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Anatomically Shaped Autogenous Engineered Bone Graft for TMJ Condyle Reconstruction: Mid-Point Analysis
Oral Abstract Track 3 timization of resorbable cements that are amenable to the addition of growth factors. References: D. Olton et al., Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency Biomaterials 28, 1267 (Feb, 2007). P. Kumta, C. Sfeir, D.H. Lee, D. Olton and D. Choi, Nanostructured calcium phosphates for biomedical applications: novel synthesis and characterization, Acta Biomater 1 (2005) (1), pp. 65– 83
Anatomically Shaped Autogenous Engineered Bone Graft for TMJ Condyle Reconstruction: Mid-Point Analysis S. Bhumiratana, D. M. Alfi: Columbia University College of Dental Medicine, K. Yeager, R. Eton, J. Bernhard, J. Bova, F. Shah, J. Gimble, M. Lopez, S. Eisig, G. Vunjak-Novakovic Statement of the Problem: Maxillofacial surgeons must reconstruct complex deformities that require functional and esthetic precision. Ideal grafts must be predictable and exactly match 3D form unique to every patient’s defect in order to restore function. Current standards of practice require lengthy procedures, secondary surgical sites, immense resources, but still yield compromised results. With the advancements in tissue engineering through regulation of osteogenic differentiation and functional assembly of stem cells, we are able to produce autogenous bone grafts engineered in vitro. Here we report our experience with custom-made bone grafts of the TMJ condyle and ramus with the use of autogenous adipose stem cells (ASCs) in a large-animal study. Materials and Methods: Yucatan minipigs were randomly divided into 3 groups: (i) condylectomy (n⫽2), (ii) scaffold implantation (n⫽6), and (iii) autogenous engineered bone graft implantation (n⫽6). The process for engineering anatomically shaped TMJ condyle for implantation was adapted from previous work.1 In brief, facial skeletons of each pig were CT scanned and reconstructed in 3D. Condyle/ramus units were chosen for reconstruction (Fig. 1A). Anatomically shaped scaffolds for each pig were fabricated from trabecular bone of adult bovine knees based on previously reported methods.1 To engineer the bone grafts, scaffolds were seeded with autogenous ASCs isolated from subcutaneous fat of each pig.2 The grafts were cultured in osteogenic medium in specially designed perfusion bioreactors [Fig. 1B] for 3 weeks prior to implantation to facilitate stem cell growth, osteogenic differentiation, and bone matrix deposition. Condylectomies to include a portion of the ramus were planned virtually and carried out under general anesthesia. The pigs were reconstructed with a scaffold alone or autogenous engineered bone graft. All grafts were rigidly fixated using 2.0 mm titanium miniAAOMS • 2012
Figure 1. Mid-point results of tissue engineered reconstruction of TMJ in Yucatan minipig. (A) 3D reconstructed pig mandible with selected TMJ condyle for reconstruction; (B) Perfusion bioreactor for cultivation of autogenous bone graft; (C) Engineered graft and extracted condyle; 3-month post-implantation CT image of pigs receiving (D) scaffold implantation and (E) autogenous tissue engineered bone implantation.
plates. The planned duration of study is 6 months. We report here the mid-point analysis in which 2 pigs from each implantation group were sacrificed 3 months postimplantation. CT scans were conducted after surgery, at 6 weeks, and 3 months. TMJ condyles were harvested after sacrifice and assessed for graft remodeling in terms of compactness, integration, and resorption. Results: Bone grafts were successfully fabricated with the exact shape and size unique to each condyle [Fig. 1C]. Cell seeding of scaffolds and cultivation resulted in fully cellularized engineered bone tissue. All pigs survived the surgery without complications. At 3 months, pigs with untreated condylectomies regenerated incomplete ramus-condyle units (RCU). Pigs with scaffold implantation showed incomplete regeneration with significant scaffold resorption [Fig. 1D]. In contrast, pigs with autogenous engineered bone displayed regeneration as well as integration of the RCU [Fig. 1E]. The harvested condyles at 3 months demonstrated clear differences between the groups, with regeneration of a rigid and functional mandible in the autogenous engineered bone graft group. The scaffold-only group failed to obtain comparable results, and healed with graft resorption and fibrous ingrowth. Conclusions: Based on the mid-point results of the large animal model, autogenous engineered bone grafts maintain tissue volume and enhance regeneration, yielding a promising treatment for facial bone reconstruction. References: Grayson WL et.al., Engineering anatomically shaped human bone grafts, Proc. Natl. Acad. Sci.; 2010, 107(8):3299-304. Williams KJ et.al., Isolation and characterization of porcine adipose tissue-derived adult stem cells, Cells Tissues Organs, 2008; 188:251258.
