PTH Treatment Increases Cortical Bone Mass More in Response to Compression than Tension in Mice.

Parathyroid hormone (PTH) is an anabolic osteoporosis treatment that increases bone mass and reduces fracture risk. Clinically, the effects of PTH are site-specific, increasing bone mass more at the spine than the hip and not increasing bone mass at the radius. Differences in local loading environment between the spine, hip, and radius may help explain the variation in efficacy, as PTH and mechanical loading have been shown to synergistically increase bone mass. We hypothesized that differences in loading mode might further explain these variations. Owing to the curvature of the mouse tibia, cyclic compression of the hindlimb causes bending at the tibial midshaft, placing the anterior surface under tension and the posterior surface under compression. We investigated the combination of PTH treatment and tibial loading in an osteoblast-specific estrogen receptor-alpha knockout mouse model of low bone mass (pOC-ERαKO) and their littermate controls (LCs) and analyzed bone morphology in the tensile, compressive, and neutral regions of the tibial midshaft. We also hypothesized that pretreating wild-type C57Bl/6J (WT) mice with PTH prior to mechanical loading would enhance the synergistic anabolic effects. Compression was more anabolic than tension, and PTH enhanced the effect of loading, particularly under compression. PTH pretreatment maintained the synergistic anabolic effect for longer durations than concurrent treatment and loading alone. Together these data provide insights into more effective physical therapy and exercise regimens for patients receiving PTH treatment. © 2022 American Society for Bone and Mineral Research (ASBMR).

Molecular Identification of Spatially Distinct Anabolic Responses to Mechanical Loading in Murine Cortical Bone.

Osteoporosis affects over 200 million women worldwide, one-third of whom are predicted to suffer from an osteoporotic fracture in their lifetime. The most promising anabolic drugs involve administration of expensive antibodies. Because mechanical loading stimulates bone formation, our current data, using a mouse model, replicates the anabolic effects of loading in humans and may identify novel pathways amenable to oral treatment. Murine tibial compression produces axially varying deformations along the cortical bone, inducing highest strains at the mid-diaphysis and lowest at the metaphyseal shell. To test the hypothesis that load-induced transcriptomic responses at different axial locations of cortical bone would vary as a function of strain magnitude, we loaded the left tibias of 10-week-old female C57Bl/6 mice in vivo in compression, with contralateral limbs as controls. Animals were euthanized at 1, 3, or 24 hours post-loading or loaded for 1 week (n = 4-5/group). Bone marrow and cancellous bone were removed, cortical bone was segmented into the metaphyseal shell, proximal diaphysis, and mid-diaphysis, and load-induced differential gene expression and enriched biological processes were examined for the three segments. At each time point, the mid-diaphysis (highest strain) had the greatest transcriptomic response. Similarly, biological processes regulating bone formation and turnover increased earlier and to the greatest extent at the mid-diaphysis. Higher strain induced greater levels of osteoblast and osteocyte genes, whereas expression was lower in osteoclasts. Among the top differentially expressed genes at 24-hours post-loading, 17 had known functions in bone biology, of which 12 were present only in osteoblasts, 3 exclusively in osteoclasts, and 2 were present in both cell types. Based on these results, we conclude that murine tibial loading induces spatially unique transcriptomic responses correlating with strain magnitude in cortical bone. © 2022 American Society for Bone and Mineral Research (ASBMR).

Bone mass and adaptation to mechanical loading are sexually dimorphic in adult osteoblast-specific ERα knockout mice.

