Formation Of Hydroxyapatite Crystals Research Articles

Uraemia-associated cardiovascular and lung injury

Sir, Dr Yuan recently reported the interesting case of a 23-year-old dialysis patient with extensive calcifications, including lung calcifications [1]. We similarly encountered a young patient who had developed severe lung calcifications, which proved reversible after intensifying the dialysis regimen and optimization of the calcium–phosphate regulation. This patient is a 29-year-old woman with a history of end-stage renal disease (ESRD) due to primary focal segmental glomerulosclerosis. She had recently started haemodialysis three times weekly for 3 h via a central venous catheter. Because of progressive dyspnoea, a chest X-ray was performed which showed extensive nodular opacities in both lungs (Figure 1A). Chest computed tomography revealed ground-glass nodules with a centrilobular distribution and consolidations with a heterogeneous aspect suggestive of calcium deposition (Figure 2). In the clinical context of ESRD, severely deregulated calcium–phosphate homeostasis (serum phosphorus 3.55 mmol/L (11.0 mg/dL); parathyroid hormone 170 pmol/L (1600 pg/mL)) and after exclusion of other plausible explanations, these lesions were considered ESRD-related lung calcifications. Fig. 1. Chest X-rays. Diffuse nodular opacities can be seen in both lungs (A; arrows). One year after intensifying the dialysis regimen and optimization of the calcium–phosphate regulation, a significant reduction of the opacities can be seen (B). Fig. 2. Chest computed tomography shows centrilobular ground-glass nodules (arrows) and consolidations with a heterogeneous aspect (open arrows). We therefore increased the dialysis dose by multiple interventions and improved calcium–phosphate homeostasis. Dialysis duration and frequency were increased to 4 h, four times weekly. Furthermore, an arteriovenous fistula was created and low-flux haemodialysis was changed to haemodiafiltration. In addition, her calcium–phosphate regulation was optimized by medication and compliance counselling. In the next year, her weekly Kt/V increased from 3.45 to 9.3 and her calcium–phosphate regulation improved. The dyspnoea disappeared and the chest X-ray showed a significant reduction in pulmonary calcifications (Figure 1B). Lung calcification is a frequent complication of ESRD, found at postmortem examination in up to 80% of the patients with ESRD [2]. Pulmonary calcifications can be divided into metastatic pulmonary calcifications (MPC), dystrophic calcifications and calcific uraemic arteriolopathy (CUA). Dystrophic calcification refers to hydroxyapatite crystal formation in previously damaged tissue and is not specifically associated with ESRD. CUA, also known as calciphylaxis, involves hydroxyapatite crystal formation in small vessels which results in tissue ischaemia and necrosis in reaction to tissue damage or inflammation. In contrast, MPC manifests without ischaemic or necrotic changes, consists of amorphous whitlockite instead of hydroxyapatite, and is not related to an inciting event [2–4]. Both CUA and MPC are well-known complications of ESRD. Although the chest computed tomography pictures suspected of MPC, we could not rule out the co-existence of CUA. Therapy for both CUA and MPC includes optimization of the calcium–phosphate regulation and increasing dialysis doses [4]. Although the reports on reversible lung calcifications are limited, our case demonstrates the potential reversibility of pulmonary calcifications by improving dialysis and drug treatment.

The mineralization inducing peptide derived from dentin sialophosphoprotein for bone regeneration

