Dose response of bone-targeted enzyme replacement for murine hypophosphatasia

Authors

Manisha C. Yadav, Isabelle Lemire, Pierre Leonard, Guy Boileau, Laurent Blond, Martin Beliveau, Esther Cory, Robert L. Sah, Michael P. Whyte, Philippe Crine and José Luis Millán

Abstract

Hypophosphatasia (HPP) features rickets or osteomalacia from tissue-nonspecific alkaline phosphatase (TNSALP) deficiency due to deactivating mutations within the ALPL gene. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP (sALP-FcD10, renamed ENB-0040) prevents manifestations of HPP when initiated at birth in TNSALP knockout (Akp2−/−) mice. Here, we evaluated the dose–response relationship of ENB-0040 to various phenotypic traits of Akp2−/− mice receiving daily subcutaneous (SC) injections of ENB-0040 from birth at 0.5, 2.0, or 8.2 mg/kg for 43 days. Radiographs, μCT, and histomorphometric analyses documented better bone mineralization with increasing doses of ENB-0040. We found a clear, positive correlation between ENB-0040 dose and prevention of mineralization defects of the feet, rib cage, lower limbs, and jaw bones. According to a dose–response model, the ED80 (the dose that prevents bone defects in 80% of mice) was 3.2, 2.8 and 2.9 mg/kg/day for these sites, respectively. Long bones seemed to respond to lower daily doses of ENB-0040. There was also a positive relationship between ENB-0040 dose and survival. Median survival, body weight, and bone length all improved with increasing doses of ENB-0040. Urinary PPi concentrations remained elevated in all treatment groups, indicating that while this parameter is a good biochemical marker for diagnosing HPP, it may not be a good follow up marker for evaluating response to treatment when administering bone-targeted TNSALP. These dose–response relationships strongly support the pharmacological efficacy of ENB-0040 for HPP, and provide the experimental basis for the therapeutic range of ENB-0040 chosen for clinical trials. We find a positive correlation between ENB-0040 dose and prevention of mineralization defects in a murine model of hypophosphatasia. We found a positive relationship between ENB-0040 dose and survival. Urinary pyrophosphate is not a good follow up marker for evaluating response when using bone-targeted TNSALP treatment. These studies help set the therapeutic range of ENB-0040 to be chosen for clinical trials.

Link to Article

http://dx.doi.org/10.1016/j.bone.2011.03.770

Vitamin D deficiency promotes growth of MCF-7 human breast cancer in a rodent model of osteosclerotic bone metastasis

Authors

Li Laine Ooi, Yu Zheng, Hong Zhou, Trupti Trivedi, Arthur D. Conigraveb, Markus J. Seibel, Colin R. Dunstan

Abstract

Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice. The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels. In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)2D3 inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49±0.08mm2 versus 1.68±0.15mm2, P<0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81±0.13mm2 versus 0.52±0.11mm2 in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth. Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.

Link to Article

http://dx.doi.org/10.1016/j.bone.2010.07.012

Quantitative Histomorphometric Assessment of Regenerate Cellularity and Bone Quality in Mandibular Distraction Osteogenesis After Radiation Therapy

Authors

Alero F. Inyang, Daniel Schwarz, Ameen M. Jamali, Steven R. Buchman

Abstract

Background: The use of mandibular distraction osteogenesis (MDO) for tissue replacement after oncologic resection in head and neck cancer could have immense therapeutic ramifications. We have previously demonstrated significantly decreased mechanical and microdensitomeric metrics of our MDO regenerate after 36-Gy radiation. Quantitative histomorphometry, a third metric, would permit objective investigation of the effects of radiation on tissue and cellular composition. Our hypothesis is that radiation-induced cellular depletion and diminution in function impair optimal bone regeneration. Five rats received radiation to the left mandible; 5 received none. All animals underwent surgical placement of external fixators, creation of mandibular osteotomies, distraction to a 5.1-mm gap width, and consolidation. Point counting and color thresholding were performed. There was a significant increase in empty lacunae and a corresponding diminution in osteocytes after radiation. Whereas the volume fraction of mineralized, mature bone was not different, that of nonmineralized, immature osteoid was significantly increased in the radiated group compared with that in the nonradiated group. Our findings confirm our prior 2 metrics. Actually, all 3 diverse metrics-microdensitometry, biomechanical analysis, and histomorphometry-corroborate our hypothesis of cellular depletion and diminution of function as the potential mechanism of radiation-induced attenuation in the distracted regenerate. Furthermore, our findings of tissue and cellular changes in the irradiated regenerate elucidate the pathophysiology of decreased bone quality when amalgamated with our previous results. Therapeutic agents may now be introduced, and their effects on the irradiated regenerate critically measured, so that MDO may be used as a viable reconstructive option in patients with head and neck cancer.

