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Osteomyelitis remains one of the greatest risks in orthopaedic surgery. Although many organisms are linked to skeletal infections, Staphylococcus aureusremains the most prevalent and devastating causative pathogen. Important discoveries have uncovered novel mechanisms of S. aureuspathogenesis and persistence within bone tissue, including implant-associated biofilms, abscesses and invasion of the osteocyte lacuno-canalicular network. However, little clinical progress has been made in the prevention and eradication of skeletal infection as treatment algorithms and outcomes have only incrementally changed over the past half century. In this Review, we discuss the mechanisms of persistence and immune evasion in S. aureusinfection of the skeletal system as well as features of other osteomyelitis-causing pathogens in implant-associated and native bone infections. We also describe how the host fails to eradicate bacterial bone infections, and how this new information may lead to the development of novel interventions. Finally, we discuss the clinical management of skeletal infection, including osteomyelitis classification and strategies to treat skeletal infections with emerging technologies that could translate to the clinic in the future.

Background: Conventional bacterial cultures frequently fail to identify the dominant pathogen in polymicrobial foot infections, in which Staphylococcus aureusis the most common infecting pathogen. Previous work has shown that species-specific immunoassays may be able to identify the main pathogen in musculoskeletal infections. We sought to investigate the clinical applicability of a S. aureusimmunoassay to accurately identify the infecting pathogen and monitor its infectivity longitudinally in foot infection. We hypothesized that this species-specific immunoassay could aid in the diagnosis of S. aureusand track the therapeutic response in foot infections.Methods: From July 2015 to July 2019, 83 infected foot ulcer patients undergoing surgical intervention (debridement or amputation) were recruited and blood was drawn at 0, 4, 8, and 12 weeks. Whole blood was analyzed for S. aureus–specific serum antibodies (mix of historic and new antibodies) and plasmablasts were isolated and cultured to quantify titers of newly synthesized antibodies (NSAs). Anti–S. aureusantibody titers were compared with culture results to assess their concordance in identifying S. aureusas the pathogen. The NSA titer changes at follow-ups were compared with wound healing status to evaluate concordance between evolving host immune response and clinically resolving or relapsing infection.Results: Analysis of serum for anti–S. aureusantibodies showed significantly increased titers of 3 different anti–S. aureusantibodies, IsdH (P= .037), ClfB (P= .025), and SCIN (P= .005), in S. aureusculture-positive patients compared with culture-negative patients. Comparative analysis of combining antigens for S. aureusinfection diagnosis increased the concordance further. During follow-up, changes of NSA titers against a single or combination of S. aureusantigens significantly correlated with clinically resolving or recurring infection represented by wound healing status.Conclusion: In the management of foot infection, the use of S. aureus–specific immunoassay may aid in diagnosis of the dominant pathogen and monitoring of the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay could detect recurrent foot infection, which may guide a surgeon’s decision to intervene.Level of Evidence: Level II, prospective comparative study.

Prior research establishing that bone interacts in coordination with the bone marrow microenvironment (BMME) to regulate hematopoietic homeostasis was largely based on analyses of individual bone-associated cell populations. Recent advances in intravital imaging has suggested that the expansion of hematopoietic stem cells (HSCs) and acute myeloid leukemia cells is restricted to bone marrow microdomains during a distinct stage of bone remodeling. These findings indicate that dynamic bone remodeling likely imposes additional heterogeneity within the BMME to yield differential clonal responses. A holistic understanding of the role of bone remodeling in regulating the stem cell niche and how these interactions are altered in age-related hematological malignancies will be critical to the development of novel interventions. To advance this understanding, herein, we provide a synopsis of the cellular and molecular constituents that participate in bone turnover and their known connections to the hematopoietic compartment. Specifically, we elaborate on the coupling between bone remodeling and the BMME in homeostasis and age-related hematological malignancies and after treatment with bone-targeting approaches. We then discuss unresolved questions and ambiguities that remain in the field.

Background: Although treatment options and algorithms for rheumatoid arthritis (RA) have improved remarkably in recent decades, there continues to be no definitive cure or pharmacologic intervention with reliable long-term efficacy. For this reason, the combination of medications and healthy lifestyle modifications are essential for controlling joint disease, and extra-articular manifestations of RA, such as interstitial lung disease (ILD) and other lung pathologies, which greatly impact morbidity and mortality. Generally, exercise has been deemed beneficial in RA patients, and both patients and clinicians are motivated to incorporate effective non-pharmacologic interventions. However, there are limited evidence-based and specific exercise regimens available to support engagement in such activities for RA patients. Here, we provided the continuous opportunity for exercise to mice and implemented automated recording and quantification of wheel running behavior. This allowed us to describe the associated effects on the progression of inflammatory-erosive arthritis and ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA. Methods: Wild-type (WT; males, n=9; females, n=9) and TNF-Tg (males, n=12; females, n=14) mice were singly housed with free access to a running wheel starting at 2 months until 5 to 5.5 months of age. Measures of running included distance, rate, length, and number of run bouts, which were derived from continuously recorded data streams collected automatically and in real-time. In vivo lung, ankle, and knee micro-computed tomography (micro-CT), along with terminal micro-CT and histology were performed to examine the association of running behaviors and disease progression relative to sedentary controls. Results: TNF-Tg males and females exhibited significantly reduced running distance, rate, length, and number of run bouts compared to WT counterparts by 5 months of age (p<0.0001). Compared to sedentary controls, running males and females showed increased aerated lung volumes (p<0.05) that were positively correlated with running distance and rate in female mice (WT: Distance, ρ=0.705/rate, ρ=0.693 (p<0.01); TNF-Tg: ρ=0.380 (p=0.06)/ρ=0.403 (p<0.05)). Talus bone volumes were significantly reduced in running versus sedentary males and negatively correlated with running distance and rate in TNF-Tg mice (male: ρ=−903/ρ=−0.865; female: ρ=−0.614/ρ=−0.594 (p<0.001)). Histopathology validated the lung and ankle micro-CT findings. Conclusions: Implementation of automated wheel running behavior metrics allows for evaluation of longitudinal activity modifications hands-off and in real-time to relate with biomarkers of disease severity. Through such analysis, we determined that wheel running activity increases aerated lung volumes, but exacerbates inflammatory-erosive arthritis in TNF-Tg mice. To the end of a clinically relevant model, additional functional assessment of these outcomes and studies of pain behavior are warranted.

