- Email: [email protected]
Structural evaluation of the bone formed during endochondral ossification of young rats treated with bisphosphonates
S112
Abstracts
densitometric (DEXA) method. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of mandible were estimated. Pearson's correlation coefficient (r) was determined between all the investigated variables and P b 0.05 was considered as statistically significant. Obtained results showed significant positive correlation between body weight and mandible weight, lenght, Bvol, Cd, BMD, BMC, Wy and Wf (P b 0.05). All the investigated parameters of the mandible were found to be positively correlated. However, statistically insignificant correlations of MvBMD and body weight, mandible weight, mandible length and Bvol were stated. Furthermore, Bvol and Cd were not found to be significantly correlated (P N 0.05). In conclusion, this study showed numerous positive correlations between final body weight, densitometric, morphometric and mechanical properties of mandible. This bone of pigs may be used as an attractive model for further investigations on metabolic response of skeleton to physiological, nutritional, toxicological and pharmacological factors influencing bone tissue metabolism. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.342
PP154 Detection of osteoadherin during intramembranous and endochondral ossification by colloidal gold immunocytochemistry and Western blotting D.S. Janonesa,⁎, T.A.G. Donatoa, V. Bradaschia-Corrêaa, F. Bleicherb, V.E. Arana-Chaveza a Biomaterials and Oral Biology, Universaty of São Paulo, São Paulo, Brazil b Laboratoire Odontoblast et régénération dentinaire, University of Lyon, Lyon, France Abstract: Osteoadherin (OSAD) is a keratan sulfate proteoglycan firstly isolated from bovine bone. Although the distribution of OSAD in mineralized tissues had been described suggesting a role in biomineralization, its specific role remains poorly understood. The present study compared the appearance of OSAD during both intramembranous and endochondral ossification and correlated it with early stages of mineralization. Parietal bone of 17, 18 and 21 day-old embryos and mandibular condyle of 30 day-old Wistar rats were removed. Some specimens were frozen in liquid nitrogen while others were fixed in 0.1% glutaraldehyde + 4% formaldehyde under microwave (MW) irradiation. Some specimens were left undecalcified, while others were decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White resin. The expression of OSAD was analyzed by immunocytochemistry and Western blotting. Ultrathin sections were incubated with a polyclonal rabbit antibody against rat OSAD. In both intramembranous and endochondral ossification the gold particles were always uniformly distributed in the cytoplasm of osteoblasts but they only appeared in the mineralizing matrix when the fibrilar stage was taking place, remaining as a component of the mineralized bone matrix. Western blots carried out with the same antibody revealed that 17-day-old embryos contained slightly less OSAD than 18-day-old embryos, while immunoreactivity was weak in 21 day-old embryos. The results suggest that OSAD play a role in collagen fibril mineralization maybe as an organizer of matrix assembly or by retaining the mineral crystals into the matrix, besides exerting binding activities among bone matrix components. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
