A composite construct comprising of a bone mesenchymal stem cell (BMSC) sheet, plasmid DNA, encoding human bone morphogenic protein-2 (hBMP-2), and poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) sponge was design...A composite construct comprising of a bone mesenchymal stem cell (BMSC) sheet, plasmid DNA, encoding human bone morphogenic protein-2 (hBMP-2), and poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) sponge was designed and employed in the restoration of rat calvarial defects. To improve gene transfection efficiency, a cationic chitosan derivative, N,N,N,-trimethyl chitosan chloride (TMC), was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 13% in rat BMSCs, resulting in heterogeneous hBMP-2 expression in a 10-d culture period in vitro. In vivo culture of the composite constructs was performed by implantation into rat full-thickness calvarial defects, using constructs lacking pDNA-hBMP-2 or BMSC sheets as controls. Significantly higher heterogeneous expression of hBMP-2 was detected in vivo at 2 weeks for the cell sheet/DNA complex/scaffold constructs, compared with the constructs lacking pDNA-hBMP-2 or BMSC sheets. New bone formation was evident as early as 4 weeks in the experimental constructs. At 8 weeks, partial bridging of calvarial defects was observed in the experimental constructs, which was significantly better than the constructs lacking pDNA-hBMP-2 or BMSC sheets. Therefore, the combination of the PLGA/HA scaffold with BMSC sheets and gene therapy vectors is effective at enhancing bone formation.展开更多
Background The initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we develope...Background The initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate （ACP）/poly（L-lactic acid） （PLLA） material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated. Methods A porous ACP/PLLA composite and PLLA scaffold （as a control） were incubated in fetal bovine serum （FBS） containing phosphate-buffered saline （PBS）, and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy （SEM）. The expression levels of the genes encoding integrin subunits αl, α5, αv, β1, focal adhesion kinase （FAK）, and glyceraldehyde-3-phosphate dehydrogenase （GAPDH） were determined using real-time reverse transcription polymerase chain reaction （RT-PCR）. Results The ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits αl, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite. Conclusions The ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits αl, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.展开更多
基金supported by the National Natural Science Foundation of China (20934003)the Science Technology Program of Zhejiang Province (2009C14003, 2009C13020)the National Basic Research Program of China (2011CB606203)
文摘A composite construct comprising of a bone mesenchymal stem cell (BMSC) sheet, plasmid DNA, encoding human bone morphogenic protein-2 (hBMP-2), and poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) sponge was designed and employed in the restoration of rat calvarial defects. To improve gene transfection efficiency, a cationic chitosan derivative, N,N,N,-trimethyl chitosan chloride (TMC), was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 13% in rat BMSCs, resulting in heterogeneous hBMP-2 expression in a 10-d culture period in vitro. In vivo culture of the composite constructs was performed by implantation into rat full-thickness calvarial defects, using constructs lacking pDNA-hBMP-2 or BMSC sheets as controls. Significantly higher heterogeneous expression of hBMP-2 was detected in vivo at 2 weeks for the cell sheet/DNA complex/scaffold constructs, compared with the constructs lacking pDNA-hBMP-2 or BMSC sheets. New bone formation was evident as early as 4 weeks in the experimental constructs. At 8 weeks, partial bridging of calvarial defects was observed in the experimental constructs, which was significantly better than the constructs lacking pDNA-hBMP-2 or BMSC sheets. Therefore, the combination of the PLGA/HA scaffold with BMSC sheets and gene therapy vectors is effective at enhancing bone formation.
基金The present study was supported by grants from the Natural Science Foundation of China （No. 81201414 and No. 81101377）, the Doctoral Fund of the Ministry of Education of China （No. 20100101120132）, and the Natural Science Grants of the Zhejiang Province （No. LQ14H060001）.
文摘Background The initial osteoblastic adhesion to materials characterizes the first phase of cell-material interactions and influences all the events leading to the formation of new bone. In a previous work, we developed a novel amorphous calcium phosphate （ACP）/poly（L-lactic acid） （PLLA） material that demonstrated morphologic variations in its microstructure. The aim of this study was to investigate the initial interaction between this material and osteoblastic cells. Cellular attachment and the corresponding signal transduction pathways were investigated. Methods A porous ACP/PLLA composite and PLLA scaffold （as a control） were incubated in fetal bovine serum （FBS） containing phosphate-buffered saline （PBS）, and the protein adsorption was determined. Osteoblastic MG63 cells were seeded on the materials and cultured for 1, 4, 8, or 24 hours. Cell attachment was evaluated using the MTS method. Cell morphology was examined using scanning electron microscopy （SEM）. The expression levels of the genes encoding integrin subunits αl, α5, αv, β1, focal adhesion kinase （FAK）, and glyceraldehyde-3-phosphate dehydrogenase （GAPDH） were determined using real-time reverse transcription polymerase chain reaction （RT-PCR）. Results The ACP/PLLA material significantly increased the protein adsorption by 6.4-fold at 1 hour and 2.4-fold at 24 hours, compared with the pure PLLA scaffold. The attachment of osteoblastic cells to the ACP/PLLA was significantly higher than that on the PLLA scaffold. The SEM observation revealed a polygonal spread shape of cells on the ACP/ PLLA, with the filopodia adhered to the scaffold surface. In contrast, the cells on the PLLA scaffold exhibited a spherical or polygonal morphology. Additionally, real-time RT-PCR showed that the genes encoding the integrin subunits αl, αv, β1, and FAK were expressed at higher levels on the ACP/PLLA composite. Conclusions The ACP/PLLA composite promoted protein adsorption and osteoblastic adhesion. The enhanced cell adhesion may be mediated by the binding of integrin subunits αl, αv, and β1, and subsequently may be regulated through the FAK signal transduction pathways.