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve “target-and-release” drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (hydroxybisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.

Although clinical outcomes for patients with rheumatoid arthritis (RA) have greatly improved with the use of biologic and conventional DMARDs, approximately 40% of patients do not achieve primary clinical outcomes in randomized trials, and only a small proportion achieve lasting remission. Over the past decade, studies in murine models point to the critical role of the lymphatic system in the pathogenesis and therapy of inflammatory-erosive arthritis, presumably by the removal of catabolic factors, cytokines and inflammatory cells from the inflamed synovium. Murine studies demonstrate that lymphatic drainage increases at the onset of inflammatory-erosive arthritis but, as inflammation progresses to a more chronic phase, lymphatic clearance declines and both structural and cellular changes are observed in the draining lymph node. Specifically, chronic damage to the lymphatic vessel from persistent inflammation results in loss of lymphatic vessel contraction followed by lymph node collapse, reduced lymphatic drainage, and ultimately severe synovitis and joint erosion. Notably, clinical pilot studies in patients with RA report lymph node changes following treatment, and thus draining lymphatic vessels and nodes could represent a potential biomarker of arthritis activity and response to therapy. Most importantly, targeting lymphatics represents an innovative strategy for therapeutic intervention for RA.

While the 21stcentury has seen major advances in evidence-based medicine to improve health, athletic performance, and injury prevention, our inability to implement these best practices across underserved American communities has limited the impact of these breakthroughs in Sports Medicine. Rochester, New York is stereotypical of American communities in which an economically challenged racially diverse urban center with grossly underperforming public schools is surrounded by adequately resourced predominantly Caucasian state-of-the-art education systems. As these great disparities perpetuate and further degrade our society in the absence of interventions, the need for community engagement initiatives is self-evident. However, what interventions could have a positive effect, and how they could be administered remain open questions. Here we describe the Center for Human Athleticism, Musculoskeletal Performance and Prevention (CHAMPP) pilot program at the University of Rochester, whose goal is improved health for all participants through Fitness Science. Current efforts are focused on adding psychosocial and nutritional wellness components, as well as providing wearable devices to enhance participation and engagement offering a unique data analytics platform.

Background: Lymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls. Methods: Single-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations. Results: Fluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrbexpression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod. Conclusions: Single-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

Interleukin-27 is a pleiotropic cytokine whose functions during bacterial infections remain controversial, and its role in patients with S. aureusosteomyelitis is unknown. To address this knowledge gap, we completed a clinical study and observed elevated serum IL-27 levels (20-fold higher, P< 0.05) in patients compared with healthy controls. Remarkably, IL-27 serum levels were 60-fold higher in patients immediately following septic death than in uninfected patients (P< 0.05), suggesting a pathogenic role of IL-27. To test this hypothesis, we evaluated S. aureusosteomyelitis in WT and IL-27Rα−/−mice with and without exogenous IL-27 induction by intramuscular injection of rAAV-IL-27p28 or rAAV-GFP, respectively. We found that IL-27 was induced at the surgical site within 1 day of S. aureusinfection of bone and was expressed by M0, M1 and M2 macrophages and osteoblasts but not by osteoclasts. Unexpectedly, exogenous IL-27p28 (~2 ng·mL−1in serum) delivery ameliorated soft tissue abscesses and peri-implant bone loss during infection, accompanied by enhanced local IL-27 expression, significant accumulation of RORγt+neutrophils at the infection site, a decrease in RANK+cells, and compromised osteoclast formation. These effects were not observed in IL-27Rα−/−mice compared with WT mice, suggesting that IL-27 is dispensable for immunity but mediates redundant immune and bone cell functions during infection. In vitro studies and bulk RNA-seq of infected tibiae showed that IL-27 increased nos1, nos2, il17a, il17f, and rorcexpression but did not directly stimulate chemotaxis. Collectively, these results identify a novel phenomenon of IL-27 expression by osteoblasts immediately following S. aureusinfection of bone and suggest a protective role of systemic IL-27 in osteomyelitis.

