Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
Bone formation and regeneration therapies continue to require optimization and improvement because many skeletal disorders remain undertreated. Clinical solutions to nonunion fractures and osteoporotic vertebral compression fractures, for example, remain suboptimal and better therapeutic approaches must be created. The widespread use of recombinant human bone morphogenetic proteins (rhBMPs) for spine fusion was recently questioned by a series of reports in a special issue of The Spine Journal, which elucidated the side effects and complications of direct rhBMP treatments. Gene therapy—both direct (in vivo) and cell-mediated (ex vivo)—has long been studied extensively to provide much needed improvements in bone regeneration. In this article, we review recent advances in gene therapy research whose aims are in vivo or ex vivo bone regeneration or formation. We examine appropriate vectors, safety issues, and rates of bone formation. The use of animal models and their relevance for translation of research results to the clinical setting are also discussed in order to provide the reader with a critical view. Finally, we elucidate the main challenges and hurdles faced by gene therapy aimed at bone regeneration as well as expected future trends in this field.
Gene therapy; Bone regeneration; Tissue engineering; Viral vectors; Nonviral vectors
Mesenchymal stem cell (MSC) transplantation has shown tremendous promise as a therapy for repair of various tissues of the musculoskeletal, vascular, and central nervous systems. Based on this success, recent research in this field has focused on complex tissue damage, such as that which occurs from traumatic spinal cord injury (TSCI). As the critical event for successful exogenous, MSC therapy is their migration to the injury site, which allows for their anti-inflammatory and morphogenic effects on fracture healing, neuronal regeneration, and functional recover. Thus, there is a need for a cost-effective in vivo model that can faithfully recapitulate the salient features of the injury, therapy, and recovery. To address this, we review the recent advances in exogenous MSC therapy for TSCI and traumatic vertebral fracture repair and the existing challenges regarding their translational applications. We also describe a novel murine model designed to take advantage of multidisciplinary collaborations between musculoskeletal and neuroscience researchers, which is needed to establish an efficacious MSC therapy for TSCI.
The incidence of low back pain is extremely high and is often linked to intervertebral disc (IVD) degeneration. The mechanism of this disease is currently unknown. In this study, we have investigated the role of β-catenin signaling in IVD tissue function.
β-catenin protein levels were measured in disc samples derived from patients with disc degeneration and normal subjects by immunohistochemistry (IHC). To generate β-catenin conditional activation (cAct) mice, Col2a1-CreERT2 transgenic mice were bred with β-cateninfx(Ex3)/fx(Ex3) mice. Changes in disc tissue morphology and function were analyzed by micro-CT, histology and real-time PCR assays.
We found that β-catenin protein was up-regulated in disc tissues from patients with disc degeneration. To assess the effects of increased β-catenin on disc tissue we generated β-catenin cAct mice. Overexpression of β-catenin in disc cells led to extensive osteophyte formation in 3- and 6-month-old β-catenin cAct mice which were associated with significant changes in the cells and extracellular matrix of disc tissues and growth plate. Gene expression analysis demonstrated that activation of β-catenin could enhance Runx2-dependent Mmp13 and Adamts5 expression. Moreover, genetic ablation of the Mmp13 or Adamts5 under β-catenin cAct background, or treatment of β-catenin cAct mice with a specific MMP13 inhibitor, ameliorated the mutant phenotype.
β-catenin signaling pathway plays a critical role in disc tissue function.
CD23+CD21highCD1dhigh B cells (Bin cells) accumulate in the LNs draining inflamed joints of the TNFα transgenic (TNFtg) mouse model of rheumatoid arthritis, and are primarily involved in the significant histological and functional LN alterations that accompany disease exacerbation in this strain. Here we investigate the origin and function of Bin cells. We show that adoptively transferred GFP+ sorted mature follicular B (FoB) cells home preferentially to inflamed LNs of TNFtg mice where they rapidly differentiate into Bin cells, with a close correlation with the endogenous Bin fraction. Bin cells are also induced in wild-type (WT) LNs after immunization with T-dependent antigens, and display a germinal center phenotype at higher rates compared to FoB cells. Furthermore, we show that Bin cells can capture and process antigen immune complexes in a CD21-dependent manner more efficiently than FoB cells, and express higher levels of MHCII and costimulatory antigens CD80 and CD86. We propose that Bin cells are a previously unrecognized inflammation-induced B cell population with increased antigen capture and activation potential, which may facilitate normal immune responses but may contribute to autoimmunity when chronic inflammation causes their accumulation and persistence in affected LNs.