Link to Article

http://dx.doi.org/10.1097/SCS.0b013e3181ec693f

Osteoblast-specific expression of MEF induces osteopenia through downregulation of osteoblastogenesis and upregulation of osteoclastogenesis

Authors

Keyung-Jo Seul, Hye-Sim Cho, Sun-Hee Heo, Wook-Young Baek, Jung-Eun Kim, Eui Kyun Park, Je-Yong Choi, Hyun-Mo Ryoo, Je-Yoel Cho

Abstract

In bone remodeling, various transcriptional factors are involved, and the deficiency or overexpression of some of these factors results in bone defects. Myeloid elf-1-like factor (MEF) is one of the Ets transcription factors that control the expression of genes that are critical for biologic processes such as cell proliferation, differentiation, and death. Previously, we reported that MEF promotes cell proliferation and functions as a negative regulator of osteogenic differentiation by interacting directly with Runx2 and suppressing its transcriptional activity. To investigate the in vivo function of MEF in bone formation and bone remodeling in vivo, we generated transgenic mice that overexpress MEF in osteoblasts under the control of the 2.3-kb Col1α1 promoter, named Col1α1-MEF. Femoral bone in Col1α1-MEF transgenic mice exhibited low bone mass with fewer trabecular bones and thinner and less developed cortical bones. The mineralized volume fraction (BV/TV) and bone-forming rate (BFR) were remarkably decreased to about 63% and 40%, respectively, in 6-week-old MEF transgenic mice compared with wild-type mice. In addition, reduced bone mineral density was observed in lumbar vertebrae of Col1α1-MEF transgenic mice. The number of TRACP+ osteoclasts was increased in Col1α1-MEF transgenic mice and MEF-overexpressing MC3T3-E1 cells. All these in vivo results suggest that MEF suppresses bone formation by osteoblasts and facilitates bone resorption by activating osteoclasts indirectly.

Link to Article

http://dx.doi.org/10.1002/jbmr.208

Establishment of a streptozotocin-induced diabetic domestic pig model and a systematic evaluation of pathological changes in the hard and soft tissue over a 12-month period

Authors

Cornelius Von Wilmowsky, Philipp Stockmann, Philipp Metzler, Igor Alexander Harsch, Kerstin Amann, Karl Andreas Schlegel

Abstract

The number of diabetic patients in need of medical treatment is growing steadily. Therefore, a diabetic animal model with high degree of similarities with humans, which is suitable for the systematic evaluation of biomaterials and medical devices, is needed. Twenty domestic pigs were used for the study. Fifteen received Streptozotocin (STZ) to induce diabetes mellitus. Internal parameters were measured and bone as well as soft tissues biopsies were taken after 0, 6 and 12 months and evaluated qualitatively and quantitatively by means of scanning electronic microscopy, light microscopy and microradiography. The results of the clinical internal parameters, determined by the American Diabetes Association for the definition of diabetes mellitus could be fulfilled. Pathological changes of the skin vasculatures were already visible after 6 months with a significant wall thickening in the diabetic group. The bone mineralization was lower in the diabetic group after 6 months and with a significant difference after 12 months. From the present results, it can be concluded that a STZ dosage of 90 mg/kg body weight in the domestic pig is suitable for the induction of an apparent diabetes, leading to histolopathological changes in the hard and soft tissues already after 6 months. The high degree of similarities with humans makes it an interesting diabetic animal model for biomaterial research in a compromised animal model. To cite this article: von Wilmowsky C, Stockmann P, Metzler P, Harsch IA, Amann K, Schlegel KA. Establishment of a streptozotocin-induced diabetic domestic pig model and a systematic evaluation of pathological changes in the hard and soft tissue over a 12-month period.

Link to Article

http://dx.doi.org/10.1111/j.1600-0501.2010.01914.x

The effect of combined delivery of recombinant human bone morphogenetic protein-2...

Title

The effect of combined delivery of recombinant human bone morphogenetic protein-2 and recombinant human vascular endothelial growth factor 165 from biomimetic calcium-phosphate-coated implants on osseointegration

Authors

Mustafa Ramazanoglu, Rainer Lutz, Celaletdin Ergun, Cornelius von Wilmowsky, Emeka Nkenke, Karl Andreas Schlegel

Abstract

The delivery of growth factors for enhanced osseointegration depends on the effectiveness of the carrier systems at the bone–implant interface. This study evaluated the effect of solo and dual delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF165) from biomimetically octacalcium phosphate-coated implants on osseointegration. Biomimetic implants, bearing either a single growth factor (BMP or VEGF) or their combination (BMP+VEGF), were established, and compared with acid-etched (AE, control) and biomimetic implants without growth factor (CAP). Implants were placed into frontal skulls of nine domestic pigs. The quality of osseointegration was evaluated using microradiographic and histomorphometric analysis of bone formation inside four defined bone chambers of the experimental implant at 1, 2 and 4 weeks. Biomimetic implants, either with or without growth factor, showed enhanced bone volume density (BVD) values after 2 and 4 weeks. This enhancement was significant for the BMP and BMP+VEGF group compared with the control AE group after 2 weeks (P<0.05). All biomimetic calcium-phosphate (Ca-P) coatings exhibited significantly enhanced bone–implant contact (BIC) rates compared with the uncoated control surface after 2 weeks (P<0.05). However, the combined delivery of BMP-2 and VEGF did not significantly enhance BIC at the final observation period. It was concluded that the combined delivery of BMP-2 and VEGF enhances BVD around implants, but not BIC. Therefore, it may be assumed that changes in the surface characteristics should be considered when designing growth factor-delivering surfaces.

Link to Article

http://dx.doi.org/10.1111/j.1600-0501.2010.02133.x