Osteoclast Density Research Articles

Intratumoral Heterogeneity Assessment of the Extracellular Bone Matrix and Immune Microenvironment in Osteosarcoma Using Digital Imaging to Predict Therapeutic Response

The assessment of chemotherapy response in osteosarcoma (OS) based on the average percentage of viable cells is limited, as it overlooks the spatial heterogeneity of tumor cell response (foci of resistant cells), immune microenvironment, and bone microarchitecture. Despite the resulting positive classification for response to chemotherapy, some patients experience early metastatic recurrence, demonstrating that our conventional tools for evaluating treatment response are insufficient. We studied the interactions between tumor cells, immune cells (lymphocytes, histiocytes, and osteoclasts), and bone extracellular matrix (ECM) in 18 surgical resection samples of OS using multiplex and conventional immunohistochemistry (IHC: CD8, CD163, CD68, and SATB2), combined with multiscale characterization approaches in territories of good and poor response (GRT/PRT) to treatment. GRT and PRT were defined as subregions with <10% and ≥10% of viable tumor cells, respectively. Local correlations between bone ECM porosity and density of immune cells were assessed in these territories. Immune cell density was then correlated to overall patient survival. Two patterns were identified for histiocytes and osteoclasts. In poor responder patients, CD68 osteoclast density exceeded that of CD163 histiocytes but was not related to bone ECM load. Conversely, in good responder patients, CD163 histiocytes were more numerous than CD68 osteoclasts. For both of them, a significant negative local correlation with bone ECM porosity was found (P < .01). Moreover, in PRT, multinucleated osteoclasts were rounded and intermingled with tumor cells, whereas in GRT, they were elongated and found in close contact with bone trabeculae. CD8 levels were always low in metastatic patients, and those initially considered good responders rapidly died from their disease. The specific recruitment of histiocytes and osteoclasts within the bone ECM, and the level of CD8 represent new features of OS response to treatment. The associated prognostic signatures should be integrated into the therapeutic stratification algorithm of patients after surgery.

Increased nerve growth factor expression and osteoclast density are associated with subchondral bone marrow lesions in osteoarthritic knees

ObjectivesSubchondral bone marrow lesions (BMLs) detected on magnetic resonance imaging in knee osteoarthritis (OA) are associated with knee pain, though the mechanisms remain unknown. Increased nerve growth factor (NGF) expression and osteoclast density in subchondral bone appear to be the key features associated with bone pain in knee OA. Therefore, we aimed to identify associations among NGF, osteoclasts, and BMLs in knee OA. MethodsTwenty tibial plateaus were obtained from patients undergoing total knee arthroplasty for medial knee OA with BMLs at the medial tibial plateau (MTP). Osteochondral tissue samples from the weight-bearing part of the MTP, with and without BML, and from the weight-bearing part of the lateral tibial plateau (LTP), without BML, were collected. NGF expression and density of osteoclasts were compared among the three osteochondral tissue types. ResultsMTP bone with BMLs exhibited significantly higher NGF expression in bone marrow space and osteochondral channel, and higher osteoclast density than MTP bone without BML and LTP bone. The mean differences in NGF-positive area in the bone marrow space and the percentage of NGF-positive channels between MTP bones with and without BML were 9.0% (95% confidence interval [CI]: 5.9–12.1%) and 23.1% (95% CI: 11.3–35.0%), respectively. The difference in osteoclast density between MTP bones with and without BML was 0.6 osteoclasts per mm (95% CI: 0.3–0.9 osteoclasts per mm). ConclusionsIncreased NGF expression and osteoclast density are associated with subchondral BMLs in knee OA, contribute to understanding the mechanisms underlying BML-related bone pain in knee OA.

Suppression of bone resorption in ovariectomized mice using estrogen-immobilized polyphosphodiesters

Estrogens play a crucial role in bone remodeling. In this study, we synthesized two types of bone-targeted polyphosphodiesters (PPDEs): poly(diethyl sodium phosphate) (PEP•Na) and β-estradiol 17-valerate terminated PEP•Na (E2V-PEP•Na), and determined the inhibitory effect of the terminal estrogen on ovariectomy-induced bone resorption. We first assessed the impact of these polymers on the differentiation of osteoblasts and osteoclasts in vitro. Both PPDEs effectively promoted the differentiation of mouse osteoblastic MC3T3-E1 cells, with E2V-PEP•Na significantly enhancing the mineralization of these cells. Conversely, the density of osteoclasts formed from bone marrow mononuclear cells (BMNCs) significantly decreased when exposed to E2V-PEP•Na. Additionally, the formation of resorption pits on calcium phosphate-coated plates was reduced after treating BMNCs with both PPDEs. We then investigated the role of these polymers in treating osteoporosis using ovariectomized (OVX) mice. PEP•Na or E2V-PEP•Na was separately injected into the tail vein of the mice three times at 2-week intervals, starting 1 day after OVX treatment. Two weeks after the final injection, we observed that E2V-PEP•Na was distributed in bone tissue in a dose-dependent manner. The injection of E2V-PEP•Na significantly reduced bone resorption, and all bone parameters of OVX mice treated with E2V-PEP•Na were superior to those of sham mice. In contrast, PEP•Na did not affect bone mineral metabolism. In conclusion, our study is the first to demonstrate that E2V-PEP•Na can inhibit bone resorption in vivo, offering a promising new polymeric drug for preventing osteoporosis.