Estrogen receptor-alpha (ERα) regulates bone mass and is implicated in bone tissue's response to mechanical loading. The effects of ERα deletion in mice depend on sex, anatomical location, and the cellular stage at which ERα is removed. Few studies have investigated the effect of age on the role of ERα in skeletal maintenance and functional adaptation. We previously demonstrated that bone mass and adaptation to loading were altered in growing 10-week-old female and male mice lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO). Here our goal was to determine the effects of ERα and mechanical loading in skeletally-mature adult mice. We subjected 26-week-old skeletally-mature adult pOC-ERαKO and littermate control (LC) mice of both sexes to two weeks of in vivo cyclic tibial loading. ERα deletion in male mice did not alter bone mass or the response to loading. Adult female pOC-ERαKO mice had reduced cancellous and cortical bone mass and increased adaptation to high-magnitude mechanical loading compared to LC mice. Thus, ERα deletion from mature osteoblasts reduced the bone mass and increased the mechanoadaptation of adult female but not male mice. Additionally, compared to our previous work in young mice, adult female mice had greatly reduced mechanoadaptation and adult male mice retained most of their mechanoadaptation with age.

Low bone mass resulting from impaired estrogen signaling in bone increases severity of load-induced osteoarthritis in female mice.

OBJECTIVE: Reduced subchondral bone mass and increased remodeling are associated with early stage OA. However, the direct effect of low subchondral bone mass on the risk and severity of OA development is unclear. We sought to determine the role of low bone mass resulting from a bone-specific loss of estrogen signaling in load-induced OA development using female osteoblast-specific estrogen receptor-alpha knockout (pOC-ERαKO) mice. METHODS: Osteoarthritis was induced by cyclic mechanical loading applied to the left tibia of 26-week-old female pOC-ERαKO and littermate control mice at peak loads of 6.5N, 7N, or 9N for 2 weeks. Cartilage damage and thickness, osteophyte development, and joint capsule fibrosis were assessed from histological sections. Subchondral bone morphology was analyzed by microCT. The correlation between OA severity and intrinsic bone parameters was determined. RESULTS: The loss of ERα in bone resulted in an osteopenic subchondral bone phenotype, but did not directly affect cartilage health. Following two weeks of cyclic tibial loading to induce OA pathology, pOC-ERαKO mice developed more severe cartilage damage, larger osteophytes, and greater joint capsule fibrosis compared to littermate controls. Intrinsic bone parameters negatively correlated with measures of OA severity in loaded limbs. CONCLUSIONS: Subchondral bone osteopenia resulting from bone-specific loss of estrogen signaling was associated with increased severity of load-induced OA pathology, suggesting that reduced subchondral bone mass directly exacerbates load-induced OA development. Bone-specific changes associated with estrogen loss may contribute to the increased incidence of OA in post-menopausal women.

Correction to: Mechanically Induced Periprosthetic Osteolysis: A Systematic Review.

[This corrects the article DOI: 10.1007/s11420-018-9641-5.].

Disruption of the Gut Microbiome Increases the Risk of Periprosthetic Joint Infection in Mice.

BACKGROUND: Periprosthetic joint infection (PJI) is one of the most devastating complications of total joint arthroplasty. Given the mortality and morbidity associated with PJI and the challenges in treating it, there has been increased interest in risk factors that can be modified before surgery. In this study, we used a novel mouse model to consider the role of the gut microbiome as a risk factor for PJI. QUESTIONS/PURPOSES: (1) Does the state of the gut microbiota before surgery influence the likelihood of developing an established infection in a mouse model of PJI? (2) How does the state of the gut microbiota before surgery influence the local and systemic response to the presence of an established infection in a mouse model of PJI? METHODS: Male C57Bl/6 mice were divided into two groups: those with modified microbiome [INCREMENT]microbiome (n = 40) and untreated mice (n = 42). In [INCREMENT]microbiome mice, the gut flora were modified using oral neomycin and ampicillin from 4 weeks to 16 weeks of age. Mice received a titanium tibial implant to mimic a joint implant and a local inoculation of Staphylococcus aureus in the synovial space (10 colony forming units [CFUs]). The proportion of animals developing an established infection in each group was determined by CFU count. The local and systemic response to established infection was determined using CFU counts in surrounding joint tissues, analysis of gait, radiographs, body weight, serum markers of inflammation, and immune cell profiles and was compared with animals that received the inoculation but resisted infection. RESULTS: A greater proportion of animals with disrupted gut microbiota had infection (29 of 40 [73%]) than did untreated animals (21 of 42 [50%]; odds ratio, 2.63, 95% CI, 1.04-6.61; p = 0.035). The immune response to established infection in mice with altered microbiota was muted; serum amyloid A, a marker of systemic infection in mice, was greater than in mice with disrupted gut microbiota with infection (689 µg/dL; range, 68-2437 µg/dL, p < 0.05); infection associated increases in monocytes and neutrophils in the spleen and local lymph node in untreated mice but not were not observed in mice with disrupted gut microbiota. CONCLUSIONS: The findings from this in vivo mouse model suggest that the gut microbiota may influence susceptibility to PJI. CLINICAL RELEVANCE: These preclinical findings support the idea that the state of the gut microbiome before surgery may influence the development of PJI and justify further preclinical and clinical studies to develop appropriate microbiome-based interventions.

