Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy
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RESEARCH
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Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy Cheng-Li Lin1,2,3, Yi-Ting Kuo4, Che-Hao Tsao4, Yan-Jye Shyong4,5, Shu-Hsien Shih1 and Ting-Yuan Tu3,4,6*
Abstract Background: Ligamentum flavum hypertrophy (LFH) is among the most crucial factors in degenerative lumbar spinal stenosis, which can cause back pain, lower extremity pain, cauda equina syndrome and neurogenic claudication. The exact pathogenesis of LFH remains elusive despite extensive research. Most in vitro studies investigating LFH have been carried out using conventional two-dimensional (2D) cell cultures, which do not resemble in vivo conditions, as they lack crucial pathophysiological factors found in three-dimensional (3D) LFH tissue, such as enhanced cell proliferation and cell cluster formation. In this study, we generated ligamentum flavum (LF) clusters using spheroid cultures derived from primary LFH tissue. Results: The cultured LF spheroids exhibited good viability and growth on an ultra-low attachment 96-well plate (ULA 96-plate) platform according to live/dead staining. Our results showed that the 100-cell culture continued to grow in size, while the 1000-cell culture maintained its size, and the 5000-cell culture exhibited a decreasing trend in size as the culture time increased; long-term culture was validated for at least 28 days. The LF spheroids also maintained the extracellular matrix (ECM) phenotype, i.e., fibronectin, elastin, and collagen I and III. The 2D culture and 3D culture were further compared by cell cycle and Western blot analyses. Finally, we utilized hematoxylin and eosin (H&E) staining to demonstrate that the 3D spheroids resembled part of the cell arrangement in LF hypertrophic tissue. Conclusions: The developed LF spheroid model has great potential, as it provides a stable culture platform in a 3D model that can further improve our understanding of the pathogenesis of LFH and has applications in future studies. Keywords: Lumbar spinal stenosis, Ligamentum flavum, Ligamentum flavum hypertrophy, 3D cell culture, Spheroid
Introduction Ligamentum flavum hypertrophy (LFH) is among the main components of lumber spinal stenosis (LSS). The ligamentum flavum (LF) is located between the upper and lower vertebrae lamina, extends from the cervical vertebra to the sacral vertebra, and provides protection and stability to the spinal column. Recent findings * Correspondence: [email protected] 3 Medical Device Innovation Center (MDIC), National Cheng Kung University, Tainan 70101, Taiwan 4 Department of Biomedical Engineering, National Cheng Kung University, Tainan 70101, Taiwan Full list of author information is available at the end of the article
suggest that the factors leading to LFH include agerelated degeneration [1, 2] inflammation [3, 4] mechanical strain [5, 6] elastic degradation and fibrosis [7, 8]. However, the current treatments for LFH are primarily limited to invasive surgical resection, underscoring the need to explore new alternatives for novel trea
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