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Factors influencing bone formation capacity of human embryonic stem cell-derived osteoblast-like cells
e16 Abstracts Protein-7 (BMP-7). An intraperitoneal fluorochrome bone labeling was performed. Computed tomography was performed in vivo every 4 weeks. The animals were sacrificed 14 weeks after surgery. Specimens were evaluated by Environmental Scanning Electron Microscopy and by fluorescence microscopy. Osteoclasts were evaluated by a TRAP staining. Results: Newly formed bone was seen in all implants, even in absence of BMP. Bone formation (p = 0.001) and ceramic degradation (p = 0.001) was enhanced in presence of BMP-7 in the HA and in the HA60 group. In those groups osteoclasts were observed. BMP-7 did not show any statistical effect on ceramic degradation after 14 weeks on TCP, no osteoclasts were observed. Conclusions: All studied ceramics were osteoinductive. HA and HA/TCP ceramics are suitable carriers for BMP-7. BMP accelerates bone resorption, while bone formation will be enhanced. The fast breakdown of TCP is interpreted as a result of degradation as no osteoclasts were seen. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1064 39 Tridimentional collagen:elastin matrices as scaffold for soft tissue reconstruction: matrix:tissue integration study D. Parreira 1 , G. Goissis 2 , S. Suzigan 3 , J.V. Maniglia 4 1
Ear, Nose & Throat Department, University of Brasilia Medical School, UNB, Brasilia, Brazil 2 Chemistry Institute, University of São Paulo, São Carlos, Brazil 3 Anatomy Department, Brazil 4 Ear, Nose & Throat and Head and Neck Surgery Department, São José do Rio Preto Medical School, FAMERP, São José do Rio Preto, Brazil
Introduction: This work studied the integration of acellular polyanionic collagen:elastin matrices derived of bovine pericardium (BP) with variable negative charge. Objective: The purpose of this work was to evaluate the use of modified collagen matrices for soft tissue reconstruction. Materials and methods: Negative charges were introduced in the material by selective hydrolysis of carboxamide side chain groups from Asn and Gln present in the primary structure of the protein. Hydrolyzed materials after 24 and 48 h of treatment, respectively with 46± and 87± extra negative charges, were studied. Implants were placed in the subcutaneous of rats for periods of 14, 60, 120 and 180 days. Materials were characterized by differential scanning calorimetry, SEM and TEM, and explants analysed by optical microscopy (H.E., Masson tricromic and Verhoeff stains). Results: Differently from native tissue (BP), the biological response of polyanionic collagen:elastin matrices after 14 days from implantation was characterized by a progressive decrease in fibrosis, but most important, no characteristic cells of a chronic inflammatory response were observed. After 180 days, most of the implants were integrated to the implant region. Conclusion: The results suggest that acellular collagen:elastin matrices prepared by devitalization of natural tissue due to their high degree of biocompatibility and integration may be potentially useful as a scaffold for soft tissue reconstruction. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1065
40 Hedgehog signaling pathway modulates osteogenic differentiation in novel oxysterol-conditioned rabbit bone marrow stromal cells S.C. Sorice 1,∗ , A. Hokugo 1 , K. Fan 1 , V. Meliton 2 , P. Zuk 1 , W. Huang 3 , T. Miller 1,3 , F. Parhami 2 , R. Jarrahy 1 1
Plastic Surgery, USA Medicine, David Geffen School of Medicine at UCLA, USA 3 Plastic Surgery, Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, CA, USA 2
Purpose: To determine the osteogenic capacity of a novel growth factor on rabbit bone marrow stromal cells. Background: Current reconstructive techniques for complex craniofacial osseous defects are associated with significant morbidity profiles. Tissue engineering solutions offer an alternative to these techniques. Bone morphogenetic protein (BMP) is an effective osteoinductive growth factor, but its clinical application is limited by exorbitant cost and undesirable side effects. Oxysterols are naturally occurring cholesterol oxidation products that are capable of inducing osteogenic differentiation. The effects of oxysterol on rabbit-bone marrow stromal cells (BMSC) have not been described. Methods: Rabbit BMSCs were incubated with various concentrations of a novel oxysterol or BMP-2. Alkaline phosphatase (ALP) activity, expression of markers of osteogenic differentiation, and in vitro calcification assays were performed. To determine the mechanism of action of oxysterol, rabbit BMSC conditioned with oxysterol were incubated with various concentrations of the Hedgehog (Hh) inhibitor cyclopamine and ALP activity was measured. Results: ALP activity in rabbit BMSCs treated with oxysterol was significantly higher than in controls and was equivalent to activity in cells treated with BMP-2. Gene expression of osteogenic markers in BMSCs treated with oxysterol was significantly higher than in control groups. Addition of cyclopamine to cultures negated the positive effect of oxysterol on ALP activity. Conclusion: Oxysterols can induce osteogenesis in vitro in rabbit BMSCs with an efficacy similar to BMP-2. This is in part mediated through the Hh signaling pathway. Oxysterols may represent a viable alternative to BMP-2 in bone tissue engineering models. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1066 41 Factors influencing bone formation capacity of human embryonic stem cell-derived osteoblast-like cells P. Arpornmaeklong 1,∗ , M.J. Pressler 2 , P.H. Krebsbach 2 1
Oral and Maxillofacial Surgery, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Thailand 2 Biologic and Materials Sciences, School of Dentistry, The University of Michigan, Ann Arbor, MI, USA To apply human embryonic stem cells (hESCs) as a source of osteoblast-like cells (hESC-OS), factors influencing bone formation potential of hESC-OS need to be evaluated. Aims: The study aimed to investigate effects of implantation sites and biomaterials on bone formation capacity of hESC-OS.
