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Macrophages are important regulators of bone remodeling, and M1 polarization is observed in the setting of medication-related osteonecrosis of the jaws (MRONJ). Here, we characterize the phenotype of macrophages during early stages of MRONJ development in zoledronate (ZA)–treated mice with periodontal disease and explore the role of rosiglitazone, a drug that has been reported to lower the M1/M2 macrophage ratio, in MRONJ burden. Mice received ZA, and experimental periodontal disease (EPD) was induced around their second left maxillary molar. The mice were euthanized 1, 2, or 4 wk later. Micro–computed tomography and histologic and immunohistochemical analyses were carried out. In a separate experiment, mice were treated with ZA in the absence or presence of rosiglitazone, EPD was induced for 5 wk, and the MRONJ burden was assessed. An M1 predilection was noted in ZA versus vehicle (Veh) mice at 1, 2, or 4 wk after ligature placement. M1 cells were found to be positive for MMP-13, and their presence coincided with disruption of the surrounding collagen network in ZA mice. Rosiglitazone caused a reversal in the M1/M2 polarization in Veh and ZA mice. Rosiglitazone did not cause significant radiographic changes 5 wk after EPD in Veh or ZA animals. Importantly, percentage osteonecrosis and bone exposure were decreased in the rosiglitazone-treated versus nontreated ZA sites 5 wk after EPD. Our data point to an important role of M1 macrophage polarization with an overexpression of MMP-13 in the early phases of MRONJ development and provide insight into the use of interventional approaches promoting an M2 phenotype as a preventative means to alleviate MRONJ burden. [ABSTRACT FROM AUTHOR]

Osteonecrosis of the jaws (ONJ), a severe side effect of antiresorptive medications, is characterized by exposed, nonhealing bone in the oral cavity. Treatment options for ONJ range from management of symptomology to surgical resection of the affected area. Antiresorptive discontinuation, often termed a "drug holiday," has been used for managing ONJ patients. Antiresorptives can be discontinued prior to oral surgical procedures, such as tooth extraction, to prevent ONJ development or in patients with established ONJ to accelerate healing. Here, our objective was to test these clinical scenarios using the potent bisphosphonate, zoledronic acid (ZA), and the denosumab surrogate for rodents, OPG-Fc, in a rat model of ONJ. Animals were pretreated with antiresorptives or saline, after which we induced ONJ using periapical disease and tooth extraction. In our first experimental design, antiresorptives were discontinued 1 wk prior to tooth extraction, and animals were evaluated 4 wk later for clinical, radiographic, and histologic features of ONJ. In the second experiment, ONJ was established and antiresorptives were discontinued for 4 wk. Discontinuation of OPG-Fc, but not ZA, prior to tooth extraction ameliorated subsequent ONJ development. In contrast, discontinuation of either ZA or OPG-Fc in rats with established ONJ did not lead to ONJ resolution. In conclusion, our findings suggest that antiresorptive discontinuation is dependent on both the type of antiresorptive and the timing of discontinuation. [ABSTRACT FROM AUTHOR]

Osteonecrosis of the jaws (ONJ) is a rare but severe complication of antiresorptive medications, such as bisphosphonates, used in the treatment of bone malignancy or osteoporosis. Tooth extraction and dental disease have been strongly associated with ONJ development. Here, we investigated molecular and cellular markers of socket healing after extraction of healthy or teeth with experimental periodontitis (EP) in Wistar-Han rats treated with zoledronic acid (ZA). We included 4 experimental groups: vehicle-treated animals with extraction of healthy teeth or teeth with ligature-induced EP and ZA-treated animals with extraction of healthy teeth or teeth with EP. Animals were pretreated with vehicle or ZA for a week, and EP was induced. Four weeks later, the second maxillary molars were extracted; sockets were allowed to heal for 4 wk; animals were euthanized; and maxillae were isolated. Radiographically, extraction sockets in groups 1, 2, and 3 demonstrated normal healing. Contrary incomplete socket healing was noted after extraction of teeth with EP in ZA-treated rats of group 4. Histologically, persistent inflammation and extensive osteonecrosis were seen in group 4. Disorganization of the collagen network, collagen type III predominance, and lack of collagen fiber insertion in the necrotic bone were associated with impaired socket healing. Cells positive for MMP-9, MMP-13, and α-SMA expression were present at the areas of epithelial invagination and adjacent to osteonecrotic bone. Importantly, human biopsies from patients with ONJ showed similar findings. Our data emphasize the importance of dental disease and tooth extraction in ONJ pathogenesis and help delineate an altered profile in wound-healing markers during ONJ development. [ABSTRACT FROM AUTHOR]

