding the physiological modulation of WBV on other organ systems and tissues. Vibration-induced modulation of systemic hormones may provide a mechanism by which skeletal tissue is enhanced. Lastly, the most appropriate frequencies, durations, and amplitudes of vibration necessary for a beneficial response are unknown, and the type of vibratory signal (e.g., sinusoidal) is often not reported. This review summarizes the physiological responses of several organ systems in an attempt to link the global influence of WBV. Further, we report findings focused on subject populations that may benefit most from such a therapy (i.e., the elderly, postmenopausal women, etc.) in hopes of eliciting multidisciplinary scientific inquiries into this potentially therapeutic aid which presumably has global ramifications.

Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth

Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.

Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation

Mesenchymal stem cells give rise to osteoprogenitors that proliferate and differentiate into identifiable preosteoblasts, osteoblasts, bone lining cells and osteocytes. To identify and establish a molecular profile for the more primitive and uncharacterized cells in the lineage, relatively rare (<1%) osteoprogenitors present in primary cultures of fetal rat calvaria cell populations were identified by a replica plating technique. Since the cell number was limited in each colony sampled, we used global amplification PCR to analyze the repertoire of genes expressed in osteoprogenitors. We established a molecular fingerprint and a developmental sequence based on simultaneous expression patterns for both known osteoblast-associated markers (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, PTH1R and osteocalcin) and potential regulatory molecules (i.e. FGFR1, PDGF-Rα and PTHrP). By analysis of 99 osteoprogenitor and osteoblast colonies captured by replica plating at different developmental stages, we found: (1) a recognizable cohort of cells considered more primitive than committed osteoprogenitors; (2) a cohort of early progenitors transiently expressing bone sialoprotein; and (3) that mRNAs for FGF-R1, PDGF-Rα and PTH1R were expressed earlier than other markers and tended to increase and decrease in relative concert with the osteoblast-specific markers. The observations suggest that within the osteoblast differentiation sequence both discrete stages and continua of changing marker expression levels occur with variation in expression for any given marker. This combined approach of replica plating and global amplification PCR allows molecular fingerprinting of definitive primitive osteoprogenitors and will aid in identifying novel developmental stages and novel differentiation stage-specific genes as these cells progress through their differentiation sequence.

Serum bone GLA-protein is not a sensitive marker of bone turnover in Paget's disease of bone.

SummarySerum bone Gla-protein (sBGP) was measured in 32 patients with untreated Pagets disease of bone. Despite clinical and biological symptoms of active disease in all patients, sBGP was normal in 13/32 patients (41%). There was a striking discrepancy between the moderate increase of sBGP above normal values (11.4±4.5 vs 6.0±2.1 ng/ml) and the marked increase of both serum alkaline phosphatase (sAP) and urinary hydroxyproline (uOHP). sBGP was weakly correlated with sAP (r=0.50, p<0.01), uOHP (r=0.48, p<0.01) and with the extension of the disease (r=0.48, p<0.01), We conclude that sBGP is not a sensitive marker of bone turnover in patients with Pagets disease of bone, and should be interpreted with caution in this condition.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta‐adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrβ2R and the absence of Adrβ1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (−52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Youngs Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrβ2R‐deficient mice, suggest that low doses of β‐blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues. J. Cell. Physiol. 217: 819–827, 2008.

Intermittent PTH(1–84) is osteoanabolic but not osteoangiogenic and relocates bone marrow blood vessels closer to bone-forming sites†

Intermittent parathyroid hormone (PTH) is anabolic for bone. Our aims were to determine (1) whether PTH stimulates bone angiogenesis and (2) whether vascular endothelial growth factor (VEGF A) mediates PTH‐induced bone accrual. Male Wistar rats were given PTH(1–84) daily, and trabecular bone mass increased 150% and 92% after 30 and 15 days, respectively. The vascular system was contrasted to image and quantify bone vessels with synchrotron radiation microtomography and histology. Surprisingly, bone vessel number was reduced by approximately 25% and approximately 40% on days 30 and 15, respectively. PTH redistributed the smaller vessels closer to bone‐formation sites. VEGF A mRNA expression in bone was increased 2 and 6 hours after a single dose of PTH and returned to baseline by 24 hours. Moreover, anti‐VEGF antibody administration (1) blunted the PTH‐induced increase in bone mass and remodeling parameters, (2) prevented the relocation of bone vessels closer to bone‐forming sites, and (3) inhibited the PTH‐induced increase in mRNA of neuropilin 1 and 2, two VEGF coreceptors associated with vascular development and function. In conclusion, PTH(1–84) is osteoanabolic through VEGF‐related mechanism(s). Further, PTH spatially relocates blood vessels closer to sites of new bone formation, which may provide a microenvironment favorable for growth.

Ex Vivo bone formation in bovine trabecular bone cultured in a dynamic 3D bioreactor is enhanced by compressive mechanical strain.

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Youngs modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

Brain and bone damage in KARAP/DAP12 loss-of-function mice correlate with alterations in microglia and osteoclast lineages.

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KDelta75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KDelta75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KDelta75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitro microglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitro osteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KDelta75 mice.