Characterization of the bone phenotype and fracture repair in osteopetrotic Incisors absent rats

Authors

Michelle M. McDonald, Alyson Morse, Lauren Peacock, Kathy Mikulec, Aaron Schindeler, David G. Little

Abstract

Osteopetrotic patients possess a genetic condition that leads to a deficiency in osteoclast number or function. Patients have a high bone density and suffer from an increased risk of fracture. The lack of normal osteoclast activity has the potential to impede repair by complicating orthopedic fixation and/or by affecting the biology of fracture healing. The naturally occurring incisors absent (ia/ia) rat was adopted as a rodent model of congenital osteopetrosis. A detailed phenotypic analysis of the ia/ia rat indicated that some functional recovery occurred between 7 and 9 weeks. Consequently a fracture repair study was undertaken using 5-week-old rats. Closed femoral fractures were generated in ia/ia rats and control ia/+ and +/+ rats using an Einhorn apparatus. Fracture healing was examined radiologically and histologically at 1–3 weeks. No difference was seen in bridging between ia/ia and control rats at any time point. The ia/ia rats showed no delay in cartilage removal but showed a significant delay in hard callus remodeling. This is consistent with an essential role for osteoclasts in only the latter stages of endochondral bone repair. This delay in hard callus remodeling was offset by an increase in moment of inertia.

Link to Article

http://dx.doi.org/10.1002/jor.21293

The skeletal effects of the tyrosine kinase inhibitor nilotinib

Authors

Susannah O'Sullivan, Jian-Ming Lin, Maureen Watson, Karen Callon, Pak Cheung Tong, Dorit Naot, Anne Horne, Opetaia Aati, , Fran Porteous, Greg Gamble, Jillian Cornish, Peter Browett, and Andrew Grey

Abstract

Nilotinib is a tyrosine kinase inhibitor (TKI) developed to manage imatinib-resistance in patients with chronic myeloid leukemia (CML). It inhibits similar molecular targets to imatinib, but is a significantly more potent inhibitor of Bcr-Abl. Nilotinib exhibits off-target effects in other tissues, and of relevance to bone metabolism, hypophosphataemia has been reported in up to 30% of patients receiving nilotinib. We have assessed the effects of nilotinib on bone cells in vitro and on bone metabolism in patients receiving nilotinib for treatment of CML. We firstly investigated the effects of nilotinib on proliferating and differentiating osteoblastic cells, and on osteoclastogenesis in murine bone marrow cultures and RAW264.7 cells. Nilotinib potently inhibited osteoblast proliferation (0.01–1 uM), through inhibition of the platelet-derived growth factor (PDGFR). There was a biphasic effect on osteoblast differentiation such that it was reduced by lower concentrations of nilotinib (0.1–0.5 uM), with no effect at higher concentrations (1 uM). Nilotinib also potently inhibited osteoclastogenesis, predominantly by stromal-cell dependent mechanisms. Thus, nilotinib decreased osteoclast development in murine bone marrow cultures, but did not affect osteoclastogenesis in RAW264.7 cells. Nilotinib treatment of osteoblastic cells increased expression and secretion of OPG and decreased expression of RANKL. In 10 patients receiving nilotinib, levels of bone turnover markers were in the low-normal range, despite secondary hyperparathyroidism, findings that are similar to those in patients treated with imatinib. Bone density tended to be higher than age and gender-matched normal values. These data suggest that nilotinib may have important effects on bone metabolism. Prospective studies should be conducted to determine the long-term effects of nilotinib on bone density and calcium metabolism.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.04.014

Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species

Authors:

Hongkang Zhou, America B. Newnum, Joseph R. Martin, Ping Li, Mark T. Nelson, Akira Moh, Xin-Yuan Fu, Hiroki Yokota and Jiliang Li

Abstract:

Signal transducers and activators of transcription 3 (Stat3) is a transcription factor expressed in many cell types including osteoblasts, osteocytes, and osteoclasts. STAT3 mutations cause a rare human immunodeficiency disease that presents reduced bone mineral density and recurrent pathological fractures. To investigate the role of Stat3 in load-driven bone metabolism, two strains of osteoblast/osteocyte-selective Stat3 knockout (KO) mice were generated. Compared to age-matched littermate controls, this selective inactivation of Stat3 significantly lowered bone mineral density (7–12%, p < 0.05) as well as ultimate force (21–34%, p < 0.01). In ulna loading (2.50–2.75 N with 120 cycles/day at 2 Hz for 3 consecutive days), Stat3 KO mice were less responsive than littermate controls as indicated by reduction in relative mineralizing surface (rMS/BS, 47–59%, p < 0.05) and relative bone formation rate (rBFR/BS, 64–75%, p < 0.001). Furthermore, inactivation of Stat3 suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Taken together, the results support the notion that the loss-of-function mutation of Stat3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.

