Adenovirus-mediated bone morphogenetic protein-2 gene transfection of bone marrow mesenchymal stem cells combined with nano-hydroxyapatite to construct bone graft material in vitro

Figures & data

Figure 1. Nano-hydroxyapatite artificial bone under inverted microscope.

Figure 2. Nano-hydroxyapatite artificial bone under scanning electron microscope.

Table

Main reagents and apparatus
Main reagents and apparatus	Source
Ad-BMP-2 and adenovirus Ad-GFP	Constructed and preserved by the Second People's Hospital of Shenzhen
Trizol Reagent	Invitrogen Corporation
DMEM-LG and fetal bovine serum, Percoll separating solution, trypsin, Double Antibody	Hyclon Corporation
Dexamethasone, β-glyceronephosphate, ascorbic acid, MTT solution and dimethyl sulfoxide	Sigma Corporation
Mouse-anti-human I antibody and goat-anti-mouse II antibody IgG -HRP	Santa Cruz Corporation
S-P hypersensitivity reagent kit	Fuzhou Maixin Biotechnology Corporation
Universal ECL western blot	GENMED Corporation
Chemiluminescence solution

Table I. MTT analysis of bone marrow mesenchymal stem cell proliferation (_x ± s, n = 12, A value).

Group	2 d	4 d
Nano-hydroxyapatite	0.135 ± 0.009	0.274 ± 0.013
Control	0.133 ± 0.011	0.269 ± 0.015
t	0.487	0.873
P	> 0.05	> 0.05
Group	6 d	8 d
Nano-hydroxyapatite	0.379 ± 0.011	0.489 ± 0.012
Control	0.383 ± 0.013	0.482 ± 0.015
t	0.975	1.262

Figure 3. Primary cells covered the bottom of culture flask, orderly arranged along long axis of cells body.

Figure 4. Osteogenic induction for 21 d, alizarin red staining of calcium nodules were red.

Figure 5. Cells transfected by adenovirus-green fluorescent protein 48 hour were almost green fluorescent.

Figure 6. Transfection 72 hour, bone morphogenetic protein-2 immunohistochemical staining showed positive expression in cytoplasm.

Figure 7. Untransfected cells were negative for bone morphogenetic protein-2 immunohistochemical staining.

Figure 8. Western blot detection of bone morphogenetic protein-2 expression.

Figure 9. Compound for 3 d, transfected bone marrow mesenchymal stem cells adhere and extend good in the lacunae and loophole of nano-hydroxyapatite material.

Figure 10. Compound for 5 d, transfected bone marrow mesenchymal stem cells fully spread and extend pseudopodia on the surface of nano-hydroxyapatite A material, calcium deposit can be seen around the cells.

Figure 11. Western blot detection of bone morphogenetic protein-2 expression in cells.