Orthopaedic and trauma device-related infection (ODRI) remains one of the major complications in modern trauma and orthopaedic surgery.Despite best practice in medical and surgical management, neither prophylaxis nor treatment of ODRI is effective in all cases, leading to infections that negatively impact clinical outcome and significantly increase healthcare expenditure.The following review summarises the microbiological profile of modern ODRI, the impact antibiotic resistance has on treatment outcomes, and some of the principles and weaknesses of the current systemic and local antibiotic delivery strategies.The emerging novel strategies aimed at preventing or treating ODRI will be reviewed. Particular attention will be paid to the potential for clinical impact in the coming decades, when such interventions are likely to be critically important.The review focuses on this problem from an interdisciplinary perspective, including basic science innovations and best practice in infectious disease.Cite this article: Moriarty TF, Kuehl R, Coenye T, et al. Orthopaedic device related infection: current and future interventions for improved prevention and treatment. EFORT Open Rev2016;1:89-99. DOI: 10.1302/2058-5241.1.000037.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P< 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

ABSTRACTObesity and diabetes are among the greatest risk factors for infection following total joint arthroplasty. However, the underlying mechanism of susceptibility is unclear. We compared orthopedic implant-associated Staphylococcus aureusinfections in type 1 (T1D) versus type 2 (T2D) diabetic mouse models and in patients with S. aureusinfections, focusing on the adaptive immune response. Mice were fed a high-fat diet to initiate obesity and T2D. T1D was initiated with streptozotocin. Mice were then given a trans-tibial implant that was precoated with bioluminescent Xen36 S. aureus. Although both mouse models of diabetes demonstrated worse infection severity than controls, infection in T2D mice was more severe, as indicated by increases in bioluminescence, S. aureusCFU in tissue, and death within the first 7 days. Furthermore, T2D mice had an impaired humoral immune response at day 14 with reduced total IgG, decreased S. aureus-specific IgG, and increased IgM. These changes were not present in T1D mice. Similarly, T2D patients and obese nondiabetics with active S. aureusinfections had a blunted IgG response to S. aureus. In conclusion, we report the first evidence of a humoral immune deficit, possibly due to an immunoglobulin class switch defect, in obesity and T2D during exacerbated S. aureusinfection which may contribute to the increased infection risk following arthroplasty in patients with T2D and obesity.

Micro-computed tomography (μCT) is a valuable imaging modality for longitudinal quantification of bone volumes to identify disease or treatment effects for a broad range of conditions that affect bone health. Complex structures, such as the hindpaw with up to 31 distinct bones in mice, have considerable analytic potential, but quantification is often limited to a single bone volume metric due to the intensive effort of manual segmentation. Herein, we introduce a high-throughput, user-friendly, and semi-automated method for segmentation of murine hindpaw μCT datasets.

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (μCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The μCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin(Oc/Bglap) and integrin binding sialoprotein(Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

To investigate the efficacy of endocrine parathyroid hormone treatment on tissue-engineered bone regeneration, massive femoral defects in C57Bl/6 mice were reconstructed with either 100:0 or 85:15 poly-lactic acid (PLA)/beta-tricalcium phosphate (β-TCP) scaffolds (hereafter PLA or PLA/βTCP, respectively), which were fabricated with low porosity (<30%) to improve their structural rigidity. Experimental mice were treated starting at 1 week postop with daily subcutaneous injections of 40 μg/kg teriparatide until sacrifice at 9 weeks, whereas control mice underwent the same procedure but were injected with sterile saline. Bone regeneration was assessed longitudinally using planar X-ray and quantitative microcomputed tomography, and the reconstructed femurs were evaluated at 9 weeks either histologically or biomechanically to determine their torsional strength and rigidity. Teriparatide treatment increased bone volume and bone mineral content significantly at 6 weeks and led to enhanced trabeculated bone callus formation that appeared to surround and integrate with the scaffold, thereby establishing union by bridging bone regeneration across the segmental defect in 30% of the reconstructed femurs, regardless of the scaffold type. However, the bone volume and mineral content in the PLA reconstructed femurs treated with teriparatide was reduced at 9 weeks to control levels, but remained significantly increased in the PLA/βTCP scaffolds. Further, bridged teriparatide-treated femurs demonstrated a prototypical brittle bone torsion behavior, and were significantly stronger and stiffer than control specimens or treated specimens that failed to form bridging bone union. Taken together, these observations suggest that intermittent, systemic parathyroid hormone treatment can enhance bone regeneration in scaffold-reconstructed femoral defects, which can be further enhanced by mineralized (βTCP) particles within the scaffold.

While various problems with bone healing remain, the greatest clinical change is the absence of an effective approach to manage large segmental defects in limbs and craniofacial bones caused by trauma or cancer. Thus, nontraditional forms of medicine, such as gene therapy, have been investigated as a potential solution. The use of osteogenic genes has shown great potential in bone regeneration and fracture healing. Several methods for gene delivery to the fracture site have been described. The majority of them include a cellular component as the carrying vector, an approach known as cell-mediated gene therapy. Yet, the complexity involved with cell isolation and culture emphasizes the advantages of direct gene delivery as an alternative strategy. Here we review the various approaches of direct gene delivery for bone repair, the choice of animal models, and the various outcome measures required to evaluate the efficiency and safety of each technique. Special emphasis is given to noninvasive, quantitative, in vivomonitoring of gene expression and biodistribution in live animals. Research efforts should aim at inducing a transient, localized osteogenic gene expression within a fracture site to generate an effective therapeutic approach that would eventually lead to clinical use.

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2−/−mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2−/−mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2−/−callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2−/−mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9expression in the COX-2−/−fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2−/−mice.