Twenty male turkeys were randomly divided into 2 weight-matched groups (N = 10 per group) at the age of 28 days of life. The first group consisted of control birds that received placebo orally. The second group (AKG group) received oral administration with AKG (0.4 g/kg b.w./d). The birds were kept under standard rearing conditions and slaughtered at the age of 108 days of life to analyse left tibia in terms of bone morphology (weight (W), length (L), cross-sectional area (A), second moment of inertia (Ix), mean relative wall thickness (MRWT), cortical index (CI)), volumetric bone mineral density (vBMD), as well mechanical properties (maximum elastic strength (Wy) and ultimate strength (Wf)). Using computed tomography technique (Somatom Emotion, Siemens), vBMD of the trabecular and the cortical bone of tibia was measured. Mechanical properties of tibia were estimated using three-point bending test and an INSTRON 3367 apparatus (Instron, USA). Statistical analysis was performed with a use of Student t-test and P b 0.05 was considered as statistically significant. Obtained results showed significantly increased weight, length, Ix, trabecular bone mineral density, cortical bone mineral density and Wy of tibia by 14.4%, 6.2%, 23.9%, 6.1%, 8.7%, and 31.8%, respectively, when compared to the controls (all P b 0.05). All these results indicate that long-term oral administration with AKG in geese improves vBMD of the trabecular and cortical bone compartments as well as morphological and mechanical properties of tibia in growing geese. Thus, the study have shown for the first time that AKG administration may be considered as an effective nutrient improving skeletal system quality in geese. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.344
PP156 Alfacalcidols effect to shoulder fracture consolidation period at the patient with osteoporosis E. Solod⁎, A. Lazarev, S. Rodionova Central Institute of Traumatology and Orthopedics, Moscow, Russian Federation Abstract: Unstable fractures (classified Neer CS, 1970) in patients with systemic osteoporosis are characterized by the highest incidence of longer seam. Objective: Comparative assessment of the impact of alfacalcidols in the treatment of humerus fractures. Materials and methods: The study included 62 patients (mean age 69.7 years) with the proximal humerus fractures and a confirmed diagnosis of osteoporosis (senile and postmenopausal). All patients completed the operation, closed nailing tight V-spoke. The study group (48 patients) was receiving a daily alfacalcidol dose of 0.75–1.00 μg and 1000 mg of calcium carbonate in the postoperative period, the control group (14 patients) — only calcium carbonate. Patients were observed for 1 year. To control the formation of a full callus in the area surgical intervention and dynamics of the BMD at the spine (L1–L4) were performed at 6 and 12 months after surgery. Results: In the group receiving alfacalcidol the fracture was consolidated for 138 days and the group receiving only calcium carbonate — 188 days (p b 0.05). After 12 months BMD at the spine (L1–L4) in the study group was increased with an average of 2%. At the control group, BMD decreases about 2.5%. At the control group only 3 patients had a new compression fracture of the vertebral body. Conclusion: Alfacalcidols use in the postoperative period after minimally invasive osteosynthesis V-spoke shortens the formation of a full callus and prevents the progression of bone loss in the axial skeleton. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None Declared. doi:10.1016/j.bone.2012.02.345
doi:10.1016/j.bone.2012.02.343
PP155 Beneficial effects of long-term alpha-ketoglutarate (AKG) administration on tibia properties in domestic geese D. Niedzielaa,⁎, M.R. Tataraa, W. Krupskib, B. Tymczynac, I. Luszczewska-Sierakowskad a Department of Animal Physiology, University of Life Sciences, Lublin, Poland b II Department of Radiology, Lublin, Poland c Departament of Conservative Dentistry, Medical University of Lublin, Lublin, Poland d Department of Animal Anatomy, University of Life Sciences, Lublin, Poland Abstract: Alpha-ketoglutarate (AKG) is a natural bioactive substance positively influencing bone tissue metabolism. The aim of the study was to investigate the effects of oral administration with AKG on skeletal system properties in geese during growth.