Impact of Long-Term Swimming Exercise on Rat Femur Bone Quality.

Considering the conflicting evidence regarding the potential long-term detrimental effect of swimming during growth on femur quality and fracture risk, our aim was to investigate the effect of eight months of swimming on femur quality. Twenty male eight-week-old Wistar rats were assigned into a swimming (SW; n = 10; 2 h/day, 5 days/week) or active control group (CG; n = 10, housed with running wheel) for eight months. Plasma osteocalcin and C-terminal telopeptide of type I collagen concentrations (ELISA) were assessed at baseline, four, and eight months of protocol. Femur structure (micro-computed tomography), biomechanical properties (three-point bending), and cellular density (histology) were determined after the protocol. SW displayed a lower uncoupling index, suggesting higher bone resorption, lower empty lacunae density, cortical and trabecular femur mass, femur length and cortical thickness, and higher cortical porosity than CG (p < 0.05). Although both biomarkers' concentrations decreased in both groups throughout the experiment (p < 0.001), there were no significant differences between groups (p > 0.05). No differences were also found regarding biomechanical properties, bone marrow adiposity, and osteocyte and osteoclast densities (p > 0.05). Long-term swimming was associated with unbalanced bone turnover and compromised femur growth, lower femur mass, and deteriorated cortical bone microarchitecture. However, femur trabecular microarchitecture and biomechanical properties were not affected by swimming.

Catalpol attenuates osteoporosis in ovariectomized rats through promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis

BackgroundCatalpol, a major active component of the Chinese herb Rehmannia glutinosa, possesses various pharmacological benefits, including anti-inflammatory, antidiabetic, and antitumor properties. Recent studies have reported that catalpol can attenuate bone loss and enhance bone formation. Nevertheless, the molecular mechanisms underlying its effects on osteoporosis pathogenesis remain unclear. PurposeWe investigated whether catalpol had a protective effect against postmenopausal osteoporosis (PMOP) and explored its exact mechanism of action. MethodsSeventy-two rats were randomly divided into six groups: sham, model, low-dose catalpol (5 mg/kg/day), medium-dose catalpol (10 mg/kg/day), high-dose catalpol (20 mg/kg/day), and positive control (alendronate, 2.5 mg/kg). In this experiment, a ovariectomy was performed to establish a female rat model of PMOP. After 12 weeks of gavage, micro-computed tomography (micro-CT) and histochemical staining were performed to evaluate bone mass, bone microstructure and histological parameters. Furthermore, RAW 264.7 cells were induced by RANKL to form mature osteoclasts to investigate the effect of catalpol on osteoclast differentiation and apoptosis in vitro. Additionally, the osteoclast apoptosis-related proteins of Sirt6, ERα, FasL, NFATc1, cleaved-caspase 8, cleaved-caspase 3, and Bax were assessed using western blotting. The expressions of NFATc1, Ctsk, Oscar, and Trap were quantified using RT-qPCR. The apoptotic rate of the osteoclasts was determined using flow cytometry. Sirt6 knockdown was performed using siRNA gene silencing in experiments to investigate its role in catalpol-mediated osteoclast apoptosis. The deacetylation of ERα in osteoclasts was tested via co-immunoprecipitation. ResultsCatalpol (10 and 20 mg/kg) and alendronate (2.5 mg/kg) could significantly improve bone mineral density (BMD) and microstructure and decrease osteoclast density in ovariectomized (OVX) rats. In addition, catalpol (10 and 20 mg/kg) upregulated the expression of Sirt6, ERα, FasL, cleaved-caspase 8, cleaved-caspase 3, Bax, and downregulated the expression of NFATc1, Ctsk, Oscar, Trap both in vivo and in vitro. Catalpol also promoted ERα deacetylation and stabilized ERα protein to enhance the expression of FasL. In addition, Sirt6 knockdown by siRNA prevented ERα deacetylation and eliminated catalpol-mediated osteoclast apoptosis. ConclusionsThe present study demonstrated that catalpol prevents estrogen deficiency-induced osteoporosis by promoting osteoclast apoptosis via the Sirt6-ERα-FasL axis. These findings revealed a novel molecular mechanism underpinning the impact of catalpol in the progression of osteoporosis and provided novel insights into the treatment of osteoporosis.