Dentin sialophosphoprotein (DSPP) has been shown to play a primary role in the formation and growth of hydroxyapatite crystals in an extracellular matrix of hard tissue such as bone and teeth. We hypothesized that the mineralization ability of DSPP might depend on a specific domain within it. Three peptides, which have hydroxyapatite (HA) binding affinity, denoted as mineralization inducing peptide (MIP1, MIP2, and MIP3) were identified from DSPP. The both of MIP2 and MIP3 had HA nucleation activity demonstrated by XRD. Among three MIPs, MIP3 significantly supported the human bone marrow stromal cell differentiation into osteoblastic cells. An immunoblot with antibodies specific for the phosphorylated forms of ERK was conducted with cells treated by MIP3. MIP3 transduced intracellular signals via the ERK pathways and was able to induce osteoblastic differentiation, as seen by high expression of ALP, type 1 collagen, OC, OPN, and Runx2 in accordance with applied MIP3 concentration. The Asp, Glu, and Ser residues in MIP3 play important roles for the affinity of calcium in HA bone mineral. Further animal experiment with MIP3 in combination with hydroxyapatite mineral induced marked new bone formation for 4 weeks at rabbit calvarial defect model. The new bone area was much higher in test group, implying that the peptide modified group had excellent biocompatibility when compared with the unmodified group. Taken together, the MIP from DSPP has potential to enhance mineralization followed by to enhance osteoblastic differentiation and bone regeneration.

Correction: Altered Bone Development and an Increase in FGF-23 Expression in Enpp1−/−Mice

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1 mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1 mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1 mice (P,0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1 tibiae and femurs from 22-week-old mice (P,0.05). Circulating phosphate and calcium levels were reduced (P,0.05) in the Enpp1 null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P,0.05) in Enpp1 mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1 mice at 22 weeks of age (P,0.05). Fgf23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1 mice compared to controls. These results indicate that Enpp1 mice are characterized by severe disruption to the architecture and mineralization of longbones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization. Citation: Mackenzie NCW, Zhu D, Milne EM, van ’t Hof R, Martin A, et al. (2012) Altered Bone Development and an Increase in FGF-23 Expression in Enpp1 Mice. PLoS ONE 7(2): e32177. doi:10.1371/journal.pone.0032177 Editor: Marlon R. Schneider, University of Munich, Germany Received October 11, 2011; Accepted January 22, 2012; Published February 16, 2012 Copyright: 2012 Mackenzie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an Institute Strategic Programme Grant and Institute Career Path Fellowship funding from the Biotechnology and Biological Sciences Research Council (BBSRC; www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vicky.macrae@roslin.ed.ac.uk

Monitoring ceramic hydroxyapatite media degradation using dynamic image analysis and uniaxial confined bulk compression

Ceramic hydroxyapatite (CHT) is a multimodal chromatographic medium widely used in the pharmaceutical industry for the purification of biomolecules. CHT is a sintered form of hydroxyapatite crystals with moderate stability at acidic conditions. This moderate stability may lead to underperformance of CHT packed bed lifetime, especially under acidic conditions, which should be monitored by diagnostic tools to design optimal buffer systems for the step. This study presents the application of dynamic image analysis (DIA) and uniaxial confined bulk compression (UCBC) to monitor CHT particle degradation as a function of buffer composition. DIA was used to evaluate changes in solidity and morphology, while UCBC was used to evaluate changes in resistance to uniaxial compression. All properties were studied as a function of bed position and operational parameters. Results show that when CHT is exposed to acidic pH, adding phosphate and/or calcium at concentrations of 1 mM minimizes changes in particle solidity and mechanical strength. Changes in CHT morphological properties (i.e., convexity, aspect ratio) are also affected by the presence of calcium and/or phosphate in the inlet buffers. Furthermore, calcium and phosphate have a positive effect on the mechanical behavior of CHT, which is related to changes in the CHT particle solidity.

