Fourier Transform Infrared Microspectroscopy Combined with Principal Component Analysis and Artificial Neural Networks for the Study of the Effect of β-Hydroxy-β-Methylbutyrate (HMB) Supplementation on Articular Cartilage.

Izabela Świetlicka,Siemowit Muszyński,Marta Arczewska,Ewa Tomaszewska,Marcin B Arciszewski,Piotr Dobrowolski,Hauke Clausen-Schaumann,Mariusz Gagoś,Katarzyna Kras,Carina Prein,Tomasz Blicharski,Attila Aszodi,Michał Świetlicki

doi:10.3390/ijms22179189

Abstract

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary β-hydroxy-β-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young’s modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology.

Highlights

Articular cartilage (AC) is a non-vascular type of connective tissue covering the end of bones forming synovial joints to facilitate the distribution of loads across articular surfaces with a low frictional coefficient and buffer vibration [1]
Since the mechanism of hydroxy-β-methyl butyrate (HMB) action remains unknown, it is hypothesized that HMB could affect protein metabolism acting on several levels, e.g., inhibiting proteolysis, attenuating depression in protein synthesis, metabolic inhibitors and promoting protein synthesis [43,44,45,46], which is manifested by modifications in skeletal muscle structure as well as by changes in the mechanical and morphological properties of bone [18,20,40,42,47] and teeth [41]
This is the first report combining FTIR imaging with multivariate analysis to study the effect of the HMB supplementation on the structure and properties of articular cartilage

Summary

Introduction

Articular cartilage (AC) is a non-vascular type of connective tissue covering the end of bones forming synovial joints to facilitate the distribution of loads across articular surfaces with a low frictional coefficient and buffer vibration [1]. The mature, non-calcified AC tissue demonstrates zonal heterogeneity due to the orientation of the collagen fibrils and molecular composition, it is subdivided into three histological zones (superficial zone (SZ), middle zone (MZ) and deep zone (DZ), of which the latter is separated from the calcified AC zone by the tidemark) [4]. SZ with the lowest PGs content [5] but a high level of collagen, collagen fibrils are oriented in parallel to the AC surface. Such an arrangement is responsible for most of the tensile stiffness of cartilage. In the MZ (ca. 60% of total AC thickness), collagen fibrils are mostly randomly organized, while in the DZ they are aligned perpendicular to the AC surface [5,6]

Methods

Results

Conclusion