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws. [ABSTRACT FROM PUBLISHER]

Background: The prostaglandins (PG) E2 and PGF2α are important cytokines in periodontal physiology and pathology. PGE2 and PGF2α alter cell function by binding and activating the plasmamembrane G-protein-coupled PG receptors. In this study, we examined the PGE2 and PGF2α effects on the immortalized cementoblastic OCCM cells.Methods: Confluent OCCM cells were treated with PGE2 , PGF2α , specific activators/inhibitors of the EP prostanoid receptors, a specific activator of the FP prostanoid receptor, and direct activators/inhibitors of the protein kinase C (PKC) signaling pathway. Mineral nodule formation was assessed by the von Kossa stain.Results: PGE2 and PGF2α significantly increased mineralization of OCCM cells. The EP1 and EP3 PG receptor activators 16,16-dimethyl-prostaglandin E2 and sulprostone, also increased mineralization. In contrast, specific activators of the EP2 or the EP2/EP3/EP4 receptors did not have any effect. Fluprostenol, a specific activator of the FP receptor, significantly increased mineralization of OCCM cells. FP and EP (1 or 3) receptors signal through activation of the protein kinase C (PKC) pathway. Indeed, phorbol 12-myristate 13-acetate (PMA), a direct activator of the PKC pathway, significantly increase OCCM mineralization, while pre-treatment of OCCM cells with the PKC inhibitor GF109203× (bisindolylmaleimide) significantly decreased mineralization.Conclusion: We conclude that PGE2 and PGF2α exert an anabolic effect on OCCM mineralization through activation of PKC signaling. J Periodontol 2005;76:303-309. [ABSTRACT FROM AUTHOR]

Aim: To investigate the influence of diabetes mellitus (DM) in a murine model of peri-implantitis (PI)., Materials and Methods: Twenty-seven 4-week-old C57BL/6J male mice had their first and second maxillary left molars extracted. Eight weeks later, one machined implant was placed in each mouse. Four weeks after osseointegration, the mice were divided into three groups: (a) control (C), (b) PI and (c) DM + PI. DM was induced by streptozotocin (STZ) administration. After DM induction, PI was induced using ligatures for 2 weeks. The hemimaxillae were collected for micro-CT and histological analyses. The primary outcomes consisted of linear (mm) and volumetric (mm 3 ) bone loss. Secondary outcomes were based on histological analysis and included inflammatory infiltrate, osteoclastic activity, matrix organization, composition and remodelling. Data are presented as means ± SEM. Statistical analyses were performed using one-way ANOVA, followed by Tukey's test., Results: Gingival tissue oedema was detected in the PI and DM + PI groups. Micro-CT showed significantly increased linear and volumetric bone loss in the DM + PI group compared to the C and PI groups. H&E staining showed greater inflammatory response and bone resorption in the PI and DM + PI groups than in the C group. The DM + PI group had significantly higher osteoclast numbers than the C and PI groups. Picrosirius red stained less for types I and III collagen in the PI and DM + PI groups than in the C group. There was a significant increase in monocyte/macrophage (CD-11b) counts and matrix metalloproteinases (MMP-2 and MMP-8) marker levels and a significant decrease in the matrix metalloproteinases inhibition marker (TIMP-2) levels in the DM + PI group compared to the C and PI groups., Conclusions: DM exacerbates PI-induced soft-tissue inflammation, matrix degradation and bone loss., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Medication related osteonecrosis of the jaws (MRONJ), a rare side-effect of antiresorptive medications, is described as exposed bone in the oral cavity that lasts for at least 8 weeks. Most studies report a female predilection for MRONJ; these findings could be due to the increased use of antiresorptives in females, or due to inherent differences between male versus female patients., Purpose: The purpose of this study was to measure and compare the incidence and severity of osteonecrosis of the jaws (ONJ) between male and female mice., Study Design, Setting, Sample: We designed a randomized in-vivo animal study utilizing male and female mice treated with zoledronic acid (ZA). Experimental periodontitis was induced in 24 male and 24 female mice using a silk ligature following administration of saline or a potent bisphosphonate. After 8 weeks, animals were evaluated radiographically and histologically., Independent Variable: The independent variables were sex (male vs female) and treatment group (ZA vs saline control). Treatment was randomly assigned with balanced distribution between male and female animals., Main Outcome Variable: The main outcome variable was ONJ status coded as present or absent. ONJ was defined as present if there was histologic contact between the ligature and the alveolar bone. Secondary outcomes of interest were radiographic and histologic parameters., Analysis: Statistical differences were analyzed using a two-way