Proteomic Identification of a Novel Hsp90-Containing Protein–Mineral Complex Which Can Be Induced in Cells in Response to Massive Calcium Influx

Fetuin-A is known for limiting the expansion and formation of hydroxyapatite crystals from calcium phosphate aggregates in circulation by forming a soluble fetuin-mineral complex. This study was aimed to uncover potential proteins involved in the regulation of calcium phosphate precipitation within cells. We found that a novel protein-mineral complex (PMC) can be generated after introduction of calcium chloride and sodium phosphate into the porcine brain protein extract prepared in Tris-HCl buffer. Selectively enriched proteins in the pellet were confirmed by immunoblotting, including heat shock protein 90 (Hsp90), annexin A5, calreticulin, nucleolin, and other proteins. In addition, purified native Hsp90 directly bound both amorphous calcium phosphate and hydroxyapatite and underwent conformational changes and oligomerization in the presence of excess calcium and phosphate. The morphology of the PMC prepared from Hsp90, calcium, and phosphate was distinctly different from that of hydroxyapatite under transmission electron microscope observation. When cultured SiHa cells were treated with a calcium ionophore or damaged by scratch to induce the massive calcium influx, a complex was formed and observed at discrete sites near the plasma membrane as revealed by antibodies against Hsp90, annexin A5, calreticulin, nucleolin, and other proteins. This complex could also be probed in situ with fetuin-A suggesting the existence of calcium phosphate aggregates in this complex. Inhibition of the complex formation by bisphosphonates hindered cell recovery from A23187 assault. Our results show that following membrane damage amorphous calcium phosphate develops at sites near membrane rupture where saturated calcium phosphate concentration is achieved. As a result, Hsp90 and other proteins are recruited, and the cytosolic PMC is formed. Inhibition of the cytosolic PMC formation may in part contribute to the cellular toxicity and in vivo side effects of bisphosphonates, particularly in cells prone to membrane damage under physiological conditions such as gastrointestinal epithelial and oral cavity epithelial cells.

Enzyme replacement therapy (ENB-0040) in hypophosphatasia improves functional outcome and decreases TNSALP substrates in adolescents and adults

Hypophosphatasia (HPP), a heritable metabolic bone disease, results from inactivating mutation(s) in the gene for the tissue non-specific isoenzyme of alkaline phosphatase (TNSALP). The natural substrates that accumulate in HPP include inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5′ phosphate (PLP), the principal circulating form of vitamin B6. PPi excess blocks hydroxyapatite crystal formation causing rickets in growing children and osteomalacia in adults. There is no approved therapy. ENB-0040, a bone-targeted, recombinant, human TNSALP given subcutaneously has improved severely affected infants and children with HPP. Objective: Evaluate TNSALP substrate levels following 24 weeks of ENB-0040 treatment (Rx) and six-minute walk test (6MWT) function in adolescents and adults with HPP. The 6MWT is a performance-based measure of functional exercise capacity. Methods: 6 adolescents and 13 adults with HPP (mean age 42 yrs [14–68]) were randomized in an open-label, multicenter, multinational, dose-ranging, concurrent control study of the safety, efficacy, and pharmacokinetics of ENB-0040. Patients received 0, 2.1, or 3.5 mg/kg/wk of ENB-0040. Results: At baseline (BL), patients averaged 349 m during the 6MWT (6–620 m), but 10 required assistive devices. The differences walked for the Rx vs no Rx groups were +26 vs −14 m at week 24. Among those with functional impairment (n=12) whose BL 6MWT was 25%N75% of normal, 9 improved (8/9 Rx, and 1/3 no Rx). The mean improvements for the 2.1 and 3.5 mg/kg/wk groups were +35.4 m (n=5) and +43.5 m (n=4), respectively. Injection site reactions occurred in 7 patients, but did not cause discontinuation of treatment. Six serious adverse events (SAEs) were reported among 3 patients, two observation patients had 4 of the SAEs. No SAEs were from ENB-0040. Serum PLP levels decreased in both groups, from 324 and 602 ng/mL at BL to 69 ng/mL and 38 ng/L at week 24, respectively (p=0.009). Conclusion: ENB-0040 significantly decreases TNSALP substrates and improves the 6MWT in adolescents and adults with HPP. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: M. Whyte grant/research support from Enobia, consulting fees from Enobia, C. Greenberg grant/research support from Enobia, Advisory Board Membership of Enobia, consulting fees from Enobia, P. Kishnani grant/research support from Enobia, Advisory Board Membership of Enobia, consulting fees from Enobia, K. Madson grant/research support from Enobia, A. Mhanni grant/research support from Enobia, T. Weber: none declared, K. Mack grant/research support from Enobia, A. Reeves grant/research support from Enobia, H. Plotkin employee of Enobia, N. Kreher employee of Enobia, H. Landy employee of Enobia. doi:10.1016/j.bone.2012.02.101 OC31 (recipient of a 2012 ECTS Travel Award) Detection of autoantibodies to osteoprotegerin in patients with severe idiopathic osteoporosis P. Riches⁎, T. Gilchrist, W.D. Fraser, S.H. Ralston Rheumatology Department, Institute of Genetics and Molecular Medicine, Edinburgh, UK Faculty of Medicine and Health Sciences, University of East Anglia,

Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice.

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1−/− mice compared to controls. These results indicate that Enpp1−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.

Genetics and Mechanisms of Crystal Deposition in Calcium Pyrophosphate Deposition Disease

Calcium pyrophosphate deposition (CPPD) disease (common in older adults) can be asymptomatic, associated with osteoarthritis, or can present as acute/chronic inflammatory arthritis. Due to the phenotypic complexity of CPPD, the European League Against Rheumatism (EULAR) recently made recommendations on terminology, diagnosis, and management based on available research evidence and expert consensus. There are no disease-modifying treatments for CPPD disease, and therapy remains nonspecific with the use of anti-inflammatory and analgesic drugs. For years, it has been known that inorganic phosphate and pyrophosphate regulate the formation of CPP or hydroxyapatite crystals. The discovery of ANKH (human homologue of progressive ankylosis) mutations in familial CPPD disease confirmed the importance of phosphate/pyrophosphate homeostasis in CPPD, with ANKH being a regulator of inorganic pyrophosphate transport. Despite progress in our understanding of the function of ANKH, much remains to be investigated. This review summarizes the genetic basis of this disease and focuses on the challenges of research in this area.

Preparation of a porous scaffold based on polypropylene grafted with monomethylitaconate as potential bone graft

Based on polypropylene (PP) grafted with different percentages of maleic anhydride (PP-g-MA) or monomethylitaconate (PP-g-MMI) a new porous scaffold was prepared with porosities in the range of 50–200 μm. The swelling capacity was analyzed, and morphological, mechanical, and elemental analyses of these scaffolds were carried out. In vitro swelling in the simulated body fluid (SBF), chitosan (CHI) of low (70 kDa) and high (350 kDa) molecular weight, and chitosan-hydroxyapatite solutions were assayed at 37 °C from 24 h to 4 weeks. The swelling degree (SD) of these scaffolds was in the range of 25%–125%. The highest SD value was found in the low-molecular weight (LMW) chitosan solution. The PP-g-MMI and PP-g-MA with 0.7% and 1% of grafting, respectively, showed the highest SD values in the CHI solution. The in vitro treatment of the scaffold was performed by immersion in LMW chitosan and/or the double ionic diffusion (DID) method. The pore structure of the scaffolds was unaltered after these treatments, as revealed by scanning electron microscopy (SEM). Mechanical properties, that is, fracture resistance and deformation of the porous scaffolds depended on the percentage of grafting. Scaffolds with a smaller pore size showed higher mechanical properties. Energy Dispersive Spectroscopy (EDS) measurements of PP-g-MMI with 0.7% of grafting after in vitro treatment revealed the formation of hydroxyapatite (HA) crystals with different morphologies on the porous scaffold. It was concluded that the porous scaffold based on PP-g-MMI could be used as a potential prototype bone graft. Open image in new window

Efficacy and safety of 2-year etidronate treatment in a child with generalized arterial calcification of infancy