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Oral Abstract Track 3 Acknowledgements The work was supported by BioAcellerate funding of the New York City Partnership Foundation (grant CU11-1915 to GVN).
Zhang Y et al. cDNA microarray analysis of gene expression in human fibroblast cells irradiated with red light J Invest Dermatol 2003, 120(5):849-57.
Deregulation of Specific Sets of Genes Detected by Microarray Analysis of Marrow Stromal Fibroblast Cells Stimulated by IR and VR Light
Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) on a Collagen Ceramic Sponge (CCS) for Mandibular Continuity Defects in NonHuman Primates
S. Yen: University of Southern California, J. Guo, D. Wai, Q. Wang, Y. Wang, C. Au Yeung Conflicting reports have questioned whether low level laser treatment can have an effect on bone. Purpose:The purpose of this study was to examine how light affects gene expression. Methods: Clones of marrow fibroblast stem cells were isolated from human bone marrow and used for primary cell cultures. The cells were grown without light as a control and with light under eight different conditions: two wavelengths of light(830-IR and 633nmVR) at four different energy levels(0.5.1.0,1.5 and 2.0 joules /cm2). Results: Light stimulated some cultures to proliferate up to 40% faster depending on the type of light stimulation using BrdU markers. For the light-stimulated cultures, western blot data for osteocalcin, alkaline phosphatase and RUNX 2, markers of osteoblast differentiation and bone formation, did not show a pattern of increased or early gene expression compared to control cultures. Affymetrix Human exon microarrays of 22,000 protein-encoding genes (40 markers per gene) showed distinct sets of genes being deregulated for each of the eight experimental conditions. A two-fold screen for gene expression differences between control and light-stimulated cultures revealed a complex network of gene expression interactions involving dose response and silencing of inhibitor genes for candidate genes such as RANKL, IL-1A and MMP10. Pathway analysis showed the top VR biological gene networks were skeletal and muscular system development and function, tissue development and amino acid metabolism, consistent with bone turnover. The top biological functions associated with IR stimulation are skin condition, genetic disorders, cancer, immune response. Conclusion: This study provides strong evidence for light stimulating different sets of genes according to wavelength and energy level that can alter bone turnover. The data provides a molecular explanation for the conflicting reports in the current literature. References: Wu YH et al. Effects of low-level irradiation on mesenchymation stem cell proliferation: a microarray analysis. Lasers Med Sci, 2011(epub, ahead of print).
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R. Falke: Loma Linda University, A. Buxton, A. Herford, M. Lu, R. Tandon Purpose: In the surgical bony defect site frequently seen in oral and maxillofacial surgery, it is necessary to have a graft material that will maintain the space of the soft tissue periosteal envelope. If the material is not able to maintain this space during the crucial 3- to 8-week postoperative period, the periosteal envelope will collapse and a minimal amount of bone will be regenerated. The aim of our study was to examine osseous repair of a full continuity defect of the mandible, facilitated by rhBMP-2 released from a collagen ceramic sponge (CCS). In this study, rhBMP-2/CCS treatment was explored at two concentrations: 0.75 mg/cc and 1.3 mg/cc, stabilized with a titanium reconstruction plate. Materials and Methods: Six male non-human primates (Rhesus Macaques) were used in this bilateral study, allowing for 12 investigational sites. Animals underwent general anesthesia and were then prepared for surgical mandibular resection by extraction of the posterior mandibular teeth from canine through 2nd molar, leaving the 3rd molar teeth and the incisors intact to maintain occlusal interarch distance and to afford a natural tooth occlusion for postoperative diet. The maxillary canines were also removed to prevent damage to the soft tissue of the mandible. The alveolar sites were allowed to heal after tooth extraction for a minimum of 3 months. Following healing of the soft tissue, animals received a staged, bilateral hemimandibulectomy using a submandibular approach. There was a 5-week delay between each mandibulectomy received by the same primate. The mandibular ostectomy produced a complete discontinuity defect including the mandibular inferior border with the resected area measuring in excess of 2.5 cm in antero–posterior length. The defect was filled with rhBMP-2/CCS at 0.75 or 1.3 mg/cc, stabilized with a titanium reconstruction plate, and the tissues were closed in layers. Tetracycline labels were given 3 weeks after resection/reconstruction to measure new bone growth and 16 weeks later in order to label bone turnover. The animals were maintained on a soft diet and observed for 6 months post final reconstruction. Animals were then euthanized with intravenous pentobarbital, underwent cannulation of bilateral carotid arteries and were perfused with 10% formalin. The manAAOMS • 2012
