A skeleton in a huff: insights in etiologies of osteosclerosis.

A 30-yr-old man developed right lower leg pain and a palpable solid mass. Radiographic imaging revealed a periosteal reaction with an exostotic mass arising from the right distal fibula. Generalized skeletal osteosclerosis with periosteal reaction was discovered on a radiographic skeletal survey. A biopsy of the right fibular mass revealed reactive woven bone. The patient was referred to a metabolic bone disease clinic, where laboratory values were consistent with secondary hyperparathyroidism and increased bone turnover. A DXA bone density scan revealed high bone density, with an L1-4 spine Z-score of +9.3, a left femoral neck Z-score of +8.5, and a total hip Z-score of +6.5. A dental exam revealed generalized gingival inflammation, teeth mobility, generalized horizontal alveolar bone loss and widening of the periodontal ligament space, increased bone density around the teeth, and thickening of the radicular lamina dura. An extensive evaluation was performed, with the result of a single test revealing the diagnosis. The differential diagnoses of osteosclerosis affecting the skeleton, teeth, and oral cavity are discussed.

A 30-yr-old man developed, over a short period, pain in his lower right leg accompanied by a hard mass. He also reported weight loss and night sweats for the past 6 months. After evaluation by his primary physician, an X-ray was ordered that reported a bony mass arising from the right fibula bone. A biopsy was performed of the mass, but no evidence of cancer or any other specific abnormality was found. The patient was then referred to a bone disease specialty clinic. Laboratory tests revealed a large increase in how quickly the patient's skeleton was remodeling, affecting the balance of bone formation and removal involved in maintaining a healthy skeleton. A bone density scan reported that the patient had very dense bones. Other unusual changes were also discovered in a dental exam, suggesting bone thickening. After an extensive evaluation, a single blood test revealed the cause of the fibular bone mass and dense bones.

Silver Diamine Fluoride and Progression of Incipient Approximal Caries in Permanent Teeth: A Retrospective Study.

Purpose: The purpose of this study was to evaluate the progression of incipient approximal caries lesions in permanent teeth of children and adolescents with and without silver diamine fluoride (SDF) application. Methods: A retrospective analysis of dental records and radiographs was performed. Baseline and follow-up bitewing radiographs were evaluated and scored using International Caries Classification and Management System (ICCMS) criteria to assess lesion progression. Results: A total of 131 lesions from 68 subjects (mean age equals 9.6 years) were evaluated radiographically and followed for up to 41 months (mean time equals 13.61±6.8 months); 23.6 percent of lesions in the SDF group progressed compared to 38.1 percent in the control group (P<0.001). On average, lesions in the control group increased more per month compared to the study group (P<0.001). The odds of lesion progression in the control group were 2.76 times the odds of progression in the study group. There was a statistically significant difference in lesion progression based on application method; lesions where SDF was applied with Superfloss progressed more per month, on average, versus microbrush application. Conclusions: Silver diamine fluoride may be an effective therapy to slow caries progression of incipient approximal lesions in permanent teeth in high caries-risk populations. Future studies are needed to detect differences in application methods.

Articular cartilage protection in Ctsk-/- mice is associated with cellular and molecular changes in subchondral bone and cartilage matrix.

Osteoarthritis (OA) is a degenerative disease and a major cause of chronic disability in aging individuals. Cathepsin K (CatK), encoded by the Ctsk gene, has been implicated in the pathogenesis of pycnodysostosis and osteoporosis. The use of a selective inhibitor of CatK was recently shown to delay OA progression in rabbits. However, the cellular mechanisms underlying these protective effects remain unexplored. We examined articular cartilage maintenance and joint bone remodeling using Ctsk null mice (Ctsk-/- ) which underwent destabilization of the medial meniscus (DMM). We found that Ctsk-/- mice displayed delayed remodeling of subchondral and calcified cartilage by osteoclasts and chodroclasts respectively in DMM-induced osteoarthritis. While WT mice displayed a more severe OA phenotype than Ctsk-/- mice at 16 weeks, higher subchondral bone volume and lower trabecular spacing were also observed in surgically-induced OA joints of Ctsk-/- mice. However, no differences were seen in non-surgical controls. During OA progression, TRAP+ osteoclast numbers were increased in both WT and Ctsk-/- mice. However, Ctsk-/- mice had fewer physis-derived chondroclasts than WT when OA was present. These data suggest that CatK may differentially regulate chondroclastogenesis in the growth plate. Targeted PCR arrays of RNA harvested from laser captured osteoclasts in the subchondral bone and chondroclasts in the growth plate demonstrated differential expression of Atp6v0d2, Tnfrsf11a, Ca2, Calcr, Ccr1, Gpr68, Itgb3, Nfatc1, and Syk genes between WT and Ctsk-/- mice at 8- and 16-weeks post-DMM. Our data provide insight into the cellular mechanisms by which cathepsin K deletion delays OA progression in mice.

