PCA-Assisted Raman Analysis of Osteonecrotic Human Femoral Heads

Abstract

Osteonecrosis of the femoral head (ONFH) occurs frequently in adolescents and young adults and causes progressive deformation and destruction of the hip joint and impairs standing and walking, resulting in a significant decrease in the quality of life of patients. In addition, studies have shown that a history of corticosteroid administration and heavy alcohol consumption are closely related to the occurrence of ONFH. However, the detailed mechanism by which steroid administration and alcohol consumption are associated with the development of the disease is still unknown. With many researches still ongoing and without a clear biological pathway for osteonecrosis, effective preventive measures cannot be taken. Therefore, the current focus of ONFH treatment is to establish an early diagnosis and treatment strategy. We obtained the femoral heads of four patients with steroidal ONFH and three patients with alcoholic ONFH. We then compared the femoral heads of steroidal and alcoholic osteonecrosis by analyzing them at the molecular level by Raman spectroscopy. Crystallographic changes (deformations) in the mineral phase and fraction of organic material respect to the total mass were then plotted as a function. We found that changes in bone composition in ONFH were different in steroidal and alcoholic ONFH. We conclude that this suggests that the developmental mechanisms of steroidal and alcoholic ONFH may follow different paths. We also noticed that while steroid seem to lead to a more marked degradation of the tissue, alcohol seem to affect also the quality of the healthy tissue.

Keywords: PCA; Raman spectroscopy; alcohol; bone tissue; osteonecrosis of the femoral head (ONFH); steroid.

Figure 1

Preparation of the samples: (a) an anterior view of the femoral head removed after surgery, (b) cutting line, (c) front cross-section.

Figure 2

Optical and Raman imaging of the surface of Sample No7: (a) optical image of an osteonecrotic area, (b) Raman imaging of the same area, (c) optical image of a healthy area, (d) Raman imaging of the same area.

Figure 3

Representative Raman spectra of three different regions of Sample No7: healthy in red, the boundary in blue, and osteonecrotic in yellow. For each set of data, the average spectra and the limits of the 90% confidence interval are presented in different tones. Phenylalanine is marked with an “*” and an unknown band, marked with a “#”, appears at slightly lower Raman Shifts.

Figure 4

Graphs showing the relationship between the intensity ratios (I960cm⁻¹/I1243–1269cm⁻¹, I960cm⁻¹/I1450cm⁻¹ and I960cm⁻¹/I1660cm⁻¹) and the FWHM of the band located at 960 cm⁻¹ for the healthy, boundary and ON regions of all samples (a–c). Symbols and colors are as follows: △ healthy, □ osteonecrosis and ○ boundary tissue; green, purple, pink and black for steroid, red, blue and dark blue for alcohol.

Figure 5

Graphs (a–c) of the relationship between the each of intensity ratio I960cm⁻¹/I1450cm⁻¹, I960cm⁻¹/I1243–1269cm⁻¹, I960cm⁻¹/I1660cm⁻¹ and FWHM of the band located at 960 cm⁻¹. Different trends between alcoholic necrosis and steroidal necrosis regions are shown.

Figure 6

(a) Principal Components Analysis of the different samples, divided into four groups depending on the origin and health status and (b) Principal components PC1 and PC2 with their score and an osteonecrotic spectrum for comparison.