EARLY PHYSICAL REHABILITATION FOLLOWING FRACTURE INCREASES OSTEOCHONDRAL CALLUS FORMATION IN A PRECLINICAL MODEL OF DISUSE

Patients with bone and muscle weakness from disuse have higher risk of fracture and worse post-injury mortality rates. The goal of this current study was to better inform post-fracture rehabilitation strategies by investigating if physical remobilization following disuse by hindlimb unloading improves osteochondral callus formation compared to continued disuse by hindlimb suspension (HLS). We hypothesized that continued HLS would impair callus bone and cartilage formation and that physical rehabilitation after HLS would increase callus properties.All animal procedures were approved by the VCU IACUC. Skeletally mature, male and female C57BL/6J mice (18 weeks) underwent HLS for 3 weeks. Mice then had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Mice were then either randomly assigned to continued HLS or allow normal physical weight-bearing remobilization (HLS + R). Mice allowed normal cage activity throughout the experiment served as controls (GC). All mice were sacrificed 14-days following fracture with 4-8 mice (male and female) per treatment. Data analyzed by respective ANOVA with Tukey post-hoc (*p&lt; 0.05; # p &lt; 0.10)Male and female mice showed conserved and significant decreases in hindlimb callus bone formation from continued HLS versus HLS + R. Combining treatment groups regardless of mouse sex, histological analyses using staining on these same calluses demonstrated that HLS resulted in trends toward decreased cartilage cross-sectional area and increased osteoclast density in woven bone versus physically rehabilitated mice.In support of our hypothesis, physical remobilization increases callus bone formation following fracture compared to continued disuse potentially due to increased endochondral ossification and decreased bone resorption. In all, partial weight-bearing exercise immediately following fracture may improve callus healing compared to delayed rehabilitation regimens that are frequently used.

The effect of postmenopausal osteoporosis on subchondral bone pathology in a rat model of knee osteoarthritis

This study aimed to investigate the additional effect of ovariectomy-induced osteoporosis (OP) on the pathology of knee osteoarthritis (OA) in a rat meniscectomized model, particularly focusing on subchondral bone changes and pain behaviour. Rats were divided into four groups, sham, OP, OA, OP plus OA, and assessed for histology, osteoclast activity, subchondral bone microstructure, and pain-related behaviour. Rats with OP plus OA had significantly increased calcified cartilage and subchondral bone damage scores, increased densities of subchondral osteoclasts in the weight-bearing area, and more porous subchondral trabecular bone compared with rats with OA. Loss of tidemark integrity was observed most frequently in rats with OP plus OA. The density of subchondral osteoclasts correlated with the calcified cartilage and subchondral bone damage score in rats with OA (OA and OP plus OA). No significant differences in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) expression ratio in subchondral bone and pain-related behavioural tests were observed between rats with OA and rats with OP plus OA. In rats with OA, coexisting OP potentially aggravated OA pathology mainly in calcified cartilage and subchondral trabecular bone by increasing subchondral osteoclast activity.

Bone regeneration after marginal bone resection in two-stage treatment of chronic long bone infection - a combined histopathological and clinical pilot study

IntroductionIn chronic bone infection, marginal bone resection avoids large and difficult to reconstruct bone defects. However, there is still a lack of knowledge on bone regeneration during chronic bone infection and bone healing capability after marginal bone resection. Therefore, the purpose of this study was to investigate the clinical and histopathological outcomes after marginal bone resection in chronic long bone infection. We hypothesized that there is a regenerative bone healing potential after marginal bone resection that results in an acceptable clinical outcome and improved pathohistological bone healing parameters during treatment. Materials and methodsNine patients were treated for chronic bone infections in a two-stage manner with marginal bone resection of the infected area and the placement of an antibiotic-loaded polymethyl methacrylate (PMMA) spacer at stage one followed by bone reconstruction at stage two combined with systemic antibiotic therapy. Comparable bone samples were harvested at the border region between vital and necrotic bone area during stage one and the identical location during stage two. Control bone samples were harvested from five healthy patients without bone infection. Clinical outcome in terms of infection eradication and bone consolidation were assessed. The phenotypic changes of osteocyte and morphological changes of lacunar-canalicular network were investigated by histological and immunohistochemical staining between the two observation periods. Furthermore, expression levels of major bone formation and resorption markers were investigated by immunohistochemical and tartrate-resistant acid phosphatase (TRAP) staining. ResultsThe clinical results with a follow-up of 12.9 months showed that eight of nine patients (88.9%) achieved bone consolidation after a planned two-stage procedure of marginal resection of necrotic bone and consecutive reconstruction. In four of the nine patients (44.4%), additional marginal debridements after stage two had to be performed. After marginal resection at stage one, the improved bone formation ability at stage two was demonstrated by significantly lower percentage of empty lacunae, significantly more mature osteocytes and higher BMP-2 positive cell density, whereas decreased resorption was indicated by significantly lower osteoclast density and RANKL/OPG ratio. In patients requiring additional debridement compared to patients without additional debridements, a significantly higher percentage of empty lacunae was found at stage one. ConclusionMarginal bone resection combined with local and systemic antibiotic therapy is a feasible treatment option to avoid large bone defects as bone from the marginal resection area seems to have good regenerative potential. Despite a high revision rate of 44.4%, this technique avoids large bone resection and revisions can be done by further marginal debridements.

