Reliability of new poly (lactic-co-glycolic acid) membranes treated with oxygen plasma plus silicon dioxide layers for pre-prosthetic guided bone regeneration processes.

BACKGROUND: The use of cold plasmas may improve the surface roughness of poly(lactic-co-glycolic) acid (PLGA) membranes, which may stimulate the adhesion of osteogenic mediators and cells, thus accelerating the biodegradation of the barriers. Moreover, the incorporation of metallic-oxide particles to the surface of these membranes may enhance their osteoinductive capacity. Therefore, the aim of this paper was to evaluate the reliability of a new PLGA membrane after being treated with oxygen plasma (PO2) plus silicon dioxide (SiO2) layers for guided bone regeneration (GBR) processes. MATERIAL AND METHODS: Circumferential bone defects (diameter: 11 mm; depth: 3 mm) were created on the top of eight experimentation rabbits' skulls and were randomly covered with: (1) PLGA membranes (control), or (2) PLGA/PO2/SiO2 barriers. The animals were euthanized two months afterwards. A micromorphologic study was then performed using ROI (region of interest) colour analysis. Percentage of new bone formation, length of mineralised bone, concentration of osteoclasts, and intensity of ostheosynthetic activity were assessed and compared with those of the original bone tissue. The Kruskal-Wallis test was applied for between-group com Asignificance level of a=0.05 was considered. RESULTS: The PLGA/PO2/SiO2 membranes achieved the significantly highest new bone formation, length of mineralised bone, concentration of osteoclasts, and ostheosynthetic activity. The percentage of regenerated bone supplied by the new membranes was similar to that of the original bone tissue. Unlike what happened in the control group, PLGA/PO2/SiO2 membranes predominantly showed bone layers in advanced stages of formation. CONCLUSIONS: The addition of SiO2 layers to PLGA membranes pre-treated with PO2 improves their bone-regeneration potential. Although further research is necessary to corroborate these conclusions in humans, this could be a promising strategy to rebuild the bone architecture prior to rehabilitate edentulous areas.

Cephalometric assessment of craniofacial dysmorphologies in relation with Msx2 mutations in mouse.

OBJECTIVES: To determine the role of Msx2 in craniofacial morphology and growth, we used a mouse model and performed a quantitative morphological characterization of the Msx2 (-/-) and the Msx2 (+/-) phenotype using a 2D cephalometric analysis applied on micrographs. MATERIALS AND METHODS: Forty-four three-and-a-half-month-old female CD1 mice were divided into the following three groups: Msx2 (+/+) (n = 16), Msx2 (+/-) (n = 16), and Msx2 (-/-) (n = 12). Profile radiographs were scanned. Modified cephalometric analysis was performed to compare the three groups. RESULTS: Compared with the wild-type mice, the Msx2 (-/-) mutant mice presented an overall craniofacial size decrease and modifications of the shape of the different parts of the craniofacial skeleton, namely the neurocranium, the viscerocranium, the mandible, and the teeth. In particular, dysmorphologies were seen in the cochlear apparatus and the teeth (taurodontism, reduced incisor curvature). Finally contrary to previous published results, we were able to record a specific phenotype of the Msx2 (+/-) mice with this methodology. This Msx2 (+/-) mouse phenotype was not intermediate between the Msx2 (-/-) and the wild-type animals. CONCLUSION: Msx2 plays an important role in craniofacial morphogenesis and growth because almost all craniofacial structures were affected in the Msx2(-/-) mice including both intramembranous and endochondral bones, the cochlear apparatus, and the teeth. In addition, Msx2 haploinsufficiency involves a specific phenotype with subtle craniofacial structures modifications compared with human mutations.

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.

Matrix therapy in regenerative medicine, a new approach to chronic wound healing.

Nonhealing wounds remain a major health problem whose treatment is challenging and costly. Treatments based on cells or growth factors are still not very effective. We developed an entirely novel strategy consisting in treatment of the wound-tissue matrix with biopolymers engineered to mimic heparan sulfates called OTR4120. This compound was dextran polymer with sulfated and carboxymethyl groupments. After binding to matrix proteins, the heparan-sulfate-mimicking polymer protects the microenvironment, maintaining the normal production of signals and growth factors needed for healing to occur. Here, we show that a specific biopolymer accelerates ulcer closure and improves re-epithelialization and dermal-matrix-component remodeling. OTR4120 treatment was associated with faster maturation of epidermal structures, most notably regarding the number of epithelial-cell layers, and with an appearance that more closely resembled normal skin. Treatment had also a main effect on collagen I and III expression. Necrotic skin ulcers induced in mice with doxorubicin recovered normal collagen levels and organization, with no evidence of fibrosis. Thus, appropriate polymer-based matrix therapy is a valid and simple alternative to regenerative medicine.

