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Titanium induced osteoclast recruitment and activation resulting in enhanced bone resorption: A human in vitro study
ORTHOPAEDIC SURGERY phosphatase (TRAP) and cathepsin K (CATK), and chemokines (CCL17 and 22) were measured. The effects of Ti(IV) on osteoclastic activity and differentiation were also evaluated by measuring the enzymatic activity of TRAP using ELF97, and the amount of secreted chemokines using ELISA assay. Additionally, functional evidence of osteoclastic bone resorptive activity was determined by a lacunar resorption assay.
Endogenous bone engineering using solid scaffolds with time-released porosity Alexander C Allori MD, MPH, Clarence D Lin MA, Alexander M Sailon BA, Elizabeth Clark MA, Cornelia Cretiu-Vasiliu MS, James E Smay PhD, John L Ricci PhD, Stephen M Warren MD New York University Medical Center, New York, NY
RESULTS: After Ti(IV) treatment monocytes showed an increased expression of TRAP and CATK. Detection and quantification of intracellular TRAP activity revealed a significant increase of TRAPpositive cells in treated monocytes. Ti(IV) treated monocytes became functional bone resorbing cells, increasing significantly their osteoresorptive activity. Additionally, Ti(IV) treated osteoclasts showed significantly increased CCL17 and 22 expression and secretion.
INTRODUCTION: While scaffolds employed in skeletal repair today have excellent osteoconductivity, their porous structure permits soft-tissue ingrowth. To address these problems, a novel hydroxyapatite (HA) and tri-calcium phosphate (TCP) scaffold was designed with filled internal porosity for in vitro and in vivo testing. METHODS: A 3D microprinting process was used to create microstructural lattices (11mm diameter, 3mm thickness, 250um struts, 200um pore size, HA:TCP ratio 60/40 or 15/85) that were either left porous or filled with calcium sulfate/chitosan. To assess for scaffold dissolution over time, porous and solid scaffolds were placed in DMEM⫹10%FBS and weighed daily for 64 days. In vivo, porous and solid scafffolds were implanted in a rabbit critical-size calvarial defect model for 8 and 16 weeks and analyzed by micro-CT, histology, and SEM.
CONCLUSIONS: This study provides strong support that Ti(IV) ions induce differentiation of monocytes towards mature and functional osteoclasts, and activate the expression and secretion of chemokines in mature osteoclasts. These results suggest enhanced recruitment of osteoclast precursors from the blood circulation and induced osteoclastic differentiation, which may well contribute to the pathomechanism of aseptic loosening.
RESULTS: Porous scaffolds retained 97⫾2% of their weight at 64 days. When filled by calcium sulfate, mass decreased linearly (0.44%/ day, R2⫽0.92), correlating with gradual centripetal exposure of the internal porosity at 1mm/wk. In contrast, chitosan filling of the scaffolds was irregular in its dissolution. By 8 weeks in vivo, scaffolds conducted bone across critical defects (85⫾18%) compared to nonhealed controls (p⬍0.05). Bony ingrowth corresponded to the degradation rate of the calcium sulfate. Scaffold filling also prevented ingrowth of soft tissue. Sixteen week samples are being processed.
Dynamic cell culture for vascularized bone engineering Alexander C Allori MD, MPH, Alexander M Sailon BA, Elizabeth Clark MA, Cornelia Cretiu-Vasiliu MS, James Smay PhD, John L Ricci PhD, Stephen M Warren MD New York University Medical Center, New York, NY INTRODUCTION: Bone lacuno-canalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. A novel flow-perfusion bioreactor was used to provide chemotransportation through a thick 3D scaffold in vitro. We hypothesize that flow perfusion will improve cellular distribution and viability throughout the construct and that osteoblasts will form meaningful osteoblastic networks.
CONCLUSIONS: Filling with calcium sulfate successfully limited bloody infiltration and soft tissue ingrowth but retained osteoconductivity. Present work is focused on improving scaffold osteoinductivity by using these fillers to encase bioactive molecules for ratecontrolled release.
METHODS: 3D bioprinted cylindrical hydroxyapatite-tricalciumphosphate scaffolds (24x6 mm, 200x200 m pore size) were seeded with MC3T3-E1 cells (4x106 cells/cm3) and subjected to dynamic cell culture (0.4 dyn/cm2) in osteogenic medium. Control scaffolds remained in static culture. A dye-transfer technique using diI and calcein AM was used to label cells to check for gap junction formation. Samples were harvested at 2, 4, 6, 10, 14, and 21 days, plasticembedded, and analyzed by confocal microscopy and scanning electron microscopy.
Titanium induced osteoclast recruitment and activation resulting in enhanced bone resorption: A human in vitro study Dieter Cadosch MD,* E Chan PhD, O P Gautschi MD, L Filgueira MD, R Zellweger MD, FACS University of Western Australia, Perth, Western Australia, Australia INTRODUCTION: There is increasing evidence that titanium ions are released from orthopedic implants resulting in 1M concentration in surrounding tissues and blood, and playing a role in aseptic loosening. Our aim was to investigate, whether Ti(IV) ions induce recruitment and differentiation of monocytes into osteo-resorptive multinucleated cells and influence the activation and function of in vitro generated osteoclasts.
RESULTS: Histologic analysis confirmed uniform cell distribution after seeding and that ⬎90% of cells survived the time allotted for cellular adherence. By 6 days in static culture, 80% of cells in the core of the scaffolds were non-viable, while 90% of those at the peripheral crust survived. In contrast, scaffolds subjected to flow perfusion demonstrated uniform cell density and 95% viability throughout the construct at all time points. Osteoblasts assumed characteristic dendritic phenotype and extended podocytes toward other cells, with evidence of gap junction formation.