analysis of variance with Tukey's post hoc test using a P value of 0.05 for significance., Results: The final sample was composed of 24 vehicle treated and 24 ZA treated animals. In vehicle treated animals, 8% of female and 8% of male animals developed ONJ. In ZA treated animals, 83% of female and 92% of male animals developed ONJ. Sex was not associated with the risk (measured as incidence of disease) for developing ONJ or in the radiographic or histologic parameters that were assessed (P values >.1)., Conclusions: Sex does not appear to affect the incidence of MRONJ or the severity of the disease as assessed by the radiographic and histologic parameters., (Copyright © 2024 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to assess CBCT scans of patients with medication related osteonecrosis of the jaws (MRONJ), osteoradionecrosis (ORN), osteomyelitis (OM) and jaw metastatic disease (JM), evaluate the presence and extent of radiologic findings, identify radiologic parameters that may distinguish the four entities and last, introduce a new modified radiographic index (CRIm), in order to contribute to the diagnosis of these conditions., Methods: Τwo major databases were retrospectively searched for fully documented and diagnosed CBCT scans of MRONJ, ORN, OM and JM from 2006 to 2019. 335 CBCT scans met the inclusion criteria and were assessed under standardized viewing conditions blindly by 2 observers. The CRIm index proposed in this study evaluates: lytic changes, sclerosis, periosteal bone formation, sequestration, non-healing extraction sockets and other findings which included: sinus implication, inferior alveolar canal implication and jaw fracture. Lytic changes, sclerosis, periosteal bone formation, sequestration and non-healing extraction sockets were scored as: absent (0), localized/single (1) and extensive/multiple (2). Each one of other findings were scored individually as: absent (0) and present (1). For statistical analysis t-test, Pearson's r correlation coefficient, one-way ANOVA and Bonferonni were performed., Results: Extensive lytic changes were the most common finding, especially for ORN, where it occurred in all CBCT scans (100%). The mean value of the CRIm index differs significantly between CBCT scans with MRONJ and JM, as well as between those with OM and JM (Bonferroni p < 0.001)., Conclusions: The new modified Composite Radiographic Index introduced in this study, appears to have improved an objective approach to the previously used Composite Radiographic Index by means of cumulative radiologic features. Τhe predominance of certain radiologic features in one or more of these entities may lead the diagnostician towards the correct diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious adverse effect of antiresorptive medications administered for control of osseous malignancy, osteoporosis, or other bone metabolic diseases. Despite being reported in the literature two decades ago, MRONJ etiology, pathophysiology, and progression remain largely unknown, and current nonoperative or operative treatment strategies are mostly empirical. Several hypotheses that attempt to explain the mechanisms of MRONJ pathogenesis have been proposed. However, none of these hypotheses alone is able to capture the complex mechanistic underpinnings of the disease. In this minireview, we aim to highlight key findings from clinical and translational studies and propose a unifying model for the pathogenesis and progression of MRONJ. We also identify aspects of the disease process that require further investigation and suggest areas for future research efforts toward calibrating methodologic approaches and validating experimental findings. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., Competing Interests: The authors declare that they do not have any real or perceived conflicts with the content of the manuscript. SLR is a consultant for Amgen. MRA has an active MTA with Amgen and research grants from MBX Biosciences. Neither of these is for work related to the contents of the current article., (© 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Introduction: Root resorption is a destructive process that compromises tooth structure and can result in tooth loss. Often it remains asymptomatic and is an incidental finding on radiographic examinations. The purpose of this study was to determine prevalence and characteristics of root resorption in patients referred for cone-beam computed tomography (CBCT) imaging for a variety of indications., Methods: The study included CBCT scans of 1086 consecutive patients referred for CBCT imaging over an 18-month period. A total of 1148 scans were acquired. Data were abstracted from radiology reports, and prevalence estimates of resorption were computed for the aggregate sample and also across specific indications., Results: Resorption was identified in 171 patients (15.7%, 95% CI: 13.6%-17.9%) and in 249 teeth with a prevalence range of 2.6%-92.3% across specific indications. An 18.7% of the patients had 2 resorption sites whereas 