Combined exposure to big endothelin-1 and mechanical loading in bovine sternal cores promotes osteogenesis

Authors

Luisa A. Meyer, Michael G. Johnson, Diane M. Cullen, Juan F. Vivanco, Robert D. Blank, Heidi-Lynn Ploeg, Everett L. Smith

Abstract

Increased bone formation resulting from mechanical loading is well documented; however, the interactions of the mechanotransduction pathways are less well understood. Endothelin-1, a ubiquitous autocrine/paracrine signaling molecule promotes osteogenesis in metastatic disease. In the present study, it was hypothesized that exposure to big endothelin-1 (big ET1) and/or mechanical loading would promote osteogenesis in ex vivo trabecular bone cores. In a 2 × 2 factorial trial of daily mechanical loading (−2000 με, 120 cycles daily, “jump” waveform) and big ET1 (25 ng/mL), 48 bovine sternal trabecular bone cores were maintained in bioreactor chambers for 23 days. The bone cores' response to the treatment stimuli was assessed with percent change in core apparent elastic modulus (ΔEapp), static and dynamic histomorphometry, and prostaglandin E2 (PGE2) secretion. Two-way ANOVA with a post hoc Fisher's LSD test found no significant treatment effects on ΔEapp (p = 0.25 and 0.51 for load and big ET1, respectively). The ΔEapp in the “no load + big ET1” (CE, 13 ± 12.2%, p = 0.56), “load + no big ET1” (LC, 17 ± 3.9%, p = 0.14) and “load + big ET1” (LE, 19 ± 4.2%, p = 0.13) treatment groups were not statistically different than the control group (CC, 3.3% ± 8.6%). Mineralizing surface (MS/BS), mineral apposition (MAR) and bone formation rates (BFR/BS) were significantly greater in LE than CC (p = 0.037, 0.0040 and 0.019, respectively). While the histological bone formation markers in LC trended to be greater than CC (p = 0.055, 0.11 and 0.074, respectively) there was no difference between CE and CC (p = 0.61, 0.50 and 0.72, respectively). Cores in LE and LC had more than 50% greater MS/BS (p = 0.037, p = 0.055 respectively) and MAR (p = 0.0040, p = 0.11 respectively) than CC. The BFR/BS was more than two times greater in LE (p = 0.019) and LC (p = 0.074) than CC. The PGE2 levels were elevated at 8 days post-osteotomy in all groups and the treatment groups remained elevated compared to the CC group on days 15, 19 and 23. The data suggest that combined exposure to big ET1 and mechanical loading results in increased osteogenesis as measured in biomechanical, histomorphometric and biochemical responses.

Link to Article

http://dx.doi.org/10.1016/j.bone.2016.02.001

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

Authors

Haifeng Zhang, Xiyuan Mao, Zijing Du, Wenbo Jiang, Xiuguo Han, Danyang Zhao, Dong Han & Qingfeng Li

Abstract

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing.3D printed PLA/HA,

β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1).Moreover, the biocompatibility,

bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at 4 and 8weeks after surgery, respectively. The results showed that each of the studied scaffolds own its specific merits and drawbacks. 3D printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSCs cell proliferation and differentiation to osteogenic cells. And the outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have the potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery

Link to Article

http://www.tandfonline.com/doi/pdf/10.1080/14686996.2016.1145532

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats

Authors

Subhashis Pal, Kainat Khan, Shyamsundar Pal China, Monika Mittal, Konica porwal, Richa Shrivastava, Isha Taneja, Zakir Hossain, Dhanaraju Mandalapu, Jiaur R. Gayen, Muhammad Wahajuddin, Vishnu Lal Sharma, Arun K. Trivedi, Sabyasachi Sanyal, Smrati Bhadauria, Madan M. Godbole, Sushil K. Gupta, Naibedya Chattopadhyay

Abstract

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.

Link to Article

http://dx.doi.org/10.1016/j.taap.2016.02.002

Rictor is required for optimal bone accrual in response to anti-sclerostin therapy in the mouse

