Biomineralization processes in vertebrates

Abstracts Abstract: Hypophosphatasia (HPP) is characterised by defective bone and tooth mineralisation due to deficiency of tissue non-specific alkaline phosphatase (TNSALP). It is due to loss-of-function mutations in the gene encoding TNSALP (TNSALP), heritable as autosomal dominant (AD) or autosomal recessive (AR) traits. The wide range of mutations described in TNSALP explains much of the clinical heterogeneity seen in the condition. TNSALP cleaves the extracellular substrates inorganic pyrophosphate (PPi), pyridoxal 5′phosphate (PLP) and phosphoethanolamine (PEA). Hydrolysis of PPi promotes osteoblastic mineralisation and cleavage of PLP can be crucial in maintaining central-nervous-system-localised vitamin B6 levels. HPP is highly variable in its clinical expression. In more severe cases, HPP can result in rachitic deformities, respiratory insufficiency, nephrocalcinosis and pyridoxine-responsive seizures. Other features of HPP include craniosynostosis, fractures, bone pain and, in later life, osteoarthropathy. The most severe form results in perinatal lethality. The mildest involves only early tooth loss without apparent skeletal problems (odontohypophosphatasia). Other recognised forms are described as infantile (signs aged b 6 months), childhood, adult and benign prenatal (BP-HPP). Nearly all instances of perinatal HPP and many infantile cases prove lethal. However, prenatal findings are not necessarily a good predictor of postnatal outcome. BP-HPP may constitute around 10% of all HPP cases. In almost all cases of BP-HPP the mother is affected and the rise in placental ALP through pregnancy may contribute to the observed improvements in utero. Apart from clinical and radiological examination, diagnosis of HPP is based on laboratory assays showing markedly reduced serum ALP activity for age, increased concentrations of plasma PLP and urinary PEA alongside molecular genetic studies. The incidence of severe hypophosphatasia is estimated to be between 1 in 100,000 and 1 in 300,000. The estimated incidence of AD HPP in a European population is around 1 in 6500. HPP is probably under-diagnosed. Clinicians concerned with bone disease are most commonly alert for increased alkaline phosphatase activity and awareness of the condition is low. HPP should be considered in some cases where an inflammatory process is suspected on the basis of imaging e.g. in the metaphyses of long bones. Enzyme replacement therapy with a bone-targeted, recombinant TNSALP was reported as preventing manifestations of HPP when initiated at birth in TNSALP knockout mice. Median survival, body weight, and bone length all improved with increasing doses. Recently clinical trials have been undertaken using bone-targeted TNSALP in a small number of infants and young children with severe HPP. Significant improvements in rickets and respiratory status were reported after 12 months of therapy. Improvements in functional outcomes have also been observed. There is currently no approved medical therapy for HPP. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: Conflict with named local investigator in clinical study of bonetargeted recombinant alkaline phosphatase sponsored by Enobia Pharma Inc. Author is not in receipt of any funding or support from the study sponsor. No pecuniary interests to declare. doi:10.1016/j.bone.2012.02.039

IS18 Consequences of changes in Fgf23 activity B. Lanske⁎ Developmental Biology, Harvard School of Dental Medicine, Boston, USA Learning Outcome 1: How Fgf23 regulates phosphate homeostasis and vitamin D metabolism, and how changes in Fgf23 activities affect mouse and man. Learning Outcome 2: Bone being an endocrine organ that secretes FGF23 into the circulation to target kidneys and parathyroid glands where Klotho is a required co-factor. Learning Outcome 3: Why Fgf23 ko mice have a bone mineralization defect despite high levels of mineral ions, and whether increased osteopontin levels are responsible? Abstract: Fibroblast Growth Factor 23 (FGF23) is a member of the FGF19 subfamily of fibroblast growth factors and is expressed primarily in osteoblasts, osteocytes, cementoblasts, and odontoblasts. FGF23 has been shown to be a circulating endocrine factor that inhibits renal phosphate re-absorption and 1α,25-dihydroxyvitamin D (1,25(OH)2D3) synthesis, thus playing a key role in balancing mineral ion homeostasis. FGF23 requires a co-factor, Klotho, in order to bind and activate the FGFR1(III)c receptor. Therefore the site of Klotho expression determines FGF23's target tissues. Studies have shown that Fgf23−/− and Klotho−/− mice have similar abnormal mineral ion phenotypes, confirming that these proteins are acting in the same signaling pathway. PTH is another key phosphate-regulating hormone that interacts functionally with FGF23. It has been shown to stimulate FGF23 expression via cAMP signaling and by production of the active vitamin D metabolite, calcitriol, which then stimulates Fgf23 expression in bone cells. FGF23, in turn, down-regulates PTH transcription and secretion and prevents expression of renal 1α(OH)ase in a negative feedback loop. A large number of mouse models have been generated to investigate the interactions among Fgf23, Klotho and PTH required to maintain a balanced mineral ion homeostasis and proper mineralization of the skeleton. Ffg23−/− and Klotho−/− mice were used to determine how FGF23 may or may not affect PTH functions on bone. PTH is administered therapeutically to patients with osteoporosis and it is therefore clinically important to know whether the presence of FGF23 is required for PTH to fulfill its anabolic actions. Another important question is whether FGF23 is involved in

