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Mediators of endothelial cell injury in low perfusion conditions
PLASTIC SURGERY II METHODS: ADMSCs were harvested from 3 week old female FVB mice. ADMSCs were cultured in standard osteogenic media with various concentrations of BMP-2 or retinoic acid (RA) for 3 weeks. Growth and differentiation were assessed via cell counting, Oil Red O (ORO), alkaline phosphatase (AP), and von Kossa (VK) staining. Apatite-coated poly-lactic co-glycolic acid (PLGA) scaffolds were then seeded with ADMSCs and implanted into critical sized (4mm) calvarial defects in adult male mice. Calvarial regeneration was assessed using histology and radiography.
CRANIOFACIAL/BONE/TISSUE ENGINEERING/ MICROVASCULAR/FLAPS/ OTOLARYNGOLOGY Mediators of endothelial cell injury in low perfusion conditions Julie C Philp BA, Zhen Wang MD, Ashley Chi MD, PhD, Patrick Brown PhD, Michael T Longaker MD, MBA, FACS, George Yang MD, PhD Stanford Medical School Stanford, CA
RESULTS: Only slight differences in proliferation were seen in response to BMP2, RA, or BMP2 ⫹ RA. ORO staining was dramatically decreased in treated cells. AP and von Kossa staining were induced in a synergistic fashion by BMP2 ⫹ RA. In vivo experiments revealed successful healing of calvarial defects by 12 weeks after surgery. ADMSC-seeded scaffolds produced significant intramembranous bone formation by 2 weeks, and areas of complete bony bridging by 12 weeks as demonstrated by X-ray and histology.
INTRODUCTION: During free tissue transfer operations, endothelial cells (EC) are exposed to a period of ischemia that can affect outcome. We compared gene expression profiles of ECs in normal culture conditions to serum-deprived conditions as an in vitro model of tissue stress. METHODS: ECs from multiple body sites were grown in EGM2MV with 5% fetal bovine serum (FCS). For serum-deprived samples, ECs were switched to EGM-2MV with 0.25% FCS. After 24h, ECs were harvested and mRNA was extracted. Gene expression patterns were compared between the normal and serum-deprived samples using cDNA microarray. Specific gene expression was then confirmed by quantitative PCR.
CONCLUSIONS: Our data demonstrate successful osteogenic differentiation of ADMSCs in vitro and in vivo. This novel mouse model potentiates further investigation into the mechanisms governing osteogenesis and the epigenetic manipulations that can be used to enhance this process.
RESULTS: There are 1083 genes that show more than a four fold difference in expression when comparing normal versus serum-deprived growth conditions. ECs exposed to serum deprivation up regulate inflammatory and coagulation factor genes while cell cycle genes are down regulated. Up regulated inflammatory mediators included MCP-1, interferon-alpha, TGF-alpha, colony stimulating factor 2, TNFC, and TNF receptor-associated factor 5. Coagulation factors included thrombin and the thrombin receptor. The down regulated cell cycle genes included cyclin B2 and c-myc binding protein.
Gene expression patterns during palatal shelf fusion Kenton Fong MD, David Sahar MD, Randall Nacamuli MD, Michael T Longaker MD, MBA, FACS Stanford University School of Medicine Stanford, CA INTRODUCTION: Cleft palate is a common disorder that occurs when there is failure of the palatal shelves to fuse during normal development. The genes mediating normal palatal fusion are largely unknown.
CONCLUSIONS: Our data suggest that serum deprivation induces gene expression of inflammatory mediators and coagulation factors that can lead to poor outcome during tissue transfer. Understanding the mechanism of EC injury may lead to the development of rationally designed therapies aimed at minimizing complications and improving outcomes.
METHODS: We harvested mRNA and performed microarray (⬃40,000 element) analysis of mice palatal shelves at timepoints before, during, and after palatal fusion (E13.5, E14.5, and E15.5). IHC was performed using standard techniques. RESULTS: Replicates clustered together by time point indicating consistency between harvests. Hox-7A, a gene implicated in both mice and humans as important to cleft lip and palate formation was upregulated during palatal fusion. Several retinoic acid responsive genes such as Stra6 were also upregulated during fusion. Furthermore, we found the homeobox gene Lim-1 to be upregulated during palatal fusion. To confirm our microarray findings of differential expression of LIM-1 during palate formation, we performed immunohistochemistry. During early secondary palate formation (E12.5), as the shelves are pointing inferiorly, LIM-1 expression was maximal at the lateral wall and tip of the palate epithelia. Interestingly, minimal expression was noted in the medial palatal wall epithelia. LIM-1 expression regressed with epithelial to mesenchymal transition at the midline epithelial seam as the approaching palates fuse on E14.5. LIM-1 expression was completely abolished within the palate mesenchyme by E16.5 after completion of fusion.
Mouse adipose-derived mesenchymal stromal cells undergo osteogenic differentiation in vitro and in vivo Yun-Ying Shi BS, Randall Nacamuli MD, Ali Salim MD, Oliver Aalami MD, Catherine Cowan MS, Michael T Longaker MD, MBA, FACS Stanford University Stanford, CA INTRODUCTION: Although the multilineage potential of human processed lipoaspirate has previously been demonstrated, few studies exist utilizing mouse adipose-derived mesenchymal cells (ADMSCs) in vitro or in vivo. Here, we develop a method for osteogenic differentiation of ADMSCs in vitro and utilize them to repair calvarial defects in vivo.
