Estrogen withdrawal transiently increased bone turnover without affecting the bone balance along the tooth socket in rats.

BACKGROUND: Estrogen withdrawal causes marked bone loss in the appendicular skeleton but slightly affects mandibular cancellous bone; in contrast, little is known of its effects on alveolar wall turnover associated with tooth drift. In this study, we assessed short-term changes in alveolar wall turnover after an ovariectomy and compared it to other bone sites exhibiting different levels of turnover. METHODS: Forty Sprague-Dawley rats were ovariectomized or sham operated. Right mandibles and femurs were processed without demineralization for bone histomorphometry in three different sites: the alveolar wall around the first molar buccal root, apical interradicular bone, and femoral metaphysis. Bone changes were assessed 14 and 28 days after the ovariectomy. Data were compared using non-parametric statistics. RESULTS: At 14 days, on the resorption side of the alveolar wall, resorption parameters were higher in the ovariectomized rats (P <0.01), whereas the formation was lower (P <0.05); on the formation side, the daily mineral apposition rate increased (P <0.01). The root resorption was higher in ovariectomized rats (P <0.05). In the periodontal ligament, the numbers of osteoclast precursors were significantly higher. At 28 days, the drift slowed down in both the sham and ovariectomized groups. The ovariectomy had no effect on interradicular bone turnover, whereas bone loss and numbers of osteoclasts were strongly increased in the femur as soon as 14 days after the ovariectomy. CONCLUSIONS: Estrogen withdrawal had transient repercussions on alveolar wall turnover. The different reactivities of the three envelopes studied suggest that a response to an ovariectomy in the short term is related to initial basal turnover.

Mast cell activation and degranulation occur early during induction of periosteal bone resorption.

We have previously postulated that mast cells participate in the cellular network involved in osteoclastic resorption, probably through histamine release. In this study, we examined mast cell activation and histamine release during origination of resorption. Groups of 10 rats were killed 0, 0.5, 1, 1.5, 3, 6, 9, 12 and 18 h after induction of resorption in a synchronized model of cortical resorption along the mandible. The total number of mast cells was transiently decreased by about one-third at 1 and 9 h. Mast cell activation was monitored by Alcian blue-safranin staining. Early after induction, mast cells started to release their mediator stores; complete release led to the apparent disappearance of the cells with the staining technique used. Histamine immunostaining confirmed the release of histamine and its diffusion in the extracellular environment. Massive degranulation was observed at 1.5 and 9 h with toluidine blue staining. Cell recovery, assessed in terms of histidine decarboxylase expression, occurred gradually. The number of ED1+ osteoclast precursors strongly increased from 12 h up to 18 h. Most parameters had returned to baseline at 18 h, except the ED1+ cells. H2 receptor inhibition with famotidine strongly decreased ED1+ osteoclast precursors at 12 h and subsequently osteoclasts at the peak of resorption. These data support a role of mast cells in resorption origination. They show an early and transient intervention of mast cells in the events regulating the recruitment of circulating osteoclast precursors and ultimately of resorption. Mast cell activation and degranulation induce the release of mediators, particularly histamine acting through its H2 receptors, which are likely involved in these reactions.

Chemical sympathectomy-induced changes in TH-, VIP-, and CGRP-immunoreactive fibers in the rat mandible periosteum: influence on bone resorption.

The expression of neurotransmitter receptors by bone cells supports the concept that the nervous system is a regulator of bone metabolism. The discrimination of the respective roles of the sensory and sympathetic nervous systems requires evidence of topographic relationships between the corresponding fibers and the cells involved in bone turnover, in vivo. In this study, the influence of the sympathetic system on bone resorption was assessed by using a synchronized model of cortical resorption along the mandible. The sympathetic system was destroyed by daily injections of guanethidine (40 mg/kg) for 25 days; a resorption wave was induced on day 21. The distribution of periosteal tyrosine-hydroxylase (TH)-, vasoactive intestinal polypeptide (VIP)-, and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) fibers was studied by compartmentalizing the periosteum. Most fibers were located in the distal, non-osteogenic compartment. TH-IR fibers were located perivascularly, VIP-IR fibers were gathered at the boundary with the osteogenic compartment, and CGRP-IR fibers were scattered. Sympathectomy decreased the number of TH- and VIP-IR fibers and increased the number of CGRP-IR fibers, without changing their topography. After the injection of Fast blue, a retrograde fluorescent marker, over the periosteum, fluorescent neuronal cell bodies were found in the superior cervical ganglion (SCG). Many neurons were TH-IR and very few were VIP-IR. Sympathectomy decreased the numbers of fluorescent and TH-IR cell bodies. It also decreased the number of preosteoclasts and osteoclasts, which had a drastic effect on the cortical bone surface, as assessed by scanning electron microscopy. These data indicate that VIP-IR fibers have a strategic position close to the most peripheral and less differentiated, osteogenic cells, pointing to a functional relationship. As poorly differentiated osteogenic cells support preosteoclast differentiation, VIP-IR fibers may be involved in this process, as suggested by the smaller number of preosteoclasts in sympathectomized rats. Although VIP is predominantly a parasympathetic mediator, it seemed to be conveyed by sympathetic fibers, as shown by the marked effect of guanethidine treatment. Nevertheless, these fibers did not originate from the SCG, contrary to TH-IR fibers.

Osteoclasts differentiate from resident precursors in an in vivo model of synchronized resorption: a temporal and spatial study in rats.