Bone marrow macrophages support prostate cancer growth in bone.

Resident macrophages in bone play important roles in bone remodeling, repair, and hematopoietic stem cell maintenance, yet their role in skeletal metastasis remains under investigated. The purpose of this study was to determine the role of macrophages in prostate cancer skeletal metastasis, using two in vivo mouse models of conditional macrophage depletion. RM-1 syngeneic tumor growth was analyzed in an inducible macrophage (CSF-1 receptor positive cells) ablation model (MAFIA mice). There was a significant reduction in tumor growth in the tibiae of macrophage-ablated mice, compared with control non-ablated mice. Similar results were observed when macrophage ablation was performed using liposome-encapsulated clodronate and human PC-3 prostate cancer cells where tumor-bearing long bones had increased numbers of tumor associated-macrophages. Although tumors were consistently smaller in macrophage-depleted mice, paradoxical results of macrophage depletion on bone were observed. Histomorphometric and micro-CT analyses demonstrated that clodronate-treated mice had increased bone volume, while MAFIA mice had reduced bone volume. These results suggest that the effect of macrophage depletion on tumor growth was independent of its effect on bone responses and that macrophages in bone may be more important to tumor growth than the bone itself. In conclusion, resident macrophages play a pivotal role in prostate cancer growth in bone.

Polarization of prostate cancer-associated macrophages is induced by milk fat globule-EGF factor 8 (MFG-E8)-mediated efferocytosis.

Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells.

Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone.

Cellular subpopulations in the bone marrow play distinct and unexplored functions in skeletal homeostasis. This study delineated a unique role of osteal macrophages in bone and parathyroid hormone (PTH)-dependent bone anabolism using murine models of targeted myeloid-lineage cell ablation. Depletion of c-fms(+) myeloid lineage cells [via administration of AP20187 in the macrophage Fas-induced apoptosis (MAFIA) mouse model] reduced cortical and trabecular bone mass and attenuated PTH-induced trabecular bone anabolism, supporting the positive function of macrophages in bone homeostasis. Interestingly, using a clodronate liposome model with targeted depletion of mature phagocytic macrophages an opposite effect was found with increased trabecular bone mass and increased PTH-induced anabolism. Apoptotic cells were more numerous in MAFIA versus clodronate-treated mice and flow cytometric analyses of myeloid lineage cells in the bone marrow showed that MAFIA mice had reduced CD68(+) cells, whereas clodronate liposome-treated mice had increased CD68(+) and CD163(+) cells. Clodronate liposomes increased efferocytosis (clearance of apoptotic cells) and gene expression associated with alternatively activated M2 macrophages as well as expression of genes associated with bone formation including Wnt3a, Wnt10b, and Tgfb1. Taken together, depletion of early lineage macrophages resulted in osteopenia with blunted effects of PTH anabolic actions, whereas depletion of differentiated macrophages promoted apoptotic cell clearance and transformed the bone marrow to an osteogenic environment with enhanced PTH anabolism. These data highlight a unique function for osteal macrophages in skeletal homeostasis.

Parathyroid hormone-related protein drives a CD11b+Gr1+ cell-mediated positive feedback loop to support prostate cancer growth.

In the tumor microenvironment, CD11b(+)Gr1(+) bone marrow-derived cells are a predominant source of protumorigenic factors such as matrix metalloproteinases (MMP), but how distal tumors regulate these cells in the bone marrow is unclear. Here we addressed the hypothesis that the parathyroid hormone-related protein (PTHrP) potentiates CD11b(+)Gr1(+) cells in the bone marrow of prostate tumor hosts. In two xenograft models of prostate cancer, levels of tumor-derived PTHrP correlated with CD11b(+)Gr1(+) cell recruitment and microvessel density in the tumor tissue, with evidence for mediation of CD11b(+)Gr1(+) cell-derived MMP-9 but not tumor-derived VEGF-A. CD11b(+)Gr1(+) cells isolated from mice with PTHrP-overexpressing tumors exhibited relatively increased proangiogenic potential, suggesting that prostate tumor-derived PTHrP potentiates this activity of CD11b(+)Gr1(+) cells. Administration of neutralizing PTHrP monoclonal antibody reduced CD11b(+)Gr1(+) cells and MMP-9 in the tumors. Mechanistic investigations in vivo revealed that PTHrP elevated Y418 phosphorylation levels in Src family kinases in CD11b(+)Gr1(+) cells via osteoblast-derived interleukin-6 and VEGF-A, thereby upregulating MMP-9. Taken together, our results showed that prostate cancer-derived PTHrP acts in the bone marrow to potentiate CD11b(+)Gr1(+) cells, which are recruited to tumor tissue where they contribute to tumor angiogenesis and growth.

