Poster Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction The occurrence of chondrometaplasia and calcification in hypertrophied ligamentum flavum (LF) is not uncommon. Proliferative cartilage cells with surrounding overgrowth of type II collagen were identified in 91% of the hypertrophied LF from patients with lumbar spinal stenosis (LSS). Though chondrometaplasia and calcium deposition were identified in hypertrophied LF, the etiology is not well elucidated. This study is to investigate the pathological role of elastin-derived peptides (EDPs) in chondrogenic differentiation and calcification of LF-derived cells in the process of LF hypertrophy.

Methods A total of 22 LF specimens from patients with LSS and 5 control LF specimens from patients with lumbar disc herniation (LDH) or adolescent idiopathic scoliosis (AIS) were analyzed histologically using hematoxylin and eosin (H&E), Elastica van Gieson (EVG), Alcian blue (AB) and Alizarin Red S (AR) staining. The thickness of LF on the radiographic images was measured. In the in vitro study, isolated LF-derived cells were treated with human lung peptides (hEDP) at various concentration, and viability and proliferation were analyzed. To investigate the effect of EDPs on chondrogenic differentiation, LF-derived cells were cultured in chondrogenic medium supplemented with or without transforming growth factor β-3 (TGFβ-3) and treated with or without hEDP at 100µg/ml. To investigate the effect of EDPs on calcification, LF cells were cultured in osteogenic medium and treated with or without hEDP. Cartilage specific genes markers and osteogenic gene markers were analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). To further evaluate chondrogenic differentiation of LF-derived cells, sulfated glycosaminoglycan (s-GAG) was measured in cell culture supernatant and extracellular matrix (ECM), along with cell Safranine O staining to investigate the production of cartilage matrix. Secreted matrix metalloproteinase-13 (MMP-13) in the culture medium was measured using MMP-13 enzyme-linked immunosorbent assay (ELISA) kit. On the other hand, Von-Kossa staining and ALP activity assay were conducted to evaluate the effect of EDPs on LF-derived cell calcification.

Results The LF in patients with LSS exhibited a significantly increased thickness (5.36 ± 1.11 mm) compared to the control group's LF (2.26 ± 0.70 mm, p<0.001). The hypertrophied LF showed proliferation of chondroid cells, breakdown of elastic fiber, deposition of proteoglycan and calcification. From the cell culture study, hEDP accelerated LF cell proliferation in a dose-dependent manner. Functionally, hEDP significantly increased s-GAG production in cell culture medium and worked synergically with TGFβ-3 to facilitate cartilage matrix deposition in ECM, whereas the production of MMP-13 was reduced by hEDP. The mRNA expression of cartilage specific markers, such as Col II, ACAN and SOX9, were significantly upregulated with hEDP treatment. hEDP significantly increased expression of osteogenesis-related markers and facilitated calcium deposition and promoted ALP activity in vitro.

Discussion We showed for first time that elastin degradation products accelerate chondrogenic differentiation and calcification of LF-derived cells. This phenomenon may contribute to the development of chondrometaplasia and calcification observed in hypertrophied LF. However, future studies are required to demonstrate the functional significance of elastin degradation products in LF‐derived cells differentiation and calcification in vivo as well as in LF hypertrophy clinical pathology.