Background: Our previous studies reveal that impaired draining function of the synovial lymphatic vessel (LV) contributes to the pathogenesis of inflammatory arthritis, but the cellular and molecular mechanisms involved are not fully understood. Objective: To investigate the involvement of lymphatic muscle cells (LMCs) in mediating impaired LV function in inflammatory arthritis. Methods: TNF transgenic (TNF-Tg) arthritic mice were used. The structure and function of the LVs that drained the hind limbs were examined by whole-mount immunofluorescence staining, electron microscopy, and near-infrared lymphatic imaging. Primary LMCs were treated with TNF, and the changes in proliferation, apoptosis, and functional gene expression were assessed. The roles of the herbal drug, Panax notoginseng saponins (PNS), in arthritis and LVs were studied. Results: TNF-Tg mice developed ankle arthritis with age, which was associated with abnormalities of LVs: (1) dilated capillary LVs with few branch points, (2) mature LVs with reduced LMC coverage and draining function, and (3) degenerative and apoptotic appearance of LMCs. TNF caused LMC apoptosis, reduced expression of muscle functional genes, and promoted the production of nitric oxide (NO) by lymphatic endothelial cells (LECs). PNS attenuated arthritis, restored LMC coverage and draining function of mature LVs, inhibited TNF-mediated NO expression, and reduced LMC apoptosis. Conclusion: The impaired draining function of LVs in TNF-Tg mice involves LMC apoptosis. TNF promotes LMC death directly and indirectly via NO production by LECs. PNS attenuates arthritis, improves LVs, and prevents TNF-induced LMC apoptosis by inhibiting NO production of LECs. LMCs contribute to the dysfunction of synovial LVs in inflammatory arthritis.

Proinflammatory cytokines produced in inflamed joints promote the generation and release from the bone marrow of osteoclast precursors (OCPs). These OCPs are recruited to inflamed joints where, in response to proinflammatory cytokines, they differentiate into osteoclasts and behave as immunomodulatory cells. We review the regulation of OCP formation and differentiation, and propose that they are not only sources of bone-resorbing osteoclasts, but also of immune cells that influence the progression of inflammatory bone diseases.

Inflammation-induced osteolysis is a problem in both inflammatory arthritis and total joint arthroplasty. New drug therapies have been shown to slow, halt, or even reverse the osteolysis associated with inflammatory arthritis. Unfortunately, similar advances in the medical treatment of periprosthetic osteolysis have not occurred. This review will update the state of periprosthetic osteolysis.

Smad3 deficiency accelerates chondrocyte maturation and leads to osteoarthritis. Primary chondrocytes without Smad3 lack compensatory increases of TGF‐β signaling factors, but BMP‐related gene expression is increased. Smad2 or Smad3 overexpression and BMP blockade abrogate accelerated maturation in Smad3−/−chondrocytes. BMP signaling is increased in TGF‐β deficiency and is required for accelerated chondrocyte maturation.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP‐2‐producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene‐enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.Introduction: The loss of cellular activity in a structural bone allograft markedly reduces its healing potential compared with a live autograft. To further understand the cellular mechanisms for structural bone graft healing and repair and to devise a therapeutic strategy aimed at enhancing the performance of allograft, we established a segmental femoral structural bone graft model in mice that permits qualitative and quantitative analyses of graft healing and neovascularization.Materials and Methods: Using this segmental femoral bone graft model, we transplanted live isografts harvested from Rosa 26A mice that constitutively express β‐galactosidase into their wildtype control mice. In an attempt to emulate the osteogenic and angiogenic properties of periosteum, we applied a cell‐based, adenovirus‐mediated gene therapy approach to engraft BMP‐2‐producing bone marrow stromal cells onto devitalized allografts.Results: X‐gal staining for donor cells allowed monitoring the progression of periosteal progenitor cell fate and showed that 70% of osteogenesis was attributed to cellular proliferation and differentiation of donor progenitor cells on the surface of the live bone graft. Quantitative μCT analyses showed a 3‐fold increase in new bone callus formation and a 6.8‐fold increase in neovascularization for BMP‐2/stromal cell‐treated allograft compared with control acellular allografts. Histologic analyses showed the key features of autograft healing in the BMP‐2/stromal cell‐treated allografts, including the formation of a mineralized bone callus completely bridging the segmental defects, abundant neovascularization, and extensive resorption of bone graft.Conclusions: The marked improvement of healing in these cellularized allografts suggests a clinical strategy for engineering a functional periosteum to improve the osteogenic and angiogenic properties of processed allografts.

Since the discovery of the RANK, its ligand, and osteoprotegerin as the final effectors of osteoclastogenesis and bone resorption, there has been an aggressive effort to develop a biologic antagonist of this pathway as a therapeutic for metabolic bone disease. While the early candidate osteoprotegerin molecules failed to meet clinical expectations, a human monoclonal antibody that recognizes RANKL (AMG162; Amgen, Inc., Thousand Oaks, California) is showing tremendous promise in phase II and phase III clinical trials. Here we review the trials and tribulations of RANK blockade for osteoporosis, tumor metastasis to bone, hypercalcemia of malignancy, inflammatory arthritis, and periprosthetic osteolysis.

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real‐time RT‐PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis.

Synovial fibroblasts are possible mediators of osteolysis. Fibroblasts respond directly to titanium particles and increase RANKLexpression through a COX‐2/PGE2/EP4/PKA signaling pathway. Fibroblasts pretreated with titanium or PGE2 stimulated osteoclast formation, showing the functional importance of RANKLinduction. Synovial fibroblasts and their activation pathways are potential targets to prevent osteolysis.

Genetic studies have demonstrated that osteoclasts are essential for focal erosion of bone and cartilage as a consequence of chronic pro-inflammatory cytokine production (TNF, IL-1) in inflammatory arthritis. In these inflamed joints, mature osteoclasts differentiate from circulating osteoclast precursors (OCPs) in response to local increased production of RANKL and pro-inflammatory cytokines. Once activated to resorb calcified matrix, these osteoclasts have a short lifespan (days), and must be continually replaced by bone marrow derived OCPs to achieve focal erosions. Therefore, the peripheral blood OCP frequency may directly determine the severity of bone erosion around inflamed joints. Based on the remarkable advances in our knowledge of osteoclast biology, investigators have been able to model the molecular events that control temporal and spatial regulation of osteoclastogenesis in vivo. Here we review the results of these pre-clinical and clinical studies and provide model of pathogenesis in which pro-inflammatory cytokines produced in the joint feedback on the bone marrow to produce and release elevated numbers of OCP into the circulation, which then home to the inflamed joint. Understanding the regulation of OCP generation and mobilization will provide a new strategy for development of drug targets in the treatment of inflammatory arthritis and other disorders associated with elevated peripheral OCP/myeloid progenitors.