Generalized arterial calcification of infancy (GACI, MIM#208000) is a rare autosomal recessive disorder characterized by extensive calcifications in the media of large- and medium-sized muscular arteries. Most affected children die in early infancy because of cardiac failure. GACI is linked to mutations in the ENPP1 gene, which encodes for an enzyme that generates inorganic pyrophosphate (PP(i)), a potent inhibitor of hydroxyapatite crystal formation. Treatment with bisphosphonates, which are synthetic PP(i) analogues, has been proposed as a means of reducing arterial calcifications in GACI patients, but no formalized treatment approach exists. We report on the long-term survival of a severe case of GACI linked to a novel homozygous missense mutation c.583T/C in the ENPP1 gene, diagnosed prenatally, and treated with bisphosphonates. Intravenous disodium pamidronate (three infusions at days 8, 15, and 18 of 0.25, 0.50, and 0.50 mg/kg, respectively) was changed to oral disodium etidronate (starting dose of 20 mg/kg daily, 50 mg die) at 3 weeks of age. Although the etidronate dose was maintained at 50 mg daily in our patient (corresponding to a progressive decrease from 20 to 5 mg/kg daily), the progressive resolution of arterial calcifications seen by 3 months of age was maintained until 2 years of age. Throughout the 2-year follow-up, our patient developed mild hypophosphatemia, due to renal phosphate wasting, without clinical, biochemical, or radiological sign of rickets. High-dose bisphosphonate therapy may not be necessary for an extended period of time in children with GACI.

Biomimetic growth of hydroxyapatite on super water-soluble carbon nanotube-protein hybrid nanofibers

We report here on a facile strategy for the creation of super water-soluble carbon nanotube (CNT)-protein hybrid nanofibers. Toluene-soluble CNTs were first synthesized by noncovalent functionalizing CNTs with a bifunctional linker, Z-glycine N-succinimidyl ester (Z-Gly-OSu), and then water-soluble CNT-protein hybrid nanofibers were created by phase-transferring the Z-Gly-OSu-functionalized CNTs from the toluene phase to protein aqueous solution. CNT-fibronectin (CNT-FN) and CNT-hemoglobin (CNT-HGB) hybrid nanofibers were successfully prepared in this work. Raman and XPS spectra confirmed the successful functionalization of CNTs with Z-Gly-OSu. TEM results indicated the formation of CNT-protein hybrid nanofibers. Biomimetic mineralization was conducted on the CNT-FN hybrid nanofibers and nanofiber film to explore the effects of protein and CNTs on the formation of hydroxyapatite (HA) crystals. SEM, Raman spectra, and TEM results indicated that the FN molecules promote the nucleation of crystalline nuclei and the CNT templates control the orientation of nuclei and the crystal growth of HA to form flake-like HA crystals.

Amelogenin-assisted ex vivo remineralization of human enamel: Effects of supersaturation degree and fluoride concentration

The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions, and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by field emission–scanning electron microscopy, attenuated total reflection–Fourier transformed infrared and X-ray diffraction. The concentration of fluoride and the supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP) = 10.2 ± 2.0 with 1.5 ± 0.5 mg l −1 fluoride and 40 ± 10 μg ml −1 amelogenin, pH 6.8 ± 0.4. A phase diagram summarizes the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management.

Lack of nucleotide pyrophosphatase phosphodiesterase1 activity in ENPP1-/- mice results in altered bone development and pathological mineralisation of soft tissue

