Clinical implications of linking microstructure, spatial biochemical, spatial biomechanical, and radiological features in ligamentum flavum degeneration

Abstract

Background: The ligamentum flavum (LF) degeneration is a critical factor in spinal stenosis, leading to nerve compression and pain. Even with new treatment options becoming available, it is vital to have a better understanding of LF degeneration to ensure the effectiveness of these treatments.

Objective: This study aimed to provide insight into LF degeneration by examining the connections between various aspects of LF degeneration, including histology, microstructure, chemical composition, and biomechanics.

Method: We analyzed 30 LF samples from 27 patients with lumbar vertebrae, employing magnetic resonance imaging (MRI) to link lumbar disc degeneration grades with fibrosis levels in the tissue. X-ray diffraction (XRD) analysis assessed microstructural alterations in the LF matrix component due to degeneration progression. Instrumented nanoindentation combined with Raman spectroscopy explored the spatial microbiomechanical and biochemical characteristics of the LF's ventral and dorsal regions.

Results: Our outcomes revealed a clear association between the severity of LF fibrosis grades and increasing LF thickness. XRD analysis showed a rise in crystalline components and hydroxyapatite molecules with progressing degeneration. Raman spectroscopy detected changes in the ratio of phosphate, proteoglycan, and proline/hydroxyproline over the amide I band, indicating alterations in the extracellular matrix composition. Biomechanical testing demonstrated that LF tissue becomes stiffer and less extensible with increasing fibrosis.

Discussion: Notably, the micro-spatial assessment revealed the dorsal side of the LF experiencing more significant mechanical stress, alongside more pronounced biochemical and biomechanical changes compared to the ventral side. Degeneration of the LF involves complex processes that affect tissue histology, chemical composition, and biomechanics. It is crucial to fully understand these changes to develop new and effective treatments for spinal stenosis. These findings can improve diagnostic accuracy, identify potential biomarkers and treatment targets, guide personalized treatment strategies, advance tissue engineering approaches, help make informed clinical decisions, and educate patients about LF degeneration.

Keywords: Raman spectroscopy; biomechanics; ligamentum flavum; microstructure; nanoindentation; radiology assessment.

FIGURE 1

(A) Thirty samples with varying fibrosis grades were obtained from 27 patients. (B) Statistical analysis of fiber alignment determined by the fiber dispersion. (C–E) Representative grades 1 fibrosis: H&E staining of tissue, black and white fiber orientation, and fiber alignment histogram, respectively. (F–H) Representative grades 2 fibrosis: H&E staining of tissue, black and white fiber orientation, and fiber alignment histogram, respectively. (I–K) Representative grades 3 fibrosis: H&E staining of tissue, black and white fiber orientation, and fiber alignment histogram, respectively. All representatives of H&E staining of tissues, black and white fiber orientation, and fiber alignment histogram were taken from the dorsal side of each grade. The black scale bar on H&E staining images represents 50 μm. NS, Not statistically different, *: p < 0.05.

FIGURE 2

(A) Direct visualization of LF thickness within the software interface. The red arrow highlights the LF in its characteristic butterfly shape. (B) Variation in LF thickness across different stages of fibrosis. Higher grades of fibrosis are associated with increased LF thickness. (C) Representative magnetic resonance imaging (MRI) scans of corresponding degenerative disc stages are categorized according to the Pfirrmann classification. Note the absence of Grade I Pfirrmann discs in the analyzed samples. (D) The distribution of fibrosis grades within samples classified by Pfirrmann grades demonstrates that any level of fibrosis can coexist with Pfirrmann grades II to V, suggesting independent progression patterns for these two pathologies. NS, Not statistically different, *: p < 0.05.

FIGURE 3

Fibrosis progression alters tissue microstructure and biochemical composition. (A) X‐ray diffraction (XRD) patterns demonstrate collagen/elastin and mineral presence changes in grade 2 fibrosis, with hydroxyapatite crystals appearing in grade 3 fibrosis. Each graph represents the average of four samples per fibrosis grade (B) Average Raman spectra from all tissue with different grades highlight biochemical shifts across fibrosis grades (VG1‐3, DG1‐3), including alterations in proline/hydroxyproline (peaks 1 and 2), phosphate (peak 3), proteoglycan groups (peaks 4 and 5), and amide I groups (peak 6) which became the focus of this study. DG, Dorsal Grade; VG, Ventral Grade.

FIGURE 4

Raman markers of fibrosis progression. (A) Phosphate/amide I and (B) Proteoglycan/amide I increase in the ventral and dorsal regions as fibrosis progresses. (C) Proline, Hydroxyproline/amide I show significant changes in the ventral region but remain relatively stable in the dorsal region. NS, Not statistically different. *: p < 0.05, **: p < 0.001.

FIGURE 5

Fibrosis grading across fibrosis levels. In all measures (A–D), dorsal values exceed ventral values. (A) Er steadily increases in both regions. (B) H increases sharply in both areas. (C) E′ exhibits a similar increase. (D) E″ shows a decrease in both areas, with the decline more pronounced in the dorsal region. NS, Not statistically different, *: p < 0.05, **: p < 0.001.

FIGURE 6

Spearman's rank correlation coefficient (ρ) of LF characteristics across different grades. Lower triangular: Scatter plots with confidence ellipses. Upper triangular: ρ (−1 to +1). Negative values indicate a negative correlation, 0 means no correlation, and positive values indicate a positive correlation. D E″, Dorsal Loss Modulus; D E′, Dorsal Storage Modulus; D Er, Dorsal Reduce Modulus; D H, Dorsal Hardness; D PH, Dorsal Proline/Hydroxyproline; D Pho, Dorsal Phosphate; D Prot, Dorsal Proteoglycan; FG, Fibrosis Grade; PG, Pfirrmann Grade; T, LF Thickness; V E′, Ventral Storage Modulus; V E″, Ventral Loss Modulus; V Er, Ventral Reduced Modulus; V H, Ventral Hardness; V PH, Ventral Proline/Hydroxyproline; V Pho, Ventral Phosphate; V Prot, Ventral Proteoglycan.