Abstracts Materials and methods: Human ESC-OS were seeded on various biomaterials including porous collagen type I and polycarpolactone/-tricalcium phosphate scaffolds, gelatin sponges and -TCP particles. Samples were subcutaneously transplanted in immunodeficient mice for 6 weeks. Human ESC-OS on -TCP particles were transplanted in calvarial defects of immunodeficient rats for 6 weeks as a positive control. Bone formation and mineralization were investigated using soft radiograph, micro-computer tomography, polarized light microscope, histological analysis, von Kossa and immunohistochemical staining and qRT-PCR. Oligonucleotide microarry was applied to compare expression of genes related to osteogenesis of hESC-OS with osteoblast-like cells derived from human bone marrow stromal cells (hBMSC-OS). Results: Human ESC-OS deposited bone on -TCP particles in rat calvarial defects. Only mineralization of bone matrixes on the scaffolds and expression of human osteoblast-related genes were detected in subcutaneous transplantation. In comparison to hBMSC-OS, microarray analysis demonstrated lower expression levels of genes related to osteogenesis and mineralization, such as MSX1, TGFBR1, BGLAP, IBSP, BMP4, BMP6 and wnt1 and higher levels of osteogenesis inhibitor, DKK3 of hESC-OS. Conclusion: Bone formation capacity of hESC-OS are influenced by implantation sites. Human ESC-OS required osteogenic stimuli from local host cells to enhance their bone formation capacity. Local stimuli are essential to survival, growth and differentiation of transplanted hESC-OS. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1067 42 Potential additive effect of BMP-2 and a novel oxysterol in inducing osteogenic differentiation in rabbit bone marrow stromal cells S.C. Sorice 1,∗ , A. Hokugo 1 , K. Fan 1 , P. Zuk 1 , W. Huang 2 , T. Miller 1,2 , R. Jarrahy 1 1
Plastic Surgery, David Geffen School of Medicine at UCLA, USA Plastic Surgery, Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, CA, USA
2
Purpose: To determine the osteogenic capacity of combining BMP-2 and a novel growth factor in rabbit bone marrow stromal cells. Background: Current treatments of complex craniofacial osseous defects are limited by significant morbidity profiles. Tissue engineering solutions offer an alternative to these techniques. Large quantities of BMP are necessary for clinical efficacy resulting in exorbitant cost and undesirable side effects like heterotropic ossification. Oxysterols are osteoinductive cholesterol oxidation products. Synergistic effects of BMP-2 and oxysterol
e17
on osteogenic differentiation have been described in murine mesenchymal stem cells (MSCs). Methods: Rabbit BMSCs were incubated with various concentrations of a novel oxysterol, BMP-2, or a combination at various concentrations. Alkaline phosphatase (ALP) activity was performed. To determine the combination mechanism of action of BMP-2 and oxysterol, rabbit BMSC conditioned with oxysterol and BMP-2 were incubated with a Hedgehog (Hh) inhibitor cyclopamine and ALP activity was measured. Results: ALP activity in oxysterol treated rabbit BMSCs was higher than in controls and was equivalent to those with BMP-2. ALP activity of rabbit BMSCs treated with the combination was higher than in single drug groups at all concentrations, demonstrating an additive effect of the two growth factors. Cyclopamine negated the positive effect of oxysterol on ALP, but did not decrease this to baseline. Conclusion: Oxysterols and BMP-2 appear to have an additive effect on osteogenesis in vitro in rabbit BMSCs. This osteogenic activity is in part mediated through the Hh signaling pathway. The combination may allow decreasing the necessary dosage of BMP-2 to achieve osteogenesis. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1068 Topic: TMJ and Pain Interpositional arthroplasty using iliac crest graft for TMJ ankylosis in a child: report of a case 43 ˜ Alsina ∗ , J.R. Velazquez, M.A. Fonseca Dìaz P.L. Acuna Pontificia Universidad Catolica Argentina, Buenos Aires, Argentina Reconstruction of the temporomandibular joint (TMJ) in a growing child after release of ankylosis is a challenging problem in maxillofacial surgery. One of the goals of ideal reconstruction is to maintain normal growth and development of the face using an active growth centre. This case presentation refers to a 11-year-old child, which was diagnosed with an unilateral TMJ ankylosis. After the release of ankylosis mass, the reconstruction was performed using an iliac crest bone graft that is capable of active growth due to his intrinsic growth potential. This presentation refers to the experience using this type of autogenous graft and a 2-year follow up registration of the case. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1069