Notch2 Blockade Mitigates Methotrexate Chemotherapy-Induced Bone Loss and Marrow Adiposity.

Childhood cancer methotrexate (MTX) chemotherapy often causes bone growth impairments, bone loss, and increased risks of fractures during or after treatment, for which the pathobiology is unclear and there is a lack of specific treatment. Our time course analyses of long bones from rats receiving intensive MTX treatment (mimicking a clinical protocol) found decreased trabecular bone volume, increased osteoclast formation and activity, increased adipogenesis in the expense of osteogenesis from the bone marrow stromal cells at days 6 and 9 following the first of five daily MTX doses. For exploring potential mechanisms, PCR array expression of 91 key factors regulating bone homeostasis was screened with the bone samples, which revealed MTX treatment-induced upregulation of Notch receptor NOTCH2, activation of which is known to be critical in skeletal development and bone homeostasis. Consistently, increased Notch2 activation in bones of MTX-treated rats was confirmed, accompanied by increased expression of Notch2 intracellular domain protein and Notch target genes HEY1, HES1 and HEYL. To confirm the roles of Notch2 signalling, a neutralising anti-Notch2 antibody or a control IgG was administered to rats during MTX treatment. Microcomputed tomography analyses demonstrated that trabecular bone volume was preserved by MTX+anti-Notch2 antibody treatment. Anti-Notch2 antibody treatment ameliorated MTX treatment-induced increases in osteoclast density and NFATc1 and RANKL expression, and attenuated MTX-induced bone marrow adiposity via regulating Wnt/β-catenin signalling and PPARγ expression. Thus, Notch2 signalling plays an important role in mediating MTX treatment-induced bone loss and bone marrow adiposity, and targeting Notch2 could be a potential therapeutic option.

Species-independent stimulation of osteogenic differentiation induced by osteoclasts

The coupling of bone resorption and bone formation is well-recognized in the bone remodeling process, in which osteoblasts and osteoclasts are key players. However, the anabolic effect of human primary osteoclasts has rarely been reported as mouse and cell line derived osteoclasts were mostly used in previous reports. Therefore, a comprehensive comparison of mouse and human osteoclasts and their corresponding functions is needed to study cell-cell interactions between osteoclasts and osteoblasts. Osteoclasts from mouse and human origin were generated, characterized and compared, after which their anabolic effects on the osteogenic differentiation of mouse and human MSCs were assessed. Both murine RAW264.7 derived osteoclasts (mOCs) and primary human osteoclasts (hOCs) derived from buffy coats characteristically displayed multinuclearity, marked integrin β3 expression and enhanced TRAP activity. Despite comparable cell size, mOCs showed higher osteoclast density (number of osteoclasts per cm2 culture dish) and osteoclast nuclearity (average number of nuclei per osteoclast), but lower TRAP activity compared to hOCs. Culturing primary rat and human bone marrow MSCs with the conditioned medium of mOCs or hOCs showed anabolic effects regarding the osteogenic differentiation of MSCs with superiority of hOCs over mOCs. We conclude that despite morphological and functional differences between mouse and human osteoclasts, their secretory factors evoke similar anabolic effects on MSC osteogenic differentiation.

Osteoclast density is not increased in bone adjacent to radiolucencies (cysts) in juvenile equine medial femoral condyles.

