NLRP3 is involved in long bone edification and the maturation of osteogenic cells.

Overexpression of the nucleotide-binding leucine-rich repeat protein 3 (NLRP3) inflammasome in chronic auto-immune diseases leads to skeletal anomalies, with severe osteopenia due to the activation of osteoclasts. Reproducing this phenotype in Nlrp3 knock-in mice has provided insights into the role of NLRP3 in bone metabolism. We studied the role of NLRP3 in physiological bone development using a complete Nlrp3 knock-out mouse model. We found impaired skeletal development in Nlrp3-/- mice, resulting in a shorter stature than that of Nlrp3+/+  mice. These growth defects were associated with altered femur bone growth, characterized by a deficient growth plate and an osteopenic profile of the trabeculae. No differences in osteoclast recruitment or activity were observed. Instead, Nlrp3-/- femurs showed a less mineralized matrix in the trabeculae than those of Nlrp3+/+  mice, as well as less bone sialoprotein (BSP) expressing hypertrophic chondrocytes. In vitro, primary osteoblasts lacking NLRP3 expression showed defective mineralization, together with the downregulation of BSP expression. Finally, follow-up by micro-CT highlighted the role of NLPR3 in bone growth, occurring early in living mice, as the osteopenic phenotype diminishes over time. Overall, our data suggest that NLRP3 is involved in bone edification via the regulation of hypertrophic chondrocyte maturation and osteoblast activity. Furthermore, the defect appeared to be transitory, as the skeleton recovered with aging.

Differences between inflammatory and catabolic mediators of peri-implantitis and periodontitis lesions following initial mechanical therapy: An exploratory study.

BACKGROUND AND OBJECTIVE: The aim of this study was to analyze the differences in inflammatory and catabolic mediators expressed in peri-implantitis compared to periodontitis lesions after non-surgical therapy. Peri-implantitis is associated with a faster rate of bone loss when compared with periodontitis, and peri-implant non-surgical therapy is ineffective to cure peri-implantitis. This may be due to persistent inflammation in peri-implantitis tissues after initial mechanical treatment. MATERIAL AND METHODS: Eleven patients with peri-implantitis and 10 with severe chronic periodontitis received non-surgical therapy. They were included at re-evaluation (8 weeks) if they presented pocket depth ≥6 mm with bleeding on probing, and the indication for open flap debridement surgery. Connective tissues were harvested during surgery from diseased sites. Healthy gingiva were harvested during third molar extraction in a third group of healthy patients (n=10). Explants were incubated for 24 hours in media culture and the release of cytokines, chemokines, growth factors, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand (RANKL), matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase (TIMP) in the conditioned media was analyzed by an exploratory multiplex immunoassay. When difference was found in the conditioned media, an immunohistochemistry was performed to compare expression in the tissues. RESULTS: Connective tissues from non-stabilized peri-implantitis exhibited a distinct cytokine profile compared to periodontitis lesions that did not respond to initial therapy. Indeed, TIMP-2 was significantly increased in media from peri-implantitis (P≤.05). In addition, the in situ expression of TIMP-2, interleukin-10 and RANKL was also significantly increased in peri-implantitis tissues (P≤.05). However, the ratio of RANKL/osteoprotegerin-positive cells did not vary (P≥.05). CONCLUSION: This study suggests that peri-implantitis and periodontitis connective tissues exhibit differences in response to non-surgical treatment, which may contribute to a different pattern of disease evolution.

Abnormal osteopontin and matrix extracellular phosphoglycoprotein localization, and odontoblast differentiation, in X-linked hypophosphatemic teeth.

Mutations in phosphate-regulating gene (PHEX) lead to X-linked hypophosphatemic rickets (XLH), a genetic disease characterized by impaired mineralization in bones and teeth. In human XLH tooth dentin, calcospherites that would normally merge as part of the mineralization process are separated by unmineralized interglobular spaces where fragments of matrix proteins accumulate. Here, we immunolocalized osteopontin (OPN) in human XLH teeth, in a three-dimensional XLH human dental pulp stem cell-collagen scaffold culture model and in a rat tooth injury repair model treated with acidic serine- and aspartate-rich motif peptides (ASARM). In parallel, matrix extracellular phosphoglycoprotein (MEPE) immunolocalization and alkaline phosphatase (ALP) activity were assessed in XLH teeth. OPN was expressed by odontoblasts in the XLH models, and localized to the abnormal calcospherites of XLH tooth dentin. In addition, ALP activity and MEPE localization were abnormal in human XLH teeth, with MEPE showing an accumulation in the unmineralized interglobular spaces in dentin. Furthermore, XLH odontoblasts failed to form a well-polarized odontoblast layer. These data suggest that both MEPE and OPN are involved in impaired tooth mineralization associated with XLH, possibly through different effects on the mineralization process.

