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Vertical bone formation on the CaP implant surface
Abstracts / Bone 46 (2010) S9–S83
genesis. This findings could explain, at least in part, the pathogenetic mechanisms of senile osteoporosis. doi:10.1016/j.bone.2010.01.126
124. Extracellular nucleotides regulate the expression and activity of ecto-nucleotidases by osteoblasts Isabel Orriss, Timothy Arnett Cell and Developmental BIology, University College London, London, United Kingdom Previous work has shown that extracellular nucleotides (>1 μM ATP/UTP) prevent bone nodule formation in vitro by blocking mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of mineralisation. Both PPi, a potent inhibitor of mineralisation and Pi, can be generated from extracellular nucleotides by the actions of ecto-nucleotidases. There are four families of ecto-nucleotidases: 1) E-NTPdases (ecto-nucleoside triphosphate diphosphohydrolase); 2) the E-NPPs (ecto-nucleotide pyrophosphatase/phosphodiesterase); 3) alkaline phosphatases (ALP), and 4) ecto-5′nucleotidase (E-5′N). E-NTPdases catalyse the reactions: NTP→NDP+Pi and NDP→NMP+Pi, whereas E-NPPs hydrolyse NTP→NMP+PPi or NDP→NMP+Pi. This study used qPCR and biochemical assays to determine the expression and activity of ecto-nucleotidases by primary rat osteoblasts; mRNA expression and enzyme activity were studied at days 4, 7 and 14. We found osteoblasts express mRNA for a number of ecto-nucleotidases. The rank order of mRNA expression in mature, bone forming osteoblasts was E-NTPdase 6>E-NPP1>E-NTPdase4>ALP>E-NTPdase5>ENPP3>E-5′N>E-NTPdase 3>E-NPP2>E-NTPdase1>E-NTPdase 2. We have previously shown that ATP and UTP, signalling via the P2Y2 receptor, inhibit ALP expression and activity. Bone mineralisation and ecto-nucleotidase activity was studied in osteoblasts treated with several ATP-analogues (the P2X1 and P2X3 receptor agonists α,βmeATP and β,γ-meATP and the P2X7 receptor agonist, BzATP). Osteoblast expression of P2X receptor mRNA and protein was demonstrated using qPCR, immunocytochemistry and western blotting. α,β-meATP (1 μM), β,γ-meATP (1 μM) and BzATP (100 μM) inhibited bone mineralisation by 70%, 90% and >90%, respectively. These agonists decreased osteoblast ALP activity by up to 50%, without affecting cell numbers. β,γ-meATP and BzATP increased total E-NPP activity by up to 50% and 100%, respectively. No effects of these agonists on E-NPP1 and ALP expression were observed. E-NPP1 and ALP play a key role in the regulation of PPi levels in bone. These data indicate that increased E-NPP1, as well as decreased ALP activity, leading to PPi accumulation, may contribute to the powerful inhibitory action of extracellular nucleotides on matrix mineralisation in osteoblast cultures. These observations suggest the possibility that extracllular nucleotides could act to regulate tissue mineralisation in vivo. doi:10.1016/j.bone.2010.01.127
125. Vertical bone formation on the CaP implant surface Victor Palarie, Mohamad Zahalka State University of Medicine and Pharmacy “N. Testemitanu”, Orhei, Moldova Aim: to prove the concept that an implant system with Calcium Phosphate (CaP) bioactive osteoconductive surface has the ability to
S55
guide vertical bone growth in a rabbit mandible model. Material and Methods: 10 adult white rabbits each received custom-designed dental implants (Alfa Gate. The implants from right mandible had sandblasted acid-etched surfaces (SLA), covered with CaP - test. The control served SLA implants from left side. The superior part (2 mm) of each implant was left outside the lateral aspect of posterior mandibular bone and covered by periosteum, muscle, subcutaneous tissue, and skin. After 10 weeks animals were sacrificed. Bone formation around implants was measured using micro-CT imaging and histology. RESULTS: At 10 weeks, new supracrestal bone was seen adjacent to CaP implants. The mean supracrestal bone heights achieved for CaP implants was 2.+//0-0.7 mm. Histological results showed that supracrestal bone-to-implant contact for implants with CaP surface was more with 2 mm, than SLA. Conclusions: Successful implant-guided supracrestal osteogenesis has been demonstrated in a rabbit model with use of bioactive CaP osteoconductive implant surfaces. doi:10.1016/j.bone.2010.01.128