PP157 Structural evaluation of the bone formed during endochondral ossification of young rats treated with bisphosphonates E. Rezende⁎, L.M.B. Ambrósio, V. Bradaschia-Correa, V.E. Arana-Chavez Oral Biology, University of São Paulo, São Paulo, Brazil Abstract: The bisphosphonates (BPs) are drugs with well known capacity of inhibiting bone resorption by osteoclasts. There are two major types of BPs, the nitrogenated and the non-nitrogenated, which possess different mechanisms of inhibition of the resorptive activity. The clastic cells are necessary, during the endochondral ossification, for the resorption of calcified cartilage septa and allow the invasion of osteogenic cells that secrete bone matrix over the calcified cartilage matrix. Although it has been reported that BP treatment promotes the enlargement of the cartilage zones, the effects of the both BP types on the bone formed during the endochondral ossification of long bones are not completely elucidated. The present
Abstracts study evaluated the structure of the bone present in the ossification zone from the growth plate of young rats treated with alendronate (ALN), a nitrogenated BP, and etidronate (ETN), a non-nitrogenated BP. Newborn Wistar rats were daily subcutaneously injected with 2.5 mg/kg ALN or 8 mg/kg ET during 21 days. The controls were injected with saline solution. On the day cited, the animals were anesthetized and euthanized, and their femur and tibiae epiphyses were dissected out and fixed in 2% glutaraldehyde + 2.5% formaldehyde under microwave irradiation. For scanning electron microscopy (SEM) analyses, the specimens had the organic material removed then were longitudinally fractured, dehydrated, radiographed and gold coated. The specimens for transmission electron microscopy (TEM) analysis were decalcified in 4.13% EDTA, embedded in Spurr resin, and 70 nm-thick ultrathin sections were analyzed. The ETN animals exhibited lower body weight compared to the ALN ones, while ALN body weight was lower than controls. SEM analyses of the ALN specimens revealed the presence of cartilage septa and bone trabeculae, in which resorptive lacunae were not detected. The ETN specimens presented thicker and more numerous bone trabeculae than controls; numerous resorptive lacunae were detected in both groups. TEM analyses revealed that the clastic cells of ALN specimens were not attached to the mineralized surfaces and exhibited latent phenotype; the bone trabeculae exhibited typical features of primary bone. The clastic cells of ETN group were activated and they presented resorptive activity. The present results indicate that alendronate treatment during skeletal growth may yield an osteopetrotic-like phenotype, while etidronate promotes an osteoporotic-like phenotype. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.346
PP158 Disclosing the underlying interaction between bone and peripheral nervous system: Optimization of coculture systems E.C. Netoa,b,⁎, J. Alvesa, J. Ribasc, S. Ferreiraa, N.L. Jeond, H.R. Ryue, J. Malvaf, R. Almeidaf, M. Lamgharia a INEB — Instituto de Engenharia Biomédica, Portugal b Faculdade de Medicina, Universidade do Porto, Porto, Portugal c Universidade de Coimbra, Coimbra, Portugal d Institute of Advanced Machinery and Design, Seoul, Korea, Republic of Korea e WCU Multiscale Mechanical Design, Seoul, Republic of Korea f CNC — Centro de Neurociência e Biologia Celular, Coimbra, Portugal Abstract: Several evidences were provided showing nerve fibers in the bone microenvironment. The involvement of the peripheral nervous system (PNS) in the bone homeostasis is evidenced by the expression of different neuropeptides and neurotransmitters, and its receptors, in bone cells. However this interaction is not fully understood. Therefore, the knowledge of the interaction between bone and the PNS is mandatory to understand and promote bone regeneration. This study aims to establish a coculture system, mimicking the in vivo interaction between bone and the PNS and explore the cellular mechanisms behind the functional relationships. Two coculture systems, transwells and microfluidic chambers, were established in order to provide tools to study the interaction between osteoblasts (Ob) and sensory neuronal cells. Non-contiguous cocultures were performed using transwells to allow the exchange of soluble factors without cell–cell contact. Since Ob (MC3T3-E1 cell line) and sensory neurons (ND7/23 cell line) are from different origins, we established the optimal conditions (cell density, serum concentration and time of coculture) to ensure viability and maintenance of phenotypes of two cell types. Microfluidic chambers were used as another approach for the coculture system, particularly due to its proprieties of compartmentalization. Primary cells were used: dorsal root ganglia (DRGs) and bone marrow stromal cells (BMSCs) from 7 week old mice (C57Bl/6).Cell density of BMSCs and culture of adult DRGs were optimized to ensure the success of the