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Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells.

Shen Y, Qiao H, Fan Q, Zhang S, Tang T - Biomed Res Int (2015)

Bottom Line: Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority.Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection.These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 20011, China.

ABSTRACT
Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority. We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection. Ethidium bromide (EB)/Calcein AM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicated that all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly, the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experiments demonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4 weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenic induction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

No MeSH data available.


The morphology of APA microcapsules and the viability of various microencapsulated cells at 1 week or 4 weeks after microencapsulation (EB/Calcein AM staining). (a) The morphology of APA microcapsules. (A) indicates the microcapsules without cells and (B) indicates the microcapsules containing the BMSCs. Scale: 100 μm. (b) The viability of different microencapsulated tissue-derived mesenchymal stem cells at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: BMSCs, middle: ADSCs, and right: SMSCs. Scale: 20 μm. (c) The viability of different microencapsulated cells lines at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: C3H10T1/2, middle: C2C12, and right: NIH/3T3. Scale: 20 μm.
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fig2: The morphology of APA microcapsules and the viability of various microencapsulated cells at 1 week or 4 weeks after microencapsulation (EB/Calcein AM staining). (a) The morphology of APA microcapsules. (A) indicates the microcapsules without cells and (B) indicates the microcapsules containing the BMSCs. Scale: 100 μm. (b) The viability of different microencapsulated tissue-derived mesenchymal stem cells at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: BMSCs, middle: ADSCs, and right: SMSCs. Scale: 20 μm. (c) The viability of different microencapsulated cells lines at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: C3H10T1/2, middle: C2C12, and right: NIH/3T3. Scale: 20 μm.

Mentions: The microcapsules with or without entrapped nontransfected cells generally exhibited a smooth and spherical shape with diameters of approximately 250 μm (Figure 2(a)). This uniform structure enabled the collection of results that were sufficiently reliable. Further EB/Calcein AM staining revealed that, after the microencapsulation of various cell categories, the entrapped cells were capable of surviving at least 4 weeks with no evident cell death (Figures 2(b) and 2(c)), meaning that a 4-week span of observation was able to comprehensively evaluate the transition of cytokines expressions.


Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells.

Shen Y, Qiao H, Fan Q, Zhang S, Tang T - Biomed Res Int (2015)

The morphology of APA microcapsules and the viability of various microencapsulated cells at 1 week or 4 weeks after microencapsulation (EB/Calcein AM staining). (a) The morphology of APA microcapsules. (A) indicates the microcapsules without cells and (B) indicates the microcapsules containing the BMSCs. Scale: 100 μm. (b) The viability of different microencapsulated tissue-derived mesenchymal stem cells at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: BMSCs, middle: ADSCs, and right: SMSCs. Scale: 20 μm. (c) The viability of different microencapsulated cells lines at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: C3H10T1/2, middle: C2C12, and right: NIH/3T3. Scale: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588358&req=5

fig2: The morphology of APA microcapsules and the viability of various microencapsulated cells at 1 week or 4 weeks after microencapsulation (EB/Calcein AM staining). (a) The morphology of APA microcapsules. (A) indicates the microcapsules without cells and (B) indicates the microcapsules containing the BMSCs. Scale: 100 μm. (b) The viability of different microencapsulated tissue-derived mesenchymal stem cells at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: BMSCs, middle: ADSCs, and right: SMSCs. Scale: 20 μm. (c) The viability of different microencapsulated cells lines at 1 week and 4 weeks (EB/Calcein AM staining). The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. Left: C3H10T1/2, middle: C2C12, and right: NIH/3T3. Scale: 20 μm.
Mentions: The microcapsules with or without entrapped nontransfected cells generally exhibited a smooth and spherical shape with diameters of approximately 250 μm (Figure 2(a)). This uniform structure enabled the collection of results that were sufficiently reliable. Further EB/Calcein AM staining revealed that, after the microencapsulation of various cell categories, the entrapped cells were capable of surviving at least 4 weeks with no evident cell death (Figures 2(b) and 2(c)), meaning that a 4-week span of observation was able to comprehensively evaluate the transition of cytokines expressions.

Bottom Line: Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority.Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection.These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 20011, China.

ABSTRACT
Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority. We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection. Ethidium bromide (EB)/Calcein AM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicated that all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly, the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experiments demonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4 weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenic induction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

No MeSH data available.