There is a knowledge gap about how equine MFC subchondral radiolucencies (SR) arise and evolve. Osteoclasts are believed to have a role but have not been studied in situ. To measure and compare osteoclast density and the percentage of chondroclasts in healthy and MFC SR specimens from juvenile Thoroughbreds. Cadaveric study. Medial femoral condyles (MFC) from a tissue bank of equine stifles were studied. Inclusion criteria were MFCs (≤8months old) with a computed tomography SR lesion and histological focal failure of endochondral ossification (L group). Contralateral, lesion-free, MFCs were a control group (CC). Osteochondral slabs were cut through the lesion (L), a healthy site immediately caudal to the lesion, (internal control; IC) and the contralateral, site-matched controls (CC). Histological sections were immunostained with Cathepsin K for osteoclast counting. Osteoclasts in contact with the growth cartilage (chondroclasts) were also counted. The sections were segmented into regions of interest (ROI) at different depths in the subchondral bone: ROI1 (0-1mm), ROI2 (1-3mm) and ROI3 (3-6mm). Osteoclasts were counted and the bone area was measured in each ROI to calculate their density. Chondroclasts were counted in ROI1 . Sections were studied from L and IC (n=6) and CC sites (n=5). Osteoclast density was significantly higher in ROI1 when compared with ROI3 in all groups. Although higher osteoclast density was measured in ROI1 in the L group, no significant differences were detected when compared with control ROIs. The proportion of chondroclasts in ROI1 was lower in the L sections when compared with controls but no significant differences were detected. Limited sample size. Osteoclasts are important actors in MFC subchondral bone development, digesting both growth cartilage (chondroclasts) and bone, but the pathophysiology of early MFC SRs cannot be explained solely by an increased osteoclast presence in the subchondral bone.

The effect of genetically modified platelet-derived growth factor-BB over-expressing mesenchymal stromal cells during core decompression for steroid-associated osteonecrosis of the femoral head in rabbits

BackgroundApproximately one third of patients undergoing core decompression (CD) for early-stage osteonecrosis of the femoral head (ONFH) experience progression of the disease, and subsequently require total hip arthroplasty (THA). Thus, identifying adjunctive treatments to optimize bone regeneration during CD is an unmet clinical need. Platelet-derived growth factor (PDGF)-BB plays a central role in cell growth and differentiation. The aim of this study was to characterize mesenchymal stromal cells (MSCs) that were genetically modified to overexpress PDGF-BB (PDGF-BB-MSCs) in vitro and evaluate their therapeutic effect when injected into the bone tunnel at the time of CD in an in vivo rabbit model of steroid-associated ONFH.MethodsIn vitro studies: Rabbit MSCs were transduced with a lentivirus vector carrying the human PDGF-BB gene under the control of either the cytomegalovirus (CMV) or phosphoglycerate (PGK) promoter. The proliferative rate, PDGF-BB expression level, and osteogenic differentiation capacity of unmodified MSCs, CMV-PDGF-BB-MSCs, and PGK-PDGF-BB-MSCs were assessed. In vivo studies: Twenty-four male New Zealand white rabbits received an intramuscular (IM) injection of methylprednisolone 20 mg/kg. Four weeks later, the rabbits were divided into four groups: the CD group, the hydrogel [HG, (a collagen-alginate mixture)] group, the MSC group, and the PGK-PDGF-BB-MSC group. Eight weeks later, the rabbits were sacrificed, their femurs were harvested, and microCT, mechanical testing, and histological analyses were performed.ResultsIn vitro studies: PGK-PDGF-BB-MSCs proliferated more rapidly than unmodified MSCs (P < 0.001) and CMV-PDGF-BB-MSCs (P < 0.05) at days 3 and 7. CMV-PDGF-BB-MSCs demonstrated greater PDGF-BB expression than PGK-PDGF-BB-MSCs (P < 0.01). However, PGK-PDGF-BB-MSCs exhibited greater alkaline phosphatase staining at 14 days (P < 0.01), and osteogenic differentiation at 28 days (P = 0.07) than CMV-PDGF-BB-MSCs. In vivo: The PGK-PDGF-BB-MSC group had a trend towards greater bone mineral density (BMD) than the CD group (P = 0.074). The PGK-PDGF-BB-MSC group demonstrated significantly lower numbers of empty lacunae (P < 0.001), greater osteoclast density (P < 0.01), and greater angiogenesis (P < 0.01) than the other treatment groups.ConclusionThe use of PGK-PDGF-BB-MSCs as an adjunctive treatment with CD may reduce progression of osteonecrosis and enhance bone regeneration and angiogenesis in the treatment of early-stage ONFH.