Effect of a calcium-silicate-based restorative cement on pulp repair.

In cases of pulp injury, capping materials are used to enhance tertiary dentin formation; Ca(OH)(2) and MTA are the current gold standards. The aim of this study was to evaluate the capacity of a new calcium-silicate-based restorative cement to induce pulp healing in a rat pulp injury model. For that purpose, cavities with mechanical pulp exposure were prepared on maxillary first molars of 27 six-week-old male rats, and damaged pulps were capped with either the new calcium-silicate-based restorative cement (Biodentine), MTA, or Ca(OH)(2). Cavities were sealed with glass-ionomer cement, and the repair process was assessed at several time-points. At day 7, our results showed that both the evaluated cement and MTA induced cell proliferation and formation of mineralization foci, which were strongly positive for osteopontin. At longer time-points, we observed the formation of a homogeneous dentin bridge at the injury site, secreted by cells displaying an odontoblastic phenotype. In contrast, the reparative tissue induced by Ca(OH)(2) showed porous organization, suggesting a reparative process different from those induced by calcium silicate cements. Analysis of these data suggests that the evaluated cement can be used for direct pulp-capping.

Expression of MMP-2, 9 and 13 in newly formed bone after sinus augmentation using inorganic bovine bone in human.

BACKGROUND AND OBJECTIVE: The aim of the present study was to analyse the expression of MMP-2, MMP-9 and MMP-13 in newly formed bone following maxillary sinus augmentation using inorganic bovine bone substitute, because these MMPs play a major role in bone remodeling and bone resorption. MATERIAL AND METHODS: Deproteinized bovine bone (Bio-Oss(®)) was used to fill cavities after elevating the sinus mucosa. Twenty patients with edentulous posterior maxilla were treated with 20 sinus-augmentation procedures using a two-stage technique. Forty-nine Straumann(®) endosseous implants were used to complete the implant-prosthetic rehabilitation. One cylinder-shaped bone biopsy from each patient was taken from the augmented maxillary region using trephine burs at the second stage of surgery, 8 months after grafting. A biopsy was also taken as a control from the upper molar region from six different patients who did not undergo the sinus procedure. All biopsies were subjected to biochemical analysis and staining for TRAP. RESULTS: No implant losses or failures occurred. The large number of TRAP-positive multinucleated osteoclasts in resorption lacunae indicated that the resorption was very active in all grafts, in contrast with the control group. Zymography and western blot analysis demonstrated a significantly increased expression of MMP-2, MMP-9 and MMP-13 in the newly formed bone compared with controls (p < 0.05). CONCLUSION: The quantity of osteoclastic cells and the increased expression of proteolytic enzymes suggest that 8 months after grafting, inorganic bovine bone is slowly resorbing and is the site of important remodeling of the newly formed bone by means of resorption and synthesis.

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.

Periodontitis destructions are restored by synthetic glycosaminoglycan mimetic.

Periodontitis are bacterium-driven inflammatory diseases that destroy tooth-supporting tissues whose complete restoration is not currently possible. RGTA, a new class of agents, have this capacity in an animal model. Periodontitis was induced in hamsters and, starting 8 weeks later, injected RG1503, a glycosaminoglycan synthesized from a 40 kDa dextran behaving like a heparan sulfate mimetic (1.5 mg kg(-1) w(-1)) or saline for 8 weeks. The three periodontium compartments were evaluated by immunohistochemistry and morphometry. The gingival extracellular matrix disorganized by inflammation was restoring under treatment. The collagen network was repaired and resumed its previous organization. Fibrillin-1 expression was restored so that the elastic network rebuilt at a distance from the pocket and began to reconstruct near the pocket. Apoptotic cell numbers were decreased in the pocket epithelium, and more so in the infiltrated connective tissue. The continuity and the thickness of the basement membrane were restored and testified normalization of epithelium connective tissue interaction. The amount of alveolar bone increased around the first molar, and the interradicular bone was rebuilt. The root cementum was thickened and the number of proliferating cells in the periodontal ligament was increased close to the cementum. RG1503 treatment induces potent anabolic reactions in the extracellular matrices of the different tissues of the periodontium and recruitment of progenitors. In particular, the cell proliferation close to the root surface suggests the reformation of a functional attachment apparatus. These results demonstrate that RG1503 reverses the degenerative changes induced by inflammation and favors the conditions of a regenerative process. Thus, RGTA, a known matrix component mimetic and protector, may be considered as a new therapeutic tool to regenerate the tissues destroyed by periodontitis.