Event Abstract Back to Event Lack of nucleotide pyrophosphatase phosphodiesterase1 activity in ENPP1-/- mice results in altered bone development and pathological mineralisation of soft tissue NCW Mackenzie1*, D Zhu1, EM Milne1, JL Millan2, C Farquharson1 and VE Macrae1 1 University of Edinburgh, the Roslin Institute and Royal (Dick) School of Veterinary Studies, United Kingdom 2 Sanford-Burnam Medical Research Institute, United States Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required to convert extracellular ATP into inorganic pyrophosphate (PPi), a crucial negative regulator of hydroxyapatite (HA) crystal formation. NPP1, together with bone alkaline phosphatase, is responsible for maintaining the balance between PPi and inorganic phosphate (Pi) and thereby regulating the rate of HA formation in the developing skeleton. In this present study a detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1-/-) was carried out to further the understanding of its role in skeletal and soft tissue mineralisation. Histopathological assessment of Enpp1-/- mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind limb showed evidence of hyper-mineralisation in the talocrural joint and hypo-mineralisation in the femur and tibia. Comprehensive microCT analysis of the tibia and femur examined the effects of Enpp1 ablation on trabecular architecture and bone geometry at 6 and 22 weeks of age in both male and female mice. Trabecular number, trabecular percentage bone volume, structure model index, trabecular and cortical thickness were significantly reduced (P<0.05) in tibiae and femurs from the Enpp1-/- mice compared to wild-type controls. This decreased bone mass was reflected by a significant reduction in maximum bone stiffness in Enpp1-/- tibiae and femurs from 22 week old mice (P<0.05) as determined by three-point bending. Circulating phosphate and calcium plasma levels were also reduced (P<0.05) in the Enpp1-/- null mice. Plasma levels of osteocalcin, a marker of bone formation, were significantly decreased at 6 weeks of age (P<0.05) in Enpp1-/- mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (RatlapsTM ), a marker of bone resorption, and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1-/- mice at 22 weeks of age (P<0.05). These results demonstrate that Enpp1-/- mice are characterised by severe disruption to the architecture and mineralisation of long-bones, dysregulation of calcium/phosphate homeostasis and hypercalcification in soft tissues. In summary, NPP1 is essential for normal bone development and the control of physiological bone mineralisation and pathological mineralisation in soft tissues. Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Topic: Abstracts Citation: Mackenzie N, Zhu D, Milne E, Millan J, Farquharson C and Macrae V (2011). Lack of nucleotide pyrophosphatase phosphodiesterase1 activity in ENPP1-/- mice results in altered bone development and pathological mineralisation of soft tissue. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00041 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Dr. NCW Mackenzie, University of Edinburgh, the Roslin Institute and Royal (Dick) School of Veterinary Studies, Roslin, United Kingdom, neil.mackenzie@roslin.ed.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers NCW Mackenzie D Zhu EM Milne JL Millan C Farquharson VE Macrae Google NCW Mackenzie D Zhu EM Milne JL Millan C Farquharson VE Macrae Google Scholar NCW Mackenzie D Zhu EM Milne JL Millan C Farquharson VE Macrae PubMed NCW Mackenzie D Zhu EM Milne JL Millan C Farquharson VE Macrae Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Modifications of a calcium phosphate cement with biomolecules—Influence on nanostructure, material, and biological properties

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding.

Synthesis of cellulose–nanohydroxyapatite composite in 1-n-butyl-3-methylimidazolium chloride

In this study cellulose–nanohydroxyapatite composite was fabricated for bone tissue engineering applications. In this composite a natural biopolymer was reinforced with bioactive nanohydroxyapatite for replacement or healing of bone. The ionic liquid 1- n -butyl-3-methylimidazolium chloride (BmimCl) was used for dissolution of cellulose. Nanohydroxyapatite (n-HAp) powder was characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM) and FT-infrared (FTIR) spectroscopy. Thermogravimetric analysis (TGA) as well as MTT assay of nanocomposite was conducted to assess the thermal stability and cytotoxicity of the samples. The results of XRD showed the formation of hydroxyapatite crystals and also SEM images indicated that n-HAp powder was nanocrystalline. The composite was thermally less stable than native cellulose. Also native cellulose had higher decomposition rate and mass loss. Results of biological test showed that the samples were biocompatible with no toxicity. Also, SEM observations demonstrated that human osteoblast cells can attach to the surface of the nanocomposite samples and this composite can be used as bone tissue engineering.