To address the controversy of whether TNFα can compensate for RANKL in osteoclastogenesis in vivo, we used a TNFα‐induced animal model of inflammatory arthritis and blocked RANKL/RANK signaling. TNFα increased osteoclast precursors available for RANK‐dependent osteoclastogenesis. RANK signaling is not required for the TNFα‐stimulated increase in CD11bhiosteoclast precursors but is essential for mature osteoclast formation.

Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation‐independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation.

Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)–mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris–induced bone loss in a murine model of osteolysis. Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or β-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses. Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ–transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor κB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss. OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene–induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris–associated osteolysis and aseptic loosening.

To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno-associated virus (rAAV) transduction in human articular chondrocytes. Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0–1,000 J/m²) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence-activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full-thickness explants were exposed to UV light (0–200 J/m²), infected with rAAV-eGFP, and analyzed for transduction via immunohistochemistry. Toxicity from UV exposure was observed at doses ≥500 J/m² and ≥200 J/m² in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m² increased the mean fluorescence intensity (MFI) of the transduced cells 4-fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached ~100% after exposure to 100 J/m² of UV light and 10³ MOIs for 8 days. Under the same conditions, 200 J/m² of UV light enhanced the MFI 7-fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m² of UV light increased the transduction efficiency 2-fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m² of UV light increased the transduction efficiency 3-fold (to 100%). UV light at doses of up to 200 J/m² (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV-infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV-activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.

There is a temporal coupling between the processes of bone resorption and bone formation in normal skeletal remodeling. That is, osteoblastic activity usually follows episodes of osteoclastic activity. However, what has not been universally appreciated is that there also is a spatial coupling between these processes. Bone formation only occurs in the immediate vicinity of the resorptive event. In this study, we describe a phage display technique that has been used to identify the mechanisms by which osteoblasts recognize components of the prior resorbed lacunar surface. Using a type V tartrate‐resistant acid phosphatase (TRAP) as the bait and a random peptide M13 phage display library as the probe, we have identified specific sequences that show a very high affinity for TRAP. One of these peptides, designated clone 5, has a subnanomolar Kdfor TRAP, interacts with TRAP in a Far‐Western assay, binds exclusively to TRAP within osteoclast lacunae, is present in osteoblasts, and can effectively block osteoblast binding to resorption surfaces. The clone 5 peptide shows a high homology to glypican 4 (GPC4), a proteoglycan attachment receptor found in a number of cell types.

To evaluate the potential of viral interleukin-10 (vIL-10) gene therapy as an approach to prevent wear debris–induced inflammation, osteoclastogenesis, and bone resorption as it relates to periprosthetic osteolysis in patients with total joint replacements. Replication-defective adenovirus vectors expressing vIL-10 (AdvIL-10) or LacZ (AdLacZ) target genes were used to transduce fibroblast-like synoviocytes (FLS) in vitro, and the effects of these cells on wear debris–induced proinflammatory cytokine production and receptor activator of nuclear factor κB ligand + macrophage colony-stimulating factor splenocyte osteoclastogenesis were assessed by enzyme-linked immunosorbent assay and tartrate-resistant acid phosphatase assay. The effects of AdvIL-10 administration on wear debris–induced osteolysis in vivo were analyzed using the mouse calvaria model, in which AdLacZ was used as the control. In the presence of AdLacZ-infected FLS, titanium particle–stimulated macrophages exhibited a marked increase in secretion of tumor necrosis factor α (TNFα) (6.5-fold), IL-6 (13-fold), and IL-1 (5-fold). Coculture with AdvIL-10–transduced FLS suppressed cytokine secretion to basal levels, while addition of an anti–IL-10 neutralizing antibody completely blocked this effect. The vIL-10–transduced FLS also inhibited osteoclastogenesis 10-fold in an anti–IL-10–sensitive manner. In vivo, titanium implantation resulted in a 2-fold increase in osteoclasts (P &lt; 0.05) and in a 2-fold increase in sagittal suture area (P &lt; 0.05). This increase over control levels was completely blocked in mice receiving intraperitoneal injections of AdvIL-10, all of whom had measurable serum vIL-10 levels for the duration of the experiment. Immunohistochemistry demonstrated reduced cyclooxygenase 2 and TNFα expression in AdvIL-10–infected animals. This study demonstrates that gene delivery of vIL-10 inhibits 3 processes critically involved in periprosthetic osteolysis: 1) wear debris–induced proinflammatory cytokine production, 2) osteoclastogenesis, and 3) osteolysis.

Prosthesis failure due to wear debris‐induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF‐κB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)‐induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti‐treated RANK−/− mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti‐treated were still depleted for multinucleated tartrate‐resistant acid phosphatase‐positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris‐induced osteolysis without jeopardizing osteogenesis.

The human genome is 99.9 identical between human beings. The remaining 0.1 of difference constitutes the source of inherited disease. Most of this variation is comprised of single-nucleotide polymorphisms (SNPs), or single-base differences in the genome. By examining associations between SNPs and disease, researchers hope to decipher the genetic underpinnings of disease. Most SNPs have been examined with case-control studies using the “candidate gene approach” in which a single SNP is tested for association with disease. Unfortunately, associations have generally been weak. Multigenic SNP analysis in which hundreds or thousands of SNPs are examined in relation to disease will probably be required to unveil strong, clinically useful genetic associations. Current technological breakthroughs such as sequencing of the human genome and progress in high-throughput SNP analysis predict that that SNPs will have a tremendous impact on both clinical medicine and biomedical research in the near future.