Link to Article:

http://dx.doi.org/10.1016/j.bone.2011.04.020

Crebbp haploinsufficiency in mice alters the bone marrow microenvironment leading to loss of stem cells and excessive myelopoiesis

Authors

Stephanie N. Zimmer, Qing Zhou, Ting Zhou, Ziming Cheng, Sherry L. Abboud-Werner, Diane Horn, Michael Lecocke, Ruth White, Andrei V. Krivtsov, Scott A. Armstrong, Andrew L. Kung, David M. Livingston, and Vivienne I. Rebel

Abstract

CREB binding protein (CREBBP) is important for cell-autonomous regulation of hematopoiesis, including the stem cell compartment. Here we describe that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the bone marrow (BM) microenvironment of Crebbp+/- mice was unable to properly maintain the immature stem - and progenitor pools. Instead, it stimulates myeloid differentiation that progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone, predominantly caused by increased osteoclastogenesis. Although colony-forming unit-fibroblast and the total osteoblast numbers were decreased, the bone formation rate was similar to that found in wild-type mice. At a molecular level, we found that the known hematopoietic modulators MMP9 and KITL were decreased with heterozygous levels of Crebbp. Lastly, potentially important regulatory proteins (ESAM1 and CDH5) were increased on Crebbp+/- endothelial cells. Together, our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and prevent excessive myeloproliferation.

Link to Article

http://dx.doi.org/10.1182/blood-2010-09-307942

Effect of paricalcitol and cinacalcet on serum phosphate, FGF-23, and bone in rats with chronic kidney disease

Authors

Jane L. Finch, Masanori Tokumoto, Hironori Nakamura, Wei Yao, Mohammad Shahnazari, Nancy Lane, and Eduardo Slatopolsky

Abstract

Calcimimetics activate the calcium-sensing receptor (CaR) and reduce parathyroid hormone (PTH) by increasing the sensitivity of the parathyroid CaR to ambient calcium. The calcimimetic, cinacalcet, is effective in treating secondary hyperparathyroidism in dialysis patients [chronic kidney disease (CKD 5)], but little is known about its effects on stage 3–4 CKD patients. We compared cinacalcet and paricalcitol in uremic rats with creatinine clearances “equivalent” to patients with CKD 3–4. Uremia was induced in anesthetized rats using the 5/6th nephrectomy model. Groups were 1) uremic control, 2) uremic + cinacalcet (U+Cin; 15 mg·kg−1·day−1 po for 6 wk), 3) uremic + paricalcitol (U+Par; 0.16 μg/kg, 3 × wk, ip for 6 wk), and 4) normal. Unlike U+Par animals, cinacalcet promoted hypocalcemia and marked hyperphosphatemia. The Ca × P in U+Cin rats was twice that of U+Par rats. Both compounds suppressed PTH. Serum 1,25-(OH)2D3 was decreased in both U+Par and U+Cin rats. Serum FGF-23 was increased in U+Par but not in U+Cin, where it tended to decrease. Analysis of tibiae showed that U+Cin, but not U+Par, rats had reduced bone volume. U+Cin rats had similar bone formation and reduced osteoid surface, but higher bone resorption. Hypocalcemia, hyperphosphatemia, low 1,25-(OH)2D3, and cinacalcet itself may play a role in the detrimental effects on bone seen in U+Cin rats. This requires further investigation. In conclusion, due to its effects on bone and to the hypocalcemia and severe hyperphosphatemia it induces, we believe that cinacalcet should not be used in patients with CKD without further detailed studies.

Link to Article

http://ajprenal.physiology.org/content/298/6/F1315.abstract

Characterization of Ex Vivo–Generated Bovine and Human Cartilage by Immunohistochemical, Biochemical, and Magnetic Resonance Imaging Analyses

Authors

Ashleigh E. Nugent, David A. Reiter, Kenneth W. Fishbein, Denise L. McBurney, Travis Murray, Dorota Bartusik, Sharan Ramaswamy, Richard G. Spencer, Walter E. Horton

Abstract

Osteoarthritis (OA) is a prevalent age-associated disease involving altered chondrocyte homeostasis and cartilage degeneration. The avascular nature of cartilage and the altered chondrocyte phenotype characteristic of OA severely limit the capacity for in vivo tissue regeneration. Cell- and tissue-based repair has the potential to revolutionize treatment of OA, but those approaches have exhibited limited clinical success to date. In this study, we test the hypothesis that bovine and human chondrocytes in a collagen type I scaffold will form hyaline cartilage ex vivo with immunohistochemical, biochemical, and magnetic resonance (MR) endpoints similar to the original native cartilage. Chondrocytes were isolated from 1- to 3-week-old calf knee cartilage or from cartilage obtained from human total knee arthroplasties, suspended in 2.7 mg/mL collagen I, and plated as 300 μL spot cultures with 5 × 106 each. Medium formulations were varied, including the amount of serum, the presence or absence of ascorbate, and treatments with cytokines. Bovine chondrocytes generated metachromatic territorial and interstitial matrix and accumulated type II collagen over time. Type VI collagen was confined primarily to the pericellular region. The ex vivo–formed bovine cartilage contained more chondroitin sulfate per dry weight than native cartilage. Human chondrocytes remained viable and generated metachromatic territorial matrix, but were unable to support interstitial matrix accumulation. MR analysis of ex vivo–formed bovine cartilage revealed evidence of progressively maturing matrix, but MR-derived indices of tissue quality did not reach those of native cartilage. We conclude that the collagen-spot culture model supports formation and maturation of three-dimensional hyaline cartilage from active bovine chondrocytes. Future studies will focus on determining the capacity of human chondrocytes to show comparable tissue formation.

Link to Article

http://dx.doi.org/10.1089/ten.tea.2009.0717