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Comparative Study
. 2026 Feb 16;52(1):48.
doi: 10.1007/s00068-026-03112-9.

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

Tim Niklas Bewersdorf  1   2 , Jakob Hofmann  2 , Laura Boehm  2 , Sebastian Findeisen  2 , Christian Schamberger  2 , Thomas Lingner  3 , Ulrike Sommer  2 , Gerhard Schmidmaier  2 , Tobias Grossner  4   5
Affiliations
Comparative Study

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

Tim Niklas Bewersdorf et al. Eur J Trauma Emerg Surg. .

Abstract

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.

Keywords: Atrophic non-union; Autologous bone graft; Bone-marrow mesenchymal stem cells; In-vitro; Osteogenic activity; Proliferative activity.

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Conflict of interest statement

Declarations. Ethics approval: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The experimental study was approved by the Ethics Committee board, Faculty of Medicine, Heidelberg University, Heidelberg, Germany (No. S-523/2023). Consent to participate and publish: Informed consent was obtained from all individual participants included in the study. Patients signed informed consent regarding publishing their data. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Experimental setup of the study. MSC: mesenchymal stem cells; NU: atrophic non-union; RIA: Reamer-Irrigator-Aspirator femur aspirate; 99mTc-HDP: 99mTechnetium-hydroxydiphosphonate; ALP: alkaline phosphatase
Fig. 2
Fig. 2
Line graph with error bars of mean values of WST-1 cell proliferation assay during ongoing osteogenic differentiation of 21 days in-vitro. The proliferation rates of both groups were measured on day 0, 7, 14, and 21 after 20 min incubation. Results were calculated by subtraction of reference wavelength. n = 6 donors, n = 6 samples per donor and timepoint, n = 1 technical replicate. Line: connects mean values of the groups. Whiskers: standard deviation within a specific group and timepoint. Significances are indicated by stars; stars without a bar show significance between NU and GRA within one timepoint; dashed bars under the stars indicate the compared GRA group of each significance. Solid bars above the stars indicate the compared NU group of each significance. NU: atrophic non-union derived mesenchymal stem cells. GRA: graft (RIA femur aspirate or iliac crest) derived mesenchymal stem cells. ns: not significant, *: p < 0.05, ***: p < 0.001
Fig. 3
Fig. 3
DAPI immunofluorescence microscopy cell count. a: box plot of cell counts per dish: mean cell amount per group assessed by DAPI immunofluorescence staining displayed as 104 cells. Boxes: interquartile range, whiskers: 1.5 x interquartile range, horizontal line: median, x: mean. Significances are indicated by stars; bars under the stars indicate the compared group of each significance. NU: atrophic non-union derived mesenchymal stem cells. GRA: graft (RIA femur aspirate or iliac crest) derived mesenchymal stem cells. NU-Co: non-osteogenic differentiated control group for NU, which was formed out of samples of the same donor. GRA-Co: non-osteogenic differentiated control group for GRA, which was formed out of samples of the same donor. n = 6 donors, n = 6 samples per donor, n = 1 technical replicate, n = 6 vision fields per sample. ns: not significant. b: Example immunofluorescence image of GRA (iliac crest) on day 21. c: Example immunofluorescence image of corresponding GRA-Co on day 21
Fig. 4
Fig. 4
Calcium concentration in supernatants. a: box plot of calcium concentration in supernatants: mean calcium concentration in mmol/l in supernatants per group in-vitro. b: box plot of calcium concentration in formula image. Mean calcium concentrations in supernatants were normed for 104 cells by dividing the results by average cell number of the same sample. n = 6 donors, n = 6 samples per donor, n = 1 technical replicate. Boxes: interquartile range, whiskers: 1.5x interquartile range, horizontal line: median, x: mean. Significances are indicated by stars. NU: atrophic non-union derived mesenchymal stem cells. GRA: graft (RIA femur aspirate or iliac crest) derived mesenchymal stem cells. ***: p < 0.001
Fig. 5
Fig. 5
ALP activity in supernatants. a: box plot of mean ALP activity detected in supernatants: mean ALP activity measured in U/l in supernatants per group in-vitro. b: box plot of ALP activity displayed in formula image. Mean ALP activity was normed for 104 cells by dividing the results by average cell number of the same sample n = 6 donors, n = 6 samples per donor, n = 1 technical replicate. Boxes: interquartile range, whiskers: 1.5x interquartile range, horizontal line: median, x: mean. Significances are indicated by stars. NU: atrophic non-union derived mesenchymal stem cells. GRA: graft (RIA femur aspirate or iliac crest) derived mesenchymal stem cells. ***: p < 0.001
Fig. 6
Fig. 6
99mTechnetium-hydroxydiphosphonate (99mTc-HDP) uptake. a: box plot of mean 99mTc-HDP uptake in MBq: mean 99mTc-HDP uptake per group in-vitro. b: box plot of 99mTc-HDP uptake in MBq per 104 cells. Mean 99mTc-HDP uptake per group was normed for 104 cells by dividing the results by average cell number of the same sample. n = 6 donors, NU/GRA: n = 6 samples per donor, NU-Co/GRA-Co: n = 2 samples per donor, n = 2 technical replicates (gamma count measurements). Boxes: interquartile range, whiskers: 1.5x interquartile range, horizontal line: median, x: mean. Significances are indicated by stars; bars under the stars indicate the compared group of each significance. NU: atrophic non-union derived mesenchymal stem cells. GRA: graft (RIA femur aspirate or iliac crest) derived mesenchymal stem cells. NU-Co: non-osteogenic differentiated control group for NU, which was formed out of samples from the same donor. GRA-Co: non-osteogenic differentiated control group for GRA, which was formed out of samples from the same donor. ns: not significant, ***: p < 0.001
Fig. 7
Fig. 7
Box plot of WST-1 Assay – Day 7. WST-1 cell proliferation assay of RIA and IC on day 7: mean absorbance values per group after 20 min incubation in-vitro. Results were calculated by subtraction of absorbance of reference wavelength. n = 3 donors per group, n = 6 samples per donor, n = 1 technical replicate. Boxes: interquartile range, whiskers: 1.5x interquartile range, horizontal line: median, x: mean. Significances are indicated by stars; bar under the stars indicate the compared group of each significance. RIA: femur aspirate derived mesenchymal stem cells harvested by Reamer-Irrigator-Aspirator technique. IC: Iliac crest derived mesenchymal stem cells. ns: not significant
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