8.8% had 3 or more resorption sites. The majority of affected teeth were anteriors (43.8%), followed by molars (40.6%) and premolars (14.5%). The most prevalent resorption types were external (29.3%), cervical (22.5%), infection-induced apical resorption (13.7%), internal (9.6%), and impacted tooth induced (8.8%). The majority of teeth with resorption did not have prior endodontic treatment (73.9%) and had radiographically normal periapex (69.5%). Of 249 teeth with resorption, 31% presented as incidental finding. The prevalence of incidental findings of resorption increased with age, P < .05, and was significantly lower for anterior teeth (20.2%) as compared to premolars (41.7%) and molars (36.6%), (P < .05)., Conclusion: The high proportion of incidental findings of resorption detected by CBCT suggests that resorption is not recognized/detected by conventional radiography and therefore remains underdiagnosed., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Root resorption is a destructive process that compromises tooth structure and can result in tooth loss. Often it remains asymptomatic and is an incidental finding on radiographic examinations. The purpose of this study was to determine prevalence and characteristics of root resorption in patients referred for cone-beam computed tomography (CBCT) imaging for a variety of indications., Methods: The study included CBCT scans of 1086 consecutive patients referred for CBCT imaging over an 18-month period. A total of 1148 scans were acquired. Data were abstracted from radiology reports, and prevalence estimates of resorption were computed for the aggregate sample and also across specific indications., Results: Resorption was identified in 171 patients (15.7%, 95% CI: 13.6%-17.9%) and in 249 teeth with a prevalence range of 2.6%-92.3% across specific indications. An 18.7% of the patients had 2 resorption sites whereas 8.8% had 3 or more resorption sites. The majority of affected teeth were anteriors (43.8%), followed by molars (40.6%) and premolars (14.5%). The most prevalent resorption types were external (29.3%), cervical (22.5%), infection-induced apical resorption (13.7%), internal (9.6%), and impacted tooth induced (8.8%). The majority of teeth with resorption did not have prior endodontic treatment (73.9%) and had radiographically normal periapex (69.5%). Of 249 teeth with resorption, 31% presented as incidental finding. The prevalence of incidental findings of resorption increased with age, P < .05, and was significantly lower for anterior teeth (20.2%) as compared to premolars (41.7%) and molars (36.6%), (P < .05)., Conclusion: The high proportion of incidental findings of resorption detected by CBCT suggests that resorption is not recognized/detected by conventional radiography and therefore remains underdiagnosed., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Medication-related osteonecrosis of the jaw (MRONJ) is a severe complication of antiresorptive or antiangiogenic medications, used in the treatment of bone malignancy or osteoporosis. Bone necrosis, mainly represented by osteocytic death, is always present in MRONJ sites; however, the role of osteocyte death in MRONJ pathogenesis is unknown. High mobility group box 1 (HMGB1) is a non-histone nucleoprotein that in its acetylated form accumulates in the cytoplasm, whereas non-acetylated HMGB1 localizes in the nucleus. SIRT1 deacetylase regulates cellular localization of HMGB1. Interestingly, HMGB1 is released during cell necrosis and promotes inflammation through signaling cascades, including activation of the RAGE receptor. Here, we utilized a well-established mouse MRONJ model that utilizes ligature-induced experimental periodontitis (EP) and treatment with either vehicle or zolendronic acid (ZA). Initially, we evaluated HMGB1-SIRT1 expression in osteocytes at 1, 2, and 4 weeks of treatment. Significantly increased cytoplasmic and perilacunar HMGB1 expression was observed at EP sites of ZA versus vehicle (Veh) animals at all time points. SIRT1 colocalized with cytoplasmic HMGB1 and presented a statistically significant increased expression at the EP sites of ZA animals for all time points. RAGE expression was significantly higher in the submucosal tissues EP sites of ZA animals compared with those in vehicle group. To explore the significance of increased cytoplasmic and extracellular HMGB1 and increased RAGE expression in MRONJ pathogenesis, we used pharmacologic inhibitors of these molecules. Combined HMGB1/RAGE inhibition resulted in lower MRONJ incidence with statistically significant decrease in osteonecrotic areas and bone exposure versus non-inhibitor treated ZA animals. Together, our data point to the role of HMGB1 as a central alarmin, overexpressed at early phase of MRONJ pathogenesis during osteocytic death. Moreover, HMGB1-RAGE pathway may represent a new promising therapeutic target in patients at high risk of MRONJ. © 2022 American Society for Bone and Mineral Research (ASBMR)., (© 2022 American Society for Bone and Mineral Research (ASBMR).)