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; Immune complexes
Based on its proven anabolic effects on bone in osteoporosis patients, recombinant parathyroid hormone (PTH1-34) has been evaluated as a potential therapy for skeletal repair. Research in animals has investigated the effect of PTH1-34 in various skeletal repair models such as fractures, allografting, spinal arthrodesis, and distraction osteogenesis. These studies demonstrated that intermittent PTH1-34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms, which includes effects on mesenchymal stem cells (MSC), angiogenesis, chondrogenesis, bone formation and resorption. Furthermore, PTH1-34 was demonstrated to enhance bone repair in challenging animal models of aging, inflammatory arthritis and glucocorticoid-induced bone loss. This pre-clinical success has led to off-label clinical use, and a number of case reports documenting PTH1-34 treatment of delayed-unions and non-unions have been publish. Moreover, a phase 2 clinical trial of PTH1-34 treatment of patient with a radius fracture has now been completed. Although this trial failed to achieve its primary outcome, largely due to effective healing in the placebo group, several secondary outcomes were statistically significant, highlighted several important issues about the appropriate patient population for PTH1-34 therapy for skeletal repair. Here we review our current knowledge of the effects of PTH1-34 therapy for bone healing, enumerate several critical unresolved issues (e.g. appropriate dosing regimen and indications), and discuss this drug’s long term potential as an adjuvant for endogenous tissue engineering.
Parathyroid Hormone (PTH); skeletal repair; fracture insufficiency; allograft
Chronic complex musculoskeletal injuries that are slow to heal pose challenges to physicians and researchers alike. Orthobiologics is a relatively newer science that involves application of naturally found materials from biological sources (for example, cell-based therapies), and offers exciting new possibilities to promote and accelerate bone and soft tissue healing. Platelet-rich plasma (PRP) is an orthobiologic that has recently gained popularity as an adjuvant treatment for musculoskeletal injuries. It is a volume of fractionated plasma from the patient's own blood that contains platelet concentrate. The platelets contain alpha granules that are rich in several growth factors, such as platelet-derived growth factor, transforming growth factor-β, insulin-like growth factor, vascular endothelial growth factor and epidermal growth factor, which play key roles in tissue repair mechanisms. PRP has found application in diverse surgical fields to enhance bone and soft-tissue healing by placing supra-physiological concentrations of autologous platelets at the site of tissue damage. The relative ease of preparation, applicability in the clinical setting, favorable safety profile and possible beneficial outcome make PRP a promising therapeutic approach for future regenerative treatments. However, there is a large knowledge gap in our understanding of PRPs mechanism of action, which has raised skepticism regarding its potential efficacy and use. Thus, the aim of this review is to describe the various factors proposed to contribute to the biological activity of PRP, and the published pre-clinical and clinical evidence to support it. Additionally, we describe the current techniques and technology for PRP preparation, and review the present shortcomings of this therapy that will need to be overcome if it is to gain broad acceptance.
Management of various tumor metastases to bone has dramatically improved, but this is not so for renal cell carcinoma (RCC), which is a difficult surgical problem due to its great vascularity. Furthermore, the unique mechanisms that mediate RCC vasculogenesis in bone remain unknown. To understand this process we developed a xenograft model that recapitulates highly vascular RCC vs. less vascular tumors that metastasize to bone. Human tumor cell lines of RCC (786-O), prostate cancer (PC3), lung cancer (A549), breast cancer (MDA-MB231) and melanoma (A375) were transfected with firefly luciferase (Luc), injected into the tibiae of nude mice, and differences in growth, osteolysis and vascularity were assessed by longitudinal bioluminescent imaging (BLI), micro-CT for measurement of calcified tissues and vascularity and histology. The results showed that while RCC-Luc has reduced growth and osteolytic potential vs. the other tumor lines, it displayed a significant increase in vascular volume (p<0.05). This expansion was due to 3- and 5-fold increases in small and large vessel numbers respectively. In vitro gene expression profiling revealed that RCC-Luc expresses significantly (p<0.05) more vegf-a (10-fold) and 20-30-fold less ang-1 vs. the other lines. These data demonstrate the utility of this model to study the unique vasculogenic properties of RCC bone metastases.
