gms | German Medical Science

Objectives: Postnatal bone growth relies significantly on chondrocyte proliferation and osteogenic differentiation occurring within the growth plate (GP) through the process of endochondral ossification. Despite its pivotal role, the GP is prone to injuries, impacting 15-30% of bone fractures. These injuries can result in growth discrepancies, influencing both bone length and shape and detrimentally affecting the patient's quality of life. Currently, there is a lack of available biological therapies to prevent the formation of bone bridges, as the underlying pathological repair mechanism following GP injuries (GPI) and its contribution to growth-related issues remain unidentified. Consequently, there is a pressing need for innovative GPI models that enable real-time exploration of the pathological regeneration process.

Methods: This study aimed to investigate the molecular and cellular physiological and pathophysiological regeneration following sustained GPI in an ex vivo rat femur organotypic culture (OTC) model, specifically focusing on postnatal endochondral ossification. We utilized ex vivo bone cultures that were 300 µm thick with a 2 mm long horizontal GPI. After up to 15 days of in vitro cultivation, we conducted electron microscopy, gene expression analysis, live/dead staining, histological examinations, and immunohistochemistry to analyze key markers of endochondral ossification.

Results and conclusion: In our ex vivo rat femur organotypic cultures (OTCs), the regeneration process initiated at 3 days of in vitro cultivation (DIV), marked by the infiltration of stem cells, fibroblasts, and chondrocytes at the injury site by 7 DIV. Live/dead staining revealed elongated live cells migrating from the zone of Ranvier toward the injury site at 7 DIV, forming a network spanning the entire epiphysis by 15 DIV. Additionally, we observed a disruption in the endochondral ossification process, characterized by altered expression patterns of collagen type 2 (Col2a1), aggrecan (Acan), and collagen type X (ColX). Gene expression analysis demonstrated a significant increase in Sox9 expression at day 15 due to growth plate injury (GPI). The Ihh-PTHrP feedback loop was affected, favoring chondrocyte proliferation and maturation, with Ihh levels increasing significantly on day 7 and day 15, while PTHrP was downregulated on day 7.

Interpreting these results, our findings suggest a trauma-induced alteration in the structural architecture and organization, along with a notable impairment of chondrocyte maturation in the ex vivo organotypic GPI model, mirroring observations in animal models. Consequently, this innovative GPI model holds promise as a valuable tool for furthering our understanding of both physiological and pathophysiological growth plate repair mechanisms. Furthermore, it may find applications in tissue engineering and disease studies, offering insights into potential therapeutic interventions for growth-related issues arising from growth plate injuries.