CD14, A toll-like receptor co-factor, influences osteoclast differentiation and activity, but does not alter osteoblastic potential of bone-marrow preursors in mice

Purpose: Changes in subchondral bone structure occur in osteoarthritis (OA), and are visible by imaging as subchondral sclerosis on radiographs, and bone marrow lesions (BMLs) by MRI. These changes are the result of active bone remodeling that occurs in OA. BMLs in particular are correlated with pain in OA patients. We previously demonstrated that bone remodeling was significantly reduced after joint injury in mice deficient in CD14. CD14 enhances Toll-like receptor (TLR) mediated inflammation by acting as a co-receptor for multiple TLRs, increasing TLR sensitivity to damage associated molecular patterns (DAMPS) to activate proinflammatory signaling pathways. Soluble CD14, TLRs, and DAMPs (TLR ligands) are all elevated in OA patients, and sCD14 has been implicated as a biomarker of symptom and structural severity in OA. Increasing evidence suggestions CD14/TLR signaling influences bone remodeling as well, through activation of osteoclast precursor cells. Here, we investigate the potential mechanism by which CD14 influences bone remodeling through in vitro analysis of osteoclastogenic and osteoblastic potential and function. Methods: Bone marrow (BM) hematopoietic and mesenchymal precursors were isolated from long bones of 12-week-old male wild-type C57BL/6 (WT) and CD14-deficient (CD14-/-) mice. Briefly, BM was flushed from the femurs and tibia, and cells were cultured for 24 hours to separate non-adherent from adherent population. For osteoclastic differentiation, the non-adherent cells were seeded at a density of 26,000 cells per cm2 and were cultured in presence of monocyte/macrophage colony-stimulating factor (M-CSF; 35 ng/ml) and receptor activator of nuclear factor kβ ligand (RANKL; 100 ng/ml) for 9 days. Osteoblastic differentiation was accomplished by seeding the adherent BM cells at 30,000 cells per cm2 and culturing in the presence of 10 mM β-glycerophosphate ,50 μM ascorbic acid, and 100 nM Dexamethasone for up to 19 days. Osteoclasts were identified as multinucleated cells with positive TRAP staining. The resorptive ability of osteoclasts was assayed by quantifying resorption pits formed on bone slices. For osteoblast differentiation, Alizarin red was used to detect mineralized bone matrix. At the completion of each assay RNA was isolated from cultures and Real-time quantitative PCR analysis was performed to detect changes in mRNA levels of osteoclast (i.e. calcitonin receptor/CTR and cathepsin K/Ctsk) and osteoblast (i.e. osteopontin/OPN and osteocalcin/BLAP) differentiation markers. Results: Cells from CD14-/- mice demonstrated decreased percentages of TRAP positive cells and multi-nucleated cells (p value = 0.01; Fig. 1 A-D) and reduced Ctsk mRNA (p value = 0.03; Fig. 1 E&F), compared to WT controls. CD14-/- cells showed a 50% reduction in resorption pits and area compared to WT (Fig. 1 G&I). No significant differences were observed between strains in osteoblastic potential measured by mineralization and gene expression of osteoblastic markers (Fig. 2). Conclusions: CD14-/- BM precursor cells have reduced capacity to differentiate into functional osteoclasts, while osteoblastic potential is retained. This suggests that the reduced capacity of CD14-deficient osteoclastic cells may lead to the reduced bone remodeling in response to joint injury seen in the DMM model, by disrupting the balance between resorption and formation. Future experiments will determine the influence of DAMPs on osteoclast differentiation and activity, and the density of osteoclasts in SCB after DMM injury. These experiments will lead to a better understanding of the role of CD14 and TLR pathways on bone remodeling in response to joint injury and OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The Effect of Antiosteoporosis Therapy With Risedronate on Rotator Cuff Healing in an Osteoporotic Rat Model

Background: Osteoporosis increases the revision rate of rotator cuff repair (RCR). Weak fixation might not be the only cause of high RCR failure rates. The biological mechanism associated with tendon-to-bone healing after RCR in osteoporosis should be investigated. Hypothesis: (1) Osteoporosis would impair rotator cuff healing through the high osteoclastic activity at the repaired interface. (2) Risedronate would promote rotator cuff healing by reducing osteoclastic activity at the repaired interface. Study Design: Controlled laboratory study. Methods: A total of 84 female Sprague Dawley rats were randomly treated using ovariectomy or sham surgeries to establish osteoporotic and nonosteoporotic rat models. After confirming osteoporosis, a chronic rotator cuff tear model was created and RCR was performed. Postoperatively, osteoporotic rats were randomly divided into osteoporosis (OP) and osteoporosis with risedronate administration (OP+RIS) groups. Nonosteoporotic rats were used as the control (CON) group. Osteoclastic activity was measured at 1 and 3 weeks after RCR, and histologic analysis of the tendon-to-bone interface, bone morphometric evaluation, and biomechanical tests were performed at 4 and 8 weeks. Results: At the early healing stages of 1 and 3 weeks after RCR, the OP group showed the highest osteoclast density at the repaired interface. Compared with the OP group, risedronate administration significantly decreased osteoclast density in the OP+RIS group. At 8 weeks, histologic scores were greater in the OP+RIS group than in the OP group but still lower than in the CON group. Histologic scores at 8 weeks were negatively correlated with osteoclast density at the early healing stage. Additionally, the OP+RIS group showed better bone morphometric parameters and biomechanical properties than did the OP group. Conclusion: Osteoporosis impaired rotator cuff healing, which might be related to the high osteoclast density at the repaired interface at the early healing stage. Postoperative risedronate administration decreased osteoclast density and enhanced rotator cuff healing in osteoporotic rats, although the effect was inferior to that in nonosteoporotic rats. Clinical Relevance: Postoperative risedronate administration can be considered a potential therapy to enhance rotator cuff healing in patients with postmenopausal osteoporosis. However, this needs to be verified in a clinical setting.