Objectives: This experimental study was carried out to investigate the effects of locally delivered nanoparticles (AMG-487 NP) containing a CXCR3 antagonist in inhibiting the progression of LPS-induced inflammation, osteoclastic activity, and bone resorption on a murine model., Materials and Methods: Thirty, 7-week-old C57BL/6 J male mice were used. Inflammatory bone loss was induced by Porphyromonas gingivalis-lipopolysaccharide (P.g.-LPS) injections between the first and second maxillary molars, bilaterally, twice a week for 6 weeks (n = 20). AMG-487 NP were incorporated into a liposome carrier and locally delivered on sites where P.g.-LPS was injected. Control mice (n = 10) were injected with vehicle only. Experimental groups included (1) control, (2) LPS, and (3) LPS + NP. At the end of 1 and 6 weeks, mice were euthanized, maxillae harvested, fixed, and stored for further analysis., Results: Volumetric bone loss analysis revealed, at 1 week, an increase in bone loss in the LPS group (47.9%) compared to control (27.4%) and LPS + NP (27.8%) groups. H&E staining demonstrated reduced inflammatory infiltrate in the LPS + NP group compared to LPS group. At 6 weeks, volumetric bone loss increased in all groups; however, treatment with the CXCR3 antagonist (LPS + NP) significantly reduced bone loss compared to the LPS group. CXCR3 antagonist treatment significantly reduced osteoclast numbers when compared to LPS group at 1 and 6 weeks., Conclusions: This study showed that local delivery of a CXCR antagonist, via nanoparticles, in a bone resorption model, induced by LPS injection, was effective in reducing inflammation, osteoclast numbers, and bone loss., Clinical Relevance: CXCR3 blockade can be regarded as a novel target for therapeutic intervention of bone loss. It can be a safe and convenient method for periodontitis treatment or prevention applicable in clinical practice., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Antiresorptive agents such as bisphosphonates (BP) and denosumab are commonly prescribed for the management of primary bone malignancy, bone metastasis, osteoporosis, Paget disease, or other bone disorders. Medication-related osteonecrosis of the Jaws (MRONJ) is a rare but significant complication of antiresorptive medications. Duration, dose, and antiresorptive potency as well as concomitant diseases, additional medications, and local factors affect MRONJ incidence and severity. MRONJ pathophysiology is still poorly understood. Nevertheless, decreased bone resorption due to osteoclastic inhibition along with trauma, infection/inflammation, or blood supply inhibition are considered synergistic factors for disease development. In addition, previous data research examined the effects of antiresorptive medication on immune system components and introduced potential alterations on immune response as novel elements in MRONJ pathogenesis. Considering that macrophages are the first cells in the nonspecific immune response, it is not surprising that these multifaceted players attracted increased attention in MRONJ research recently. This current review attempted to elucidate the effects of antiresorptive medications on several aspects of macrophage activity in relation to the complex inflammatory microenvironment of MRONJ. Collectively, unravelling the mode of action and extent of macrophages' potential contribution in MRONJ occurrence will provide novel insight in disease pathogenesis and potentially identify intrinsic therapeutic targets.