New Innovations in the Treatment of PJI and Biofilms-Clinical and Preclinical Topics.

PURPOSE OF REVIEW: Periprosthetic joint infection (PJI) is a devastating complication after total joint replacement. A main source for antibiotic tolerance and treatment failure is bacterial production of biofilm-a resilient barrier against antibiotics, immune system, and mechanical debridement. The purpose of this review is to explore some novel approaches to treat PJI and biofilm-related infections. RECENT FINDINGS: Innovative treatment strategies of bacterial and biofilm infections revolve around (a) augmenting current therapies, such as improving the delivery and efficiency of conventional antibiotics and enhancing the efficacy of antiseptics and (b) administrating completely new therapeutic modalities, such as using immunotherapy, nanoparticles, lytic bacteriophages, photodynamic therapy, novel antibiotics, and antimicrobial peptides. Several promising treatment strategies for PJI are available to be tested further. The next requirement for most of the novel treatments is reproducing their effects in clinically representative animal models of PJI against clinical isolates of relevant bacteria.

Vancomycin-Loaded Polymethylmethacrylate Spacers Fail to Eradicate Periprosthetic Joint Infection in a Clinically Representative Mouse Model.

BACKGROUND: Periprosthetic joint infection (PJI) remains a devastating complication following total joint arthroplasty. Current animal models of PJI do not effectively recreate the clinical condition and thus provide limited help in understanding why treatments fail. We developed a mouse model of the first-stage surgery of a 2-stage revision for PJI involving a 3-dimensionally printed Ti-6Al-4V implant and a mouse-sized cement spacer that elutes vancomycin. METHODS: Vancomycin was mixed with polymethylmethacrylate (PMMA) cement and inserted into custom-made mouse-sized spacer molds. Twenty C57BL/6 mice received a proximal tibial implant and an intra-articular injection of 3 × 10 colony-forming units of Staphylococcus aureus Xen36. At 2 weeks, 9 mice underwent irrigation and debridement of the leg with revision of the implant to an articulating vancomycin-loaded PMMA spacer. Postoperatively, mice underwent radiography and serum inflammatory-marker measurements. Following euthanasia of the mice at 6 weeks, bone and soft tissues were homogenized to quantify bacteria within periprosthetic tissues. Implants and articulating spacers were either sonicated to quantify adherent bacteria or examined under scanning electron microscopy (SEM) to characterize the biofilm. RESULTS: Vancomycin-loaded PMMA spacers eluted vancomycin for ≤144 hours and retained antimicrobial activity. Control mice had elevated levels of inflammatory markers, radiographic evidence of septic loosening of the implant, and osseous destruction. Mice treated with a vancomycin-loaded PMMA spacer had significantly lower levels of inflammatory markers (p < 0.01), preserved tibial bone, and no intra-articular purulence. Retrieved vancomycin-loaded spacers exhibited significantly lower bacterial counts compared with implants (p < 0.001). However, bacterial counts in periprosthetic tissue did not significantly differ between the groups. SEM identified S. aureus encased within biofilm on control implants, while vancomycin-loaded spacers contained no bacteria. CONCLUSIONS: This animal model is a clinically representative model of PJI treatment. The results suggest that the antimicrobial effects of PMMA spacers are tightly confined to the articular space and must be utilized in conjunction with thorough tissue debridement and systemic antibiotics. CLINICAL RELEVANCE: These data provide what we believe to be the first insight into the effect of antibiotic-loaded cement spacers in a clinically relevant animal model and justify the adjunctive use of intravenous antibiotics when performing a 2-stage revision for PJI.