A new non targeted bone remodeling model combined with an interpolation meshless method

Regardless of the bone remodeling type (i.e. targeted or non targeted), disturbances in this process can have serious repercussions in bone diseases’ progression. Therefore, research in this field has been continuous in recent decades, producing clinically relevant results. Regarding non targeted remodeling, distinct computational models were developed to describe the process. However, they only offer a time analysis, lacking mathematical models able to simulate the process in space. Thus, this work combines, for the first time, advanced numerical discretization techniques with non targeted remodeling models. The mathematical formulation used is based on Komarova’s model, correlating the remodeling activity of osteoclasts and osteoblasts with their autocrine and paracrine signaling pathways. For the numerical example, a two-dimensional bone patch was studied, allowing a spatio-temporal analysis of bone mass, cell density of osteoclasts and osteoblasts during an event of bone remodeling. A biological stimulus was applied to trigger remodeling and the obtained bone mass variation is consistent with the applied stimulus. Also, results are supported by the histomorphometric data available in the literature. The numerical methods used in this work are the Finite Element Method (FEM) and the Radial Point Interpolation Method (RPIM). The inclusion of the meshless method RPIM conveys a great advantage for future work since the model will be combined with medical images obtained with CT scans and MRI.

Associations of bone marrow lesions related subchondral osteoclastogenesis and neurogenesis with clinical symptoms in knee osteoarthritis

Purpose: To evaluate the associations of bone marrow lesions (BMLs) (including the subtypes) related subchondral osteoclastogenesis and neurogenesis with clinical symptoms in knee osteoarthritis (OA) patients, and to figure out how subchondral cyst-like lesions caused pain of joint. Methods: Samples were harvested from 33 symptomatic knee OA patients who had underwent total knee arthroplasty (TKA). These patients were separated into three groups based on magnetic resonance imaging (MRI), including group without BMLs (n=6), group has BMLs without cyst-like lesions (n=19), and group has BMLs combined with cyst (n=8). Hematoxylin and eosin (H&E) and O/fast green (S&F) staining were deployed to show subchondral structure and cartilage damage. Tartrate resistant acid phosphatase (TRAP) and protein gene product 9.5 (PGP9.5) staining were applied to detect subchondral osteoclastogenesis and neurogenesis in various groups. Pearson’s correlation coefficient and linear regression analyses were deployed to analyze the relationships between BMLs and knee symptoms, and further to assess the associations of subchondral osteoclastogenesis and neurogenesis with pain of joint. Results: Patients with BMLs combined with cyst-like lesions exhibited necrotic bone fragments in the subchondral bone and increased destruction in the cartilage. Volume of the subchondral cyst-like component itself was significantly associated with pain in OA patients (p<0.05), moreover, it was positively linearly correlated with WOMAC pain score (r2=0.5099, p=0.0466). Subchondral osteoclast density and neurogenesis were significantly increased in group with BMLs combined with cyst compared with the other two groups (p<0.05), and they were positively associated with pain in patients with BMLs combined with cyst (p<0.05). Conclusions: Subchondral cyst was an independent factor for inducing pain of knee OA patients. Subchondral cyst-like lesions related osteoclastogenesis and neurogenesis could cause pain of joint.

TLR-4 targeting contributes to the recovery of osteoimmunology in periodontitis.

