Differentially expressed microRNAs during endochondral differentiation of human bone marrow derived mesenchymal stromal cells to identify possible biomarkers for non-union fractures

Jun 30, 2022, 12:20 PM
10m
Room: S3 A

Room: S3 A

Speaker

Breulmann, Franziska (AO Research Institute Davos)

Description

"Possible biomarkers to predict the risk of healing delays are of huge clinical interest since 10% of fracture patients progress to delayed or non-union of fractures [1]. During endochondral ossification, which takes place in mechanically unstable regions with higher risk for delayed fracture healing, the bone regenerates through chondrogenic differentiation leading to a cartilage tissue before being remodeled into bone.
MicroRNAs (miRNA) are small, non-coding RNAs known to be involved in cell regulation [2] and play potential role in differentiation of human bone marrow derived mesenchymal stromal cells (hBM-MSCs). This work aims to identify miRNA differentially expressed during early differentiation of hBM-MSCs to be used as predictive biomarkers for non-union fracture healing.

hBM-MSCs were seeded and cultured for 28 days either in chondropermissive medium (CP) as negative control (DMEM 4.5 g/l glucose, 1% Pen/Strep, 1% ITS-x, 1% non-essential amino acids, 50 µg/ml ascorbic acid, 100 nM dexamethasone) or in chondrogenic medium (CH) as positive control (CP + 10 ng/ml TGF-β1) (N=4). Medium samples and pellets were collected on day 3, 7, 14, 21 and 28. Next, a multiaxial loading bioreactor is used for mechanically induced chondrogenesis of hBM-MSCs seeded in fibrin-polyurethane scaffolds, and three groups are compared (N=4): 1) scaffolds cultured in CP-medium (negative control), 2) scaffolds cultured in CH-medium (positive control), 3) scaffolds cultured in CP-medium + mechanical loading. To validate the differentiation of MSCs under chondrogenic conditions, histology with Safranin O/Fast Green staining was performed, as well as TGF-β1 ELISA and GAG analysis on medium and scaffold samples.
RNA was isolated from the pellets and scaffolds, followed by RT-qPCR to quantify miRNA and gene expression of chondrogenic marker genes, such as ACAN, SOX9, and COL2A1 during differentiation. As investigated in previous studies, miR-193a-5p is regulated chondrogenic differentiation of hBM-MSCs [3] and miR-193a-5p can be involved in regulation of TGF-β pathway [4]. We hypothesized that this miRNA has a regulatory role in chondrogenic differentiation, driven by TGF-β1 or during mechanically induced chondrogenesis.
Results miR-193a-5p are significantly downregulated in the scaffolds and appears downregulated in pellets cultured under chondrogenic conditions, confirming previous results [3]. Levels of miR-193a-5p during mechanically-induced chondrogenesis more closely resemble those in CP conditions. Since it has been reported that miR-193a-5p can be involved in bone metabolism by inhibiting the TGF-β pathway [4], we are investigating the correlation between levels of active TGF-β1 produced by mechanical stimulation with the miRNA expression at early days of differentiation processes. If there is a direct correlation, this miRNA may function as predictive markers for MSC differentiation and can be further validated for its role during endochondral ossification.

[1] Wildemann et al. 2021. doi:10.1038/s41572-021-00289-8.
[2] Lai 2002. doi:10.1038/ng865.
[3] Della Bella et al. 2020. doi:10.3390/cells9020398.
[4] Pu et al. 2016. doi:10.1007/s10585-016-9783-0."

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