GSK-3ß inhibition suppresses instability-induced osteolysis by a dual action on osteoblast and osteoclast differentiation.

Currently, there are no medications available to treat aseptic loosening of orthopedic implants. Using osteoprotegerin fusion protein (OPG-Fc), we previously blocked instability-induced osteoclast differentiation and peri-prosthetic osteolysis. Wnt/ß-catenin signaling, which regulates OPG secretion from osteoblasts, also modulates the bone tissue response to mechanical loading. We hypothesized that activating Wnt/ß-catenin signaling by inhibiting glycogen synthase kinase-3ß (GSK-3ß) would reduce instability-induced bone loss through regulation of both osteoblast and osteoclast differentiation. We examined effects of GSK-3ß inhibition on regulation of RANKL and OPG in a rat model of mechanical instability-induced peri-implant osteolysis. The rats were treated daily with a GSK-3ß inhibitor, AR28 (20 mg/kg bw), for up to 5 days. Bone tissue and blood serum were assessed by qRT-PCR, immunohistochemistry, and ELISA on days 3 and 5, and by micro-CT on day 5. After 3 days of treatment with AR28, mRNA levels of ß-catenin, Runx2, Osterix, Col1α1, and ALP were increased leading to higher osteoblast numbers compared to vehicle-treated animals. BMP-2 and Wnt16 mRNA levels were downregulated by mechanical instability and this was rescued by GSK-3ß inhibition. Osteoclast numbers were decreased significantly after 3 days of GSK-3ß inhibition, which correlated with enhanced OPG mRNA expression. This was accompanied by decreased serum levels of TRAP5b on days 3 and 5. Treatment with AR28 upregulated osteoblast differentiation, while osteoclastogenesis was blunted, leading to increased bone mass by day 5. These data suggest that GSK-3ß inactivation suppresses osteolysis through regulating both osteoblast and osteoclast differentiation in a rat model of instability-induced osteolysis.

Quantification of Peri-Implant Bacterial Load and in Vivo Biofilm Formation in an Innovative, Clinically Representative Mouse Model of Periprosthetic Joint Infection.

BACKGROUND: Periprosthetic joint infection (PJI) is a devastating complication following total joint arthroplasty. Current animal models of PJI are limited because of a lack of quantitative methods and failure to effectively recreate the periprosthetic space. We therefore developed a murine PJI model involving a 3-dimensionally printed Ti-6Al-4V implant capable of bearing weight and permitting quantitative analysis of periprosthetic bacterial load and evaluation of biofilm. METHODS: Twenty-five 12-week-old C57BL/6 mice received a unilateral proximal tibial implant and intra-articular injection of either 3 × 10 colony forming units (CFUs) of Staphylococcus aureus Xen 36 or saline solution. Postoperatively, mice underwent gait analysis, knee radiographs, and serum inflammatory marker measurements. Following euthanasia at 2 or 6 weeks, bone and soft tissues were homogenized to quantify bacteria within periprosthetic tissues. Implants were either sonicated to quantify adherent bacteria or examined under scanning electron microscopy (SEM) to characterize biofilm. RESULTS: All mice survived surgery and were not systemically septic. The control mice immediately tolerated weight-bearing and had normal inflammatory markers and radiographic signs of osseointegration. Infected mice had difficulty walking over time, exhibited radiographic findings of septic implant loosening, and had significantly elevated inflammatory markers. Periprosthetic tissues of the infected animals displayed a mean of 4.46 × 10 CFUs of S. aureus at 2 weeks and 2.53 × 10 CFUs at 6 weeks. Viable S. aureus was quantified on retrieved implant surfaces. SEM demonstrated S. aureus cocci in clusters encased within biofilm. CONCLUSIONS: This animal model is, to our knowledge, the most clinically representative PJI replication to date. It is the first that we know of to produce infection through the same method hypothesized to occur clinically, utilize a weight-bearing implant that can osseointegrate, and provide quantitative data on 8 aspects of PJI, including radiographic features, inflammatory markers, and bacterial loads. CLINICAL RELEVANCE: This novel animal model is, to our knowledge, the first to provide a load-bearing translational representation of clinical PJI that effectively recreates the periprosthetic space.