METHODS: Human monocytes and in vitro generated osteoclasts were exposed to 1M Ti(IV) ions for ten days. Thereafter, transcription of specific osteoclast genes, including tartrate-resistant acid
© 2008 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/08/$34.00
S51
Endogenous bone engineering using solid scaffolds with time-released porosity Alexander C Allori MD, MPH, Clarence D Lin MA, Alexander M Sailon BA, Elizabeth Clark MA, Cornelia Cretiu-Vasiliu MS, James E Smay PhD, John L Ricci PhD, Stephen M Warren MD New York University Medical Center, New York, NY
RESULTS: After Ti(IV) treatment monocytes showed an increased expression of TRAP and CATK. Detection and quantification of intracellular TRAP activity revealed a significant increase of TRAPpositive cells in treated monocytes. Ti(IV) treated monocytes became functional bone resorbing cells, increasing significantly their osteoresorptive activity. Additionally, Ti(IV) treated osteoclasts showed significantly increased CCL17 and 22 expression and secretion.
INTRODUCTION: While scaffolds employed in skeletal repair today have excellent osteoconductivity, their porous structure permits soft-tissue ingrowth. To address these problems, a novel hydroxyapatite (HA) and tri-calcium phosphate (TCP) scaffold was designed with filled internal porosity for in vitro and in vivo testing. METHODS: A 3D microprinting process was used to create microstructural lattices (11mm diameter, 3mm thickness, 250um struts, 200um pore size, HA:TCP ratio 60/40 or 15/85) that were either left porous or filled with calcium sulfate/chitosan. To assess for scaffold dissolution over time, porous and solid scaffolds were placed in DMEM⫹10%FBS and weighed daily for 64 days. In vivo, porous and solid scafffolds were implanted in a rabbit critical-size calvarial defect model for 8 and 16 weeks and analyzed by micro-CT, histology, and SEM.
CONCLUSIONS: This study provides strong support that Ti(IV) ions induce differentiation of monocytes towards mature and functional osteoclasts, and activate the expression and secretion of chemokines in mature osteoclasts. These results suggest enhanced recruitment of osteoclast precursors from the blood circulation and induced osteoclastic differentiation, which may well contribute to the pathomechanism of aseptic loosening.
RESULTS: Porous scaffolds retained 97⫾2% of their weight at 64 days. When filled by calcium sulfate, mass decreased linearly (0.44%/ day, R2⫽0.92), correlating with gradual centripetal exposure of the internal porosity at 1mm/wk. In contrast, chitosan filling of the scaffolds was irregular in its dissolution. By 8 weeks in vivo, scaffolds conducted bone across critical defects (85⫾18%) compared to nonhealed controls (p⬍0.05). Bony ingrowth corresponded to the degradation rate of the calcium sulfate. Scaffold filling also prevented ingrowth of soft tissue. Sixteen week samples are being processed.
Dynamic cell culture for vascularized bone engineering Alexander C Allori MD, MPH, Alexander M Sailon BA, Elizabeth Clark MA, Cornelia Cretiu-Vasiliu MS, James Smay PhD, John L Ricci PhD, Stephen M Warren MD New York University Medical Center, New York, NY INTRODUCTION: Bone lacuno-canalicular fluid flow ensures chemotransportation and provides a mechanical stimulus to cells. A novel flow-perfusion bioreactor was used to provide chemotransportation through a thick 3D scaffold in vitro. We hypothesize that flow perfusion will improve cellular distribution and viability throughout the construct and that osteoblasts will form meaningful osteoblastic networks.
CONCLUSIONS: Filling with calcium sulfate successfully limited bloody infiltration and soft tissue ingrowth but retained osteoconductivity. Present work is focused on improving scaffold osteoinductivity by using these fillers to encase bioactive molecules for ratecontrolled release.
METHODS: 3D bioprinted cylindrical hydroxyapatite-tricalciumphosphate scaffolds (24x6 mm, 200x200 m pore size) were seeded with MC3T3-E1 cells (4x106 cells/cm3) and subjected to dynamic cell culture (0.4 dyn/cm2) in osteogenic medium. Control scaffolds remained in static culture. A dye-transfer technique using diI and calcein AM was used to label cells to check for gap junction formation. Samples were harvested at 2, 4, 6, 10, 14, and 21 days, plasticembedded, and analyzed by confocal microscopy and scanning electron microscopy.
Titanium induced osteoclast recruitment and activation resulting in enhanced bone resorption: A human in vitro study Dieter Cadosch MD,* E Chan PhD, O P Gautschi MD, L Filgueira MD, R Zellweger MD, FACS University of Western Australia, Perth, Western Australia, Australia INTRODUCTION: There is increasing evidence that titanium ions are released from orthopedic implants resulting in 1M concentration in surrounding tissues and blood, and playing a role in aseptic loosening. Our aim was to investigate, whether Ti(IV) ions induce recruitment and differentiation of monocytes into osteo-resorptive multinucleated cells and influence the activation and function of in vitro generated osteoclasts.
RESULTS: Histologic analysis confirmed uniform cell distribution after seeding and that ⬎90% of cells survived the time allotted for cellular adherence. By 6 days in static culture, 80% of cells in the core of the scaffolds were non-viable, while 90% of those at the peripheral crust survived. In contrast, scaffolds subjected to flow perfusion demonstrated uniform cell density and 95% viability throughout the construct at all time points. Osteoblasts assumed characteristic dendritic phenotype and extended podocytes toward other cells, with evidence of gap junction formation.
METHODS: Human monocytes and in vitro generated osteoclasts were exposed to 1M Ti(IV) ions for ten days. Thereafter, transcription of specific osteoclast genes, including tartrate-resistant acid
© 2008 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/08/$34.00
S51