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Osteoinduction by BMP-mutants
Oral Presentations / Q39. Bone Regeneration [-0--3~
ACCURACY A N D RELIABILITY OF 3-D CT VERSUS 3-D STEREO PHOTOGRAMMETRY TISSUE A N A L Y S I S
B A S E D FACIAL S O F T
G.R.J. Swennen, E Schutyser, A. Lemaitre, C. Malevez, A. De Mey.
1Dept of Plastic Surgery, University Hospital Brugmann (ULB), Place A. Van Gehuchten 4, 1020 Brussels, Belgium; 2Medical Image Computing (Radiology - ESAT/PSI), Faculties of Medicine and Engineering, University Hospital, Gasthuisberg, Leuven, Belgium The purpose of this study is to evaluate accuracy and reliability of three-dimensional facial soft tissue measurements using 3-D CT and 3-D stereo photogrammetry. 3-D CT soft tissue measurements and 3-D stereo photogrammetry measurements were performed on a control group of 20 patients. A total of 29 angular, 62 linear measurements and 120 (40 horizontal, 40 vertical and 40 transversal) orthogonal measurements were performed on each patient by two investigators using the MaxilimTM (version 1.3.0) software. 3-D stereo photogrammetry showed a high accuracy and reliability (r2 > 0.9), except for bony related landmarks (go, zy, or). 3-D CT soft tissue measurements showed a high accuracy and reliability (r2 >0.9) except for hairline (tr), eyebrow (sci, ft) and eyelid (ps, pi) related landmarks. Combination of 3-D CT and 3-D stereo photogrammetry facial soft tissue analysis could overcome problems in bony and hair related soft tissue landmarks.
039. Bone Regeneration
73 local concentrations which are regulated by interaction with structural proteins of the extracellular matrix e.g. heparansulfate. BMP-2 provides a heparin-binding site in the N-terminal sequence of the protein. Modification of the heparin-binding site by alteration of the amino acid sequence of BMP-2 results in a change of the local retention time. T3 and T4, two mutants with increased binding capacity to heparin, and B2GDF-5 a mutant resulting from the fusion of the N-terminal amino acid sequence of BMP-2 and the C-terminal sequence of GDF-5 were assessed for their osteoinductive properties in vivo and compared with the biological activity of the wild type BMP-2. The proteins were coupled to a equinederived collagen carrier and implanted in standardized calvarial defects (critical size defects) in rats. After 28 days bone formation was evaluated radiographically and bone formation was examined histologically. T3 and T4 showed a higher osteoinductive activity than BMP-2. Less new bone formation was observed for GDF-5 and B2GDF-5 compared with the wild type BMP-2. No difference in bone formation was found for GDF-5 and B2GDF-5. Increased heparin-binding capacity enhances osteoinductive activity of BMP-2 in vivo by a longer retention period and thus better bioavailibility. Substitution of the N-terminal amino acid sequence of GDF-5 by the sequence of BMP-2 does not result in an increased osteoinductive activity. ~--~-]
N O N - V I R A L BMP-2 GENE T R A N S F E R - A NOVEL V E C T O R R E L E A S E O U T OF A P D L L A - C O A T I N G OF METALLIC S U R F A C E S TO E N H A N C E THE B O N E - I M P L A N T INTERFACE
A. Kolk 1 , C. Pautke 1 , C. Haczek 1 , H. Deppe 1 , A. Neff 1 , A. Stemberger 2,
C. Plank 2. 1Department of Oral and Maxillofacial Surgery, Technical [ - 0 - ' ~ - - ~ EVALUATION OF O S T E O G E N E S I S USING BETAT R I C A L C I U M P H O S P H A T E IN C O M B I N A T I O N WITH A P E R I O S T E A L CELL G R A F T