Mussel‐Inspired Polydopamine Coating as a Universal Route to Hydroxyapatite Crystallization

AbstractBone tissue is a complex biocomposite material with a variety of organic (e.g., proteins, cells) and inorganic (e.g., hydroxyapatite crystals) components hierarchically organized with nano/microscale precision. Based on the understanding of such hierarchical organization of bone tissue and its unique mechanical properties, efforts are being made to mimic these organic–inorganic hybrid biocomposites. A key factor for the successful designing of complex, hybrid biomaterials is the facilitation and control of adhesion at the interfaces, as many current synthetic biomaterials are inert, lacking interfacial bioactivity. In this regard, researchers have focused on controlling the interface by surface modifications, but the development of a simple, unified way to biofunctionalize diverse organic and inorganic materials remains a critical challenge. Here, a universal biomineralization route, called polydopamine‐assisted hydroxyapatite formation (pHAF), that can be applied to virtually any type and morphology of scaffold materials is demonstrated. Inspired by the adhesion mechanism of mussels, the pHAF method can readily integrate hydroxyapatites on ceramics, noble metals, semiconductors, and synthetic polymers, irrespective of their size and morphology (e.g., porosity and shape). Surface‐anchored catecholamine moieties in polydopamine enriches the interface with calcium ions, facilitating the formation of hydroxyapatite crystals that are aligned to thec‐axes, parallel to the polydopamine layer as observed in natural hydroxyapatites in mineralized tissues. This universal surface biomineralization can be an innovative foundation for future tissue engineering.

Mineralization of breast capsular tissue: changes in factors affecting apatite formation and growth

Experiments were designed to investigate isolatable factors that could either promote or inhibit formation of hydroxyapatite (HA) crystals associated with mineralization of the fibrous capsule surrounding silicone breast implants. Explanted capsular tissue was minced, filtered (0.2μm), inoculated into culture plates containing DMEM/10% FBS and kept at 37°C, 95%O2. Within 14 days, biofilm‐like and suspending (planktonic) aggregates containing HA formed to a greater extent in plates inoculated with sample from calcified (CC) than non‐calcified (NC) capsules (158 vs 4 nephelometric turbidity units, respectively). Confocal microscopy of formalin‐fixed sections of NC showed the major cell types to be macrophages and fibroblasts. These cells were immunopositive for fetuin, a blood protein. In contrast, CC were largely fibrous; fetuin labeling was sparse except for the calcific plaque itself on the surface of the capsule adjacent to the implant. Thus, a factor which affects HA formation, possibly a HA nucleator, or a modifier of nucleator activity, can be isolated from mineralized fibrous breast capsules. Localization of fetuin within NC but not CC cells is consistent with its known role in inhibiting HA crystal growth. Supported by grants from the Fetzer Foundation, NIH HL88988 and Division of Experimental Surgery, Mayo Clinic.

The Mechanism of Mineralization and the Role of Alkaline Phosphatase in Health and Disease