Ear, Nose & Throat Department, University of Brasilia Medical School, UNB, Brasilia, Brazil 2 Chemistry Institute, University of São Paulo, São Carlos, Brazil 3 Anatomy Department, Brazil 4 Ear, Nose & Throat and Head and Neck Surgery Department, São José do Rio Preto Medical School, FAMERP, São José do Rio Preto, Brazil
Introduction: This work studied the integration of acellular polyanionic collagen:elastin matrices derived of bovine pericardium (BP) with variable negative charge. Objective: The purpose of this work was to evaluate the use of modified collagen matrices for soft tissue reconstruction. Materials and methods: Negative charges were introduced in the material by selective hydrolysis of carboxamide side chain groups from Asn and Gln present in the primary structure of the protein. Hydrolyzed materials after 24 and 48 h of treatment, respectively with 46± and 87± extra negative charges, were studied. Implants were placed in the subcutaneous of rats for periods of 14, 60, 120 and 180 days. Materials were characterized by differential scanning calorimetry, SEM and TEM, and explants analysed by optical microscopy (H.E., Masson tricromic and Verhoeff stains). Results: Differently from native tissue (BP), the biological response of polyanionic collagen:elastin matrices after 14 days from implantation was characterized by a progressive decrease in fibrosis, but most important, no characteristic cells of a chronic inflammatory response were observed. After 180 days, most of the implants were integrated to the implant region. Conclusion: The results suggest that acellular collagen:elastin matrices prepared by devitalization of natural tissue due to their high degree of biocompatibility and integration may be potentially useful as a scaffold for soft tissue reconstruction. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1065
40 Hedgehog signaling pathway modulates osteogenic differentiation in novel oxysterol-conditioned rabbit bone marrow stromal cells S.C. Sorice 1,∗ , A. Hokugo 1 , K. Fan 1 , V. Meliton 2 , P. Zuk 1 , W. Huang 3 , T. Miller 1,3 , F. Parhami 2 , R. Jarrahy 1 1
Plastic Surgery, USA Medicine, David Geffen School of Medicine at UCLA, USA 3 Plastic Surgery, Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, CA, USA 2
Purpose: To determine the osteogenic capacity of a novel growth factor on rabbit bone marrow stromal cells. Background: Current reconstructive techniques for complex craniofacial osseous defects are associated with significant morbidity profiles. Tissue engineering solutions offer an alternative to these techniques. Bone morphogenetic protein (BMP) is an effective osteoinductive growth factor, but its clinical application is limited by exorbitant cost and undesirable side effects. Oxysterols are naturally occurring cholesterol oxidation products that are capable of inducing osteogenic differentiation. The effects of oxysterol on rabbit-bone marrow stromal cells (BMSC) have not been described. Methods: Rabbit BMSCs were incubated with various concentrations of a novel oxysterol or BMP-2. Alkaline phosphatase (ALP) activity, expression of markers of osteogenic differentiation, and in vitro calcification assays were performed. To determine the mechanism of action of oxysterol, rabbit BMSC conditioned with oxysterol were incubated with various concentrations of the Hedgehog (Hh) inhibitor cyclopamine and ALP activity was measured. Results: ALP activity in rabbit BMSCs treated with oxysterol was significantly higher than in controls and was equivalent to activity in cells treated with BMP-2. Gene expression of osteogenic markers in BMSCs treated with oxysterol was significantly higher than in control groups. Addition of cyclopamine to cultures negated the positive effect of oxysterol on ALP activity. Conclusion: Oxysterols can induce osteogenesis in vitro in rabbit BMSCs with an efficacy similar to BMP-2. This is in part mediated through the Hh signaling pathway. Oxysterols may represent a viable alternative to BMP-2 in bone tissue engineering models. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1066 41 Factors influencing bone formation capacity of human embryonic stem cell-derived osteoblast-like cells P. Arpornmaeklong 1,∗ , M.J. Pressler 2 , P.H. Krebsbach 2 1
Oral and Maxillofacial Surgery, Faculty of Dentistry, Prince of Songkla University, Hat Yai, Thailand 2 Biologic and Materials Sciences, School of Dentistry, The University of Michigan, Ann Arbor, MI, USA To apply human embryonic stem cells (hESCs) as a source of osteoblast-like cells (hESC-OS), factors influencing bone formation potential of hESC-OS need to be evaluated. Aims: The study aimed to investigate effects of implantation sites and biomaterials on bone formation capacity of hESC-OS.