T. Ueno, T. Kagawa, M. Kanou, Y. Sakata, N. Mizukawa, T. Sugahara.
Department of Oral and Maxillofacial Reconstructive Surgery Okayama Univesity Graduate School of Medicine and Dentistry, Japan To investigate the osteogenic potential of beta-tricalcium phosphate (beta-TCP) in combination with free grafted periosteal cells in the rat calvarial defect model. A critical-sized defect was created in the calvaria of 7-month-old female Sprague-Dawley (SD) rats. The rats were divided into 3 groups: group A (n = 12), calvarial defect was filled with beta-TCP with a periosteal cell graft; group B (n = 10), calvarial defect was filled with beta-TCP; and group C (n = 12), calvarial defect was not filled with graft. Specimens were extirpated at 35, 45 and 60 days after grafting and examined histologically and by three-dimensional radiography. The bone area of the defect was evaluated using a computer system with automated image analysis (Mac Scope; Mitani, Fukui, Japan). Group A: at 35 days after grafting, abundant proliferation of periosteal osteogenic cells was typically observed. The porous granules were densely packed with osteoprogenitor ingrowth and woven bone. At 45 days after grafting, remarkable bone formation was noted in the defect. New bone replaced TCP particles. At 60 days after grafting, newly formed bone fused to the edges of residual bone. Most newly formed bone contacted the particles directly. Group B: at 35 days after grafting, the majority of the defect was filled with beta-TCP surrounded by fibrous tissue. Little bone formation around the porous granules was observed. At 60 days after grafting, there was a little newly formed bone in the defect. Group C: no new bone formation was observed throughout the process of healing. The area of new bone formation in the defect was significantly greater in group A than in other groups (P < 0.01). Beta-TCP in combination with periosteal cells showed the highest osteogenic potential in the rat calvarial defect. The combination of beta-TCP with a periosteal cell graft appears to be a suitable material for promoting osteogenesis.
[O~
OSTEOINDUCTIONBY BMP-MUTANTS
R. Depprich 1 , J. Handschel 1 , W. Sebald 2, K. W~rzler 3, N.R. KLibler 1 .
1Department of Cranio- and Maxillofacial Surgery, Heinrich-HeineUniversity of Duesseldo~ Germany, DuesseldoE, 2Department of Physiological Chemistry II, Biocenter, University of Wuerzburg, Germany, Wuerzburg; 3Department of Cranio- and Maxillofacial Surgery, University of Wuerzburg, Germany, Wuerzburg Bone morphogenetic proteins (BMPs) play a crucial role in bone regeneration and bone formation. Biological activity of BMPs depends on their
University of Munich; 21nstitut f#r Experimentelle Qnkologie und Therapieforschung, Technical Universtiy of Munich, Germany Healing of bony defects or bone adjacent to implants can be improved by morphogenic proteins. Animal studies have shown viral BMP-2 gene transfer to be a promising and safe new bone regeneration method, but this application is not transferable to humans due to the high risks related to viral vectors. In contrast, non-viral gene transfer of BMP-2, not related to such risks, has not been performed in vivo up to date, because among other reasons no suitable carrier system has been found allowing continuous vector release directly into bony defects. The purpose of this study was to develop a novel carrier system for the non-viral gene transfer, integrated as a special coating into a biodegradable polylactid carrier for continuous release out of metallic surfaces. For non-viral gene transfer BMP-2 cDNA, was cloned in an expression plasmid under the control of the CMV promotor (plasmid pB-BMP2). This plasmid was complexed with polyethylenimine followed by incubation with protective copolymers to generate copolymer-protected gene vectors (COPROGs). These COPROGs were integrated in a biodegradable polylactid carrier (Poly-D, L-Laktid R 203, Resomer ®, Boehringer Ingelheim, Germany). Different metallic surfaces (aluminium and titanium foils) were coated with the COPROGs/PDLLA in different concentrations. For the non-viral gene transfer in vitro we used human H293 cells which were seeded onto the foils. The BMP-2 expression was analysed by a BMP-2 Elisa. Coating of different metallic surfaces was possible resulting in a continuous vector release in vitro. The effect of non-viral BMP-2 gene transfer was dependent on the COPROGs/PDLLA concentration with best results regarding the coating stability and the extent of BMP-2 expression at the level of 0.8% (w/w) COPGROGs:PDLLA. A maximum BMP-2 expression was found on day 2 with app. 4.500 pg BMP-2 per ml medium and found BMP-2 expression persisted for at least 25 days. To our knowledge, this is the first presentation of a non-viral gene transfer of BMP-2 out of a biodegradable carrier. Using this approach, it is possible to perform a non-viral gene transfer of BMP-2 directly in vivo. Contrary to the viral gene transfer, non-viral gene transfer can be transferred into humans without the risks related to the virus itself. Furthermore, we addressed the problem of a continuous vector release from metallic surfaces to enhance the interface or directly into a bony defect.