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.

Kinetic study of early regenerative effects of RGTA11, a heparan sulfate mimetic, in rat craniotomy defects.

We previously reported that RGTA, a synthetic heparan sulfate mimetic, induces almost complete closure of craniotomy defects one month after surgery in adult rats. RGTA-treated wounds showed features suggesting unusual cell and matrix interactions reminiscent of developmental events. As healing success or failure is determined shortly after wounding, we examined early events in RGTA-treated wounds. Collagen plasters soaked in a solution of RGTA11 (1.5 Microg per piece) or saline (control) were implanted in rat craniotomy defects. Seven control and seven treated rats were killed daily from days 1 to 7 after surgery. The lesions and adjacent tissues were sampled and processed for morphometry. A layer of type III collagen along the dura mater (DM) thickened up to day 5 in RGTA-treated wounds (p < 0.05 vs day 1), but became thinner in control wounds. Alkaline phosphatase-positive osteoprogenitor cells were detected on day 1 in this layer. Their number increased, and they migrated toward the mid-sagittal sinus and to connective tissue adjacent to the sinus, where they aggregated and differentiated into osteoblasts, forming bone nodules on day 6. These features were not seen in control wounds. Angiogenesis was significantly enhanced in RGTA-treated wounds, especially near the sinus. In vitro, bovine bone endothelial (BBE) cell proliferation was inhibited by RGTA11 in a concentration-dependent manner. In contrast, RGTA11 strongly enhanced the effect of fibroblast growth factor-2 on BBE cell proliferation. These results show that RGTA11, possibly by interacting with heparin-binding growth factors, elicits vascular reactions accompanying the recruitment of a large pool of committed osteoprogenitors from the DM. The DM and the sinus appear to be important centers of organization for craniotomy defect healing. RGTA probably creates an environment that starts a program of directing healing towards bone formation and defect closure.

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.

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.

Short-term prevention of osteoclastic resorption and osteopenia in ovariectomized rats treated with the H(2) receptor antagonist cimetidine.

Ovariectomy rapidly induces strong osteoclast differentiation, leading to a marked loss of cancellous bone in the rat appendicular skeleton. As we found that histamine inhibition prevented periosteal bone resorption in rats, we tested the hypothesis that cimetidine, an H(2) receptor antagonist, prevents the osteoclastic burst and subsequent trabecular bone loss in this setting. Forty female Sprague-Dawley rats were ovariectomized (ovx) or sham-operated. Rats from each group received daily intramuscular injections of cimetidine (125 mg/kg per day) or vehicle. The animals were killed 14 days after surgery, and their femora were processed for morphometry. Cimetidine had no effect on serum estradiol levels in the control and ovx rats. BV/TV was reduced by 36% in the ovx rats, and by 10% in the cimetidine treated rats (p < 0.01). Tb.N and Tb.Wi were significantly reduced by 30% in the ovx rats and by 15% ovx-treated ones. OcS/BS did not change in the treated ovx rats, but increased 3.7-fold in the untreated ovx ones (p < 0.001). The N.Oc/TBPm increased markedly in the ovx rats (2.6-fold, p < 0.0001 vs. controls), but only slightly in the cimetidine-treated animals (+18%, p < 0.05 vs. controls), with a significant difference between the cimetidine-treated and -untreated ovx animals (p < 0.001). Cimetidine had no effect on these parameters in sham-operated animals. These results show that histamine inhibition by an H(2) receptor antagonist partially prevents the consequences of castration on cancellous bone, possibly by an action on osteoclast differentiation. Interestingly, cimetidine had no effect on basal resorption along trabecular bone. Histamine inhibition by H(2) blockers warrants further investigation in this model of osteopenia.

Time-course of mast cell accumulation in rat bone marrow after ovariectomy.