Cyclooxygenases are the rate-limiting enzymes in the production of prostaglandins. Cyclooxygenases have long been recognized as important during inflammatory processes, and inhibitors are among the most commonly prescribed and used therapeutic agents in the world. They are commonly used to treat arthritic conditions and are frequently used to manage pain from musculoskeletal conditions, including soft tissue injury, fracture, and after both bone and soft tissue surgeries. Since the discovery of a second isoform of cyclooxygenase (cyclooxygenase-2, or Cox-2), selective agents designed to inhibit a single isoform have been developed.

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-β, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-β stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-β decreased the expression of type X collagen. TGF-β isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-β were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-β, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-β significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-β signaling molecule, Smad 3, mimicked the effect of TGF-β (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-β. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-β, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-β, but only partially blocked the effect of TGF-β on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-β through Smad mediated signaling events. © 2001 Wiley-Liss, Inc.

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo‐oxygenase 2 (COX‐2) and mice that lack either COX‐1 (COX‐1−/−) or COX‐2 (COX‐2−/−) to show that COX‐2, but not COX‐1, plays an important role in wear debris‐induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild‐type and COX‐1−/− mice. COX‐2 and prostaglandin E2(PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2production, and osteolysis. In comparison with wild‐type and COX‐1−/− mice, COX‐2−/− mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX‐2 is an important mediator of wear debris‐induced osteolysis and suggests that COX‐2 inhibitors are potential therapeutic agents for the prevention of wear debris‐induced osteolysis.

A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor α (TNF‐α) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle‐induced osteolysis. Etanercept is a soluble inhibitor of TNF‐α, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agent's ability to prevent wear debris‐induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)‐stimulated macrophages. Using a quantitative in vivo model of wear debris‐induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF‐α (hTNF‐α) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF‐α is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF‐α inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.

Mutant BMP receptors were transfected into cultured embryonic upper sternal chrondrocytes using retroviral vectors to determine if BMP signaling is required for chondrocyte maturation and the expression of a key regulatory molecule, Indian hedgehog (Ihh). Chondrocytes infected with replication competent avian retroviruses (RCAS) viruses carrying constitutive active (CA) BMPR‐IA and BMPR‐IB had enhanced expression of type X collagen and Ihh mRNA. Addition of PTHrP, a known inhibitor of chondrocyte maturation, abolished the expression of type X collagen, BMP‐6, and Ihh mRNAs in control cells. In contrast, PTHrP treated cultures infected with of CA BMPR‐IA or CA BMPR‐IB had low levels of BMP‐6 and type X collagen, but high levels of Ihh expression. Although dominant negative (DN) BMPR‐IA had no effect, DN BMPR‐IB inhibited the expression of type X collagen and BMP‐6, and decreased alkaline phosphatase activity, even in the presence of exogenously added BMP‐2 and BMP‐6. DN BMPR‐IB also completely blocked Ihh expression. Overall, the effect of DN BMPR‐IB mimicked the effects of PTHrP. To determine if there is an autocrine role for the BMPs in chondrocyte maturation, the cultures were treated with noggin and follistatin, molecules that bind BMP‐2/‐4 and BMP‐6/‐7, respectively. While noggin and follistatin inhibited the effects of recombinant BMP‐2 and BMP‐6, respectively, they had only minimal effects on the spontaneous maturation of chondrocytes in culture, suggesting that more than one subgroup of BMPs regulates chondrocyte maturation. The results demonstrate that: (i) BMP signaling stimulates chondrocyte maturation; (ii) BMP signaling increases Ihh expression independent of maturational effects; and (iii) BMP signaling can partially overcome the inhibitory effects of PTHrP on maturation. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Individuals who suffer from severe joint destruction caused by the various arthritidies often undergo total joint arthroplasty. A major limitation of this treatment is the development of aseptic loosening of the prosthesis in as many as 20% of patients. The current paradigm to explain aseptic loosening proposes that wear debris generated from the prosthesis initiates a macrophage‐mediated inflammatory response by resident macrophages, leading to osteoclast activation and bone resorption at the implant interface. No therapeutic interventions have been proved to prevent or inhibit aseptic loosening. The development of therapeutic strategies is limited due to the absence of a quantitative surrogate in which drugs can be screened rapidly in large numbers of animals. We have previously described a model in which titanium particles implanted on mouse calvaria induce an inflammatory response with osteolysis similar to that observed in clinical aseptic loosening. Here, we present new methods by which the osteolysis in this model can be quantified. We determined that 6–8‐week‐old mice in normal health have a sagittal suture area of 50 (±6) μm2, which contains approximately five osteoclasts. As a result of the titanium‐induced inflammation and osteolysis, the sagittal suture area increases to 197 (±27) μm2, with approximately 30 osteoclasts, after 10 days of treatment. The sagittal suture area and the number of osteoclasts in the calvaria of sham‐treated mice remained unchanged during the 10 days. We also determined the effects of pentoxifylline, a drug that blocks the responses of tumor necrosis factor‐α to wear debris, and the osteoclast inhibitor alendronate. We found that both drugs effectively block wear debris‐induced osteolysis but not osteoclastogenesis. In conclusion, we found the measurements made with this model to be reproducible and to permit quantitative analysis of agents that are to be screened for their potential to prevent aseptic loosening.