Def6 Restrains Osteoclastogenesis and Inflammatory Bone Resorption.

Inflammatory bone resorption mediated by osteoclasts is a major cause of morbidity and disability in many inflammatory disorders, including rheumatoid arthritis (RA). The mechanisms that regulate osteoclastogenesis and bone resorption in inflammatory settings are complex and have not been well elucidated. In this study, we identify the immunoregulator differentially expressed in FDCP 6 homolog (Def6) as a novel inhibitor of osteoclastogenesis in physiological and inflammatory conditions. Def6 deficiency in Def6-/- mice enhanced the sensitivity of osteoclast precursors to the physiological osteoclastogenic inducer receptor activator for NF-κB ligand, and Def6-/- osteoclasts formed actin rings. Furthermore, Def6 deficiency markedly increased TNF-α-induced osteoclastogenesis in vitro and in vivo and enhanced bone resorption in an inflammatory osteolysis mouse model. TNF-α serum levels correlated negatively with Def6 expression levels in osteoclast precursors obtained from RA patients, and the osteoclastogenic capacity of the osteoclast precursors was significantly inversely correlated with their Def6 expression levels, indicating that Def6 functions as an inhibitor of excessive osteoclast formation and bone destruction in RA. Mechanistically, Def6 suppressed osteoclastogenesis and the expression of key osteoclastogenic factors NFATc1, B lymphocyte-induced maturation protein-1, and c-Fos by regulating an endogenous IFN-ß-mediated autocrine feedback loop. The Def6-dependent pathway may represent a novel therapeutic target to prevent pathological bone destruction.

Developing a Clinically Representative Model of Periprosthetic Joint Infection.

➤The poor treatment outcomes for periprosthetic joint infection (PJI) reflect the limited understanding that currently exists regarding the pathogenesis of this devastating clinical problem.➤Current animal models of PJI are limited in their translational nature primarily because of their inability to recreate the periprosthetic environment.➤A greater mechanistic understanding of the musculoskeletal and immune systems of small animals, such as mice and rats, provides a more robust platform for modeling and examining the pathogenesis of PJI.➤A clinically representative PJI model must involve an implant that recreates the periprosthetic space and be amenable to methodologies that identify implant biofilm as well as quantify the peri-implant bacterial load.

Transcriptional profiling of cortical versus cancellous bone from mechanically-loaded murine tibiae reveals differential gene expression.

Mechanical loading is an anabolic stimulus that increases bone mass, and thus a promising method to counteract osteoporosis-related bone loss. The mechanism of this anabolism remains unclear, and needs to be established for both cortical and cancellous envelopes individually. We hypothesized that cortical and cancellous bone display different gene expression profiles at baseline and in response to mechanical loading. To test this hypothesis, the left tibiae of 10-week-old female C57Bl/6 mice were subjected to one session of axial tibial compression (9N, 1200cycles, 4Hz triangle waveform) and euthanized 3 and 24h following loading. The right limb served as the contralateral control. We performed RNA-seq on marrow-free metaphyseal samples from the cortical shell and the cancellous core to determine differential gene expression at baseline (control limb) and in response to load. Differential expression was verified with qPCR. Cortical and cancellous bone exhibited distinctly different transcriptional profiles basally and in response to mechanical loading. More genes were differentially expressed with loading at 24h with more genes downregulated at 24h than at 3h in both tissues. Enhanced Wnt signaling dominated the response in cortical bone at 3 and 24h, but in cancellous bone only at 3h. In cancellous bone at 24h many muscle-related genes were downregulated. These findings reveal key differences between cortical and cancellous genetic regulation in response to mechanical loading. Future studies at different time points and multiple loading sessions will add to our knowledge of cortical and cancellous mechanotransduction with the potential to identify new targets for mouse genetic knockout studies and drugs to treat osteoporosis.