Histamine mediates osteoclastic resorption only during the acute phase of bone loss in ovariectomized rats.

Short-term studies have shown that histamine is involved, via its H2 receptors (H2R), in the mediator network regulating trabecular bone loss in long bones of ovariectomized (OVX) rats. It is not known whether this effect of histamine persists over time or involves other skeletal sites. In this study, rats were maintained for 6 months postOVX and treated daily with saline or famotidine (10 mg kg(-1)), an H2R antagonist. At the end of the experimental period, femur trabecular bone mass was markedly decreased in OVX rats, whether or not they were treated with famotidine. In contrast, in the fourth lumbar vertebra, where bone loss starts later than in the femur, famotidine treatment attenuated the decline in trabecular bone volume, protected the trabecular architecture, maintained the thickness of the cortices and reduced the numbers of osteoclasts and tartrate-resistant acid phosphatase-positive preosteoclasts, whereas it had no influence on bone formation parameters. In vertebral bone marrow of OVX rats, the numbers of mast cells (MCs) and non-MC histamine-producing cells increased, while famotidine treatment significantly diminished both cell populations. These data show that H2R antagonism does not protect trabecular bone mass in the long term, and that short-term protection involves all bones. Histamine is involved during the early phase of strong osteoclastic resorption but not during the late phase of slower resorption, suggesting that different mediator networks control the two phases of destruction. Histamine would be part of the network mediating the early phase.

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.

Effects of essential fatty acid deficiency on periodontal tissue adaptation to spontaneous tooth migration.

Essential fatty acids (EFAs) play a significant role in bone metabolism. Herein we studied the adaptation of alveolar bone to physiologic tooth drift in young rats deprived of essential fatty acids from birth. Reductions in femur size and trabecular bone volume reflected body growth impairment. Along the alveolar wall, osteoclastic resorption and bone formation were depressed, disrupting the adaptive deformation of the tooth socket to ongoing migration. As a result, the periodontal ligament narrowed considerably, and further adaptation was achieved through root resorption. Essential fatty acid deficiency (EFAD), did not affect precursor recruitment or differentiation in the periodontal ligament (PDL), but caused redistribution of nonspecific-esterase (NSE)-positive osteoclast precursors and tartrate-resistant acid phosphatase (TRAP)-positive pre-osteoclasts between the bone compartment (which was depleted) and the root compartment (which was enriched). EFAD had also a marked effect on the PDL vasculature; the number of vessels was reduced, whereas their size was markedly increased. As a whole, our results show that EFAD disturbs alveolar bone adaptation to drift, but that a reaction (detrimental to root integrity) prevents root collision with the bone surface, thereby preserving the PDL as a source of precursor cells for bone and cementum homeostasis. Moreover, our results confirm that although alveolar bone resorption is arachidonic acid-dependent, the factors activating root resorption are different.

Histamine participates in the early phase of trabecular bone loss in ovariectomized rats.

We have previously reported that cimetidine, a reference H2 receptor antagonist, attenuates the initial osteoclastic burst and subsequent trabecular bone loss induced by ovariectomy (ovx) in rats. This study was designed to determine whether these effects are specific to H2 antagonism. To this end, we compared the effects of two H2 receptor antagonists, cimetidine and famotidine. In addition, we analyzed the response of histamine-producing cells to these inhibitors. Seventy-two 90-day-old female Sprague-Dawley rats were ovariectomized or sham-operated, and received single daily intramuscular injections of cimetidine (125 mg/kg), famotidine (10 mg/kg), or vehicle. The animals were killed 14 days after surgery and their femurs were processed for histomorphometry. Trabecular bone volume was reduced by 30% in ovx rats and by 15% in cimetidine- and famotidine-treated rats. Architectural parameters were reduced by about 20% in ovx rats. Cimetidine and famotidine attenuated these consequences of ovx by about 50%. Trabecular connectivity was deteriorated by ovx, while cimetidine and famotidine attenuated this effect. Resorption parameters were increased by ovx, while cimetidine and famotidine prevented this increase. Kinetic bone formation parameters were increased by ovx, while cimetidine and famotidine had no influence. Neither cimetidine nor famotidine had any observable effect in sham-treated rats. Mast cell numbers increased by 250% in ovx rats and by only 40% in H2 antagonists-treated ovx rats. A resident histamine-positive, non-mast cell, population found in bone marrow was increased by 25% by ovx. Interestingly, cimetidine and famotidine reduced this population in both sham-operated and ovx rats, famotidine being more potent than cimetidine. These results show that H(2) receptor blockade partially prevents the consequences of castration on cancellous bone resorption in female rats, and strongly suggest that histamine participates in the mediator network regulating estrogen deficiency induced bone resorption. A large population of histamine-producing cells, which differ morphologically from mast cells and belong to an immature marrow population, may be a source of histamine in this model. The H(2) blockers targeted this population, and this effect appeared to explain the anti-resorptive action of the two drugs.