coculture. The optimization of the coculture showed that 250 sensory neuronal cells/cm2, 10000 Ob cells/cm2, 0.5% serum and 24 h of coculture were the ideal conditions for the maintenance of the viability and phenotype of OB and sensory neurons in non-contiguous coculture system. Besides the regular interaction between these two cell types, this system might be useful to study several drugs in the context of bone regeneration. The results of the microfluidic chambers' coculture system showed that the Ob and DRGs were able to survive and interact with each other. Using these devices, as a coculture system, direct contact between axons and Ob can be studied separately from the molecular reactions occurring in the cell soma. Through this work, a functional coculture system between Ob and sensory neuronal cells was provided which will allow further studies comprising drugs to promote Ob and neuronal cells differentiation and, ultimately, bone regeneration. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: E. Neto Grant/research support from this work was funded by FCT project: PTDC/SAU-OSM/101469/2008, J. Alves: none declared, J. Ribas: none
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declared, S. Ferreira: none declared, N. L. Jeon: none declared, H. Ryu: none declared, J. Malva: none declared, R. Almeida: none declared, M. Lamghari Grant/research support from FCT project: PTDC/SAU-OSM/101469/2008. doi:10.1016/j.bone.2012.02.347
PP159 Clinical and histological evaluation of post-extraction platelet-rich fibrin socket filling: A prospective randomized controlled study F. Hausera,b,⁎, N. Gaydarovb, I. Badoudc, L. Vazquezb, J.-P. Bernardb, P. Ammannc a Centre Médico-Dentaire Balexert, Switzerland b Departement of Stomatology and Oral Surgery, School of Dental Medicine, University of Geneva, Switzerland c Division of Bone Disease, Departement of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland Abstract: Different surgical procedures using platelet-rich fibrin (PRF) as healing or grafting material have been described, but with contradictory results. We investigated whether the use of PRF membranes for socket filling could improve the microarchitecture and the intrinsic bone tissue quality of the alveolar bone after premolar extraction and assessed the influence of the surgical procedure prior to implant placement. Twenty-three patients who required extraction of a nonrestorable premolar followed by implant placement were randomized to 3 groups: PRF group with simple extraction and socket filling with PRF; PRF-flap group with extraction with a mucosal flap and socket filling with PRF; and a control group with simple extraction without socket filling. Implant placement was performed at week 8 and a bone biopsy was obtained from the centre of the socket for histomorphometric analysis. Loss of the alveolar crest width was clinically measured and superimposable radiographs were performed to measure the vertical alveolar bone loss prior to extraction and implant placement. μCT analysis was performed to investigate volume and microarchitecture of the new formed bone. Table 1 Results
μCT BV/TV (1) Tb.N (1/mm) Tb.Sp (mm) Alveolar crest width mean loss (mm)
Control
PRF
PRF-flap
0.249 ± 0.037 4.34 ± 0.34 0.234 ± 0.019 − 0.43**
0.281 ± 0.037 5.56 ± 0.37* 0.173 ± 0.016* − 0.06
0.197 ± 0.027 4.55 ± 0.32 0.219 ± 0.012 − 0.42
Results are bone volume to total volume (BV/TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp). Values are mean ± SEM. *p b 0.05, compared to control. ** compared to PRF. A significant effect was also observed on intrinsic bone tissue quality: elastic modulus (+17.42%, p b 0.05), hardness (+10.01%) and working energy (+11.05%, p b 0.05) were increased in the PRF group compared to PRF-flap group. Socket filling with PRF without mucosal flap showed better bone healing with improvement of the bone microarchitecture and intrinsic bone tissue quality, and a better preservation of the alveolar crest width. The influence of the surgical procedure on bone healing seemed as important as the grafting material. The flap represented an invasive procedure for the soft tissue with a negative impact on bone healing inside the socket that could neutralize the advantages of the PRF. These results support the use of a minimally traumatic procedure for tooth extraction and socket filling with PRF to achieve preservation of the hard tissue. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.348
PP160 Calcified cartilage in mature rat cortical bone F.L. Bach-Gansmoa,⁎, A. Brüelb, J.S. Thomsenb, H. Birkedalc a iNano b Department of Biomedicine, Aarhus University, Aarhus, Denmark c Department of Chemistry, Aarhus University, Aarhus, Denmark Abstract: Bone remodeling of cortical bone in humans involves Haversian remodeling. However, this mode of remodeling is very scarce in rodents such as rats [1, 2]. Indeed, why Haversian remodeling is used in some animals but not in others remains an unresolved question.