126. Identification and characterization of 2 missense mutations in the LRP4 gene causing increased bone mineral density Elke Piters1, Olivier Leupin2, Eveline Boudin1, Fenna De Freitas1, Manuel Bueno3, Feliciano Ramos3, Peter Itin4, Michaela Kneissel2, Wim Van Hul1 1 Department of Molecular Genetics, University Antwerp, Edegem, Belgium 2 Novartis Biologics Center, Novartis Pharma AG, Basel, Switzerland 3 Departmento de Traumatología, Facultad de Medicina, Zaragoza, Spain 4 Department of Dermatology, University Hospital Basel, Basel, Switzerland Recently, some of us reported the identification of the low density lipoprotein-related protein (LRP)-4 gene as an interaction partner for sclerostin, a potent inhibitor of bone formation secreted by osteocytes (Leupin et al. JBMR 24, Suppl 1, 1250 (2009)). Functional studies indicated that LRP4 acts as a facilitator of sclerostin-mediated inhibition of bone formation. We therefore hypothesized that loss of function mutations in LRP4 could result in a sclerosing bone phenotype similarly to the loss of function mutations in the SOST gene encoding sclerostin which cause sclerosteosis. We sequenced all 38 coding exons of LRP4 and their exon-intron boundaries in an extended set of cases diagnosed with different sclerosing bone dysplasias. This resulted in the identification of 2 missense mutations in LRP4, both absent in a large number of control individuals of mixed European origin. The first was a homozygous mutation found in a Greek female patient with sclerosteosis, displaying severely increased BMD, facial asymmetry and both finger and nail dysplasia with syndactyly. In a young Spanish male subject, we found a heterozygous mutation. This patient was also diagnosed with sclerosteosis again showing increased BMD, facial palsy and syndactyly. Both mutated residues are highly conserved in multiple species and occur adjacent to each other at the surface of an extracellular β-propeller domain, which was shown to be required for the sclerostin-facilitator activity. According to both Polyphen and SIFT prediction algorithms, the two substitutions are not tolerable for the functioning of the protein. Furthermore, functional analyses indicated that both mutations impair the interaction with sclerostin and the concomitant sclerostin-facilitator effect. In conclusion, with these genetic studies, we were able to corroborate the previous findings that interaction between sclerostin and LRP4 is required to mediate sclerostin inhibitory function on bone formation, thus indicating an important role for LRP4 in bone. doi:10.1016/j.bone.2010.01.129
genesis. This findings could explain, at least in part, the pathogenetic mechanisms of senile osteoporosis. doi:10.1016/j.bone.2010.01.126
124. Extracellular nucleotides regulate the expression and activity of ecto-nucleotidases by osteoblasts Isabel Orriss, Timothy Arnett Cell and Developmental BIology, University College London, London, United Kingdom Previous work has shown that extracellular nucleotides (>1 μM ATP/UTP) prevent bone nodule formation in vitro by blocking mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of mineralisation. Both PPi, a potent inhibitor of mineralisation and Pi, can be generated from extracellular nucleotides by the actions of ecto-nucleotidases. There are four families of ecto-nucleotidases: 1) E-NTPdases (ecto-nucleoside triphosphate diphosphohydrolase); 2) the E-NPPs (ecto-nucleotide pyrophosphatase/phosphodiesterase); 3) alkaline phosphatases (ALP), and 4) ecto-5′nucleotidase (E-5′N). E-NTPdases catalyse the reactions: NTP→NDP+Pi and NDP→NMP+Pi, whereas E-NPPs hydrolyse NTP→NMP+PPi or NDP→NMP+Pi. This study used qPCR and biochemical assays to determine the expression and activity of ecto-nucleotidases by primary rat osteoblasts; mRNA expression and enzyme activity were studied at days 4, 7 and 14. We found osteoblasts express mRNA for a number of ecto-nucleotidases. The rank order of mRNA expression in mature, bone forming osteoblasts was E-NTPdase 6>E-NPP1>E-NTPdase4>ALP>E-NTPdase5>ENPP3>E-5′N>E-NTPdase 3>E-NPP2>E-NTPdase1>E-NTPdase 2. We have previously shown that ATP and UTP, signalling via the P2Y2 receptor, inhibit ALP expression and activity. Bone mineralisation and ecto-nucleotidase activity was studied in osteoblasts treated with several ATP-analogues (the P2X1 and P2X3 receptor agonists α,βmeATP and β,γ-meATP and the P2X7 receptor agonist, BzATP). Osteoblast expression of P2X receptor mRNA and protein was demonstrated using qPCR, immunocytochemistry and western blotting. α,β-meATP (1 μM), β,γ-meATP (1 μM) and BzATP (100 μM) inhibited bone mineralisation by 70%, 90% and >90%, respectively. These agonists decreased osteoblast ALP activity by up to 50%, without affecting cell numbers. β,γ-meATP and BzATP increased total E-NPP activity by up to 50% and 100%, respectively. No effects of these agonists on E-NPP1 and ALP expression were observed. E-NPP1 and ALP play a key role in the regulation of PPi levels in bone. These data indicate that increased E-NPP1, as well as decreased ALP activity, leading to PPi accumulation, may contribute to the powerful inhibitory action of extracellular nucleotides on matrix mineralisation in osteoblast cultures. These observations suggest the possibility that extracllular nucleotides could act to regulate tissue mineralisation in vivo. doi:10.1016/j.bone.2010.01.127