Renal Cell Carcinoma (RCC); vasculogenesis; bone metastasis; xenograft
Clinical management of critical bone defects remains a major challenge. Despite pre-clinical work demonstrating teriparatide (PTH1–34) effectiveness in small animals, inconclusive data from clinical trials have raised questions of dose and regimen. To address this, we completed a comprehensive study in the murine femoral allograft model, to assess the effects of dose (0.4, 4, and 40 μg/kg/day) and various treatment regimens on radiographic, histologic and biomechanical healing at 2, 4, and 9 weeks. Only the high dose (40 μg/kg) of PTH1–34 demonstrated significant effects when given daily over 9 weeks. Remarkably, equivalent biomechanical results were obtained with delayed, short treatment from 2 to 6 weeks that did not induce a significant increase in endochondral bone formation and callus volume. In contrast, PTH1–34 treatment from 1 to 5 weeks post-op demonstrated similar osteogenic effects as immediate-daily treatment for 9 weeks, but failed to achieve a significant increase in biomechanics at 9 weeks. Micro-CT and histologic analyses demonstrated that the 2-week delay in treatment allowed for timely completion of the endochondral phase, such that the prominent effects of PTH1–34 were enhanced intramembranous bone formation and remodeling at the graft-host junction. These findings support the potential use of PTH1–34 as an adjuvant therapy for massive allograft healing, and suggest that there may be an ideal treatment window in which a short course is administered following the endochondral phase to promote osteoblastic bone formation and remodeling to achieve superior union with modest callus formation.
Bone; Allograft; Parathyroid Hormone (PTH); Biomechanics; Micro-Computed Tomography (micro-CT)
Osteoclasts (OC) are bone-resorbing, multinucleated cells that are generated via fusion of OC precursors (OCP). The frequency of OCP is elevated in patients with erosive inflammatory arthritis and metabolic bone diseases. Although many cytokines and cell surface receptors are known to participate in osteoclastogenesis, the molecular mechanisms underlying the regulation of this cellular transformation are poorly understood. Herein, we focused our studies on the dendritic cell-specific transmembrane protein (DC-STAMP), a seven-pass-transmembrane receptor-like protein known to be essential for cell-to-cell fusion during osteoclastogenesis. We identified an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of DC-STAMP, and developed an anti-DC-STAMP monoclonal antibody 1A2 that detected DC-STAMP expression on human tumor giant cells, blocked OC formation in vitro, and distinguished four patterns of human PBMC with a positive correlation to OC potential. In freshly isolated monocytes, DC-STAMPhigh cells produced a higher number of OC in culture than DC-STAMPlow cells and the surface expression of DC-STAMP gradually declined during osteoclastogenesis. Importantly, we showed that DC-STAMP is phosphorylated on its tyrosine residues and physically interacts with SHP-1 and CD16, an SH2-domain-containing tyrosine phosphatase and an ITAM-associated protein, respectively. Taken together, these data show that DC-STAMP is a potential OCP biomarker in inflammatory arthritis. Moreover, in addition to its effect on cell fusion, DC-STAMP dynamically regulates cell signaling during osteoclastogenesis.
DC-STAMP; osteoclast; signaling; ITIM; ITAM; SHP-1; OCP; biomarker; Ps; PsA; CD16
Light-activated gene transduction (LAGT) is an approach to localize gene therapy via preactivation of cells with UV light, which facilitates transduction by recombinant adeno-associated virus vectors. Prior studies demonstrated that UVC induces LAGT secondary to pyrimidine dimer formation, while UVA induces LAGT secondary to reactive oxygen species (ROS) generation. However, the empirical UVB boundary of these UV effects is unknown. Thus, we aimed to define the action spectra for UV-induced LAGT independent of DNA damage, and determine an optimal wavelength to maximize safety and efficacy. Results: UV at 288, 311 and 320nm produced significant dose-dependent LAGT effects, of which the maximum (800-fold) was observed with 4kJ/m2 at 311nm. Consistent with its robust cytotoxicity, 288nm produced significantly high levels of DNA damage at all doses tested, while 311, 320 and 330nm did not generate pyrimidine dimers and produced low levels of DNA damage detected by comet assay. While 288nm failed to induce ROS, the other wavelengths were effective, with the maximum (10-fold) effect observed with 30 kJ/m2 at 311nm. An in vivo pilot study assessing 311nm-induced LAGT of rabbit articular chondrocytes demonstrated a significant 6.6-fold (p<0.05) increase in transduction with insignificant cytotoxicity. Conclusion: 311nm was found to be the optimal wavelength for LAGT based on its superior efficacy at the peak dose, and its broad safety range that is remarkably wider than the other UV wavelengths tested.