The aim of this study was to determine the potential role of TLR-4 in the osteoimmunological imbalance of periodontitis. Although current evidence supports that TLR-4 plays an important role in the inflammatory response of periodontal tissues triggered by microorganisms, little information is available regarding the function of TLR-4 in the osteoimmune regulation of homeostasis in periodontitis. Human gingival epithelial cells (HGEC) were isolated from the gingival tissues of 3 healthy volunteers and the expression of osteoclastogenic cytokines was evaluated by ELISA and real time RT-PCR. In addition, 30 C57BL/6 mice were used and randomly divided into three groups: control group, periodontitis group (CP) and periodontitis+TAK-242 (a specific inhibitor of TLR-4) group (TAK-242) and the expression of osteoclastogenic cytokines and the osteoclast density in the periodontal tissue were evaluated by immunohistochemical staining and tartrate resistant acid phosphatase staining. Moreover, micro-computed tomography (Micro-CT) was used to assess bone resorption. The in vitro results showed that TAK-242 blocked the overproduction of IL-1, IL-6, TNF-α and RANKL in HGEC treated with LPS. The in vivo results revealed that TAK-242 also effectively decreased these osteoclastogenic cytokines in periodontal tissue of mice with periodontitis. More importantly, Micro-CT analysis showed a significant reduction of the alveolar bone loss in the TAK-242 group compared with the CP group. Furthermore, the TRAP staining showed a significant lower density of osteoclasts in the alveolar bone area of the TAK-242 group. TLR-4 inhibition decreased the differentiation of osteoclast through the inhibition of the overproduction of osteoclastogenic cytokines and the prevention of the alveolar bone absorption in mouse periodontitis models. Therefore, the use of TAK-242 might contribute to the recovery of the osteoimmunological homeostasis and might provide a potential strategy to treat periodontal diseases.

β-Catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats.

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the β-catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once-daily injections, 0.75 mg/kg) with/without β-catenin inhibitor indocyanine green (ICG)-001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX-treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX-induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis-related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX-induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of β-catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c-Kit+ hematopoietic progenitor cell contents. Thus, β-catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy.

MEK1/2 blockade results in altered inflammatory response and delayed alveolar bone repair

Abstract M1 and M2 macrophages play an important modulating role in tissue repair. In alveolar bone repair, the determinant these different phenotypes acquisition and its impact in repair process remains undefined, with a current assumption that assuming M2 conversion contributes to repair resolution. Considering MEK1/2 signaling pathway as a determinant for M2 polarization, the objective of this project was to evaluate the effect of MEK1/2 pharmacological inhibition in the local host response and repair outcome. C57Bl/6 (WT) mice, treated with MEK1/2i inhibitor PD0325901 (10mg/kg/24h/IP), which underwent extraction of the right upper incisor and, subsequently analyzed by computed microtomography (uCt) and histomorphometric analysis. uCt analysis, performed at 0,3,7 and 14 days periods revealed an overall smaller bone volume in MEK1/2i group, with a statistical difference in 14 days (p&amp;lt;0,05 MEK1/2i versus Control). These results, when combined with the histomorphometric analysis, reveals a delay in the alveolar bone repair process in the MEK1/2i group, characterized at 7 days by a higher density of connective tissue parameters, specifically collagen fibers, blood vessels, inflammatory cells and blood clot, when compared with control group. Additionally, a lower density of bone tissue parameters, specifically osteoblasts, osteoclasts and bone matrix, where observed upon MEK1/2i inhibition. Therefore, partially, the results show that MEK1/2 inhibitory effect results in delayed alveolar bone repair, initially involving the control of inflammatory cell migration and subsequent bone formation. Supported by grants from FAPESP (2018/10177-9 and 2015/24637-3)

Brain-Derived Acetylcholine Maintains Peak Bone Mass in Adult Female Mice.

Preclinical and clinical data support a role of the sympathetic nervous system in the regulation of bone remodeling, but the contribution of parasympathetic arm of the autonomic nervous system to bone homeostasis remains less studied. In this study, we sought to determine whether acetylcholine (ACh) contributes to the regulation of bone remodeling after peak bone mass acquisition. We show that reduced central ACh synthesis in mice heterozygous for the choline transporter (ChT) leads to a decrease in bone mass in young female mice, thus independently confirming the previously reported beneficial effect of ACh signaling on bone mass accrual. Increasing brain ACh levels through the use of the blood brain barrier (BBB)-permeable acetylcholinesterase inhibitor (AChEI) galantamine increased trabecular bone mass in adult female mice, whereas a peripheral increase in ACh levels induced by the BBB-impermeable AChEI pyridostigmine caused trabecular bone loss. AChEIs did not alter skeletal norepinephrine level, and induced an overall increase in osteoblast and osteoclast densities, two findings that do not support a reduction in sympathetic outflow as the mechanism involved in the pro-anabolic effect of galantamine on the skeleton. In addition, we did not detect changes in the commitment of skeletal progenitor cells to the osteoblast lineage in vivo in AChEI-treated mice, nor a direct impact of these drugs in vitro on the survival and differentiation of osteoblast and osteoclast progenitors. Last, ChT heterozygosity and galantamine treatment triggered bone changes in female mice only, thus revealing the existence of a gender-specific skeletal response to brain ACh level. In conclusion, this study supports the stimulatory effect of central ACh on bone mass accrual, shows that it also promotes peak bone mass maintenance in adult mice, and suggests that central ACh regulates bone mass via different mechanisms in growing versus sexually mature mice. © 2020 American Society for Bone and Mineral Research.