Biomineralization is the process by which hydroxyapatite is deposited in the extracellular matrix. Physiological mineralization occurs in hard tissues, whereas pathological calcification occurs in soft tissues. The first step of mineralization is the formation of hydroxyapatite crystals within matrix vesicles that bud from the surface membrane of hypertrophic chondrocytes, osteoblasts, and odontoblasts. This is followed by propagation of hydroxyapatite into the extracellular matrix and its deposition between collagen fibrils. Extracellular inorganic pyrophosphate, provided by NPP1 and ANKH, inhibits hydroxyapatite formation. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization. Inorganic pyrophosphate, pyridoxal phosphate, and phosphoethanolamine are thought to be the physiologic substrates of TNAP. These accumulate in the event of TNAP deficiency, e.g., in cases of hypophosphatasia. The gene encoding TNAP is mapped to chromosome 1, consists of 12 exons, and possesses regulatory motifs in the 5'-untranslated region. Inhibition of TNAP enzymatic activity suppresses TNAP mRNA expression and mineralization in vitro. Hypophosphatasia is an inherited systemic bone disease characterized by hypomineralization of hard tissues. The phenotype of hypophosphatasia is varied. To date, more than 200 mutations in the TNAP gene have been reported. Knockout mice mimic the phenotypes of severe hypophosphatasia. Among the mutations in the TNAP gene, c.1559delT is frequent in the Japanese population. This frameshift mutation results in the expression of an abnormally long protein that is degraded in cells. DNA-based prenatal diagnosis using chorionic villus sampling has been developed, but requires thorough genetic counseling. Although hypophosphatasia is untreatable at present, the recent success of enzyme replacement therapy offers promise. The problems presented by impaired mineralization in age-related chronic diseases, such as pathologic calcification and decreasing physiological mineralization are growing in importance. Strategies for preventing pathologic calcification using TNAP and NPP1 are in development. A nutrigenomic approach, based on the relationship between TNAP gene polymorphism and bone mineral density, is also discussed.

Effect of Surface-activated PLLA Scaffold on Apatite Formation in Simulated Body Fluid

Surface-activated poly(L-lactic acid) (PLLA) films and scaffolds were investigated for their effect on the formation of hydroxyapatite (HA) in simulated body fluid (SBF). PLLA samples were treated with plasma discharge in oxygen gas; the activated polymer surfaces were subjected to in situ grafting acrylic acid (AA) monomer. The obtained PLLA-PAA was converted to PLLA-PAA-HA in SBF. The formation of HA crystals was identified by surface analyses and the size and distribution by scanning electron miscroscopy. The major elements of HA surface-modified PLLA were confirmed by electron spectroscopy for chemical analysis and attenuated total reflectance-Fourier transform infrared spectra. Fibroblast, chondrocyte, and osteoblast cells were seeded in scaffolds and cultivated in vitro; the total cellularity was higher in the PLLA-PAA-HA scaffolds than the PLLA and PLLA-HA. Histological staining of the cells was denser in the cell-seeded PLLA-PAA-HA constructs. The introduction of specific functionality on the polymer surface significantly improved apatite nucleation and growth. Thus, HA-formed PLLA scaffolds are potentially useful in musculoskeletal tissue engineering.

Preparation and Characterization of Homogeneous Hydroxyapatite/Chitosan Composite Scaffolds via In-Situ Hydration

Hydroxyapatite(HAP)/Chitosan(CS) composite is a biocompatible and bioactive material for tissue engineering. A novel homogeneous HAP/CS composite scaffold was developed via lyophilization and in situ hydration. A model CS solution with a Ca/P atom ratio of 1.67 was prepared through titration and stirring so as to attain a homogeneous dispersion of HAP particles. After lyophilization and in situ hydration, rod-shaped HAP particles (5 μm in diameter) within the CS matrix homogeneously scattered at the pore wall of the CS scaffold. X-ray diffraction (XRD) and Fouri-er-Transformed Infrared spectroscopy (FTIR) confirmed the formation of HAP crystals. The compressive strength in the composite scaffold indicated a significant increment over a CS-only scaffold. Bioactivity in vitro was completed by immersing the scaffold in simulated body fluid (SBF), and the result suggested that there was an increase in apatite formation on the HAP/CS scaffolds. Biological in vivo cell culture with MC 3T3-E1 cells for up to 7 days demonstrated that a homogeneous incorporation of HAP particles into CS scaffold led to higher cell viability compared to that of the pure CS scaffold or the HAP/CS scaffold blended. The results suggest that the homogeneous composite scaffold with better strength, bioactivity and biocompatibility can be prepared via in vitro hydration, which may serve as a good scaffold for bone tissue engineering.