Authors

Weiwei Sun, Yu Shi, Wen-Chih Lee, Seung-Yon Lee, Fanxin Long

Abstract

Wnt signaling has emerged as a major target pathway for the development of novel bone anabolic therapies. Neutralizing antibodies against the secreted Wnt antagonist sclerostin (Scl-Ab) increase bone mass in both animal models and humans. Because we have previously shown that Rictor-dependent mTORC2 activity contributes to Wnt signaling, we test here whether Rictor is required for Scl-Ab to promote bone anabolism. Mice with Rictor deleted in the early embryonic limb mesenchyme (Prx1-Cre;Rictorf/f, hereafter RiCKO) were subjected to Scl-Ab treatment for 5 weeks starting at 4 months of age. In vivo micro–computed tomography (μCT) analyses before the treatment showed that the RiCKO mice displayed normal trabecular, but less cortical bone mass than the littermate controls. After 5 weeks of treatment, Scl-Ab dose-dependently increased trabecular and cortical bone mass in both control and RiCKO mice, but the increase was significantly blunted in the latter. Dynamic histomorphometry revealed that the RiCKO mice formed less bone than the control in response to Scl-Ab. In addition, the RiCKO mice possessed fewer osteoclasts than normal under the basal condition and exhibited lesser suppression in osteoclast number by Scl-Ab. Consistent with the fewer osteoclasts in vivo, bone marrow stromal cells (BMSC) from the RiCKO mice expressed less Rankl but normal levels of Opg or M-CSF, and were less effective than the control cells in supporting osteoclastogenesis in vitro. The reliance of Rankl on Rictor appeared to be independent of Wnt-β-catenin or Wnt-mTORC2 signaling as Wnt3a had no effect on Rankl expression by BMSC from either control or RICKO mice. Overall, Rictor in the limb mesenchymal lineage is required for the normal response to the anti-sclerostin therapy in both bone formation and resorption.

Link to Article

http://dx.doi.org/10.1016/j.bone.2016.01.013

Inhibition of Transforming Growth Factor β Activation Diminishes Tumor Progression and Osteolytic Bone Disease in Mouse Models of Multiple Myeloma

Authors

Ailing Lu, Manuel Antonio Pallero, Weiqi Lei, Huixian Hong, Yang Yang, Mark J. Suto, Joanne E. Murphy-Ullrich

Abstract

Transforming growth factor (TGF)-β supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-β to its biologically active form is a major regulatory node in controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-β. TSP1 is increased in myeloma; TSP1–TGF-β activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-β activity in myeloma and antagonizing TSP1–TGF-β axis may inhibit myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-β that block TSP1–TGF-β activation were used to determine the role of the TSP1–TGF-β pathway in three mouse models of myeloma. TSP1 bound to human myeloma cells and activated latent TGF-β produced by human and mouse myeloma cell lines. Treatment with antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced tumor burden, mouse interleukin-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. Antagonists reduced TGF-β signaling (phospho-Smad2) in bone sections and reduced tumor burden in the immune competent 5TGM1 model of mouse myeloma. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-β activation as a therapeutic strategy for targeted inhibition of TGF-β in myeloma.

Link to Article

http://dx.doi.org/10.1016/j.ajpath.2015.11.003

Growth and repair factors, osteoactivin, matrix metalloproteinase and heat shock protein 72, increase with resolution of inflammation in musculotendinous tissues in a rat model of repetitive grasping

Authors

Nagat Frara, Samir M. Abdelmagid, Michael Tytell, Mamta Amin, Steven N. Popoff, Fayez F. Safadi and Mary F. Barbe

Abstract

Expression of the growth factor osteoactivin (OA) increases during tissue degeneration and regeneration, fracture repair and after denervation-induced disuse atrophy, concomitant with increased matrix metalloproteinases (MMPs). However, OA’s expression with repetitive overuse injuries is unknown. The aim of this study was to evaluate: 1) OA expression in an operant rat model of repetitive overuse; 2) expression of MMPs; 3) inflammatory cytokines indicative of injury or inflammation; and 4) the inducible form of heat shock protein 70 (HSPA1A/HSP72) as the latter is known to increase during metabolic stress and to be involved in cellular repair. Young adult female rats performed a high repetition negligible force (HRNF) food retrieval task for up to 6 weeks and were compared to control rats.

Flexor digitorum muscles and tendons were collected from 22 young adult female rats performing a HRNF reaching task for 3 to 6 weeks, and 12 food restricted control (FRC) rats. OA mRNA levels were assessed by quantitative polymerase chain reaction (qPCR). OA, MMP-1, -2, -3, and -13 and HSP72 protein expression was assayed using Western blotting. Immunohistochemistry and image analysis was used to evaluate OA and HSP72 expression. ELISA was performed for HSP72 and inflammatory cytokines.

Flexor digitorum muscles and tendons from 6-week HRNF rats showed increased OA mRNA and protein expression compared to FRC rats. MMP-1, -2 and -3 progressively increased in muscles whereas MMP-1 and -3 increased in tendons with HRNF task performance. HSP72 increased in 6-week HRNF muscles and tendons, compared to controls, and co-localized with OA in the myofiber sarcolemma. IL-1alpha and beta increased transiently in tendons or muscles in HRNF week 3 before resolving in week 6.

The simultaneous increases of OA with factors involved in tissue repair (MMPs and HSP72) supports a role of OA in tissue regeneration after repetitive overuse.

Link to Article

http://dx.doi.org/10.1186/s12891-016-0892-3