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the catabolic functions of PTH. Moreover, recent studies using Fgf23/PTH and Klotho/ PTH double knockout mice have revealed new and unexpected findings which suggest that Fgf23 and Klotho might function or interact with PTH in an independent manner. It has been shown that the defective bone mineralization in mice lacking either Fgf23 or Klotho is accompanied by an increase in expression of mineralization inhibitors such as osteopontin (Opn), dentin matrix protein 1 (Dmp1) and matrix gla protein (Mgp). PTH appears to play a key role in this regulation. More importantly, it could be demonstrated that the mineralization defect in these mice is independent of their systemic mineral ion homeostasis. Further studies are required to better understand the functional interactions between Fgf23, Klotho and PTH. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

doi:10.1016/j.bone.2012.02.040

IS19 PTH replacement therapy of hypoparathyroidism L. Rejnmark⁎ Dept of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus, Denmark Learning Outcome 1: Complications to and adverse effects of conventional treatment of hypoparathyroidism. Learning Outcome 2: Effects of PTH replacement therapy in hypoparathyroidism on bone metabolism and calcium homeostasis. Learning Outcome 3: Current limitations to use of PTH replacement therapy. Abstract: Hypoparathyroidism (HypoPT) is characterised by hypocalcaemia due to inappropriate low plasma PTH levels. For many years, the only treatment option has been calcium and (active) vitamin D supplements. Despite a normalization of plasma calcium levels, patients on conventional treatment have a number of complaints including fatigue, muscle weakness and paresthesias in fingers and feet with a reduced quality of life (QoL). Bone mass is high with an abnormal low bone turnover. Renal calcium excretion is often very high due to use of high doses of calcium supplements and lack of PTH stimulated renal tubular calcium reabsorption. In recent studies, PTH replacement therapy (PTH-RT) has been investigated as an alternative treatment option. Daily subcutaneous injections with either intact PTH (1–84) or N-terminal PTH (1–34) have been shown to abolish (or reduce) the need for calcium and vitamin D while maintaining plasma calcium within normal levels. Bone turnover is markedly increased. In cortical bone, porosity is increased causing a decreased BMD, whereas a high BMD seems to be sustained in trabecular bone. The duration of the studies performed so far have been three years or less, during which bone turnover has been increased above normal. However, it seems that the increased bone turnover may start to level off after 2.5 years of treatment. Urinary calcium has not been shown, consistently, to be normalized despite a substantial reduction in use of calcium supplements. Most likely, calcium accumulated in bone during the course of HypoPT is released due to the increased bone turnover thereby causing a sustained increased renal calcium excretion for a prolonged time period. It seems plausible, that urinary calcium may normalize during long term treatment, but more studies are needed to document this. In most instances, HypoPT is caused by surgery to the neck region with unintended removal of, or damage to, the parathyroid glands. More rarely, HypoPT is caused by autoantibodies or genetic mutations affecting PTH biosynthesis, secretion, or action. Autosomal dominant hypocalcemia (ADH) caused by an activating mutation in the calcium sensing receptor (CaSR) also causes hypocalcemia with inappropriate low PTH levels. The PTH dose needed to maintain normal plasma calcium levels vary according to the etiology. On averages, patients with ADH need twice the dose of PTH than patients with postsurgical HypoPT. Also, in postsurgical HypoPT the PTH dose needed varies largely between patients. As hypercalcemia may occur following PTH injections, a dosing regimen with two daily injections may be more feasible than a higher dose administered once-a-day. Patients treated with PTH often report a significantly improved wellbeing, but data from large scaled studies on effects of PTH-RT on QoL and symptom relief have not yet been published. Overall, PTH-RT seems to be a promising new therapeutic option in the treatment of HypoPT, if dose is titrated carefully according to the need of the individual patient. However, long term effects on bone, renal calcium excretion, and QoL need further investigations. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

doi:10.1016/j.bone.2012.02.041

IS20 Biomineralization processes in vertebrates S. Weiner⁎, J. Mahamid, L. Addadi Structural Biology, Weizmann Institute, Rehovot, Israel

