In-vitro evaluation of the proliferative and osteogenic activity of atrophic non-union derived mesenchymal stem cells compared to autologous bone graft derived mesenchymal stem cells.

PURPOSE: This study compares the proliferative and osteogenic activity of human bone marrow derived mesenchymal stem cells (MSCs) obtained from atrophic non-unions and from healthy autologous bone graft tissue (harvested from iliac crest or femoral canal) of the same patient in an in-vitro setting utilizing a matched control study design.

METHODS

MSCs underwent osteogenic differentiation over 3 weeks in-vitro (n = 6 donors; n = 36 samples/group) and the proliferative activity was accessed using DAPI-based immunofluorescence microscopy and WST-1 assay. All results regarding the osteogenic activity were normalized to 104 cells to eliminate a proliferation bias. The late osteogenic activity was evaluated by radioactive 99mTechnetium-hydroxydiphosphonate labelling of depleted hydroxyapatite, while early osteogenic markers (calcium concentration and alkaline phosphatase activity) were analysed in supernatants of cell culture media.

RESULTS

The early and late-stage osteogenic activity of atrophic non-union MSCs was significantly higher compared to healthy control graft MSCs on day 21 of osteogenic differentiation in-vitro, both in absolute numbers (early/late: p < 0.001) and after normalization to 104 cells (early/late: p < 0.001). After lower proliferative activity during the first week, non-union MSC regained good proliferative activity during the second week resulting in comparable absolute cell counts to healthy control graft MSCs after three weeks.

CONCLUSION

The results emphasize that the in-vitro osteogenic and proliferative activity of atrophic non-union MSCs is not impaired as clinically assumed but the osteogenic potential of atrophic non-union MSCs is in fact significantly higher compared to graft derived MSCs. This might be an important basic-science insight for the optimization of clinical non-union therapies.