A new approach to treat tissue destruction in periodontitis with chemically modified dextran polymers.

Periodontitis are diseases of the supportive tissues of the teeth provoked by bacteria and characterized by gingival inflammation and bone destruction. We have developed a new strategy to repair tissues by administrating agents (RGTA) that mimic heparan sulfates by protecting selectively some of the growth factors naturally present within the injured tissue and interfering with inflammation. After periodontitis induction in hamsters, the animals were left untreated or received weekly i.m. injections of RGTA1507 at a dose of 100 microg/kg, 400 microg/kg, 1.5 mg/kg, or 15 mg/kg for 4 wk. RGTA treatment significantly reduced gingival tissue inflammation, thickened the pocket epithelium by increasing cell proliferation, and enhanced collagen accumulation in the gingiva. A marked reduction in bone loss was observed, resulting from depression of osteoclasia and robust stimulation of bone formation at the dose of 1.5 mg/kg. RGTA treatment for 8 wk at this dose reversed macroscopic bone loss, sharply contrasting with the extensive bone destruction in the untreated animals. RGTA treatment decreased gelatinase A (MMP-2) and B (MMP-9) pro-forms in gingival tissues. Our data indicate that a 4 wk treatment dose-dependently attenuated gingival and bone manifestations of the disease, whereas a longer treatment restored alveolar bone close to controls. By modulating and coordinating host responses, RGTA has unique therapeutic properties and is a promising candidate for the treatment of human periodontitis.

Impact of the timing of indomethacin treatment in a model of synchronized bone remodelling in rats.

Prostaglandins (PGs) promote both bone resorption and formation in vitro and in vivo. In a synchronised model of bone remodelling, indomethacin, an inhibitor of PG synthesis, given from the start of the sequence, transiently impaired bone resorption. In this study we further explored the involvement of PGs in this model by treating rats with indomethacin (7.5 mg x kg(-1) x day(-1)) for 6 days from the peak of resorption (day 4 after activation in this model) or during reversal (day 6 after activation). In rats treated from day 4, the resorption surface (Oc.S/BS) and the number of osteoclasts (N.Oc/BPm) were higher on day 10 (+69 %, P < 0.01, and +60 %, P < 0.02 compared with controls, respectively); no effect on cell resorptive activity was observed. The bone formation surface (OS/BS) was reduced (-50 %, P < 0.01). The inactive surface (In/BS) was not modified. In rats treated from day 6, the Oc.S/BS was also higher than in controls (P < 0.02), as was the N.Oc/BPm (P < 0.05). Osteoclast activity appeared to be increased, as the osteoclast-bone interface was larger (P < 0.02), but the mean lacuna area was reduced (-23 %, P < 0.05). Bone formation was also strongly affected: the OS/BS was decreased (-66 %, P < 0.01), as was the osteoid seam thickness (-24 %, P < 0.05). The In/BS was increased 1.5-fold (P < 0.05). These data indicate that PGs intervene at various stages of this remodelling sequence, as both resorption and formation were affected by indomethacin. Although resorption resumed in the two treatment groups despite treatment continuation, the timing of treatment was clearly important. Only inhibition of PG synthesis at the peak of resorption delayed all phases of the remodelling sequence. In contrast, inhibition during the reversal phase prevented activation of a significant part of the bone surface usually involved at this stage of remodelling; this treatment schedule reduced the resorptive capacity of the system, and depressed osteoblast activity.

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.

RGTA11, a new healing agent, triggers developmental events during healing of craniotomy defects in adult rats.