Abstracts Materials and methods: Human ESC-OS were seeded on various biomaterials including porous collagen type I and polycarpolactone/-tricalcium phosphate scaffolds, gelatin sponges and -TCP particles. Samples were subcutaneously transplanted in immunodeficient mice for 6 weeks. Human ESC-OS on -TCP particles were transplanted in calvarial defects of immunodeficient rats for 6 weeks as a positive control. Bone formation and mineralization were investigated using soft radiograph, micro-computer tomography, polarized light microscope, histological analysis, von Kossa and immunohistochemical staining and qRT-PCR. Oligonucleotide microarry was applied to compare expression of genes related to osteogenesis of hESC-OS with osteoblast-like cells derived from human bone marrow stromal cells (hBMSC-OS). Results: Human ESC-OS deposited bone on -TCP particles in rat calvarial defects. Only mineralization of bone matrixes on the scaffolds and expression of human osteoblast-related genes were detected in subcutaneous transplantation. In comparison to hBMSC-OS, microarray analysis demonstrated lower expression levels of genes related to osteogenesis and mineralization, such as MSX1, TGFBR1, BGLAP, IBSP, BMP4, BMP6 and wnt1 and higher levels of osteogenesis inhibitor, DKK3 of hESC-OS. Conclusion: Bone formation capacity of hESC-OS are influenced by implantation sites. Human ESC-OS required osteogenic stimuli from local host cells to enhance their bone formation capacity. Local stimuli are essential to survival, growth and differentiation of transplanted hESC-OS. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1067 42 Potential additive effect of BMP-2 and a novel oxysterol in inducing osteogenic differentiation in rabbit bone marrow stromal cells S.C. Sorice 1,∗ , A. Hokugo 1 , K. Fan 1 , P. Zuk 1 , W. Huang 2 , T. Miller 1,2 , R. Jarrahy 1 1
Plastic Surgery, David Geffen School of Medicine at UCLA, USA Plastic Surgery, Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, CA, USA
2
Purpose: To determine the osteogenic capacity of combining BMP-2 and a novel growth factor in rabbit bone marrow stromal cells. Background: Current treatments of complex craniofacial osseous defects are limited by significant morbidity profiles. Tissue engineering solutions offer an alternative to these techniques. Large quantities of BMP are necessary for clinical efficacy resulting in exorbitant cost and undesirable side effects like heterotropic ossification. Oxysterols are osteoinductive cholesterol oxidation products. Synergistic effects of BMP-2 and oxysterol
e17
on osteogenic differentiation have been described in murine mesenchymal stem cells (MSCs). Methods: Rabbit BMSCs were incubated with various concentrations of a novel oxysterol, BMP-2, or a combination at various concentrations. Alkaline phosphatase (ALP) activity was performed. To determine the combination mechanism of action of BMP-2 and oxysterol, rabbit BMSC conditioned with oxysterol and BMP-2 were incubated with a Hedgehog (Hh) inhibitor cyclopamine and ALP activity was measured. Results: ALP activity in oxysterol treated rabbit BMSCs was higher than in controls and was equivalent to those with BMP-2. ALP activity of rabbit BMSCs treated with the combination was higher than in single drug groups at all concentrations, demonstrating an additive effect of the two growth factors. Cyclopamine negated the positive effect of oxysterol on ALP, but did not decrease this to baseline. Conclusion: Oxysterols and BMP-2 appear to have an additive effect on osteogenesis in vitro in rabbit BMSCs. This osteogenic activity is in part mediated through the Hh signaling pathway. The combination may allow decreasing the necessary dosage of BMP-2 to achieve osteogenesis. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1068 Topic: TMJ and Pain Interpositional arthroplasty using iliac crest graft for TMJ ankylosis in a child: report of a case 43 ˜ Alsina ∗ , J.R. Velazquez, M.A. Fonseca Dìaz P.L. Acuna Pontificia Universidad Catolica Argentina, Buenos Aires, Argentina Reconstruction of the temporomandibular joint (TMJ) in a growing child after release of ankylosis is a challenging problem in maxillofacial surgery. One of the goals of ideal reconstruction is to maintain normal growth and development of the face using an active growth centre. This case presentation refers to a 11-year-old child, which was diagnosed with an unilateral TMJ ankylosis. After the release of ankylosis mass, the reconstruction was performed using an iliac crest bone graft that is capable of active growth due to his intrinsic growth potential. This presentation refers to the experience using this type of autogenous graft and a 2-year follow up registration of the case. Conflict of interest: None declared. doi:10.1016/j.ijom.2011.07.1069