ACCURACY A N D RELIABILITY OF 3-D CT VERSUS 3-D STEREO PHOTOGRAMMETRY TISSUE A N A L Y S I S
B A S E D FACIAL S O F T
G.R.J. Swennen, E Schutyser, A. Lemaitre, C. Malevez, A. De Mey.
1Dept of Plastic Surgery, University Hospital Brugmann (ULB), Place A. Van Gehuchten 4, 1020 Brussels, Belgium; 2Medical Image Computing (Radiology - ESAT/PSI), Faculties of Medicine and Engineering, University Hospital, Gasthuisberg, Leuven, Belgium The purpose of this study is to evaluate accuracy and reliability of three-dimensional facial soft tissue measurements using 3-D CT and 3-D stereo photogrammetry. 3-D CT soft tissue measurements and 3-D stereo photogrammetry measurements were performed on a control group of 20 patients. A total of 29 angular, 62 linear measurements and 120 (40 horizontal, 40 vertical and 40 transversal) orthogonal measurements were performed on each patient by two investigators using the MaxilimTM (version 1.3.0) software. 3-D stereo photogrammetry showed a high accuracy and reliability (r2 > 0.9), except for bony related landmarks (go, zy, or). 3-D CT soft tissue measurements showed a high accuracy and reliability (r2 >0.9) except for hairline (tr), eyebrow (sci, ft) and eyelid (ps, pi) related landmarks. Combination of 3-D CT and 3-D stereo photogrammetry facial soft tissue analysis could overcome problems in bony and hair related soft tissue landmarks.
039. Bone Regeneration
73 local concentrations which are regulated by interaction with structural proteins of the extracellular matrix e.g. heparansulfate. BMP-2 provides a heparin-binding site in the N-terminal sequence of the protein. Modification of the heparin-binding site by alteration of the amino acid sequence of BMP-2 results in a change of the local retention time. T3 and T4, two mutants with increased binding capacity to heparin, and B2GDF-5 a mutant resulting from the fusion of the N-terminal amino acid sequence of BMP-2 and the C-terminal sequence of GDF-5 were assessed for their osteoinductive properties in vivo and compared with the biological activity of the wild type BMP-2. The proteins were coupled to a equinederived collagen carrier and implanted in standardized calvarial defects (critical size defects) in rats. After 28 days bone formation was evaluated radiographically and bone formation was examined histologically. T3 and T4 showed a higher osteoinductive activity than BMP-2. Less new bone formation was observed for GDF-5 and B2GDF-5 compared with the wild type BMP-2. No difference in bone formation was found for GDF-5 and B2GDF-5. Increased heparin-binding capacity enhances osteoinductive activity of BMP-2 in vivo by a longer retention period and thus better bioavailibility. Substitution of the N-terminal amino acid sequence of GDF-5 by the sequence of BMP-2 does not result in an increased osteoinductive activity. ~--~-]
N O N - V I R A L BMP-2 GENE T R A N S F E R - A NOVEL V E C T O R R E L E A S E O U T OF A P D L L A - C O A T I N G OF METALLIC S U R F A C E S TO E N H A N C E THE B O N E - I M P L A N T INTERFACE
A. Kolk 1 , C. Pautke 1 , C. Haczek 1 , H. Deppe 1 , A. Neff 1 , A. Stemberger 2,
C. Plank 2. 1Department of Oral and Maxillofacial Surgery, Technical [ - 0 - ' ~ - - ~ EVALUATION OF O S T E O G E N E S I S USING BETAT R I C A L C I U M P H O S P H A T E IN C O M B I N A T I O N WITH A P E R I O S T E A L CELL G R A F T