RGTA are chemically defined compounds which proved to be very potent healing agents in various tissue repair models including skin, muscle and nerve. These chemicals are believed to protect endogenously released heparin-binding growth factors and enhance their bioavailability during healing. In craniotomy defects that do not heal spontaneously in adults, RGTA promoted dose-dependent skull closure. The aim of this work was to characterize, in the same model, the events associated with wound closure by studying the expression of the osteoblastic phenotype and the distribution of some matrix proteins during RGTA11-induced bone healing. Craniotomy defects in rats were implanted with collagen plasters soaked in a solution of RGTA11 (1.5 micrograms per piece). The skulls were removed 30 days after wounding, a stage of almost complete bone filling in treated samples. Bone formed only at the edges of the defect in controls, while it formed also at the center in the form of nodules in the treated samples. RGTA11 modified the amount and distribution of the tissues including bone in the wounds. In some RGTA11-treated samples, skull closure by bone occurred and the median suture was restored. In the treated defects, alkaline phosphatase-positive (osteoprogenitor) cells were far more numerous and were distributed differently. Type I and III collagen and fibronectin deposition was markedly enhanced in the bone compartment of the wounds. Secretory osteoblasts released type III collagen. Osteocalcin expression was enhanced by RGTA11. RGTA11 thus modified the healing pattern by increasing both the cellularity and the synthesis of a bone-competent extracellular matrix, thereby restoring the original anatomy of the skull. Flat bone regeneration can be triggered in adults through developmental events (i.e. nodule formation, secretion of type III collagen by osteoblasts, suture restoration...) that are no longer operative in the wounds of mature individuals.

Multinucleated giant cells elicited around hydroxyapatite particles implanted in craniotomy defects are not osteoclasts.

BACKGROUND: The nature of the multinucleated giant cells (MNGC) elicited in contact with implantable biomaterials is still indecisive. METHOD: In Wistar rats the MNGC recruited after the implantation of hydroxyapatite (HA) particles in standardized skull defects were examined morphologically (at both the light and electron microscope levels), enzymatically (tartrate-resistant acid phosphatase and non-specific esterase), and after a challenge with salmon calcitonin. RESULTS: The MNGC were of great size and contained abundant mitochondria, vacuoles, and vesicles throughout the cytoplasm; they were either tightly apposed to the HA surface or had long and thin processes penetrating the material. When processed for tartrate-resistant acid phosphatase, only a few cells were weakly stained. The staining was totally suppressed when samples were pretreated with cyanuric chloride in the MNGC but not in the host osteoclasts. Calcitonin induced the withdrawal of the host osteoclasts from the bone surface while the MNGC remained in contact with the HA material. CONCLUSION: The MNGC recruited to HA particles did not exhibit the morphologic, enzymatic and functional characteristics of the osteoclasts, and consequently must be regarded as macrophage polykaryons.

Leukotriene inhibition in hamster periodontitis. A histochemical and morphometric study.

The effects of leukotriene (LT) inhibition on gingival and adjacent bone compartments were assessed by using phenidone (100 mg/kg/d) and ketoconazole (50 mg/kg/d) given for 4 weeks to periodontitis-affected hamsters. In the gingiva the two agents significantly decreased PMNL recruitment and migration and increased the vascular lumen. At the bone level, they reduced significantly preosteoclast and osteoclast numbers but did not affect osteoclast activity. Phenidone had no action on periodontitis induced inhibition of bone formation; in contrast ketoconazole enhanced formation. As both phenidone and ketoconazole are unspecific LT inhibitors it cannot be ascertained that the effects observed were actually due to LT inhibition. However, phenidone and ketoconazole induced changes different from indomethacin used in previous studies to inhibit the cyclooxygenase pathway. These discrepancies suggest that LT inhibition occurred in the present study and that they participate in gingival inflammation and osteoclastic destruction during hamster periodontitis.

Identification of osteoclasts and their mononuclear precursors. A comparative histological and histochemical study in hamster periodontitis.

Quantification of osteoclast resorption, a good index of periodontitis destruction, is primarily based on osteoclast identification. As their identification is sometimes dubious, we compared osteoclastic counts in hamster specimens processed for either routine histology or tartrate-resistant acid phosphatase (TRAP) staining. No difference was found between the two approaches concerning the number of osteoclasts. However the mean bone-osteoclast interface was higher in the TRAP-stained specimens (+30%, p less than 0.02). As osteoclast precursors are also TRAP+ cells, they were quantified too. Compared with controls, there was a dramatic increase (p less than 0.0001) in periodontitis-affected animals. Precursors were strongly correlated to active osteoclasts (r = 0.97). Our data suggest that precursors are recruited only when the disease is active in a given site.