Mesenchymal Stem Cell Upregulates Osteoblast Gene Expression in a Direct Co-Culture System
Thursday, October 10, 2013
Thiago de Santana Santos DDS, Msc, Oral and Maxillofacial Surgery, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil., Ribeirão Preto, Brazil
Fabiola Singaretti de Oliveira Msc, PhD, Oral and Maxillofacial Surgery, Biomolecular Laboratory Ribeirão Preto Dental School, Ribeirão Preto, Brazil
Marcio Mateus Beloti DDS, MSc, PhD, Morphology, Physiology and Basic Pathology, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil., Ribeirão Preto, Brazil
Adalberto Luiz Rosa DDS, MSc, PhD, Oral and Maxillofacial Surgery, School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil., Ribeirão Preto, Brazil
Cell therapy has been a feasible alternative to repair bone being used in the treatment of chronic non-union fractures and bone metabolic diseases with very promising results. Considering that both mesenchymal stem cells (MSCs) and osteoblasts (OB) play a role in bone repair, here we investigated the effect of direct co-culture of MSCs and OB on the gene expression of key bone markers. Rat bone marrow cells were harvested and cultured in either growth medium to obtain MSCs or in osteogenic medium (growth medium supplemented with ascorbic acid, beta-glycerophosphate, and dexamethasone) to obtain OB. After reaching subconfluence, cells were subcultured in 24-well plates for 13 days in osteogenic medium at a final concentration of 2x10
4 cells/well combining MSCs+OB in different ratios (1:1, 1:2 and 2:1). OB cultured in osteogenic medium were used as control. The gene expression of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), bone sialoprotein (Bsp) and osteocalcin (Oc) was evaluated by real-time PCR. Data were compared using the Kruskal-Wallis test, followed by Fischer test and the level of significance was set at 0.05. The results showed in the Table indicated higher expression (p<0.05) of Alp for MSCs+OB (1:1); Bsp for MSCs+OB (2:1); and Runx2, Osx and Oc for MSCs+OB (1:2) compared with OB. It shows that MSCs have a stimulatory effect on OB, that resulted in a noticeable increase in the expression of bone markers suggesting that cell therapy using a combination of MSCs and OB could improve bone repair.
Table 1. Osteoblast gene expression of association of mesenchymal stem cells (MSc) and osteoblasts (OB) after 13 days.
Gene
|
|
MSc + OB
|
Anova p-value
|
OB
|
1:1
|
1:2
|
2:1
|
Alp
|
1.007a
|
4.444b
|
3.150c
|
3.113c
|
0.01
|
Bsp
|
1.007a
|
1.789b
|
3.136c
|
5.754d
|
0.01
|
Col
|
1.035a
|
1.959b
|
7.193c
|
2.804d
|
0.01
|
Oc
|
1.005a
|
0.881a,c
|
3.430b
|
0.817c
|
0.05
|
Osx
|
1.645a
|
1.541a
|
6.855b
|
6.208b
|
0.01
|
Runx2
|
1.012a
|
2.053b
|
2.362c
|
1.024a
|
0.01
|
Lowercase different letters indicated statistical significant difference.
References
A.L. Rosa, P.T. de Oliveira, M.M. Beloti. Macroporous scaffolds associated with cells to construct a hybrid biomaterial for bone tissue engineering. Expert Rev Med Devices, 5 (2008), pp. 719–728.
M.M. Beloti, L.G. Sicchieri, P.T. de Oliveira, A.L. Rosa. The Influence of Osteoblast Differentiation Stage on Bone Formation in Autogenously Implanted Cell-Based Poly(Lactide-Co-Glycolide) and Calcium Phosphate Constructs. Tissue Eng, 18 (2012), pp. 999-1005.
Financial support: FAPESP (Grants # 2011/00919-9 and 2011/00617-2).