Anatomically Shaped Autogenous Engineered Bone Graft for TMJ Condyle Reconstruction: Mid-Point Analysis S. Bhumiratana, D. M. Alfi: Columbia University College of Dental Medicine, K. Yeager, R. Eton, J. Bernhard, J. Bova, F. Shah, J. Gimble, M. Lopez, S. Eisig, G. Vunjak-Novakovic Statement of the Problem: Maxillofacial surgeons must reconstruct complex deformities that require functional and esthetic precision. Ideal grafts must be predictable and exactly match 3D form unique to every patient’s defect in order to restore function. Current standards of practice require lengthy procedures, secondary surgical sites, immense resources, but still yield compromised results. With the advancements in tissue engineering through regulation of osteogenic differentiation and functional assembly of stem cells, we are able to produce autogenous bone grafts engineered in vitro. Here we report our experience with custom-made bone grafts of the TMJ condyle and ramus with the use of autogenous adipose stem cells (ASCs) in a large-animal study. Materials and Methods: Yucatan minipigs were randomly divided into 3 groups: (i) condylectomy (n⫽2), (ii) scaffold implantation (n⫽6), and (iii) autogenous engineered bone graft implantation (n⫽6). The process for engineering anatomically shaped TMJ condyle for implantation was adapted from previous work.1 In brief, facial skeletons of each pig were CT scanned and reconstructed in 3D. Condyle/ramus units were chosen for reconstruction (Fig. 1A). Anatomically shaped scaffolds for each pig were fabricated from trabecular bone of adult bovine knees based on previously reported methods.1 To engineer the bone grafts, scaffolds were seeded with autogenous ASCs isolated from subcutaneous fat of each pig.2 The grafts were cultured in osteogenic medium in specially designed perfusion bioreactors [Fig. 1B] for 3 weeks prior to implantation to facilitate stem cell growth, osteogenic differentiation, and bone matrix deposition. Condylectomies to include a portion of the ramus were planned virtually and carried out under general anesthesia. The pigs were reconstructed with a scaffold alone or autogenous engineered bone graft. All grafts were rigidly fixated using 2.0 mm titanium miniAAOMS • 2012
Figure 1. Mid-point results of tissue engineered reconstruction of TMJ in Yucatan minipig. (A) 3D reconstructed pig mandible with selected TMJ condyle for reconstruction; (B) Perfusion bioreactor for cultivation of autogenous bone graft; (C) Engineered graft and extracted condyle; 3-month post-implantation CT image of pigs receiving (D) scaffold implantation and (E) autogenous tissue engineered bone implantation.
plates. The planned duration of study is 6 months. We report here the mid-point analysis in which 2 pigs from each implantation group were sacrificed 3 months postimplantation. CT scans were conducted after surgery, at 6 weeks, and 3 months. TMJ condyles were harvested after sacrifice and assessed for graft remodeling in terms of compactness, integration, and resorption. Results: Bone grafts were successfully fabricated with the exact shape and size unique to each condyle [Fig. 1C]. Cell seeding of scaffolds and cultivation resulted in fully cellularized engineered bone tissue. All pigs survived the surgery without complications. At 3 months, pigs with untreated condylectomies regenerated incomplete ramus-condyle units (RCU). Pigs with scaffold implantation showed incomplete regeneration with significant scaffold resorption [Fig. 1D]. In contrast, pigs with autogenous engineered bone displayed regeneration as well as integration of the RCU [Fig. 1E]. The harvested condyles at 3 months demonstrated clear differences between the groups, with regeneration of a rigid and functional mandible in the autogenous engineered bone graft group. The scaffold-only group failed to obtain comparable results, and healed with graft resorption and fibrous ingrowth. Conclusions: Based on the mid-point results of the large animal model, autogenous engineered bone grafts maintain tissue volume and enhance regeneration, yielding a promising treatment for facial bone reconstruction. References: Grayson WL et.al., Engineering anatomically shaped human bone grafts, Proc. Natl. Acad. Sci.; 2010, 107(8):3299-304. Williams KJ et.al., Isolation and characterization of porcine adipose tissue-derived adult stem cells, Cells Tissues Organs, 2008; 188:251258.