The effects of zoledronic acid in the bone and vasculature support of hematopoietic stem cell niches.

Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.

The multifaceted actions of PTHrP in skeletal metastasis.

PTHrP, identified during the elucidation of mediators of malignancy-induced hypercalcemia, plays numerous roles in normal physiology as well as pathological conditions. Recent data support direct functions of PTHrP in metastasis, particularly from tumors with strong bone tropism. Bone provides a unique metastatic environment because of mineralization and the diverse cell populations in the bone marrow. PTHrP is a key regulator of tumor-bone interactions and regulates cells in the bone microenvironment through proliferative and prosurvival activities that prime the 'seed' and the 'soil' of the metastatic lesion. This review highlights recent findings regarding the role of PTHrP in skeletal metastasis, including direct actions in tumor cells, as well as alterations in the bone microenvironment and future perspectives involving the potential roles of PTHrP in the premetastatic niche, and tumor dormancy.

Cyclophosphamide creates a receptive microenvironment for prostate cancer skeletal metastasis.

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

Roles of bone marrow cells in skeletal metastases: no longer bystanders.

Bone serves one of the most congenial metastatic microenvironments for multiple types of solid tumors, but its role in this process remains under-explored. Among many cell populations constituting the bone and bone marrow microenvironment, osteoblasts (originated from mesenchymal stem cells) and osteoclasts (originated from hematopoietic stem cells) have been the main research focus for pro-tumorigenic roles. Recently, increasing evidence further elucidates that hematopoietic lineage cells as well as stromal cells in the bone marrow mediate distinct but critical functions in tumor growth, metastasis, angiogenesis and apoptosis in the bone microenvironment. This review article summarizes the key evidence describing differential roles of bone marrow cells, including hematopoietic stem cells (HSCs), megakaryocytes, macrophages and myeloid-derived suppressor cells in the development of metastatic bone lesions. HSCs promote tumor growth by switching on angiogenesis, but at the same time compete with metastatic tumor cells for occupancy of osteoblastic niche. Megakaryocytes negatively regulate the extravasating tumor cells by inducing apoptosis and suppressing proliferation. Macrophages and myeloid cells have pro-tumorigenic roles in general, suggesting a similar effect in the bone marrow. Hematopoietic and stromal cell populations in the bone marrow, previously considered as simple by-standers in the context of tumor metastasis, have distinct and active roles in promoting or suppressing tumor growth and metastasis in bone. Further investigation on the extended roles of bone marrow cells will help formulate better approaches to treatment through improved understanding of the metastatic bone microenvironment.

Inhibitory effects of megakaryocytic cells in prostate cancer skeletal metastasis.

Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients.

Influence of leucite content on slow crack growth of dental porcelains.

OBJECTIVES: To determine the stress corrosion susceptibility coefficient, n, of seven dental porcelains (A: Ceramco I; B: Ceramco-II; C: Ceramco-III; D: d.Sign; E: Cerabien; F: Vitadur-Alpha; and G: Ultropaline) after aging in air or artificial saliva, and correlate results with leucite content (LC). METHODS: Bars were fired according to manufacturers' instructions and polished before induction of cracks by a Vickers indenter (19.6N, 20s). Four specimens were stored in air/room temperature, and three in saliva/37 degrees C. Five indentations were made per specimen and crack lengths measured at the following times: approximately 0; 1; 3; 10; 30; 100; 300; 1000 and 3000 h. The stress corrosion coefficient n was calculated by linear regression analysis after plotting crack length as a function of time, considering that the slope of the curve was [2/(3n+2)]. Microstructural analysis was performed to determine LC. RESULTS: LC of the porcelains were 22% (A and B); 6% (C); 15% (D); 0% (E and F); and 13% (G). Except for porcelains A and D, all materials showed a decrease in their n values when stored in artificial saliva. However, the decrease was more pronounced for porcelains B, F, and G. Ranking of materials varied according to storage media (in air, porcelain G showed higher n compared to A, while in saliva both showed similar coefficients). No correlation was found between n values and LC in air or saliva. SIGNIFICANCE: Storage media influenced the n value obtained for most of the materials. LC did not affect resistance to slow crack growth regardless of the test environment.