Osteoclasts differentiate from mononucleated precursors expressing monocyte markers, which gradually evolve to preosteoclasts expressing the osteoclast phenotype. Although the role of osteogenic cells in these changes has been well documented in vitro, their contribution in vivo has not been established. In this study, a synchronized wave of resorption was activated along the mandibular periosteum. The periosteum adjacent to the bone surface studied was separated by a computer-assisted technique into an osteogenic alkaline phosphatase-positive compartment and an outer nonosteogenic compartment. Specific markers (nonspecific esterase [NSE], tartrate-resistant acid phosphatase [TRAP], and ED1 antibody, a marker of the monocyte-macrophage lineage) were used to follow osteoclast differentiation quantitatively as a function of time after activation of resorption, from day 0 to day 4 (peak of resorption in this model). Local cell proliferation was assessed in parallel. Between day 0 and day 3, the thickness of the osteogenic compartment decreased by 50% (p < 0.0002). In the osteogenic compartment, proliferating cell numbers fell by 80% at 12 day, NSE(+) cells (located farthest from the bone surface) increased 3. 9-fold on day 4 vs. day 0 (p < 0.005), ED1(+) cells decreased between day 0 and day 2 (p < 0.02) before returning to their initial value, and TRAP(+) cells increased 2.7-fold between day 1 and day 3 (p < 0.0005). Resorption was absent in the site studied on day 0, but on day 4 there were 20.5 osteoclast nuclei per millimeter of bone surface. The cell ratio changed from 30.3 NSE(+) and ED1(+) (some of which were also TRAP(+)) cells per millimeter on day 0 to 37.6 mononucleated cells plus 20.5 osteoclast nuclei on day 4. In the nonosteogenic compartment, an entry of ED1(+)/NSE(-) was observed on 12 day (+23 cells, p < 0.02 vs. day 0). This was followed by a return of ED1(+) cell numbers to the control level on day 1, and a transient increase in NSE(+) cells (+47% on day 2 vs. day 1, p < 0.02). TRAP(+) cells were never seen in this compartment. Proliferating cell numbers did not change throughout the study. Our results strongly suggest that the osteoclasts present on day 4 differentiated from the pool of TRAP(+), ED1(+), and NSE(+) cells present at the site on day 0. The osteogenic compartment was gradually replenished by cells migrating from the nonosteogenic compartment, which was supplemented by ED1(+) cells recruited from the circulation early after activation. Moreover, osteogenic cells appeared to be as crucial in vivo for the acquisition of the TRAP phenotype as previously shown in vitro.

Effects of capsaicin-induced sensory denervation on osteoclastic resorption in adult rats.

Many recent findings suggest that the nervous system has efferent effects on bone. A putative role of the sensory innervation has been assessed by using a synchronised rat model of bone resorption after treating adult animals with the neurotoxin capsaicin. Fourteen days after capsaicin treatment (50 mg kg-1) the right maxillary molars were extracted to activate a wave of resorption along the mandibular cortex. The rats were killed 4 days later (i.e. at the peak of resorption in this model), and their right mandibles were processed for histometric evaluation of resorption along the cortex and of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) fibres in the dental pulp. CGRP-IR and SP-IR fibres were significantly reduced in numbers by the capsaicin treatment (by 58 and 49%, respectively), confirming the success of sensory denervation. The resorption surface was significantly reduced (P < 0.005) versus the sham-treated animals. Although the size of the osteoclast population recruited in the site was not modified, the number of actively resorbing osteoclasts was significantly reduced (P < 0.03). However, the activity of the resorbing cells was not modified. Non-specific esterase-positive osteoclast precursors were also significantly few after capsaicin treatment. These data show that the sensory nervous system is involved in the control of bone resorption at two different levels: (1) in the recruitment of osteoclast precursors, and (2) in regulating the access of recruited cells to the bone surface.

Chemical sympathectomy impairs bone resorption in rats: a role for the sympathetic system on bone metabolism.

The possibility that the nervous system may control bone metabolism has been raised, as neuromediators physiologically conveyed by sympathetic fibers (eg, vasoactive intestinal peptide) influence bone resorption in vitro. In this study, the sympathetic system was inactivated by treating rats with guanethidine (40 mg/kg/day), a sympathetic neurotoxic, for 21 days, after which a wave of osteoclastic resorption was induced along the mandibular buccal cortex. The effects of denervation were assessed 4 days later (corresponding to the peak of resorption in this model). The rats exhibited ptosis soon after starting guanethidine, proving the success of the sympathectomy. This was associated with a significant increase in calcitonin gene-related peptide- (+54%, p < 0.02) and substance P-immunoreactive sensory fibers (+29%,p < 0.02), a known effect of sympathectomy. For the quantitation of the bone parameters, the study zone was divided into a juxta-osseous alkaline phosphatase-positive osteogenic compartment and a nonosteogenic compartment. In the osteogenic compartment, the resorption surface was reduced by 56% (p < 0.001) in the treated animals, together with a fall in the number of osteoclasts (-25%,p < 0.05) and impaired osteoclast access to the bone surface. Tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear preosteoclasts were found only in this compartment; they were reduced by 43% (p < 0.05) by the sympathectomy. No change in non-specific esterase (NSE)+ osteoclast precursors was found. In the nonosteogenic compartment, vasodilation was the only effect of sympathectomy (+80%,p < 0.05); in particular, the number of NSE+ cells was not modified. Our results indicate that: (1) interactions of NSE+ precursors with osteogenic cells are required for their differentiation into TRAP+ preosteoclasts; (2) the sympathetic nervous system is not involved in osteoclast precursor recruitment; but (3) has a significant effect on resorption by inhibiting preosteoclast differentiation and disturbing osteoclast activation. These data suggest that depletion of sympathetic mediators may disturb osteogenic cell-mediated osteoclast differentiation.