125. Vertical bone formation on the CaP implant surface Victor Palarie, Mohamad Zahalka State University of Medicine and Pharmacy “N. Testemitanu”, Orhei, Moldova Aim: to prove the concept that an implant system with Calcium Phosphate (CaP) bioactive osteoconductive surface has the ability to
S55
guide vertical bone growth in a rabbit mandible model. Material and Methods: 10 adult white rabbits each received custom-designed dental implants (Alfa Gate. The implants from right mandible had sandblasted acid-etched surfaces (SLA), covered with CaP - test. The control served SLA implants from left side. The superior part (2 mm) of each implant was left outside the lateral aspect of posterior mandibular bone and covered by periosteum, muscle, subcutaneous tissue, and skin. After 10 weeks animals were sacrificed. Bone formation around implants was measured using micro-CT imaging and histology. RESULTS: At 10 weeks, new supracrestal bone was seen adjacent to CaP implants. The mean supracrestal bone heights achieved for CaP implants was 2.+//0-0.7 mm. Histological results showed that supracrestal bone-to-implant contact for implants with CaP surface was more with 2 mm, than SLA. Conclusions: Successful implant-guided supracrestal osteogenesis has been demonstrated in a rabbit model with use of bioactive CaP osteoconductive implant surfaces. doi:10.1016/j.bone.2010.01.128
126. Identification and characterization of 2 missense mutations in the LRP4 gene causing increased bone mineral density Elke Piters1, Olivier Leupin2, Eveline Boudin1, Fenna De Freitas1, Manuel Bueno3, Feliciano Ramos3, Peter Itin4, Michaela Kneissel2, Wim Van Hul1 1 Department of Molecular Genetics, University Antwerp, Edegem, Belgium 2 Novartis Biologics Center, Novartis Pharma AG, Basel, Switzerland 3 Departmento de Traumatología, Facultad de Medicina, Zaragoza, Spain 4 Department of Dermatology, University Hospital Basel, Basel, Switzerland Recently, some of us reported the identification of the low density lipoprotein-related protein (LRP)-4 gene as an interaction partner for sclerostin, a potent inhibitor of bone formation secreted by osteocytes (Leupin et al. JBMR 24, Suppl 1, 1250 (2009)). Functional studies indicated that LRP4 acts as a facilitator of sclerostin-mediated inhibition of bone formation. We therefore hypothesized that loss of function mutations in LRP4 could result in a sclerosing bone phenotype similarly to the loss of function mutations in the SOST gene encoding sclerostin which cause sclerosteosis. We sequenced all 38 coding exons of LRP4 and their exon-intron boundaries in an extended set of cases diagnosed with different sclerosing bone dysplasias. This resulted in the identification of 2 missense mutations in LRP4, both absent in a large number of control individuals of mixed European origin. The first was a homozygous mutation found in a Greek female patient with sclerosteosis, displaying severely increased BMD, facial asymmetry and both finger and nail dysplasia with syndactyly. In a young Spanish male subject, we found a heterozygous mutation. This patient was also diagnosed with sclerosteosis again showing increased BMD, facial palsy and syndactyly. Both mutated residues are highly conserved in multiple species and occur adjacent to each other at the surface of an extracellular β-propeller domain, which was shown to be required for the sclerostin-facilitator activity. According to both Polyphen and SIFT prediction algorithms, the two substitutions are not tolerable for the functioning of the protein. Furthermore, functional analyses indicated that both mutations impair the interaction with sclerostin and the concomitant sclerostin-facilitator effect. In conclusion, with these genetic studies, we were able to corroborate the previous findings that interaction between sclerostin and LRP4 is required to mediate sclerostin inhibitory function on bone formation, thus indicating an important role for LRP4 in bone. doi:10.1016/j.bone.2010.01.129