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints, whose etiology is largely unknown. Recent studies in mice demonstrated alterations in lymphatics from affected joints precede flares. Thus, we aimed to develop novel methods for measuring lymph node pressure and lymph viscosity in limbs of mice. Pressure measurements were performed by inserting a glass micropipette connected to a pressure transducer into popliteal lymph nodes (PLN) or axillary lymph nodes (ALN) of mice and determined that the lymphatic pressures were 9 and 12 cm of water, respectively. We are also developing methods for measuring lymph viscosity in lymphatic vessels afferent to PLN, which can be measured by multi-photon fluorescence recovery after photobleaching (MP-FRAP) of FITC-BSA injected into the hind footpad. These results demonstrate the potential of lymph node pressure and lymph viscosity measurements, and warrant future studies to test these outcomes as biomarkers of arthritic flare.
Rheumatoid Arthritis; Lymph Node; Flare; Lymphatic Pressure; Lymph Viscosity
Recent studies have elucidated unanticipated connections between the immune and skeletal systems, and this relationship has led to the development of a new field known as osteoimmunology. The goal of research in this field is to: 1) further understand how the bone microenvironment influences immune cell ontogeny and subsequent effector functions, and 2) translate basic science findings in bone biology to clinical applications for autoimmune diseases that target the skeleton such as rheumatoid arthritis. In this review, we will examine the recent findings of the interplay between the immune and skeletal systems. This discussion will focus on the cells and signaling pathways in osteoimmune interactions and how innate and adaptive immune effector cells as well as cytokines and chemokines play a role in the maintenance and dysregulation of skeletal-immune homeostasis. We will also discuss how immunomodulatory biologic drugs, which specifically target these cells and effector molecules, have transformed the treatment of autoimmune mediated inflammatory diseases (IMIDs) and metabolic bone diseases such as osteoporosis.
Osteoimmunology; receptor-activator of nuclear factor kappa B (RANK); RANK-ligand; osteoprotegerin (OPG); arthritis; osteoporosis
While bone marrow edema (BME) is diagnostic of spondyloarthropathy, its nature remains poorly understood. In contrast, BME in ankylosing spondylitis is caused by TNF-induced vascular and cellular changes. To investigate the relationship between chronic compression and TNF signaling in compression induced BME we utilized a tail vertebrae compression model with WT, TNF-Tg and TNFR1&2−/− mice to evaluate: 1) healing following release of chronic compression, 2) induction of BME in the absence of TNFR, and 3) efficacy of anti-TNF therapy. Compression-induced normalized marrow contrast enhancement (NMCE) in WT was significantly decreased 3-fold (p<0.01) within 2 weeks of release, while the NMCE values in TNF-Tg vertebrae remained elevated, but had a significant decrease (p<0.05) by 6 weeks after the release of compression. TNFR1&2−/− mice were resistant to compression-induced BME. Anti-TNF therapy did not affect NMCE vs. placebo. Histological examination revealed that NMCE values significantly correlated with marrow vascularity and cellularity (p<0.05), which account for 76% of the variability of NMCE. Collectively, these data demonstrate a critical role for TNF in the induction of chronic compression-induced BME, but not in its maintenance. Amelioration of BME is achieved through biomechanical stability, but is not affected by anti-TNF therapy.
Modic Changes; CE-MRI; Bone Marrow Edema; Anti-TNF Therapy
Bone is a highly dynamic organ that interacts with a wide array cells and tissues. Recent studies have unveiled unanticipated connections between the immune and skeletal systems and this relationship led to the development a new field, osteoimmunology. This field will enable investigators to translate basic science findings in bone biology to clinical applications for inflammatory joint diseases such as psoriatic arthritis (PsA). In this review, we will examine the disruption of bone homeostasis in PsA and discuss the pivotal role of osteoclasts and osteoblasts as well as signaling pathways in the altered remodeling observed in this inflammatory arthritis. We will also discuss the effects of TNF inhibition on both bone resorption and new bone formation in PsA.
