Background and Objectives
Mesenchymal stem cells (MSCs) are bone marrow populating cells, which posses an extensive proliferation potential. In isolation and expansion protocols for clinical scale production of MSCs, fetal bovine serum (FBS) is used as a supplement with potential risk for infections as well as immunological reactions. Autologous platelet gel is made from a natural component of the patient’s own blood. Activated platelets release growth factors are mitogenic for MSCs. In vitro studies have indicated that concentration of growth factor varies according to platelet concentration, methods of preparation and mechanism of platelet growth factors release. The aim of our study was to investigate the effect of platelet growth factors on the proliferation and differentiation of human mesenchymal stem cells.
Materials and Methods
Mononuclear cells of bone marrow were collected in 10% FBS growth medium. The expanded cells were characterized by flow cytometric analysis of specific surface antigens. Analysed markers included CD45, CD34, CD166, CD105, CD90, and CD44. The gel is formed by adding calcium and thrombin to platelet rich plasma (PRP). Treated PRP was incubated for 30 min, 6,24,48 and 72 hours in incubator. Growth factors concentrations in supernatants were determined by ELISA. Human mesenchymal stem cells were cultured in the complete medium that supplemented with 10% FBS or Platelet growth factors for 8 days. The rate of proliferation was evaluated by MTT assay. Expanded cells were seeded on calcium phosphate scaffold. Cells growth and morphology on scaffold were analyzed by SEM.
Isolation and expansion of MSCs in the complete medium supplemented with platelet growth factors were successful and morphology of cells was compatible with that of FBS. Cells were highly positive for CD90, CD166, CD44 and CD105 and negative for CD34, CD45. There was no significant difference between expression of markers on cells expanded with platelet growth factors and FBS. We demonstrated that platelet growth factors provide a significantly higher proliferative effect on MSCs than those of FBS. MSCs cultured in the presence of growth factors maintain their osteogenic differentiation properties. Osteogenic differentiation was indicated by deposition of mineralized matrix stained with Alizarin red and increased expression alkaline phosphates.
Platelet growth factors can be used in place of FBS to provide a safer and more effective culture condition to expand MSC for clinical purposes. MSCs cultured in the presence of platelet growth factors maintain their osteogenic properties.
Key words : Mesenchymal Stem Cells, Osteogenesis, Tissue Scaffolds