Christian S. Thudium

Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength

Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption. To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks. Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for µCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. µCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone. In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.

A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2T3 was treated with 50% of CM or non-CM for 12days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation.

Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores osteoclast function in infantile malignant osteopetrosis.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.

Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice

The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplanted hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclastogenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell function by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97–98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knockout recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice.

Biomarker of extracellular matrix remodelling C1m and proinflammatory cytokine interleukin 6 are related to synovitis and pain in end-stage knee osteoarthritis patients.

Abstract Little is known about local and systemic biomarkers in relation to synovitis and pain in end-stage osteoarthritis (OA) patients. We investigated the associations between the novel extracellular matrix biomarker, C1M, and local and systemic interleukin 6 (IL-6) with synovitis and pain. Serum C1M, plasma, and synovial fluid IL-6 (p-IL-6, sf-IL-6) were measured in 104 end-stage knee OA patients. Contrast-enhanced magnetic resonance imaging was used to semiquantitatively assess an 11-point synovitis score; pain was assessed by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Neuropathic Pain Questionnaire (NPQ). Linear regression was used to investigate associations between biomarkers and synovitis, and biomarkers and pain while controlling for age, sex, and body mass index. We also tested whether associations between biomarkers and pain were confounded by synovitis. We found sf-IL-6 was associated with synovitis in the parapatellar subregion (B = 0.006; 95% confidence interval [CI] 0.003-0.010), and no association between p-IL-6 and synovitis. We also observed an association between C1M and synovitis in the periligamentous subregion (B = 0.013; 95% CI 0.003-0.023). Furthermore, sf-IL-6, but not p-IL-6, was significantly associated with pain, WOMAC (B = 0.022; 95% CI 0.004-0.040), and NPQ (B = 0.043; 95% CI 0.005-0.082). There was no association between C1M and WOMAC pain, but we did find an association between C1M and NPQ (B = 0.229; 95% CI 0.036-0.422). Lastly, synovitis explained both biomarker-NPQ associations, but not the biomarker-WOMAC association. These results suggest that C1M and IL-6 are associated with synovitis and pain, and synovitis is an important confounding variable when studying biomarkers and neuropathic features in OA patients.

Novel targets for the prevention of osteoporosis – lessons learned from studies of metabolic bone disorders

Introduction: Osteoporosis is a major health care problem, and whereas efficacious treatments for vertebral fracture reduction are available for osteoporosis patients, these therapies are still limited with respect to capacity for restoration of bone loss, as well as efficacy on non-vertebral fractures, such as hip fractures, which are the source of morbidity and mortality. Areas covered: Studies of rare bone diseases in humans, such as osteopetrosis, sclerosteosis, pycnodysostosis and more, have shed light on a series of drug targets in bone that have the potential to result in therapies for osteoporosis with novel mechanisms of action, and the potential to improve the standard of care substantially. We focus on how they are separated from classic treatments for osteoporosis, in terms of novel modes of action, additional beneficial effects on bone turnover and importantly also safety. We focus on the status of anti-sclerostin antibodies, novel parathyroid hormone-related protein analogs, inhibitors of cathepsin K and ClC-7 in osteoclasts, all of which are currently in development. Expert opinion: There is a good possibility that the treatment of osteoporosis will be greatly improved within the coming years; however, with numerous effective and safe drugs already available careful attention to the safety of these novel candidates is crucial.

Translational Biomarkers and Ex Vivo Models of Joint Tissues as a Tool for Drug Development in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic and degenerative autoimmune joint disease that leads to disability, reduced quality of life, and increased mortality. Although several synthetic and biologic disease‐modifying antirheumatic drugs are available, there is still a medical need for novel drugs that control disease progression. As only 10% of experimental drug candidates for treatment of RA that enter phase I trials are eventually registered by the Food and Drug Administration, there is an immediate need for translational tools to facilitate early decision‐making in drug development. In this study, we aimed to determine if the inability of fostamatinib (a small molecule inhibitor of Syk) to demonstrate sufficient efficacy in phase III of a previous clinical study could have been predicted earlier in the development process.

Forced expression of human macrophage colony-stimulating factor in CD34+ cells promotes monocyte differentiation in vitro and in vivo but blunts osteoclastogenesis in vitro

Here, we tested the hypothesis that human M‐CSF (hM‐CSF) overexpressed in cord blood (CB) CD34+ cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo.

Development and use of biochemical markers in osteoarthritis: current update

Purpose of review There is an increasing demand for noninvasive and descriptive biochemical markers (biomarkers) in osteoarthritis; for enabling early drug development (including translational research), evaluating clinical trial at an early stage and for subtyping. Purpose of the review is to review and comment on current availability of such biomarkers. Recent findings Many different biomarkers have been tested in the last 18 months. The main focus has been on testing whether the biomarkers, whether is reflect joint tissue turnover or inflammatory status, can differentiate osteoarthritis patients from healthy controls or whether the biomarkers are associated with progression. Less than a handful of studies, investigate the role of the biomarkers as response markers. Thus, there is still a great need for developing biomarkers that reflect disease activity and thereby can be used for treatment response or patient phenotyping. Summary Osteoarthritis is the most common form of joint disease. This presents the osteoarthritis research community and pharmaceutical companies developing disease-modifying osteoarthritis drugs (DMOADs) with great opportunities. There are different osteoarthritis subtypes, which complicates the traditional approaches for developing new treatments. If we can identify new markers that can distinguish different subtypes, this can greatly facilitate drug development from early discovery to late clinical development.

Osteoclasts degrade bone and cartilage knee joint compartments through different resorption processes

BackgroundOsteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage—the remodeling of which is important in the osteoarthritic disease process—have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments.MethodsMature human osteoclasts were cultured on ECMs isolated from bovine knees—articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)—in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett’s T3 post hoc test, as applicable.ResultsOsteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)—similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p < 0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16).ConclusionsOur study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.