Medication related osteonecrosis of the Jaws (MRONJ) is a severe complication of antiresorptive and anti-angiogenic medications. Osteoclast inhibition is central in MRONJ pathogenesis. Here, we investigated if local application of RANKL (a key molecule in osteoclast activation) could enhance osteoclast generation and improve extraction socket healing in the presence of bisphosphonates. Thirty Wistar-Han rats received one saline or 66 μg/kg zoledronate (ZA) i.p. dose before surgery. A week later, mandibular molars were extracted bilaterally. Collagen tapes infused with water or RANKL were placed in the extraction sockets of 60 hemimandibles of veh (veh/RANKL-, veh/RANKL+) or ZA treated rats (ZA/RANKL-, ZA/RANKL+). Rats were euthanized 3 or 12 days after surgery. Animals euthanized at 12 days received two additional veh or ZA injections. Clinical, radiographic and histologic assessments were performed. Visually, at the 3-day timepoint, no sockets demonstrated complete healing. At the 12-day timepoint, sockets of veh/RANKL- and veh/RANKL+ rats showed intact mucosa, while mucosal defects were noted in ZA/RANKL- rats. Importantly, ZA/RANKL+ sockets showed absence of bone exposure. RANKL delivery increased bone healing in the ZA/RANKL+ sites 12 days after extraction compared to the ZA/RANKL- sites. Histologically, at the 3-day timepoint, ZA/RANKL- sockets demonstrated extensive bone exposure and osteonecrosis. In contrast, ZA/RANKL+ rats showed granulation tissue coverage and significantly reduced osteonecrosis, similar to the veh groups. Importantly, in the ZA/RANKL+ group, osteoclasts attached to the bone surface and osteoclast numbers were higher compared to ZA/RANKL- sites. At the 12-day timepoint, persistent osteonecrosis and bone exposure were detected in the sockets of ZA/RANKL- animals. Contrary, ZA/RANKL+ rats demonstrated socket epithelialization and reduced osteonecrosis. Significantly more total and bony attached osteoclasts persisted in the ZA/RANKL+ vs the ZA/RANKL- group. We present a novel approach towards improving socket healing, in the presence of ZA, by enhancing osteoclastic numbers and attachment through local RANKL application. Our approach is clinically applicable and could improve treatment outcomes of patients on high-dose ZA therapy., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: The aim of this study was to investigate the relationship between M1 and M2 macrophage polarization and clinical stage in patients with medication-related osteonecrosis of the jaw (MRONJ) who underwent treatment with bisphosphonates or denosumab., Materials and Methods: M1 and M2 macrophage density and expression of interleukin (IL)-6 and IL-10 were assessed on biopsies of mucosal tissues surrounding necrotic bone in 30 MRONJ patients with stages 1-3 and controls. For identification of M1 and M2 macrophages, double CD68/iNOS and CD68/CD206 immunofluorescence staining was conducted, respectively. Computer-assisted immunofluorescence quantification of markers was performed., Results: Early stage 1 MRONJ patients showed a switch toward the M2 phenotype, as indicated by the higher density of M2 macrophages, the decreased M1/M2 ratio, and the upregulation of IL-10. MRONJ patients with advanced stages 2 and 3 showed a shift toward M1-polarized macrophages, as suggested by the higher density of M1 macrophages, the increased M1/M2 ratio, and the overexpression of IL-6. The macrophage density of both M1 and M2 subsets was significantly enhanced in patients receiving bisphosphonates compared with those receiving denosumab., Conclusions: The M1-M2 macrophage polarization status in mucosal tissues bordering necrotic bone correlates with clinical stage of MRONJ. Patients with early-stage MRONJ show a switch toward M2-polarized macrophages, while MRONJ patients with advanced stage demonstrate a shift toward the M1 phenotype., Clinical Relevance: Therapeutic molecules targeting the inflammatory microenvironment via the regulation of either M1 or M2 macrophage polarization may represent a novel strategy for treatment of MRONJ.

CCN1/Cyr61 is a dynamically expressed matricellular protein that serves regulatory functions in multiple tissues. Previous studies from our laboratory demonstrated that CCN1 regulates bone maintenance. Using an osteoblast and osteocyte conditional knockout mouse model (Ccn1 OCN ), we found a significant decrease in trabecular and cortical bone mass in vivo, in part through suppression of Wnt signaling since the expression of the Wnt antagonist sclerostin (SOST) is increased in osteoblasts lacking CCN1. It has been established that parathyroid hormone (PTH) signaling also suppresses SOST expression in bone. We therefore investigated the interaction between CCN1 and PTH-mediated responses in this study. We find that loss of Ccn1 in osteoblasts leads to impaired responsiveness to anabolic intermittent PTH treatment in Ccn1 OCN mice in vivo and in osteoblasts from these mice in vitro. Analysis of Ccn1 OCN mice demonstrated a significant decrease in parathyroid hormone receptor-1 (PTH1R) expression in osteoblasts in vivo and in vitro. We investigated the regulatory role of a non-canonical integrin-binding domain of CCN1 because several studies indicate that specific integrins are critical to mechanotransduction, a PTH-dependent response, in bone. These data suggest that CCN1 regulates the expression of PTH1R through interaction with the αvβ3 and/or αvβ5 integrin complexes. Osteoblasts that express a mutant form of CCN1 that cannot interact with αvβ3/β5 integrin demonstrate a significant decrease in mRNA and protein expression of both PTH1R and αv integrin. Overall, these data suggest that the αvβ3/β5-binding domain of CCN1 is required to endow PTH signaling with anabolic activity in bone cells. © 2020 American Society for Bone and Mineral Research (ASBMR)., (© 2020 American Society for Bone and Mineral Research (ASBMR).)