psoriasis; psoriatic arthritis; osteoclast; tumor necrosis factor; osteoblast; dickkopf-1
Although osteomyelitis (OM) remains a serious problem in orthopaedics, progress has been limited by the absence of an in vivo model that can quantify the bacterial load, metabolic activity of the bacteria over time, immunity and osteolysis. To overcome these obstacles, we developed a murine model of implant-associated OM in which a stainless steel pin is coated with Staphylococcus aureus and implanted transcortically through the tibial metaphysis. X-ray and micro-CT demonstrated concomitant osteolysis and reactive bone formation, which was evident by day 7. Histology confirmed all the hallmarks of implant-associated OM, namely: osteolysis, sequestrum formation and involucrum of Gram-positive bacteria inside a biofilm within necrotic bone. Serology revealed that mice mount a protective humoral response that commences with an IgM response after one weak, and converts to a specific IgG2b response against specific S. aureus proteins by day 11 post-infection. Real-time quantitative PCR (RTQ-PCR) for the S. aureus specific nuc gene determined that the peak bacterial load occurs 11 days post-infection. This coincidence of decreasing bacterial load with the generation of specific antibodies is suggestive of protective humoral immunity. Longitudinal in vivo bioluminescent imaging (BLI) of luxA-E transformed S. aureus (Xen29) combined with nuc RTQ-PCR demonstrated the exponential growth phase of the bacteria immediately following infection that peaks on day 4, and is followed by the biofilm growth phase at a significantly lower metabolic rate (p<0.05). Collectively, these studies demonstrate the first quantitative model of implant-associated OM that defines the kinetics of microbial growth, osteolysis and humoral immunity following infection.
Osteomyelitis; Staphylococcus aureus; Osteolysis; Humoral Immunity; Bioluminescent Imaging
To investigate if enhancement of joint lymphangiogenesis by injecting VEGF-C adeno-associated virus (AAV) into joints has therapeutic efficacy in chronic inflammatory arthritis in mice.
TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Human VEGF-C was cloned into an AAV expression vector to generate AAV-VEGF-C. AAV-VEGF-C or control AAV-Luc was injected into joints of TNF-Tg mice. MRI and lymphatic imaging were used during the 4-months following injection to assess changes in synovial volume and lymph flow from joint tissues to local draining lymph nodes. Joint inflammation, bone erosion and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry.
Intra-articular administration of AAV-VEGF-C virus significantly attenuated the increase in synovial volume and increased lymphatic vessel number in joint sections compared to AAV-Luc virus during the 4-month-period. This accompanied by reduced inflammation area, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates and was significantly improved with AAV-VEGF-C treatment.
Intra-articular injection of AAV-VEGF-C increased lymphangiogenesis and improved lymphatic drainage from inflamed joints, resulting in attenuation of joint tissue damage. Thus, improvement of joint lymphatic function by local administration of lymphatic growth factors represents a new therapeutic approach for chronic inflammatory arthritis.
VEGF-C; lymphatic system; lymphangiogenesis; inflammation; arthritis
Establish quantitative outcomes for assessing murine knee arthritis and develop an Arthritis Index that incorporates multiple outcomes into a single calculation that provides enhanced sensitivity.
Using an accepted model of meniscal/ligamentous injury (MLI)-induced osteoarthritis (OA), we assessed mouse knee arthritis using several approaches. Histology-based methods were performed to visualize joint tissues including articular cartilage and subchondral bone. Accepted histologic scoring methods and histomorphometry were performed to grade cartilage degeneration and determine articular cartilage area, respectively. MicroCT was used to visualize and quantify the bony structures of the joint including osteophytes and joint bone volume. A statistical algorithm was then developed that combined histologic scores and cartilage areas into a single Arthritis Index.
MLI induced progressive, OA-like articular cartilage degeneration characterized by increasing (worsening) histologic score and decreasing cartilage area. MicroCT revealed osteophytes and increased joint bone volume between the femoral and tibial physes following MLI. Lastly, an Arthritis Index calculation was established, which incorporated histologic scoring and cartilage area. The Arthritis Index provided enhanced quantitative sensitivity in assessing the level of joint degeneration compared to either histologic scoring or cartilage area determination alone; when using the Index, between 29% and 43% fewer samples are needed to establish statistical significance in studies of murine arthritis.
Arthritis in the mouse knee can be quantitatively assessed by histologic scoring, measuring cartilage area and determining joint bone volume. Enhanced sensitivity can be achieved by performing the Arthritis Index calculation, a novel method for quantitatively assessing mouse knee arthritis.