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Abstracts

Learning Outcome 1: Wide use of transient disordered precursor mineral phases in biomineralization. Learning Outcome 2: Vertebrates also use transient disordered precursor mineral phases. Learning Outcome 3: Need to learn much more about crystallization pathways in biomineralization. Abstract: The field of biomineralization has witnessed a major paradigm shift in the last decade. Many phyla within the animal kingdom are now known to form their mineralized tissues via a highly disordered transient precursor mineral phase. This was first demonstrated in the echinoderms, but has since been found in other phyla including the mollusks and the crustaceans. In the case of the echinoderms it is also known that the first deposited disordered mineral phase occurs in vesicles inside the cells that are responsible for the formation of the mineralized tissue. This paradigm shift raised anew the question of whether vertebrates also use this strategy. It is well known in vitro that crystalline carbonate hydroxyapatite, the mineral phase of mature bone and enamel, forms via disordered so-called amorphous calcium phosphate (ACP), that first crystallizes into octacalcium phosphate (OCP) and finally into carbonate hydroxyapatite. Direct evidence for an OCP precursor phase was presented by Crane et al. [1] who used Raman spectroscopy to study bone formation in the rat cranium. Beniash et al. [2] used X-PEEM to show that ACP is a transient precursor phase in enamel formation. Our studies by Mahamid et al. [3–5] of the fin bone of the zebra fish mineralizing collagen organic matrix framework show that ACP is a precursor phase of the mature carbonate hydroxyapatite. Cryo-SEM studies of zebra fish fin bone formation, as well as forming embryonic mouse bone, document the crystallization pathway from mineral laden vesicles to mineral granules not bound by a vesicle membrane in the forming extracellular matrix and finally crystallization into plate-shaped carbonate hydroxyapatite crystals in the mature mineralized bone. We also show that the cells adjacent to the forming mouse bone contain vesicles with a mineral phase that has a Ca/P ratio of less than one, suggesting that it might be a different mineral form, possibly a calcium polyphosphate phase. We conclude that in the animal kingdom, the use of transient disordered mineral phases includes both invertebrates and vertebrates. Much now remains to be understood regarding the manner in which ions are taken up by the cells, transported to the vesicles, as well as the modes of formation of the precursor phase, its temporary stabilization and how it ultimately destabilizes and crystallizes in intimate association with type I collagen. 1. Crane, N.J., et al. Bone, 2006. 39: p. 431–433. 2. Beniash, E., et al. J. Struct. Biol., 2009. 166: p. 133–143. 3. Mahamid, J., et al. Proc. Natl. Acad. Sci. USA, 2010. 107: p. 6316–6321. 4. Mahamid, J., et al. Proc. Natl. Acad. Sci. (USA), 2008. 105: p. 12748–12753. 5. Mahamid, J., et al. J. Struct. Biol., 2011. 174: p. 527–535. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.042

IS21 Molecular determinants of extracellular matrix mineralization M.D. McKee⁎ McGill University, Montreal, Canada Learning Outcome 1: How, when and where mineralization (calcification) occurs in bones, teeth and pathologically mineralized soft tissues. Learning Outcome 2: Extracellular matrix assembly, organization and structure of mineralized tissues at the ultra-structural level. Learning Outcome 3: The specific molecules and proteins (and the enzymes that process them) that guide normal and pathologic calcification. Abstract: Progress in biomineralization research in recent years has identified, characterized and described functions for key noncollagenous extracellular matrix proteins regulating crystal growth in the skeleton and dentition. As members of the SIBLING protein family (Small Integrin-Binding Ligands and N-linked Glycoproteins), some of these same proteins normally functioning in bones and teeth are expressed in soft tissues undergoing pathologic calcification where they inhibit ectopic crystal growth. In addition to extracellular matrix proteins regulating matrix mineralization, the enzyme tissue-nonspecific alkaline phosphatase – which is highly expressed by cells in bones and teeth – cleaves pyrophosphate; an anionic small-molecule inhibitor of mineralization produced both intracellularly and extracellularly. Transport of pyrophosphate across the cell membrane and into the extracellular matrix compartment, along with an appropriate extracellular calcium concentration, all participated in maintaining the extracellular phosphate/pyrophosphate/calcium balance necessary for skeletal and dental mineralization. Additional regulation of mineralization occurs via matrix vesicles shed from the plasma membrane of mineralized tissue cells, and by SIBLING proteins such as osteopontin (OPN) and matrix extracellular phosphoglycoprotein (MEPE) and their peptides (notably, the ASARM peptide; Acidic Serine- and Aspartate-Rich Motif). Together with the required mineral ion availability necessary for crystal growth, these molecular determinants guide appropriate tissue-specific patterns of skeletal and dental mineralization within their respective extracellular