Abstracts
densitometric (DEXA) method. Using an INSTRON 3367 apparatus (Instron, USA) and three-point bending test, mechanical parameters such as maximum elastic strength (Wy) and ultimate strength (Wf) of mandible were estimated. Pearson's correlation coefficient (r) was determined between all the investigated variables and P b 0.05 was considered as statistically significant. Obtained results showed significant positive correlation between body weight and mandible weight, lenght, Bvol, Cd, BMD, BMC, Wy and Wf (P b 0.05). All the investigated parameters of the mandible were found to be positively correlated. However, statistically insignificant correlations of MvBMD and body weight, mandible weight, mandible length and Bvol were stated. Furthermore, Bvol and Cd were not found to be significantly correlated (P N 0.05). In conclusion, this study showed numerous positive correlations between final body weight, densitometric, morphometric and mechanical properties of mandible. This bone of pigs may be used as an attractive model for further investigations on metabolic response of skeleton to physiological, nutritional, toxicological and pharmacological factors influencing bone tissue metabolism. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.342
PP154 Detection of osteoadherin during intramembranous and endochondral ossification by colloidal gold immunocytochemistry and Western blotting D.S. Janonesa,⁎, T.A.G. Donatoa, V. Bradaschia-Corrêaa, F. Bleicherb, V.E. Arana-Chaveza a Biomaterials and Oral Biology, Universaty of São Paulo, São Paulo, Brazil b Laboratoire Odontoblast et régénération dentinaire, University of Lyon, Lyon, France Abstract: Osteoadherin (OSAD) is a keratan sulfate proteoglycan firstly isolated from bovine bone. Although the distribution of OSAD in mineralized tissues had been described suggesting a role in biomineralization, its specific role remains poorly understood. The present study compared the appearance of OSAD during both intramembranous and endochondral ossification and correlated it with early stages of mineralization. Parietal bone of 17, 18 and 21 day-old embryos and mandibular condyle of 30 day-old Wistar rats were removed. Some specimens were frozen in liquid nitrogen while others were fixed in 0.1% glutaraldehyde + 4% formaldehyde under microwave (MW) irradiation. Some specimens were left undecalcified, while others were decalcified in 4.13% EDTA, dehydrated in graded concentrations of ethanol and embedded in LR White resin. The expression of OSAD was analyzed by immunocytochemistry and Western blotting. Ultrathin sections were incubated with a polyclonal rabbit antibody against rat OSAD. In both intramembranous and endochondral ossification the gold particles were always uniformly distributed in the cytoplasm of osteoblasts but they only appeared in the mineralizing matrix when the fibrilar stage was taking place, remaining as a component of the mineralized bone matrix. Western blots carried out with the same antibody revealed that 17-day-old embryos contained slightly less OSAD than 18-day-old embryos, while immunoreactivity was weak in 21 day-old embryos. The results suggest that OSAD play a role in collagen fibril mineralization maybe as an organizer of matrix assembly or by retaining the mineral crystals into the matrix, besides exerting binding activities among bone matrix components. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