We previously reported that mast cells accumulate in the tibia bone marrow of ovariectomized (OVX) rats. In this study, the timing of mast cell accumulation and osteoclast generation were compared to determine whether or not mast cell accumulation preceded osteoclast recruitment after ovariectomy. This may be significant because of the number of cytokines released by mast cells that are potentially active on resorption. Sprague-Dawley rats (120) aged 12 weeks were OVX or sham-operated, and killed on days 4, 7, 14, 28, and 56 postsurgery. Ten additional intact rats were used as baseline controls. Ovariectomy was confirmed by a sharp and sustained fall in serum estradiol. The loss in trabecular bone volume (BV/TV) began on day 7, reaching 80% on day 56 (P < 0.001 vs baseline controls). The number of osteoclasts (N.OC/TBPm) increased in the OVX rats between days 4 and 7 (+130%; P < 0.001), and continued rising to day 28. During the next month, it decreased greatly (-63%, P < 0.001 on day 56 vs day 28). In the sham-treated rats, few mast cells were scattered in the bone marrow (1.9 cells/mm2 in the baseline controls). Their number fluctuated during the experimental period, but at each time-point it was lower than in the OVX rats. They were predominantly (90%) of the mucosal subtype. In the OVX rats, their number doubled between days 4 and 14 (P < 0.001), reached 8.6 cells/mm2 on day 28 (a 5.4-fold increase compared with day 4 OVX rats), and plateaued for the next 4 weeks. OVX had no effects on mast cell subtypes. In conclusion, mast cell accumulation and osteoclast differentiation are precocious and concomitant; this does not support a direct role for mast cells in osteoclast recruitment. Rather, the two cell populations may derive from a common precursor or be targeted simultaneously by estrogen depletion through common stimulator(s). Mast cell hyperplasia appears to be a significant, and usually unknown, manifestation of ovariectomy in the bone marrow environment.

Strontium ranelate inhibits bone resorption while maintaining bone formation in alveolar bone in monkeys (Macaca fascicularis).

Strontium ranelate (S12911) has previously been shown to stimulate bone formation and inhibit bone resorption in rats. To determine whether strontium ranelate affects normal bone remodeling, we studied the effect of strontium ranelate on alveolar bone in monkeys. Strontium ranelate, at dosages of 100, 275, and 750 mg/kg per day, or vehicle, were given by gavage to 31 normal adult monkeys (Macaca fascicularis) (15 males, 16 females), aged 3-4 years. Treatment for 6 months with strontium ranelate resulted in an increase in plasma strontium concentration. Histomorphometric analyses of indices of bone formation and resorption were determined in standardized areas of alveolar bone. Treatment with strontium ranelate decreased the histomorphometric indices of bone resorption (osteoclast surface and number) with a maximal significant effect at the highest dose tested. In contrast to this inhibitory effect on bone resorption, strontium ranelate maintained bone formation. Although the amount of osteoid tended to increase, strontium ranelate, even at the highest dose, had no deleterious effect on bone mineralization, as evaluated by mineral apposition rate and osteoid thickness. These findings show that strontium ranelate decreases indices of bone resorption while maintaining bone formation in the alveolar bone in monkeys.

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.

A single low dose of RGTA, a new healing agent, hastens wound maturation and enhances bone deposition in rat craniotomy defects.

RGTA, a new family of dextran-derived healing agents, promotes the repair of various tissues, including bone. In this study, we examined whether a dose of RGTA lower than in our previous studies could still modify the healing pattern in craniotomy defects. In 24 rats, two defects (3 mm diameter) were drilled on either side of the calvaria sagittal suture. The right defect was filled with a piece of collagen soaked with RGTA in phosphate-buffered saline (PBS; 4 microg/ml), and the left one with collagen soaked in PBS only. After 7, 14 and 21 days, the calvaria were removed and processed for histometry. On day 7, in contrast with the control defects, the treated sites were inflammation-free and centripetal bone plates had started to grow. By day 14, the bone filling was significantly enhanced in the treated defects (+290%, p<0.05), and isolated bone nodules had formed within the fibrous connective tissue (= fibrous hammock) joining the defect edges. The hammock had already differentiated by day 7 in all the RGTA-treated defects, and it was significantly thicker on days 14 (+190%, p<0.05) and 21 (+139%, p<0.05). The colonization of the hammock by mast cells was increased in the treated sites (+320%, p<0.05 on day 21). On day 7, most of the bony edges of the treated defects had been resorbed by osteoclasts, while the process only started in the controls. These data indicate that a low dose of RGTA modified the cascade of events occurring at the initial stages of repair, so that the tissular maturation of the treated defects was more rapid. In fact the use of RGTA in the wounds provoked a shift from a fibrous repair as seen in the controls, to a bone reconstruction favoring defect closure.