In vitro effects of recombinant adenovirus-mediated bone morphogenetic protein 2/vascular endothelial growth factor 165 on osteogenic differentiation of bone marrow mesenchymal stem cells

Figures & data

Table 1. Primer sequences for real-time PCR.

Gene	Primer sequence (5′-3′)
β-Actin	Upstream: AGGGAAATCGTGCGTGAC
	Downstream: CGCTCGTTGCCAATAGTGA
	VEGF165	Upstream: GCCTTGCTGCTCTACCTCCAC
Downstream: GCACACAGGATGGCTTGAAGATG
BMP2		Upstream: CGTCAAGCCAAACACAAACAGC
	Downstream: GAGCCACAATCCAGTCATTCCAC
	OPN	Upstream: CCTCCCGGTGAAAGTGAC
Downstream: CTGTGGCGCAAGGAGATT
OPG		Upstream: GTTCCTGCACAGCTTCACAA
	Downstream: AAACAGCCCAGTGACCATTC
	OC	Upstream: CCTTCATGTCCAAGCAGGA
Downstream: GGCGGTCTTCAAGCCATAC

Figure 1. Fluorescence microscopic observation of C3H10T1/2 cells 48 h after infection (100×). (A): Ad-VEGF165 group; (B): Ad-BMP2 group; (C): Ad-VEGF165 + Ad-BMP2 group.

Figure 2. VEGF165 and BMP2 mRNA expression levels detected by real-time PCR (x±s, n = 3).

Table 2. VEGF165 and BMP2 mRNA expression levels detected by real-time PCR (x±s, n = 3).

	VEGF165		BMP2
	Group	D2	D7	D2	D7
Control	1.032 ± 0.121	1.021 ± 0.112	1.030 ± 0.132	1.011 ± 0.058
Ad-null	1.241 ± 0.915	1.421 ± 1.015	1.331 ± 0.981	1.562 ± 1.042
Ad-VEGF165	32.343 ± 5.453^a	11.187 ± 1.236^a	5.052 ± 1.045	4.616 ± 0.887
Ad-BMP2	7.165 ± 1.027	5.231 ± 0.892	54.184 ± 7.731^b	15.453 ± 4.765^b
Ad-VEGF165 + Ad-BMP2	36.374 ± 6.165^a	9.224 ± 1.037^a	50.067 ± 8.173^b	17.711 ± 5.147^b
F value	141.977	47.122	138.544	42.376
P values	0.000	0.000	0.000	0.000

^aCompared with control, Ad-null and Ad-BMP2 groups, P < 0.05.

^bCompared with control, Ad-null and Ad-VEGF165 groups, P < 0.05.

Figure 3. Detection of osteogenic indices by Western blot. 1: Control group; 2: Ad-null group; 3: Ad-VEGF165 group; 4: Ad-BMP2 group; 5: Ad-VEGF165 + Ad-BMP2 group.

Table 3. OC, OPN, and OPG expression levels (x±s, n = 3).

Group	OC	OPN	OPG
Control	1.002 ± 0.003	1.001 ± 0.002	1.004 ± 0.132
Ad-null	1.023 ± 0.172	1.121 ± 0.015	0.953 ± 0.141
Ad-VEGF165	1.163 ± 0.113^a	1.217 ± 0.216^a	1.372 ± 0.115^a
Ad-BMP2	4.715 ± 0.470^a	3.945 ± 0.652^a	5.796 ± 0.330^a
Ad-VEGF165 + Ad-BMP2	9.024 ± 0.475^a^b	6.113 ± 0.717^a^,^b	8.727 ± 0.563^a^,^b
F value	381.747	76.956	337.244
P values	0.000	0.000	0.000

^aComparison between Ad-BMP2, Ad-VEGF165 + Ad-BMP2 groups and control, Ad-null and Ad-VEGF165 groups, P < 0.05.

^bComparison between Ad-VEGF165 + Ad-BMP2 and Ad-BMP2 groups, P < 0.05.

Figure 4. ALP activities (x±s, n = 3). a: comparison between Ad-BMP2, Ad-VEGF165 + Ad-BMP2 groups and control, Ad-null and Ad-VEGF165 groups, P < 0.05; b: comparison between Ad-VEGF165 + Ad-BMP2 and Ad-BMP2 groups, P < 0.05.

Figure 5. ALP staining results (100×). (A): Control group; (B): Ad-null group; (C): Ad-VEGF165 group; (D): Ad-BMP-2 group; (E): Ad-VEGF165 + Ad-BMP2 group.

Table 4. ALP activities (x±s, n = 3).

Group	ALP activity
Control	3884.002 ± 200.503
Ad-null	4307.323 ± 367.782
Ad-VEGF165	5604.003 ± 797.713^a
Ad-BMP2	131,107.697 ± 10,933.210^a
Ad-VEGF165 + Ad-BMP2	166081.704 ± 6364.774^a
F value	592.547
P values	0.000

^aComparison between Ad-BMP2, Ad-VEGF165 + Ad-BMP2 groups and control, Ad-null and Ad-VEGF165 groups, P < 0.05.

^bComparison between Ad-VEGF165 + Ad-BMP2 and Ad-BMP2 groups, P < 0.05.

Figure 6. Alizarin red staining results (100×). (A): Control group; (B): Ad-null group; (C): Ad-VEGF165 group; (D): Ad-BMP-2 group; (E): Ad-VEGF165 + Ad-BMP2 group.