Twenty male turkeys were randomly divided into 2 weight-matched groups (N = 10 per group) at the age of 28 days of life. The first group consisted of control birds that received placebo orally. The second group (AKG group) received oral administration with AKG (0.4 g/kg b.w./d). The birds were kept under standard rearing conditions and slaughtered at the age of 108 days of life to analyse left tibia in terms of bone morphology (weight (W), length (L), cross-sectional area (A), second moment of inertia (Ix), mean relative wall thickness (MRWT), cortical index (CI)), volumetric bone mineral density (vBMD), as well mechanical properties (maximum elastic strength (Wy) and ultimate strength (Wf)). Using computed tomography technique (Somatom Emotion, Siemens), vBMD of the trabecular and the cortical bone of tibia was measured. Mechanical properties of tibia were estimated using three-point bending test and an INSTRON 3367 apparatus (Instron, USA). Statistical analysis was performed with a use of Student t-test and P b 0.05 was considered as statistically significant. Obtained results showed significantly increased weight, length, Ix, trabecular bone mineral density, cortical bone mineral density and Wy of tibia by 14.4%, 6.2%, 23.9%, 6.1%, 8.7%, and 31.8%, respectively, when compared to the controls (all P b 0.05). All these results indicate that long-term oral administration with AKG in geese improves vBMD of the trabecular and cortical bone compartments as well as morphological and mechanical properties of tibia in growing geese. Thus, the study have shown for the first time that AKG administration may be considered as an effective nutrient improving skeletal system quality in geese. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.344
PP156 Alfacalcidols effect to shoulder fracture consolidation period at the patient with osteoporosis E. Solod⁎, A. Lazarev, S. Rodionova Central Institute of Traumatology and Orthopedics, Moscow, Russian Federation Abstract: Unstable fractures (classified Neer CS, 1970) in patients with systemic osteoporosis are characterized by the highest incidence of longer seam. Objective: Comparative assessment of the impact of alfacalcidols in the treatment of humerus fractures. Materials and methods: The study included 62 patients (mean age 69.7 years) with the proximal humerus fractures and a confirmed diagnosis of osteoporosis (senile and postmenopausal). All patients completed the operation, closed nailing tight V-spoke. The study group (48 patients) was receiving a daily alfacalcidol dose of 0.75–1.00 μg and 1000 mg of calcium carbonate in the postoperative period, the control group (14 patients) — only calcium carbonate. Patients were observed for 1 year. To control the formation of a full callus in the area surgical intervention and dynamics of the BMD at the spine (L1–L4) were performed at 6 and 12 months after surgery. Results: In the group receiving alfacalcidol the fracture was consolidated for 138 days and the group receiving only calcium carbonate — 188 days (p b 0.05). After 12 months BMD at the spine (L1–L4) in the study group was increased with an average of 2%. At the control group, BMD decreases about 2.5%. At the control group only 3 patients had a new compression fracture of the vertebral body. Conclusion: Alfacalcidols use in the postoperative period after minimally invasive osteosynthesis V-spoke shortens the formation of a full callus and prevents the progression of bone loss in the axial skeleton. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None Declared. doi:10.1016/j.bone.2012.02.345
doi:10.1016/j.bone.2012.02.343
PP155 Beneficial effects of long-term alpha-ketoglutarate (AKG) administration on tibia properties in domestic geese D. Niedzielaa,⁎, M.R. Tataraa, W. Krupskib, B. Tymczynac, I. Luszczewska-Sierakowskad a Department of Animal Physiology, University of Life Sciences, Lublin, Poland b II Department of Radiology, Lublin, Poland c Departament of Conservative Dentistry, Medical University of Lublin, Lublin, Poland d Department of Animal Anatomy, University of Life Sciences, Lublin, Poland Abstract: Alpha-ketoglutarate (AKG) is a natural bioactive substance positively influencing bone tissue metabolism. The aim of the study was to investigate the effects of oral administration with AKG on skeletal system properties in geese during growth.