Objective: The aim of this study was to investigate the effects of the long-term alendronate administration on bone healing in defects created in rat calvarias., Materials and Methods: Female Wistar rats were randomly distributed into 2 groups: Control (CTL): animals received saline solution once a week; and Alendronate (ALD): rats underwent alendronate treatment (1 mg/kg/weekly). After 120 days from the commencement of treatment, a critical size defect was created in all animals, and 10 animals from each group were sacrificed at 5, 10, 15, 20, 25, 30, 45 and 60-days after the defect creation. On the day of sacrifice, urine and blood samples were collected for determination of the serum levels of bone resorption and formation markers by enzyme linked immunosorbent assay, and the urinary concentration of deoxypyridinoline. Bone mineral density (BMD) in the femurs, descriptive histology, tartrate-resistant acid-phosphatase staining and immunohistochemical analyzes were assessed in the calvaria., Results: Alendronate group showed increased BMD compared to the test group. The concentration of C-terminal telopeptide of type I collagen and deoxypyridinoline decreased significantly, and the concentration of aminoterminal propeptide of procollagen type 1 and osteocalcin were significant lower in the alendronate group. Immunohistochemical analysis showed significant downregulation in the inducible nitric oxide synthase, runt-related transcription factor-2, cathepsin-K and receptor activator of nuclear factor kappa-B ligand expression in the alendronate group. Vascular endothelial growth factor and osteopontin were upregulated in the later periods of alendronate group., Conclusions: Our results suggest that long-term treatment with alendronate did not compromise the repair processing of critical size defects in rat., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We sought to evaluate the effects of magnesium (Mg) intake deficiency on bone metabolism in rats with induced periodontal disease (PD). Holtzman rats were randomly divided into two groups: Control - animals fed a standard diet and test - animals fed a diet with 90% Mg deficiency. After 60 days on the diets, all animals received ligature on the lower left first molars to induce PD. Animals were euthanized after 30 days following ligature placement. Blood and urine were collected for determination of serum concentrations of Mg, calcium, osteocalcin (OCN), alkaline phosphatase and parathyroid hormone (PTH) by enzyme-linked immunosorbent assay, and the urinary concentration of deoxypyridinoline (DPD). Systemic bone mineral density (BMD), bone volume and architectural bone parameters were evaluated by micro-CT in L4 lumbar vertebrae and mandible. Tartrate-resistant acid phosphatase staining and immunohistochemical (IHC) analysis of inducible nitric oxide synthase (iNOS), Runt-related transcription factor 2 (RUNX2), CD86, CD80, proliferating cell nuclear antigen, vascular endothelial growth factor, OCN and osteopontin were investigated. Reverse-transcription polymerase chain reaction was employed to assess mRNA expression of receptor-activator of nuclear factor-kB ligand, osteoprotegerin (OPG) and interleukin (IL)-6. Mg deficiency was associated with higher concentrations of PTH and DPD, and significant decrease on both systemic and mandibular BMD, as well as greater severity of alveolar and trabecular bone loss. Significant increase in osteoclasts was observed in the test group with PD. IHC analysis showed significant increase in the expression of iNOS and decreased expression of OCN and RUNX2. Increased IL-6 mRNA and decreased OPG mRNA expressions were evidenced in the test group with PD. Mg deficiency caused systemic effects indicative of altered bone metabolism in the vertebrae and affected both immune and stromal cells, aggravating inflammatory bone resorption in the ligature-induced model of periodontitis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteonecrosis of the jaw (ONJ), a rare, but potentially severe side effect of anti-resorptive medications, presents as exposed bone in the maxillofacial region lasting for at least 8 weeks. While clinical experience and animal models concur in finding that systemic antiresorptive treatment in conjunction with local risk factors, such as tooth extraction or dental disease may lead to ONJ development, the subclinical molecular changes that precede bone exposure remain poorly understood. The identification of these changes is not only important in understanding disease pathophysiology, but could provide potential for treatment development. Here, we evaluated the early stages of ONJ utilizing a model of experimental periodontitis (EP) in mice treated with two different types of antiresorptives, targeting potential changes in vasculature, hypoxia, oxidative stress, and apoptosis. Antiresorptive treatment in animals with EP increased levels of empty osteocytic lacunae and increased ONJ prevalence compared to Veh animals. The arteriole and venule network seen around EP areas was diminished in animals treated with antiresorptives. Higher levels of vascular endothelial growth factor A (VEGF-A) and vascular cell adhesion protein-1 (VCAM-1) were observed 1-week following EP in treated animals. Finally, levels of hypoxia, oxidative stress, and apoptosis remained high in antiresorptive treated animals with EP through the duration of the experiment. Together, our data point to subclinical vasculature organizational disturbances that subsequently affect levels of hypoxia, oxidative stress, and apoptosis in the area of developing ONJ., (Copyright © 2019. Published by Elsevier Inc.)