T. Ueno, T. Kagawa, M. Kanou, Y. Sakata, N. Mizukawa, T. Sugahara.
Department of Oral and Maxillofacial Reconstructive Surgery Okayama Univesity Graduate School of Medicine and Dentistry, Japan To investigate the osteogenic potential of beta-tricalcium phosphate (beta-TCP) in combination with free grafted periosteal cells in the rat calvarial defect model. A critical-sized defect was created in the calvaria of 7-month-old female Sprague-Dawley (SD) rats. The rats were divided into 3 groups: group A (n = 12), calvarial defect was filled with beta-TCP with a periosteal cell graft; group B (n = 10), calvarial defect was filled with beta-TCP; and group C (n = 12), calvarial defect was not filled with graft. Specimens were extirpated at 35, 45 and 60 days after grafting and examined histologically and by three-dimensional radiography. The bone area of the defect was evaluated using a computer system with automated image analysis (Mac Scope; Mitani, Fukui, Japan). Group A: at 35 days after grafting, abundant proliferation of periosteal osteogenic cells was typically observed. The porous granules were densely packed with osteoprogenitor ingrowth and woven bone. At 45 days after grafting, remarkable bone formation was noted in the defect. New bone replaced TCP particles. At 60 days after grafting, newly formed bone fused to the edges of residual bone. Most newly formed bone contacted the particles directly. Group B: at 35 days after grafting, the majority of the defect was filled with beta-TCP surrounded by fibrous tissue. Little bone formation around the porous granules was observed. At 60 days after grafting, there was a little newly formed bone in the defect. Group C: no new bone formation was observed throughout the process of healing. The area of new bone formation in the defect was significantly greater in group A than in other groups (P < 0.01). Beta-TCP in combination with periosteal cells showed the highest osteogenic potential in the rat calvarial defect. The combination of beta-TCP with a periosteal cell graft appears to be a suitable material for promoting osteogenesis.
[O~
OSTEOINDUCTIONBY BMP-MUTANTS
R. Depprich 1 , J. Handschel 1 , W. Sebald 2, K. W~rzler 3, N.R. KLibler 1 .
1Department of Cranio- and Maxillofacial Surgery, Heinrich-HeineUniversity of Duesseldo~ Germany, DuesseldoE, 2Department of Physiological Chemistry II, Biocenter, University of Wuerzburg, Germany, Wuerzburg; 3Department of Cranio- and Maxillofacial Surgery, University of Wuerzburg, Germany, Wuerzburg Bone morphogenetic proteins (BMPs) play a crucial role in bone regeneration and bone formation. Biological activity of BMPs depends on their
University of Munich; 21nstitut f#r Experimentelle Qnkologie und Therapieforschung, Technical Universtiy of Munich, Germany Healing of bony defects or bone adjacent to implants can be improved by morphogenic proteins. Animal studies have shown viral BMP-2 gene transfer to be a promising and safe new bone regeneration method, but this application is not transferable to humans due to the high risks related to viral vectors. In contrast, non-viral gene transfer of BMP-2, not related to such risks, has not been performed in vivo up to date, because among other reasons no suitable carrier system has been found allowing continuous vector release directly into bony defects. The purpose of this study was to develop a novel carrier system for the non-viral gene transfer, integrated as a special coating into a biodegradable polylactid carrier for continuous release out of metallic surfaces. For non-viral gene transfer BMP-2 cDNA, was cloned in an expression plasmid under the control of the CMV promotor (plasmid pB-BMP2). This plasmid was complexed with polyethylenimine followed by incubation with protective copolymers to generate copolymer-protected gene vectors (COPROGs). These COPROGs were integrated in a biodegradable polylactid carrier (Poly-D, L-Laktid R 203, Resomer ®, Boehringer Ingelheim, Germany). Different metallic surfaces (aluminium and titanium foils) were coated with the COPROGs/PDLLA in different concentrations. For the non-viral gene transfer in vitro we used human H293 cells which were seeded onto the foils. The BMP-2 expression was analysed by a BMP-2 Elisa. Coating of different metallic surfaces was possible resulting in a continuous vector release in vitro. The effect of non-viral BMP-2 gene transfer was dependent on the COPROGs/PDLLA concentration with best results regarding the coating stability and the extent of BMP-2 expression at the level of 0.8% (w/w) COPGROGs:PDLLA. A maximum BMP-2 expression was found on day 2 with app. 4.500 pg BMP-2 per ml medium and found BMP-2 expression persisted for at least 25 days. To our knowledge, this is the first presentation of a non-viral gene transfer of BMP-2 out of a biodegradable carrier. Using this approach, it is possible to perform a non-viral gene transfer of BMP-2 directly in vivo. Contrary to the viral gene transfer, non-viral gene transfer can be transferred into humans without the risks related to the virus itself. Furthermore, we addressed the problem of a continuous vector release from metallic surfaces to enhance the interface or directly into a bony defect.