# 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

## TL;DR

This study shows that mesenchymal stem cells from atrophic non-unions have higher osteogenic activity than those from healthy bone grafts in laboratory tests.

## Contribution

The study reveals that atrophic non-union MSCs have greater osteogenic potential than previously assumed, challenging clinical assumptions.

## Key findings

- Atrophic non-union MSCs showed significantly higher early and late osteogenic activity compared to graft-derived MSCs.
- Non-union MSCs regained strong proliferative activity by the second week, leading to comparable cell counts after three weeks.
- Osteogenic potential of non-union MSCs was not impaired, contradicting clinical expectations.

## Abstract

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.

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.

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.

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.