Objectives: The aim of this study was to investigate bone turnover alterations after alendronate (ALD) withdrawal and its influence on dental implants osseointegration., Materials and Methods: Seventy female Wistar rats were randomly divided in 2 groups that received on day 0 either placebo (control group-CTL; n = 10) or 1 mg/kg sodium alendronate (ALD; n = 60) once a week for 4 months. At day 120, ALD treatment was suspended for 50 animals. Then, a titanium implant was placed in the left tibia of each rat that were randomly allocated in five subgroups of ten animals each, according to the period of evaluation: day 0 (INT-0), day 7 (INT-7), day 14 (INT-14), day 28 (INT-28), and day 45 (INT-45) after ALD withdrawal. CTL group and a group that received ALD until the end of the experimental period (non-interrupted group-non-INT; n = 10) underwent implant placement on day 120. Animals were euthanized 28 days after implant surgery. Bone mineral density (BMD) of femur and lumbar vertebrae were evaluated by DXA, biochemical markers of bone turnover were analyzed by ELISA, and bone histomorphometry was performed to measure bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO)., Results: All groups receiving ALD showed higher BMD values when compared to CTL group, which were maintained after its withdrawal. Decreased concentrations in all bone turnover markers were observed in the non-INT group, and in the groups in which ALD was discontinued compared to the CTL group. The non-INT group showed lower %BIC and notably changes in bone quality, which was persistent after drug withdrawal., Conclusion: Collectively, the findings of this study demonstrated that ALD therapy decreased bone turnover and impaired bone quality and quantity around dental implants, and that its discontinuation did not reverse these findings., Clinical Relevance: The severe suppression of bone turnover caused by the prolonged use of ALD may alter the capacity of bone tissue to integrate with the implant threads impairing the osseointegration process.

Objectives: To explore whether differences exist in the clinical and radiographic presentation of oncologic vs osteoporotic patients with medication-related osteonecrosis of the jaw (MRONJ)., Methods: We retrospectively assessed panoramic radiographs and CBCT examinations of 70 MRONJ patients receiving antiresorptive medications for the management of either osteoporosis or bone malignancy. Radiographic features of MRONJ were documented and categorized according to severity. A composite radiographic index (CRI) was constructed to account for the heterogeneity in radiographic manifestations of MRONJ and further stratify extent of osseous changes., Results: Patients with osteoporosis were mostly older females and presented more frequently with Stage 2 MRONJ, while patients with malignancy were equally distributed between males and females, and presented mostly with Stage 1 MRONJ. Most MRONJ lesions in oncologic patients occurred in the mandible, whereas the maxilla and mandible were equally affected in osteoporotic patients. Patients with minimal radiographic changes (low CRI score) often presented with MRONJ in dentate areas, while most patients in medium and high CRI groups presented with MRONJ after recent tooth extraction. The low CRI group consisted of primarily oncologic patients, while osteoporotic vs oncologic patients were divided more evenly in the other CRI groups ( p = 0.083). While CRI scores increased with clinical staging, a Spearman's rank correlation coefficient of 0.49 suggests that clinical appearance does not reliably predict osseous changes., Conclusions: Our data identify differences in the MRONJ appearance of patients with osteoporosis vs malignancy and emphasize the significance of detailed radiographic assessment, in addition to the clinical appearance, in characterizing the osseous changes of the disease.