© 2004 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/04/$30.00
S62
CRANIOFACIAL/BONE/TISSUE ENGINEERING/ MICROVASCULAR/FLAPS/ OTOLARYNGOLOGY Mediators of endothelial cell injury in low perfusion conditions Julie C Philp BA, Zhen Wang MD, Ashley Chi MD, PhD, Patrick Brown PhD, Michael T Longaker MD, MBA, FACS, George Yang MD, PhD Stanford Medical School Stanford, CA
RESULTS: Only slight differences in proliferation were seen in response to BMP2, RA, or BMP2 ⫹ RA. ORO staining was dramatically decreased in treated cells. AP and von Kossa staining were induced in a synergistic fashion by BMP2 ⫹ RA. In vivo experiments revealed successful healing of calvarial defects by 12 weeks after surgery. ADMSC-seeded scaffolds produced significant intramembranous bone formation by 2 weeks, and areas of complete bony bridging by 12 weeks as demonstrated by X-ray and histology.
INTRODUCTION: During free tissue transfer operations, endothelial cells (EC) are exposed to a period of ischemia that can affect outcome. We compared gene expression profiles of ECs in normal culture conditions to serum-deprived conditions as an in vitro model of tissue stress. METHODS: ECs from multiple body sites were grown in EGM2MV with 5% fetal bovine serum (FCS). For serum-deprived samples, ECs were switched to EGM-2MV with 0.25% FCS. After 24h, ECs were harvested and mRNA was extracted. Gene expression patterns were compared between the normal and serum-deprived samples using cDNA microarray. Specific gene expression was then confirmed by quantitative PCR.
CONCLUSIONS: Our data demonstrate successful osteogenic differentiation of ADMSCs in vitro and in vivo. This novel mouse model potentiates further investigation into the mechanisms governing osteogenesis and the epigenetic manipulations that can be used to enhance this process.
RESULTS: There are 1083 genes that show more than a four fold difference in expression when comparing normal versus serum-deprived growth conditions. ECs exposed to serum deprivation up regulate inflammatory and coagulation factor genes while cell cycle genes are down regulated. Up regulated inflammatory mediators included MCP-1, interferon-alpha, TGF-alpha, colony stimulating factor 2, TNFC, and TNF receptor-associated factor 5. Coagulation factors included thrombin and the thrombin receptor. The down regulated cell cycle genes included cyclin B2 and c-myc binding protein.
Gene expression patterns during palatal shelf fusion Kenton Fong MD, David Sahar MD, Randall Nacamuli MD, Michael T Longaker MD, MBA, FACS Stanford University School of Medicine Stanford, CA INTRODUCTION: Cleft palate is a common disorder that occurs when there is failure of the palatal shelves to fuse during normal development. The genes mediating normal palatal fusion are largely unknown.
CONCLUSIONS: Our data suggest that serum deprivation induces gene expression of inflammatory mediators and coagulation factors that can lead to poor outcome during tissue transfer. Understanding the mechanism of EC injury may lead to the development of rationally designed therapies aimed at minimizing complications and improving outcomes.
METHODS: We harvested mRNA and performed microarray (⬃40,000 element) analysis of mice palatal shelves at timepoints before, during, and after palatal fusion (E13.5, E14.5, and E15.5). IHC was performed using standard techniques. RESULTS: Replicates clustered together by time point indicating consistency between harvests. Hox-7A, a gene implicated in both mice and humans as important to cleft lip and palate formation was upregulated during palatal fusion. Several retinoic acid responsive genes such as Stra6 were also upregulated during fusion. Furthermore, we found the homeobox gene Lim-1 to be upregulated during palatal fusion. To confirm our microarray findings of differential expression of LIM-1 during palate formation, we performed immunohistochemistry. During early secondary palate formation (E12.5), as the shelves are pointing inferiorly, LIM-1 expression was maximal at the lateral wall and tip of the palate epithelia. Interestingly, minimal expression was noted in the medial palatal wall epithelia. LIM-1 expression regressed with epithelial to mesenchymal transition at the midline epithelial seam as the approaching palates fuse on E14.5. LIM-1 expression was completely abolished within the palate mesenchyme by E16.5 after completion of fusion.
Mouse adipose-derived mesenchymal stromal cells undergo osteogenic differentiation in vitro and in vivo Yun-Ying Shi BS, Randall Nacamuli MD, Ali Salim MD, Oliver Aalami MD, Catherine Cowan MS, Michael T Longaker MD, MBA, FACS Stanford University Stanford, CA INTRODUCTION: Although the multilineage potential of human processed lipoaspirate has previously been demonstrated, few studies exist utilizing mouse adipose-derived mesenchymal cells (ADMSCs) in vitro or in vivo. Here, we develop a method for osteogenic differentiation of ADMSCs in vitro and utilize them to repair calvarial defects in vivo.
© 2004 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/04/$30.00
S62