Intermittent Parathyroid Hormone Enhances Cancellous Osseointegration of a Novel Murine Tibial Implant.

BACKGROUND: Long-term fixation of uncemented joint implants requires early mechanical stability and implant osseointegration. To date, osseointegration has been unreliable and remains a major challenge in cementless total knee arthroplasty. We developed a murine model in which an intra-articular proximal tibial titanium implant with a roughened stem can be loaded through the knee joint. Using this model, we tested the hypothesis that intermittent injection of parathyroid hormone (iPTH) would increase proximal tibial cancellous osseointegration. METHODS: Ten-week-old female C57BL/6 mice received a subcutaneous injection of PTH (40 µg/kg/day) or a vehicle (n = 45 per treatment group) five days per week for six weeks, at which time the baseline group was killed (n = 6 per treatment group) and an implant was inserted into the proximal part of the tibiae of the remaining mice. Injections were continued until the animals were killed at one week (n = 7 per treatment group), two weeks (n = 14 per treatment group), or four weeks (n = 17 per treatment group) after implantation. Outcomes included peri-implant bone morphology as analyzed with micro-computed tomography (microCT), osseointegration percentage and bone area fraction as shown with backscattered electron microscopy, cellular composition as demonstrated by immunohistochemical analysis, and pullout strength as measured with mechanical testing. RESULTS: Preimplantation iPTH increased the epiphyseal bone volume fraction by 31.6%. When the data at post-implantation weeks 1, 2, and 4 were averaged for the iPTH-treated mice, the bone volume fraction was 74.5% higher in the peri-implant region and 168% higher distal to the implant compared with the bone volume fractions in the same regions in the vehicle-treated mice. Additionally, the trabecular number was 84.8% greater in the peri-implant region and 74.3% greater distal to the implant. Metaphyseal osseointegration and bone area fraction were 28.1% and 70.1% higher, respectively, in the iPTH-treated mice than in the vehicle-treated mice, and the maximum implant pullout strength was 30.9% greater. iPTH also increased osteoblast and osteoclast density by 65.2% and 47.0%, respectively, relative to the values in the vehicle group, when the data at post-implantation weeks 1 and 2 were averaged. CONCLUSIONS: iPTH increased osseointegration, cancellous mass, and the strength of the bone-implant interface. CLINICAL RELEVANCE: Our murine model is an excellent platform on which to study biological enhancement of cancellous osseointegration.

Effects of Deletion of ERα in Osteoblast-Lineage Cells on Bone Mass and Adaptation to Mechanical Loading Differ in Female and Male Mice.

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed-background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell-specific ERαKO mice. Therefore, we crossed ERα floxed (ERα(fl/fl)) and osteocalcin-Cre (OC-Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC-ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro-CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC-ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC-ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC-ERαKO female L(5) vertebrae and with increased maximum moment in pOC-ERαKO male femora. Female pOC-ERαKO mice responded more to mechanical loading, whereas the response of pOC-ERαKO male animals was similar to their littermate controls.

Intermittent PTH administration and mechanical loading are anabolic for periprosthetic cancellous bone.