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Oral Abstract Track 3 Acknowledgements The work was supported by BioAcellerate funding of the New York City Partnership Foundation (grant CU11-1915 to GVN).
Zhang Y et al. cDNA microarray analysis of gene expression in human fibroblast cells irradiated with red light J Invest Dermatol 2003, 120(5):849-57.
Deregulation of Specific Sets of Genes Detected by Microarray Analysis of Marrow Stromal Fibroblast Cells Stimulated by IR and VR Light
Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) on a Collagen Ceramic Sponge (CCS) for Mandibular Continuity Defects in NonHuman Primates
S. Yen: University of Southern California, J. Guo, D. Wai, Q. Wang, Y. Wang, C. Au Yeung Conflicting reports have questioned whether low level laser treatment can have an effect on bone. Purpose:The purpose of this study was to examine how light affects gene expression. Methods: Clones of marrow fibroblast stem cells were isolated from human bone marrow and used for primary cell cultures. The cells were grown without light as a control and with light under eight different conditions: two wavelengths of light(830-IR and 633nmVR) at four different energy levels(0.5.1.0,1.5 and 2.0 joules /cm2). Results: Light stimulated some cultures to proliferate up to 40% faster depending on the type of light stimulation using BrdU markers. For the light-stimulated cultures, western blot data for osteocalcin, alkaline phosphatase and RUNX 2, markers of osteoblast differentiation and bone formation, did not show a pattern of increased or early gene expression compared to control cultures. Affymetrix Human exon microarrays of 22,000 protein-encoding genes (40 markers per gene) showed distinct sets of genes being deregulated for each of the eight experimental conditions. A two-fold screen for gene expression differences between control and light-stimulated cultures revealed a complex network of gene expression interactions involving dose response and silencing of inhibitor genes for candidate genes such as RANKL, IL-1A and MMP10. Pathway analysis showed the top VR biological gene networks were skeletal and muscular system development and function, tissue development and amino acid metabolism, consistent with bone turnover. The top biological functions associated with IR stimulation are skin condition, genetic disorders, cancer, immune response. Conclusion: This study provides strong evidence for light stimulating different sets of genes according to wavelength and energy level that can alter bone turnover. The data provides a molecular explanation for the conflicting reports in the current literature. References: Wu YH et al. Effects of low-level irradiation on mesenchymation stem cell proliferation: a microarray analysis. Lasers Med Sci, 2011(epub, ahead of print).
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R. Falke: Loma Linda University, A. Buxton, A. Herford, M. Lu, R. Tandon Purpose: In the surgical bony defect site frequently seen in oral and maxillofacial surgery, it is necessary to have a graft material that will maintain the space of the soft tissue periosteal envelope. If the material is not able to maintain this space during the crucial 3- to 8-week postoperative period, the periosteal envelope will collapse and a minimal amount of bone will be regenerated. The aim of our study was to examine osseous repair of a full continuity defect of the mandible, facilitated by rhBMP-2 released from a collagen ceramic sponge (CCS). In this study, rhBMP-2/CCS treatment was explored at two concentrations: 0.75 mg/cc and 1.3 mg/cc, stabilized with a titanium reconstruction plate. Materials and Methods: Six male non-human primates (Rhesus Macaques) were used in this bilateral study, allowing for 12 investigational sites. Animals underwent general anesthesia and were then prepared for surgical mandibular resection by extraction of the posterior mandibular teeth from canine through 2nd molar, leaving the 3rd molar teeth and the incisors intact to maintain occlusal interarch distance and to afford a natural tooth occlusion for postoperative diet. The maxillary canines were also removed to prevent damage to the soft tissue of the mandible. The alveolar sites were allowed to heal after tooth extraction for a minimum of 3 months. Following healing of the soft tissue, animals received a staged, bilateral hemimandibulectomy using a submandibular approach. There was a 5-week delay between each mandibulectomy received by the same primate. The mandibular ostectomy produced a complete discontinuity defect including the mandibular inferior border with the resected area measuring in excess of 2.5 cm in antero–posterior length. The defect was filled with rhBMP-2/CCS at 0.75 or 1.3 mg/cc, stabilized with a titanium reconstruction plate, and the tissues were closed in layers. Tetracycline labels were given 3 weeks after resection/reconstruction to measure new bone growth and 16 weeks later in order to label bone turnover. The animals were maintained on a soft diet and observed for 6 months post final reconstruction. Animals were then euthanized with intravenous pentobarbital, underwent cannulation of bilateral carotid arteries and were perfused with 10% formalin. The manAAOMS • 2012