Key Takeaways & Executive Findings
- •• Inhibition of miR-1246 and miR-335-5p enhances chondrogenic and hypertrophic differentiation in human BMSCs, but only in a subset of donors, indicating donor-specific responses. • miR-193a-5p inhibition has minimal effect on early endochondral differentiation, suggesting a limited functional role in this process. • Responder donors show significant upregulation of GDF5 and CCN5 and downregulation of SKIL upon miR-335-5p inhibition, providing potential mechanistic targets. • The findings highlight the need for personalized approaches in fracture healing prediction and therapy, as miRNA effects vary among individuals.
Abstract
Background Identification of biomarkers to predict the risk of healing delays are of huge clinical interest since 10% of fracture patients progress to delayed or non-union. During endochondral ossification, which takes place in mechanically unstable regions, the bone regenerates through a cartilage intermediate. We previously identified miR-1246, miR-335-5p and miR-193a-5p as fracture-related biomarkers in patient serum, but they appear not to have a functional role in an in vitro model of direct ossification. However, their involvement in other processes related to fracture healing cannot be ruled out and the most common healing process in fracture repair is secondary healing by way of endochondral ossification. Therefore, this study aims to explore the role of miR-1246, miR-335-5p and miR-193a-5p during in vitro endochondral differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs). Methods The activity of miR-1246, miR-335-5p, and miR-193a-5p was transiently inhibited just before pellet formation and the start of chondrogenic differentiation in human BMSCs (n=5 donors), serving as a model for early endochondral ossification. The effect of miRNA inhibition was assessed by histology (Safranin O/Fast Green), immunohistochemistry (type II and type X collagen), and gene expression analysis by bulk RNA sequencing and RT-qPCR. Results Inhibition of miR-1246 and miR-335-5p enhanced chondrogenic and hypertrophic differentiation in BMSCs from three out of five donors, while miR-193a-5p inhibition had minimal effect. Donors were categorized as “responders” or “non-responders” based on histological and gene expression profiles. RNA sequencing and RT-qPCR identified differentially expressed genes, including a 1.6 and 1.5-fold upregulation of GDF5 and CCN5 respectively (p<0.05) and downregulation of SKIL (fold change: 1.3, p=0.0563) after miR-335-5p inhibition, while the same genes were unaltered by miRNA inhibition in non-responders, suggesting donor-specific responses to miRNA inhibition during early chondrogenesis.
1. Introduction
Fracture non-union and delayed healing affect up to 10% of patients, leading to functional and psychological disability. There are several known risk factors including biological, surgical, and mechanical [1], but predicting the individual risk of developing a non-union is not yet possible.
After hematoma formation, which is a rich source of signalling molecules [2–5], bone healing can take place through intramembranous or endochondral ossification [6]. During intramembranous ossification, bone forms within a few days at the periosteal sites by direct differentiation of mesenchymal stromal cells (MSCs) into osteoblasts. On the other hand, during endochondral ossification, bone heals indirectly through formation of a cartilage intermediate. Numerous elements of these processes could be negatively influenced, causing disruptions in the healing process, and their early identification could allow a better prediction of the healing course.
Until now, there are no biomarkers predicting the risk of a fracture healing complication [1]. To enhance the management of non-unions or proactively mitigate the likelihood of fracture healing complications, it becomes imperative to identify early biomarkers capable of predicting abnormal healing. MicroRNAs (miRNAs) are small, noncoding RNAs involved in the regulation of gene expression pathways by degradation and translational repression of messenger RNA (mRNA). They influence virtually all cellular processes, including proliferation, differentiation, apoptosis, and cell migration [7–10]. miRNAs have already been discussed as predictive markers since they are indicative of cellular processes and can be assessed non-invasively in blood and other bodily fluids [11, 12]. miRNAs have also been investigated as predictive markers for individual outcomes of bone diseases [13]. They have been described to directly regulate osteoblast differentiation, including early regulation of the osteogenic factor Runx2 [14, 15].
A recently published work of our group identified miR-193a-5p, miR-1246, and miR-335-5p as differentially expressed in the serum of fracture patients.
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Franziska L. Breulmann, Silvia A. Berger, Elena Della Bella, Martin J. Stoddart (2026). Donor-dependent regulation of type II and X collagen deposition by early modulation of miR-335-5p and miR-1246 during chondrogenic commitment. Stem Cell Research & Therapy. https://doi.org/10.1186/s13287-025-04589-8
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Frequently Asked Questions
What is the main objective of this study?
The study aims to explore the role of miR-1246, miR-335-5p, and miR-193a-5p during in vitro endochondral differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs), to understand their potential as biomarkers for fracture healing complications.
How were the effects of miRNA inhibition assessed?
The effects were assessed by histology (Safranin O/Fast Green), immunohistochemistry for type II and type X collagen, and gene expression analysis using bulk RNA sequencing and RT-qPCR.
What were the key findings regarding donor variability?
Inhibition of miR-1246 and miR-335-5p enhanced chondrogenic and hypertrophic differentiation in BMSCs from three out of five donors, while miR-193a-5p inhibition had minimal effect. Donors were categorized as 'responders' or 'non-responders' based on histological and gene expression profiles, indicating donor-specific responses.
Which genes were differentially expressed after miR-335-5p inhibition in responders?
In responders, miR-335-5p inhibition led to a 1.6-fold upregulation of GDF5 and 1.5-fold upregulation of CCN5 (p<0.05), and downregulation of SKIL (fold change: 1.3, p=0.0563). These genes were unaltered in non-responders.
What is the clinical significance of this study?
The study highlights the potential of miRNAs as predictive biomarkers for fracture healing outcomes, but also emphasizes the need for personalized approaches due to donor-specific responses. This could lead to better management and proactive mitigation of fracture healing complications.
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