Osteoarthritis; Articular cartilage; Meniscal injury; Micro-computed tomography; Arthritis index
Critically sized large bone defects commonly result from trauma, radical tumor resections or infections. Currently, massive allografting remain as the clinical standard to treat these critical defects. Unfortunately, allograft healing is limited by the lack of osteogenesis and bio-integration of the graft to the host bone. Based on its widely studied anabolic effects on the bone, we have proposed that teriparatide [recombinant parathyroid hormone (PTH1–34)] could be an effective adjuvant for massive allograft healing. In support of this theory, here we review studies that have demonstrated that intermittent PTH1–34 treatment enhances and accelerates the skeletal repair process via a number of mechanisms including: effects on mesenchymal stem cells (MSC), angiogenesis, chondrogenesis, bone formation and remodeling. We also review the current literature on the effects of PTH1–34 therapy on bone healing, and discuss this drug’s long term potential as an adjuvant for endogenous tissue engineering.
Parathyroid Hormone (PTH); teriparatide; osseointegration; bio-integration; fracture healing; non-union; allograft repair; biomaterials
Based on remarkable success of PTH as an anabolic drug for osteoporosis, case reports of off-label use of teriparatide (1-34 PTH) in patients with complicated fractures and non-unions are emerging. We investigated the mechanisms underlying PTH accelerated fracture repair. Bone marrow cells from 7 days 40cg/kg of teriparatide treated or saline control mice were cultured and Osx and osteoblast phenotypic gene expression assessed by real time RT-PCR in these cells. Fractured animals injected daily with either saline or 40cg/kg of teriparatide for up to 21 days were X-rayed and histological assessment performed, as well as immunohistochemical analyses of the Osx expression in the fracture callus. Osx, Runx2 and osteoblast or chondrocyte phenotypic gene expression was also assessed in fracture calluses. Our data shows that Osx and Runx2 are up-regulated in marrow-derived MSCs isolated from mice systemically treated with teriparatide. Furthermore, these MSCs undergo accelerated osteoblast maturation compared to saline injected controls. Systemic teriparatide treatments also accelerated fracture healing in these mice concomitantly with increased Osx expression in the PTH treated fracture calluses compared to controls. Collectively, these data suggest a mechanism for teriparatide mediated fracture healing possibly via Osx induction in MSCs.
Teriparatide; PTH; Osterix/Sp7; Runx2; fractures
The receptor activator of nuclear factor-κB ligand (RANKL), its cognate receptor RANK, and its natural decoy receptor osteoprotegerin have been identified as the final effector molecules of osteoclastic bone resorption. This has provided an ideal target for therapeutic interventions in metabolic bone disease. As described in previous reviews in this supplement, RANKL signaling is required for osteoclast differentiation, activation, and survival. Furthermore, in vivo inhibition of RANKL leads to immediate osteoclast apoptosis, and there are no in vivo models of bone resorption that are refractory to RANKL inhibition. Thus, the only step remaining in the development of a clinical intervention is the generation of a safe, effective, and specific drug that can inhibit RANKL in humans. Here we review the clinical development of denosumab (formerly known as AMG 162), which is a fully human mAb directed against RANKL. This discussion includes the breadth of 21 human studies that have led to the current phase 3 clinical trials seeking approval for use of this agent to treat postmenopausal women with low bone mineral density (osteoporosis) and patients with metastatic lytic bone lesions (multiple myeloma, and prostate and breast cancer).
As a downstream product of cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2) plays a crucial role in the regulation of bone formation. It has four different receptor subtypes (EP1 through EP4), each of which exerts different effects in bone. EP2 and EP4 induce bone formation through the protein kinase A (PKA) pathway, whereas EP3 inhibits bone formation in vitro. However, the effect of EP1 receptor signaling during bone formation remains unclear. Closed, stabilized femoral fractures were created in mice with EP1 receptor loss of function at 10 weeks of age. Healing was evaluated by radiographic imaging, histology, gene expression studies, micro–computed tomographic (µCT), and biomechanical measures. EP1−/− mouse fractures have increased formation of cartilage, increased fracture callus, and more rapid completion of endochondral ossification. The fractures heal faster and with earlier fracture callus mineralization with an altered expression of genes involved in bone repair and remodeling. Fractures in EP1−/− mice also had an earlier appearance of tartrate-resistant acid phosphatase (TRAcP)–positive osteoclasts, accelerated bone remodeling, and an earlier return to normal bone morphometry. EP1−/− mesenchymal progenitor cells isolated from bone marrow have higher osteoblast differentiation capacity and accelerated bone nodule formation and mineralization in vitro. Loss of the EP1 receptor did not affect EP2 or EP4 signaling, suggesting that EP1 and its downstream signaling targets directly regulate fracture healing. We show that unlike the PGE2 receptors EP2 and EP4, the EP1 receptor is a negative regulator that acts at multiple stages of the fracture healing process. Inhibition of EP1 signaling is a potential means to enhance fracture healing. © 2011 American Society for Bone and Mineral Research.
