A novel one-pot process for near-net-shape fabrication of open-porous resorbable hydroxyapatite/protein composites and in vivo assessment

Authors

Berit Mueller, Dietmar Koch, Rainer Lutz, Karl A. Schlegel, Laura Treccani, Kurosch Rezwan

Abstract

We present a mild one-pot freeze gelation process for fabricating near-net, complex-shaped hydroxyapatite scaffolds and to directly incorporating active proteins during scaffold processing. In particular, the direct protein incorporation enables a simultaneous adjustment and control of scaffold microstructure, porosity, resorbability and enhancement of initial mechanical and handling stability. Two proteins, serum albumin and lysozyme, are selected and their effect on scaffold stability and microstructure investigated by biaxial strength tests, electron microscopy, and mercury intrusion porosimetry. The resulting hydroxyapatite/protein composites feature adjustable porosities from 50 % to 70 % and a mechanical strength ranging from 2 to 6 MPa comparable to that of human spongiosa without any sintering step. Scaffold degradation behaviour and protein release are assessed by in vitro studies. A preliminary in vivo assessment of scaffold biocompatibility and resorption behaviour in adult domestic pigs is discussed. After implantation, composites were resorbed up to 50 % after only 4 weeks and up to 65 % after 8 weeks. In addition, 14 % new bone formation after 4 weeks and 37 % after 8 weeks were detected. All these investigations demonstrate the outstanding suitability of the one-pot-process to create, in a customisable and reliable way, biocompatible scaffolds with sufficient mechanical strength for handling and surgical insertion, and for potential use as biodegradable bone substitutes and versatile platform for local drug delivery.

Link To Article

http://dx.doi.org/10.1016/j.msec.2014.05.017

Mechanical Load Increases in Bone Formation Via a Sclerostin-Independent Pathway

Authors

A Morse, MM McDonald, NH Kelly, KM Melville, A Schindeler, I Kramer, M Kneissel, MCH van der Meulen, and DG Little

Abstract

Sclerostin, encoded by the Sost gene, is an important negative regulator of bone formation that has been proposed to have a key role in regulating the response to mechanical loading. To investigate the effect of long-term Sclerostin deficiency on mechanotransduction in bone, we performed experiments on unloaded or loaded tibiae of 10 week old female Sost-/- and wild type mice. Unloading was induced via 0.5U BTX injections into the right quadriceps and calf muscles, causing muscle paralysis and limb disuse. On a separate group of mice, increased loading was performed on the left tibiae through unilateral cyclic axial compression of equivalent strains (+1200µe) at 1200 cycles/day, 5days/week. Another cohort of mice receiving equivalent loads (-9.0N) also were assessed. Contralateral tibiae served as normal load controls. Loaded/unloaded and normal load tibiae were assessed at day 14 for bone volume (BV) and formation changes. Loss of BV was seen in the unloaded tibiae of wild type mice, but BV was not different between normal load and unloaded Sost-/- tibiae. An increase in BV was seen in the loaded tibiae of wild type and Sost-/- mice over their normal load controls. The increased BV was associated with significantly increased mid-shaft periosteal MS/BS, MAR and BFR/BS, and endosteal MAR and BFR/BS. Notably, loading induced a greater increase in periosteal MAR and BFR/BS in Sost-/- mice than in wild type controls. Thus, long-term Sclerostin deficiency inhibits the bone loss normally induced with decreased mechanical load, but can augment the increase in bone formation with increased load.

Link To Article

http://dx.doi.org/10.1002/jbmr.2278

Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1-/- Mice

Authors

Manisha C. Yadav PhD., Carmen Huesa PhD., Sonoko Narisawa PhD., Marc F. Hoylaerts PhD., Alain Moreau PhD., Colin Farquharson PhD. and José Luis Millán PhD

Abstract

PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) have non-redundant functions during skeletal mineralization. While TNAP deficiency (Alpl-/- mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PPi), comparably elevated levels of PPi in Phospho1-/- mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PPi in Alpl-/- mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1-/- mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p-OPN) peptides in Phospho1-/- mice, suggesting that accumulation of p-OPN causes the skeletal abnormalities in Phospho1-/- mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1-/- as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1-/- mice at 3 months of age. Mineralization assays comparing [Phospho1-/-; Spp1-/-], Phospho1-/- and Spp1-/- chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1-/-; Spp1-/-] mice. Thus, while Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PPi and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PPi and elevated p-OPN levels causing the respective skeletal phenotypes in Alpl-/- and Phospho1-/- mice.

Link To Article

http://dx.doi.org/10.1002/jbmr.2281

Aging diminishes lamellar and woven bone formation induced by tibial compression in adult C57BL/6

Authors

Nilsson Holguin, Michael D. Brodt, Michelle E. Sanchez, Matthew J. Silva

Abstract

Aging purportedly diminishes the ability of the skeleton to respond to mechanical loading, but recent data show that old age did not impair loading-induced accrual of bone in BALB/c mice. Here, we hypothesized that aging limits the response of the tibia to axial compression over a range of adult ages in the commonly used C57BL/6. We subjected the right tibia of old (22 month), middle-aged (12 month) and young-adult (5 month) female C57BL/6 mice to peak periosteal strains (measured near the mid-diaphysis) of −2200 με and −3000 με (n = 12–15/age/strain) via axial tibial compression (4 Hz, 1200 cycles/day, 5 days/week, 2 weeks). The left tibia served as a non-loaded, contralateral control. In mice of every age, tibial compression that engendered a peak strain of −2200 με did not alter cortical bone volume but loading to a peak strain of −3000 με increased cortical bone volume due in part to woven bone formation. Both loading magnitudes increased total volume, medullary volume and periosteal bone formation parameters (MS/BS, BFR/BS) near the cortical midshaft. Compared to the increase in total volume and bone formation parameters of 5-month mice, increases were less in 12- and 22-month mice by 45–63%. Moreover, woven bone incidence was greatest in 5-month mice. Similarly, tibial loading at −3000 με increased trabecular BV/TV of 5-month mice by 18% (from 0.085 mm3/mm3), but trabecular BV/TV did not change in 12- or 22-month mice, perhaps due to low initial BV/TV (0.032 and 0.038 mm3/mm3, respectively). In conclusion, these data show that while young-adult C57BL/6 mice had greater periosteal bone formation following loading than middle-aged or old mice, aging did not eliminate the ability of the tibia to accrue cortical bone.

Link To Article

http://dx.doi.org/10.1016/j.bone.2014.05.006

RUNX3 Facilitates Growth of Ewing Sarcoma Cells

Authors

Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Xiaoke Wang, Andre J. van Wijnen, Andre M. Oliveira and Jennifer J. Westendorf

Abstract

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Link To Article

http://dx.doi.org/10.1002/jcp.24663

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

Authors

Ingo Grafe, Tao Yang, Stefanie Alexander, Erica P Homan, Caressa Lietman, Ming Ming Jiang, Terry Bertin, Elda Munivez, Yuqing Chen, Brian Dawson, Yoshihiro Ishikawa, Mary Ann Weis, T Kuber Sampath, Catherine Ambrose, David Eyre, Hans Peter Bächinger & Brendan Lee

Abstract

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations1. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms2. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap−/− mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity3, 4. Anti–TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap−/− mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

Link To Article

http://dx.doi.org/10.1038/nm.3544