Growth Plate Morphogens as Potential Modulators of Cellular Tissue Remodeling by Annulus Fibrosus Cells.

Abstract

Adolescent Idiopathic Scoliosis (AIS) is a spinal deformity of unknown etiology that begins in the intervertebral disc and emerges during pubertal growth. In the vertebral growth plate, adjacent to the disc, powerful growth plate morphogens (GPMs) modulate this growth. We evaluated whether GPMs can also alter the capacity of annulus fibrosus (AF) disc cells to proliferate and/or remodel their extracellular matrix (ECM). 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. 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. 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.