PP157 Structural evaluation of the bone formed during endochondral ossification of young rats treated with bisphosphonates E. Rezende⁎, L.M.B. Ambrósio, V. Bradaschia-Correa, V.E. Arana-Chavez Oral Biology, University of São Paulo, São Paulo, Brazil Abstract: The bisphosphonates (BPs) are drugs with well known capacity of inhibiting bone resorption by osteoclasts. There are two major types of BPs, the nitrogenated and the non-nitrogenated, which possess different mechanisms of inhibition of the resorptive activity. The clastic cells are necessary, during the endochondral ossification, for the resorption of calcified cartilage septa and allow the invasion of osteogenic cells that secrete bone matrix over the calcified cartilage matrix. Although it has been reported that BP treatment promotes the enlargement of the cartilage zones, the effects of the both BP types on the bone formed during the endochondral ossification of long bones are not completely elucidated. The present
Abstracts study evaluated the structure of the bone present in the ossification zone from the growth plate of young rats treated with alendronate (ALN), a nitrogenated BP, and etidronate (ETN), a non-nitrogenated BP. Newborn Wistar rats were daily subcutaneously injected with 2.5 mg/kg ALN or 8 mg/kg ET during 21 days. The controls were injected with saline solution. On the day cited, the animals were anesthetized and euthanized, and their femur and tibiae epiphyses were dissected out and fixed in 2% glutaraldehyde + 2.5% formaldehyde under microwave irradiation. For scanning electron microscopy (SEM) analyses, the specimens had the organic material removed then were longitudinally fractured, dehydrated, radiographed and gold coated. The specimens for transmission electron microscopy (TEM) analysis were decalcified in 4.13% EDTA, embedded in Spurr resin, and 70 nm-thick ultrathin sections were analyzed. The ETN animals exhibited lower body weight compared to the ALN ones, while ALN body weight was lower than controls. SEM analyses of the ALN specimens revealed the presence of cartilage septa and bone trabeculae, in which resorptive lacunae were not detected. The ETN specimens presented thicker and more numerous bone trabeculae than controls; numerous resorptive lacunae were detected in both groups. TEM analyses revealed that the clastic cells of ALN specimens were not attached to the mineralized surfaces and exhibited latent phenotype; the bone trabeculae exhibited typical features of primary bone. The clastic cells of ETN group were activated and they presented resorptive activity. The present results indicate that alendronate treatment during skeletal growth may yield an osteopetrotic-like phenotype, while etidronate promotes an osteoporotic-like phenotype. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.346
PP158 Disclosing the underlying interaction between bone and peripheral nervous system: Optimization of coculture systems E.C. Netoa,b,⁎, J. Alvesa, J. Ribasc, S. Ferreiraa, N.L. Jeond, H.R. Ryue, J. Malvaf, R. Almeidaf, M. Lamgharia a INEB — Instituto de Engenharia Biomédica, Portugal b Faculdade de Medicina, Universidade do Porto, Porto, Portugal c Universidade de Coimbra, Coimbra, Portugal d Institute of Advanced Machinery and Design, Seoul, Korea, Republic of Korea e WCU Multiscale Mechanical Design, Seoul, Republic of Korea f CNC — Centro de Neurociência e Biologia Celular, Coimbra, Portugal Abstract: Several evidences were provided showing nerve fibers in the bone microenvironment. The involvement of the peripheral nervous system (PNS) in the bone homeostasis is evidenced by the expression of different neuropeptides and neurotransmitters, and its receptors, in bone cells. However this interaction is not fully understood. Therefore, the knowledge of the interaction between bone and the PNS is mandatory to understand and promote bone regeneration. This study aims to establish a coculture system, mimicking the in vivo interaction between bone and the PNS and explore the cellular mechanisms behind the functional relationships. Two coculture systems, transwells and microfluidic chambers, were established in order to provide tools to study the interaction between osteoblasts (Ob) and sensory neuronal cells. Non-contiguous cocultures were performed using transwells to allow the exchange of soluble factors without cell–cell contact. Since Ob (MC3T3-E1 cell line) and sensory neurons (ND7/23 cell line) are from different origins, we established the optimal conditions (cell density, serum concentration and time of coculture) to ensure viability and maintenance of phenotypes of two cell types. Microfluidic chambers were used as another approach for the coculture system, particularly due to its proprieties of compartmentalization. Primary cells were used: dorsal root ganglia (DRGs) and bone marrow stromal cells (BMSCs) from 7 week old mice (C57Bl/6).Cell density of BMSCs and culture of adult DRGs were optimized to ensure the success of the coculture. The optimization