Abstract
Background
The molecular mechanisms underlying non-union bone fractures largely remain elusive. Recently, spatial transcriptomics approaches for musculoskeletal tissue samples have been developed requiring direct placement of histology sections on barcoded slides. However, Formalin-Fixed-Paraffin-Embedded (FFPE) bone sections have been associated with limited RNA quality and read depth compared to soft tissue. Here, we test spatial transcriptomics workflows based on transcriptomic probe transfer to characterize molecular features discriminating non-union and union bone fractures in mice.
Method
Histological sections (n=8) used for spatial transcriptomics (Visium CytAssist FFPE; 10x Genomics, n=4 on glass slides, n=4 on hydrogel-coated slides) were obtained from a fracture healing study in female 20-week-old C57BL/6J mice receiving either a femur osteotomy (0.7mm) or a segmental defect (2.4mm) (license 22/2022, Grisons CH). Sequence alignment and manual segmentation of different tissues (bone, defect region/callus, bone marrow, muscle) were performed using SpaceRanger and LoupeBrowser (10x Genomics). Differential gene expression was performed using DESeq2 (Seurat) followed by Gene-Set-Enrichment-Analysis (GSEA) of Gene Ontology (ClusterProfiler). Group comparison of quality measures was done using a Welch's t-test. Results are given as mean±standard deviation.
Result
The quality measures, mean counts, and genes per spot, were significantly ~10× higher for sections on hydrogel slides (counts: 4700±1796, genes: 2389±1170) compared to glass slides (counts: 463±415, genes: 250±223). In challenging tissues like cortical bone, we reached high counts+genes in comparison to published data.
Direct comparison of a non-union and union section showed a total of 432 differentially regulated genes, 538 in the defect region/callus. GSEA revealed differential regulation of pathways involved in muscle organ morphogenesis, cartilage development and endochondral ossification.
Conclusions
Optimized spatial transcriptomics workflows based on transcriptomic probe transfer enable for improved read depth in musculoskeletal tissue enabling the characterization of molecular features discriminating non-union and union bone fractures.
Acknowledgements
AO Foundation (AOTRAUMA), SNSF (PhD salary)
