Growth plate morphogens as potential modulators of cellular tissue remodeling by annulus fibrosus cells

Methods

AF cell responsiveness to seven GPMs was screened in monolayer culture by RT-qPCR for downstream signaling markers. GPMs and concentrations eliciting significant transcriptional responses were selected for deeper analysis. Bulk RNA sequencing (RNA-seq) was conducted on AF cells cultured in a 2.5D environment to preserve their native elongated phenotype.

Differentially expressed genes (DEGs) were identified and subjected to Gene Ontology enrichment, focusing on ECM remodeling and cell-proliferation processes. Transcriptomic findings were corroborated by assessments of cell proliferation (DNA content), sulfated glycosaminoglycan (sGAG) levels, and matrix metalloproteinase (MMP) activity. Results: BMP-2, TGF-β1, and FGF-2 significantly altered AF cell expression of downstream target genes, whereas Ihh, PTHrP, Wnt-1, and FGF-18 did not.

IGF-1 induced modest, dose-dependent gene expression changes that were not replicated by RNA-seq. BMP-2 and TGF-β1 upregulated genes associated with anabolic ECM remodeling. BMP-2 promoted a cartilage-like ECM with increased expression of type II-associated collagens, aggrecan, and hyaluronan synthesis.

TGF-β1 promoted type I collagen-associated genes, collagen cross-linking, and myofibroblast activation. In contrast, FGF-2 activated transcriptional programs related to cell-cycle progression, collagen degradation, and increased GAG turnover. Functionally, we observed higher DNA content under BMP-2 and TGF-β1 stimulation, and higher sGAG levels under BMP-2, TGF-β1, and FGF-2 treatment.

Conclusion: BMP-2, TGF-β1, and FGF-2 can modulate AF cell behavior, influencing tissue remodeling and implicating their involvement in disc wedging during AIS onset.