of the coculture showed that 250 sensory neuronal cells/cm2, 10000 Ob cells/cm2, 0.5% serum and 24 h of coculture were the ideal conditions for the maintenance of the viability and phenotype of OB and sensory neurons in non-contiguous coculture system. Besides the regular interaction between these two cell types, this system might be useful to study several drugs in the context of bone regeneration. The results of the microfluidic chambers' coculture system showed that the Ob and DRGs were able to survive and interact with each other. Using these devices, as a coculture system, direct contact between axons and Ob can be studied separately from the molecular reactions occurring in the cell soma. Through this work, a functional coculture system between Ob and sensory neuronal cells was provided which will allow further studies comprising drugs to promote Ob and neuronal cells differentiation and, ultimately, bone regeneration. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: E. Neto Grant/research support from this work was funded by FCT project: PTDC/SAU-OSM/101469/2008, J. Alves: none declared, J. Ribas: none
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declared, S. Ferreira: none declared, N. L. Jeon: none declared, H. Ryu: none declared, J. Malva: none declared, R. Almeida: none declared, M. Lamghari Grant/research support from FCT project: PTDC/SAU-OSM/101469/2008. doi:10.1016/j.bone.2012.02.347
PP159 Clinical and histological evaluation of post-extraction platelet-rich fibrin socket filling: A prospective randomized controlled study F. Hausera,b,⁎, N. Gaydarovb, I. Badoudc, L. Vazquezb, J.-P. Bernardb, P. Ammannc a Centre Médico-Dentaire Balexert, Switzerland b Departement of Stomatology and Oral Surgery, School of Dental Medicine, University of Geneva, Switzerland c Division of Bone Disease, Departement of Rehabilitation and Geriatrics, Geneva University Hospitals and Faculty of Medicine, Geneva, Switzerland Abstract: Different surgical procedures using platelet-rich fibrin (PRF) as healing or grafting material have been described, but with contradictory results. We investigated whether the use of PRF membranes for socket filling could improve the microarchitecture and the intrinsic bone tissue quality of the alveolar bone after premolar extraction and assessed the influence of the surgical procedure prior to implant placement. Twenty-three patients who required extraction of a nonrestorable premolar followed by implant placement were randomized to 3 groups: PRF group with simple extraction and socket filling with PRF; PRF-flap group with extraction with a mucosal flap and socket filling with PRF; and a control group with simple extraction without socket filling. Implant placement was performed at week 8 and a bone biopsy was obtained from the centre of the socket for histomorphometric analysis. Loss of the alveolar crest width was clinically measured and superimposable radiographs were performed to measure the vertical alveolar bone loss prior to extraction and implant placement. μCT analysis was performed to investigate volume and microarchitecture of the new formed bone. Table 1 Results
μCT BV/TV (1) Tb.N (1/mm) Tb.Sp (mm) Alveolar crest width mean loss (mm)
Control
PRF
PRF-flap
0.249 ± 0.037 4.34 ± 0.34 0.234 ± 0.019 − 0.43**
0.281 ± 0.037 5.56 ± 0.37* 0.173 ± 0.016* − 0.06
0.197 ± 0.027 4.55 ± 0.32 0.219 ± 0.012 − 0.42
Results are bone volume to total volume (BV/TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp). Values are mean ± SEM. *p b 0.05, compared to control. ** compared to PRF. A significant effect was also observed on intrinsic bone tissue quality: elastic modulus (+17.42%, p b 0.05), hardness (+10.01%) and working energy (+11.05%, p b 0.05) were increased in the PRF group compared to PRF-flap group. Socket filling with PRF without mucosal flap showed better bone healing with improvement of the bone microarchitecture and intrinsic bone tissue quality, and a better preservation of the alveolar crest width. The influence of the surgical procedure on bone healing seemed as important as the grafting material. The flap represented an invasive procedure for the soft tissue with a negative impact on bone healing inside the socket that could neutralize the advantages of the PRF. These results support the use of a minimally traumatic procedure for tooth extraction and socket filling with PRF to achieve preservation of the hard tissue. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.348
PP160 Calcified cartilage in mature rat cortical bone F.L. Bach-Gansmoa,⁎, A. Brüelb, J.S. Thomsenb, H. Birkedalc a iNano b Department of Biomedicine, Aarhus University, Aarhus, Denmark c Department of Chemistry, Aarhus University, Aarhus, Denmark Abstract: Bone remodeling of cortical bone in humans involves Haversian remodeling. However, this mode of remodeling is very scarce in rodents such as rats [1, 2]. Indeed, why Haversian remodeling is used in some animals but not in others remains an unresolved question.