EP1; FRACTURE HEALING; CHONDROCYTE; CHONDROCYTE MATURATION; MINERALIZATION; OSTEOBLASTS; OSTEOCLASTS; PGE2
Recently, methicillin-resistant Staphylococcus aureus (MRSA) has surpassed HIV as the most deadly pathogen in the United States, accounting for over 100,000 deaths per year. In orthopedics, MRSA osteomyelitis has become the greatest concern in patient care, despite the fact that improvements in surgical technique and aggressive antibiotic prophylaxis have decreased the infection rate for most procedures to less than 5%. This great concern is largely due to the very poor outcomes associated with MRSA osteomyelitis, which includes 30–50% failure rates for revision surgery. Thus, there is a need to develop additional therapeutic interventions such as passive immunization, particularly for immunocompromised patients and the elderly who are typically poor responders to active vaccines. Using a novel murine model of implant-associated osteomyelitis in which a stainless steel pin is coated with bioluminescent S. aureus and implanted transcortically through the tibial metaphysis, we discovered that mice protect themselves from this infection by mounting a specific IgG2b response against the peptidoglycan hydrolase, glucosaminidase (Gmd), an enzyme involved in cell wall digestion during binary fission. Since this subunit of S. aureus autolysin is essential for bacterial growth, and no genetic variation has been identified among clinical strains, we propose that monoclonal antibodies against this enzyme would have multiple mechanisms of action, including promotion of opsonophagocytosis and direct inhibition of enzyme function. Here we review the field of MRSA osteomyelitis and our research to date on the development of an anti-Gmd passive immunotherapy.
Osteomyelitis; Methicillin-Resistant Staphylococcus aureus (MRSA); Autolysin; Glucosaminidase; Passive immunization
Despite the remarkable healing potential of long bone fractures, traumatic injuries that result in critical defects require challenging reconstructive limb sparing surgery. While devitalized allografts are the gold standard for these procedures, they are prone to failure due to their limited osseointegration with the host. Thus, the quest for adjuvants to enhance allograft healing remains a priority for this unmet clinical need. To address this, we investigated the effects of daily systemic injections of 40 µg/kg teriparatide (recombinant human parathyroid hormone) on the healing of devitalized allografts used to reconstruct critical femoral defects (4 mm) in C57Bl/6 mice. The femurs were evaluated at 4 and 6 weeks using micro CT, histology, and torsion testing. Our findings demonstrated that teriparatide induced prolonged cartilage formation at the graft-host junction at 4 weeks, which led to enhanced trabeculated bone callus formation and remarkable graft-host integration at 6-weeks. Moreover, we observed a significant 2-fold increase in normalized callus volume (1.04 ± 0.3 vs. 0.54 ± 0.14 mm3/mm; p<0.005), and Union Ratio (0.28 ± 0.07 vs. 0.13 ± 0.09; p<0.005), compared to saline treated controls at 6-weeks. Teriparatide treatment significantly increased the torsional rigidity (585±408 versus 1175±311 N.mm2) and yield torque (6.8±5.5 versus 10.5±4.2 N.mm) compared to controls. Interestingly, the Union Ratio correlated significantly with the yield torque and torsional rigidity (R2=0.59 and R2=0.77, p<0.001, respectively). These results illustrate the remarkable potential of teriparatide as an adjuvant therapy for allograft repair in a mouse model of massive femoral defect reconstruction, and warrant further investigation in a larger animal model at longer time intervals to justify future clinical trials for PTH therapy in limb sparing reconstructive procedures.
Bone; Allograft; PTH; Biomechanics; Micro-Computed Tomography