Bone metastasis is a leading cause of morbidity in cancer patients. Bone metastasis involves a complex series of biologic events but is ultimately dependent on cancer-mediated bone resorption. Recent advances have established a critical role for resident osteoclasts in this process. Furthermore, the paracrine interactions between cancer cells, resident stromal cells and osteoblasts, and cells in the osteoclast lineage have been defined along with the biologic mediators of these important interactions. These advances have provided novel targets to inhibit tumor-mediated bone resorption and have the potential to markedly decrease the morbidity of terminal cancer.

Control of osteoclastogenesis and osteoclast activity is critical for the treatment of metabolic bone diseases. The long sought after molecules that directly control the function and fate of these specialized bone-resorbing cells have recently been identified. Here we review the discovery of these molecules (receptor activator of nuclear factor B/osteoclast differentiation and activation receptor; osteoclast differentiation factor/osteoprotegerin ligand/tumor necrosis factor–related activation-induced cytokine; and osteoprotegerin/osteoclast inhibitory factor) and the in vivopreclinical and clinical data supporting their role as dominant regulators of osteoclasts. These molecules are all members of the tumor necrosis factor receptor superfamily, which has been shown to regulate programmed cell death by either suppressing apoptosis via inducing the expression of survival genes, or initiating apoptosis through activation of the caspase cascade. In an attempt to explain the teleology of this signaling pathway and how it controls the osteoclast lifespan, we propose a model in which a loose analogy is drawn between this pathway and that of tumor necrosis factor signaling in T lymphocytes. Future experimentation to test this model will provide a better understanding of this pathway and information as to the best way to manipulate this system to treat patients with musculoskeletal disease.

Although PTHrP has been identified as a key regulator of chondrocyte differentiation in the growth plate, the factors directly regulating PTHrP expression have not been identified. Furthermore, while cells from the epiphysis are considered the physiologic source of PTHrP, the relative expression of PTHrP in epiphyseal and growth plate chondrocytes has not been defined. PTHrP expression was examined in chondrocytes isolated from 3- to 5-week-old chick long bones. The expression of PTHrP mRNA was 10-fold higher in epiphyseal chondrocytes compared to cells from the growth plate. Growth plate chondrocytes were isolated into populations with distinct maturational characteristics by countercurrent centrifugal elutriation and analyzed for PTHrP expression. The expression was highest in the least mature cells and progressively declined with the onset of maturation. The regulation of PTHrP expression was further examined in epiphyseal chondrocytes. Both TGF-β1 and cis-retinoic acid stimulation markedly increased PTHrP mRNA levels, while BMP-2 and PTHrP stimulation decreased the expression of this transcript. The effects of TGF-β1 (8.9-fold stimulation) and TGF-β3 (9.2-fold) were slightly greater than the effects of TGF-β2 (4.9-fold). The effect of TGF-β was dose-dependent and increases could be detected after 68 h of treatment. To analyze the paracrine effect of epiphyseal and growth plate chondrocytes on each other, these cells were placed in coculture and the mRNA from each of the populations was harvested separately after 24 h. Following coculture the PTHrP mRNA levels increased in the epiphyseal cells while the expression of type X collagen and Indian hedgehog transcripts decreased in growth plate chondrocytes. The results demonstrate potentially important paracrine interactions between these cell populations, possibly mediated by TGF-β and PTHrP.

Due to irreversible joint destruction caused by the various arthritides, more than 400,000 total joint arthroplasties are performed each year in the United States. As many as 20% of these require revision surgery because of aseptic loosening. The current paradigm to explain aseptic loosening is that wear debris generated from the prosthesis stimulates the release of proinflammatory cytokines (i.e., tumor necrosis factor‐α and interleukins 1 and 6) following phagocytosis by resident macrophages. These cytokines, in turn, initiate an inflammatory response, with the development of an erosive pannus that stimulates bone resorption by osteoclasts. In support of this model, we have previously shown that human monocytes produce large quantities of tumor necrosis factor‐α in response to titanium particles in vitro.In the current study, we characterized the role of tumor necrosis factor‐α/nuclear transcription factor‐κB signaling in the proinflammatory response to titanium particles in vitroand in vivo.Using the mouse macrophage cell line J774, we showed that these cells produce an amount of tumor necrosis factor‐α in response to titanium particles similar to that produced by human peripheral blood monocytes. The production of tumor necrosis factor‐α was preceded by a drop in cellular levels of inhibitory factor‐κBα protein and translocation of p50/p65 nuclear transcription factor‐κB to the nucleus 30 minutes after stimulation. Levels of tumor necrosis factor‐α and inhibitory factor‐κBα mRNA increased 30 minutes after stimulation, consistent with the activation of nuclear transcription factor‐κB. Interleukin‐6 mRNA was first seen 4 hours after the addition of the titanium particles, indicating that the production of this cytokine is secondary to the immediate nuclear transcription factor‐κB response. To test the relevance of tumor necrosis factor‐α/nuclear transcription factor‐κB signaling in response to titanium particles in vivo, we adopted an animal model in which the particles were surgically implanted on the calvaria of mice. The animals displayed a dramatic histological response to the debris, with the formation of fibrous tissue and extensive bone resorption after only 1 week. With use of immunohistochemistry and tartrate‐resistant acid phosphatase staining, tumor necrosis factor‐α and osteoclasts were readily detected at the site of inflammation and bone resorption in the calvaria of the treated mice. By testing mice that genetically over‐produce tumor necrosis factor‐α (hTNFα‐Tg), those defective in tumor necrosis factor‐α signaling (TNF‐RI‐/‐), and those that are nuclear transcription factor‐κB1‐deficient (NFκB1‐/‐), we evaluated the importance of tumor necrosis factor‐α/nuclear transcription factor‐κB signaling in the biological processes responsible for aseptic loosening. The hTNFα‐Tg mice had a grossly exaggerated response, the TNF‐RI(‐/‐) mice showed little evidence of inflammation or bone resorption, and the nuclear transcription factor‐κB1(‐/‐) mice had an inflammatory response without bone resorption. On the basis of these results, we propose a model for periprosthetic osteolysis in which wear debris particles are phagocytosed by macrophages, resulting in the activation of nuclear transcription factor‐κB and the production of tumor necrosis factor‐α. Tumor necrosis factor‐α directly induces fibroblast proliferation and tissue fibrosis and recruits or activates, or both, osteoclasts to resorb adjacent bone.

ABSTRACTApoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-?B suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-?B1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 -/-) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI -/-) mice, we find that this resistance is not mediated by IFN-ß as previously thought. While no IFNRI -/- mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals’ ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 -/-, and p65 -/- embryos, we found that NF-?B is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 -/- and p65 -/- MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-?B-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-?B has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-?B1 is required for EMCV virulence in vivo. The attenuation in p50 -/- mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.

Introduction: A pivotal effect of lymphatic vessel (LV) function in joint homeostasis was identified in the tumor necrosis factor-transgenic (TNF-Tg) mouse model of rheumatoid arthritis (RA). Specifically, loss of LV contractions is associated with progressive synovitis and erosions. Furthermore, draining lymph node expansion is a biomarker of arthritic progression, and both macrophages and lymphatic endothelial cells express inducible nitric oxide synthase (iNOS), which disrupts LV contraction and transport of immune cells to the draining lymph nodes. Therefore, to directly assess these relationships, we tested the hypothesis that TNF-Tg mice with global genetic ablation of iNOS (iNOS−/−) will show delayed draining lymph node expansion, maintained LV contractions, and decreased synovitis and erosions. Method: iNOS−/−× TNF-Tg female and male mice, and control littermates (iNOS−/−, TNF-Tg, and WT), were examined with (1) ultrasound to determine popliteal lymph node (PLN) volume and (2) near-infrared imaging (NIR) to assess popliteal LV contraction frequency, and differences between genotypes were assessed at 3, 4, 5, and 6 months of age. Knees and PLN were harvested at 4 months in females and 6 months in males, to assess synovitis, bone erosions, and cellular accumulation in PLN sinuses via histology. Results: Initially, an increase in PLN volume was observed for both female and male iNOS−/−× TNF-Tg and TNF-Tg compared to their WT and iNOS−/−counterparts at 2 and 3 months, respectively. Subsequently, TNF-Tg PLNs continue to increase in volume, while iNOS−/−× TNF-Tg did not increase in volume from the initial timepoints. WT and iNOS−/−PLN volume was unchanged throughout the experiment. LV contraction frequency was increased at 4 months in females and 5 months in males, in the iNOS−/−× TNF-Tg mice compared to the TNF-Tg. Synovitis and erosions were moderately reduced in iNOS−/−× TNF-Tg versus TNF-Tg knees in females, while no differences in knee pathology were observed in males. Conclusions: Genetic iNOS ablation maintains draining lymph node volume and LV function during TNF-induced inflammatory arthritis and is associated with moderately decreased joint inflammation and damage.

Management of foot salvage therapy (FST) for diabetic foot infections (DFI) is challenging due to the absence of reliable diagnostics to identify the etiologic agent and prognostics to justify aggressive treatments. As Staphylococcus aureusis the most common pathogen associated with DFI, we aimed to develop a multiplex immunoassay of IgG in serum and medium enriched for newly synthesized anti-S. aureusantibodies (MENSA) generated from cultured peripheral blood mononuclear cells of DFI patients undergoing FST.

To assess the utility of longitudinal ultrasound (US) to quantify volumetric changes in joint soft tissues during the progression of posttraumatic osteoarthritis (PTOA) in mice, and validate the US results with histological findings. A longitudinal cohort of 3-month-old wild-type C57BL/6 male mice received the Hulth-Telhag surgical procedure on right knee to induce PTOA, and sham surgery on their left knee as control. US scans were performed on both knees before, 2, 4, 6, and 8 weeks post-surgery. Joint space volume and Power-Doppler (PD) volume were obtained from US images via Amira software. A parallel cross-sectional cohort of mice was killed at each US time point, and knee joints were subjected to histological analysis to obtain synovial soft-tissue area and OARSI scores. The correlation between US joint space volume and histological synovial soft-tissue area or OARSI score was assessed via linear regression analysis. US images indicated increased joint space volume in PTOA joints over time, which was associated with synovial inflammation and cartilage damage by histology. These changes started from 2 weeks post-surgery and gradually became more severe. No change was detected in sham joints. Increased joint space volume was significantly correlated with increased synovial soft-tissue area and the OARSI score (P<0.001). PD signal was detected in the joint space of PTOA joints at 6 weeks post-surgery, which was consistent with the location of blood vessels that stained positively for CD31 and alpha-smooth muscle actin in the synovium. This study indicates that US is a cost-effective longitudinal outcome measure of volumetric and vascular changes in joint soft tissues during PTOA progression in mice, which positively correlates with synovial inflammation and cartilage damage.

ABSTRACTObesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureusto the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureusinfection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureusisolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitrocoagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureuswith an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfAexpression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureusexpression of clfAand infection severity. Together, these results demonstrate an adaptation by S. aureusto obesity/T2D with increased expression of clfAthat is associated with the hypercoagulable state of the host and increased virulence of S. aureus.