SUCCESSFUL ISOLATION AND OSTEOBLASTIC DIFFERENTIATION OF MINIPIG MESENCHYMAL STEM CELLS FROM BONE MARROW BUT NOT FROM PERIPHERAL BLOOD

Abstract

Background and aim: Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into osteogenic and chondrogenic pathways. MSCs are among the key repair cells in fracture healing and implant osseointegration. They are also an attractive tool of cell therapy in reconstruction procedures of bone. Minipigs are a large-animal model recommended for preclinical studies of orthopaedic bone implants. Minipigs are claimed to have bone physiology close to humans, but their MSC characteristics are poorly defined. The aim of this study was to isolate and characterize minipig bone marrow and peripheral blood derived MSCs in comparison of human MSCs.

Methods: Five male minipigs (weight 36.2 ± 2.2 kg) were subjected for experimental femoral osteotomy, which was fixed with either compression plate or intra-medullary nailing. Before surgery, bone marrow (BM) sample (2–4 ml) was aspirated from the posterior iliac crest and a peripheral blood (PB) sample (20 ml) was also collected. Mononuclear cells (MNC) were isolated by Ficoll gradient centrifugation. MSCs were cultured and selected by plastic adherence. Cell morphology was evaluated during the whole culture period and proliferation capacity was examined by determining the number of population doublings (PDs) at the end of each passage. Osteoblastic differentiation capacity was investigated by culturing MSCs in the presence of beta-glycerophosphate, dexamethasone and ascorbic acid. The lineage phenotype was studied by alkaline phosphatase and von Kossa staining.

Results: MNC were successfully isolated from all BM and PB samples. Plastic adherent cells obtained fibroblast-like morphology and proliferated over time in culture. The maximum PDs were 3.4 ± 0.7 and 4.3 ± 0.5 for BM- and PB-derived cells, respectively. The maximum PD capacity of PB-derived cells was significantly higher than that of BM-derived cells (p=0.027). However, when cultured in osteoblastic induction medium, only BM-derived cells were capable of differentiating into alkaline phosphatase positive osteoblasts with an occasional presence of von Kossa-stained mineralized bone nodules. The maximum PDs of minipig BM-derived MSCs were similar to those of human BM-MSCs isolated from young adult fracture patients.

Conclusion: We successfully isolated plastic adherent MSCs from minipig bone marrow samples, which proliferated and differentiated into cells of osteoblastic lineage. BM-derived porcine MSCs had similar morphology to human MSCs. There were marked inter-individual variations in the proliferation and differentiation capacity of minipig MSCs, resembling the observations in humans. No circulating MSCs could be detected in minipigs before surgery and this confirmed our previous observation in humans.