The purpose of this study was to determine the individual and combined effects on periprosthetic cancellous bone of intermittent parathyroid hormone administration (iPTH) and mechanical loading at the cellular, molecular, and tissue levels. Porous titanium implants were inserted bilaterally on the cancellous bone of adult rabbits beneath a loading device attached to the distal lateral femur. The left femur received a sham loading device. The right femur was loaded daily, and half of the rabbits received daily PTH. Periprosthetic bone was evaluated up to 28 days for gene expression, histology, and µCT analysis. Loading and iPTH increased bone mass by a combination of two mechanisms: (1) Altering cell populations in a pro-osteoblastic/anti-adipocytic direction, and (2) controlling bone turnover by modulating the RANKL-OPG ratio. At the tissue level, BV/TV increased with both loading (+53%, p < 0.05) and iPTH (+54%, p < 0.05). Combined treatment showed only small additional effects at the cellular and molecular levels that corresponded to a small additive effect on bone volume (+13% compared to iPTH alone, p > 0.05). This study suggests that iPTH and loading are potential therapies for enhancing periprosthetic bone formation. The elucidation of the cellular and molecular response may help further enhance the combined therapy and related targeted treatment strategies.

Female mice lacking estrogen receptor-alpha in osteoblasts have compromised bone mass and strength.

Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERα), mice with global deletion of ERα (ERαKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin-like growth factor 1 (IGF-1) serum levels, which may independently affect bone. Mice with tissue-specific ERα deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERα is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast-specific ERαKO mice (pOC-ERαKO) by crossing mice expressing a floxed ERα gene (ERα(fl/fl)) with mice transgenic for the osteocalcin-Cre promoter (OC-Cre). Having confirmed that serum levels of estrogen and IGF-1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC-ERαKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERα in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This pOC-ERαKO mouse is an important animal model that could enhance our understanding of estrogen signaling in bone cells in vivo.

Targeting the giant cell tumor stromal cell: functional characterization and a novel therapeutic strategy.

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.

Orthopedic wear debris mediated inflammatory osteolysis is mediated in part by NALP3 inflammasome activation.

Activation of myeloid cells by orthopedic particulate debris is a key event in the pathogenesis of periprosthetic osteolysis and implant loosening after total joint replacement (TJR). Several lines of evidence implicate NACHT, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome-mediated production of interleukin 1 beta (IL-1ß) in the pathogenesis of clinical disorders ascribable to foreign particulate materials, including asbestos, silica, and urate crystals. Recent reports indicate that orthopedic polymer products and metallic particulates and ions may activate the same pathway. Here, we investigated the contribution of the NALP3 inflammasome to the pathogenesis of peri-implant osteolysis. Pharmaceutical and genetic perturbations of caspase-1 and inflammasome components were used to assess the role of the NALP3 inflammasome in IL-1ß production and osteoclast formation by human monocytes and mouse macrophages in response to polymethylmethacrylate (PMMA) particle phagocytosis. The role of caspase-1 in a mouse calvarial model of particle-mediated osteolysis was assessed using µCT. Phagocytosis of PMMA particles induces caspase-1 dependent release of IL-1ß from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA-induced IL-1ß production is strictly dependent on these components. Mice lacking caspase-1, the sole effector of the NALP3 inflammasome, show reduced orthopedic wear particle-induced calvarial osteolysis compared to wild-type controls. Absence of NALP3 inflammasome components fails to alter osteoclast formation in vitro. Our findings identify the NALP3 inflammasome as a critical mediator of orthopedic wear-induced osteolysis and as a viable therapeutic target for the treatment of periprosthetic osteolysis.

Bone remodelling in inflammatory arthritis.

The inflammatory arthropathies that include rheumatoid arthritis, the seronegative spondyloarthropathies and systemic lupus erythematosus are characterised by marked alterations in the architecture and structural integrity of peri-articular bone; however, the pattern and natural history of the skeletal changes differs in these conditions. In part, this can be attributed to differences in the primary anatomical site of the inflammation, but also there is evidence that there are differences in the biological properties and products produced by inflammatory tissues. This review will focus on recent advances in the understanding of the cellular and molecular mechanisms that contribute to the differential pattern of articular bone remodelling in these prototypical inflammatory forms of arthritis.