matrices, and they also function to limit the spread of pathologic calcification seen in soft tissues such as blood vessels and kidneys. Osteopontin, in particular, is a potent calcification inhibitor that accumulates in mineralized tissues where it regulates apatitic crystal growth, and in calcified deposits during vascular calcification and nephro/urolithiasis where it limits mineralization. Additional research is required to establish the exact temporal sequence in which the molecular determinants of pathologic calcification appear relative to mineral crystal growth in different tissues, and to establish their relationship (if any) to the activation of osteogenic differentiation programs. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: Grant/research support from Enobia Pharma, Consultant for Enobia Pharma. doi:10.1016/j.bone.2012.02.043

IS22 Update on the Endocrine Society osteoporosis in men guideline R. Eastell⁎ Department of Human Metabolism, University of Sheffield, Sheffield, UK Learning Outcome 1: Guidelines for the management of osteoporosis in men have been developed that can be used as the basis for the development of local guidelines. Learning Outcome 2: Bone densitometry is central to the initial investigation and to the monitoring of treatment response. Learning Outcome 3: Treatments are effective in reducing fracture risk and are recommended in certain categories of men. Abstract: The Endocrine Society took the lead in developing guidelines for male osteoporosis and these were developed in partnership with ECTS, ASBMR, ESE, and ISCD. They used the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe the strength of recommendations and evidence quality. The Task Force concluded the following: Osteoporosis in men causes significant morbidity and mortality. We recommend testing higher risk men (aged ≥70 and men aged 50–69 who have risk factors [e.g., low body weight, prior fracture as an adult, smoking, etc.]) using central dual-energy X-ray absorptiometry (DXA). Laboratory testing should be done to detect contributing causes. Adequate calcium and vitamin D and weight-bearing exercise should be encouraged; smoking and excessive alcohol should be avoided. Pharmacologic treatment is recommended for men aged ≥50 who have had spine or hip fractures, those with T-scores of − 2.5 or below, and men at high risk of fracture based on low bone mineral density and/or clinical risk factors. Testosterone therapy should be considered for men at borderline high risk of fracture with low serum testosterone levels and an organic cause or symptoms of hypogonadism. Treatment should be monitored with serial DXA testing. The evidence base for men is weaker than for women and so there remain controversial areas. The Task Force recommended the use of a male BMD reference database for men; others have proposed the use of a female database. It recommended the use of testosterone for some indications, and this is not always included in such guidelines. These guidelines should provide a useful base for the development of local guidelines for the management of osteoporosis in men. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: Grant/research support from AstraZeneca, Unilever, Amgen, Warner-Chilcott, Unipath Limited, Novartis, Nestle Foundation, Sanofi Aventis and IDS, Consultant for Amgen, AstraZeneca, GlaxoSmithKline, Medtronics, Nastech, Nestle, Fonterra Brands, Novartis, Ono Pharma, Osteologix, Pfizer, Eli Lilly, Sanofi Aventis, Tethys, Unilever, Unipath, Inverness Medical, Johnson & Johnson, SPD, MSD, IDS, Speaker Bureau with Takeda, Eli Lilly, Amgen, Warner-Chilcott, GlaxoSmithKline Nutrition, and Roche.

doi:10.1016/j.bone.2012.02.044

IS23 IOF/ECTS working group: Framework for the development of guidelines for the management of glucocorticoid-induced osteoporosis J. Compston⁎ Department of Medicine, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK Learning Outcome 1: Risk assessment in glucocorticoid-induced osteoporosis. Learning Outcome 2: Management of glucocorticoid-induced osteoporosis. Learning Outcome 3: Pharmacological intervention for glucocorticoid-induced osteoporosis. Abstract: Osteoporosis is a common complication of glucocorticoid therapy and is associated with substantial morbidity. Although awareness of the condition has grown in recent years, it remains under-diagnosed and under-treated. Glucocorticoid-induced