Osteogenic differentiation of mesenchymal stromal cells in two-dimensional and three-dimensional cultures without animal serum.
BACKGROUND
Bone marrow-derived mesenchymal stromal cells (MSCs) have been intensely studied for the purpose of developing solutions for clinical tissue engineering. Autologous MSCs can potentially be used to replace tissue defects, but the procedure also carries risks such as immunization and xenogeneic infection. Replacement of the commonly used fetal calf serum (FCS) with human platelet lysate and plasma (PLP) to support cell growth may reduce some of these risks. Altered media could, however, influence stem cell differentiation and we address this experimentally.


METHODS
We examined human MSC differentiation into the osteoblast lineage using in vitro two- and three-dimensional cultures with PLP or FCS as cell culture medium supplements. Differentiation was followed by quantitative polymerase chain reaction, and alkaline phosphatase activity, matrix formation and matrix calcium content were quantified.


RESULTS
Three-dimensional culture, where human MSCs were grown on collagen sponges, markedly stimulated osteoblast differentiation; a fourfold increase in calcium deposition could be observed in both PLP and FCS groups. PLP-grown cells showed robust osteogenic differentiation both in two- and three-dimensional MSC cultures. The calcium content of the matrix in the two-dimensional PLP group at day 14 was 2.2-fold higher in comparison to the FCS group (p < 0.0001), and at day 21 it was still 1.3-fold higher (p < 0.001), suggesting earlier calcium accumulation to the matrix in the PLP group. This was supported by stronger Alizarin Red staining in the PLP group at day 14. In two-dimesional PLP cultures, cellular proliferation appeared to decrease during later stages of differentiation, while in the FCS group the number of cells increased throughout the experiment. In three-dimensional experiments, the PLP and FCS groups behaved more congruently, except for the alkaline phosphatase activity and mRNA levels which were markedly increased by PLP.


CONCLUSIONS
Human PLP was at least equal to FCS in supporting osteogenic differentiation of human MSCs in two- and three-dimensional conditions; however, proliferation was inferior. As PLP is free of animal components, and thus represents reduced risk for xenogeneic infection, its use for human MSC-induced bone repair in the clinic by the three-dimensional live implants presented here appears a promising therapy option.
Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. eeva.castren@helsinki.fi.
Eeva
E
Castrén
Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. tarvo.sillat@helsinki.fi.|Department of Medicine, Helsinki University Central Hospital, PO 700, 00029 HUS, Helsinki, Finland. tarvo.sillat@helsinki.fi.
Tarvo
T
Sillat
Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. sofia.oja@veripalvelu.fi.|Finnish Red Cross Blood service, Kivihaantie 7, 00310, Helsinki, Finland. sofia.oja@veripalvelu.fi.
Sofia
S
Oja
Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. ariel.noro@helsinki.fi.
Ariel
A
Noro
Finnish Red Cross Blood service, Kivihaantie 7, 00310, Helsinki, Finland. anita.laitinen@bloodservice.fi.
Anita
A
Laitinen
Department of Medicine, Helsinki University Central Hospital, PO 700, 00029 HUS, Helsinki, Finland. yrjo.konttinen@helsinki.fi.|ORTON Orthopaedic Hospital of the Invalid Foundation, PO 29, 00281, Helsinki, Finland. yrjo.konttinen@helsinki.fi.
Yrjö T
YT
Konttinen
Departments of Anatomy and Surgery, University of Oulu, Aapistie 7, 90220, Oulu, Finland. petri.lehenkari@oulu.fi.
Petri
P
Lehenkari
Institute of Biomedicine, Anatomy, Biomedicum Helsinki, University of Helsinki, PO Box 63, Helsinki, Finland. mika.hukkanen@helsinki.fi.
Mika
M
Hukkanen
Division of Hemato-Oncology and Stem cell Transplantation, Hospital of Children and Adolescents, Helsinki University Central Hospital, Helsinki, Finland. matti.korhonen@bts.redcross.fi.|Finnish Red Cross Blood service, Kivihaantie 7, 00310, Helsinki, Finland. matti.korhonen@bts.redcross.fi.
Matti
M
Korhonen
D000328:Adult; D002478:Cells, Cultured; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis; D061251:Primary Cell Culture / Q000379:methods; D044967:Serum