Image matters: Development of a novel staining method, the RGB-trichrome, as a reliable tool for the study of the musculoskeletal system

Abstract

20Morphometry and histology are essential approaches for investigation and diagnosis of musculo-21 skeletal disorders. Despite the advent of revolutionary methods of image analysis and high resolution 22 three-dimensional imaging technology, basic conventional light microscopy still provides an incisive 23 overview of the structure and tissue dynamics of the musculoskeletal system. This is crucial to both 24 preclinical and clinical research, since several clinically relevant processes, such as bone repair, 25 osteoarthritis, and metabolic bone diseases, display distinct, if not pathognomonic, histological 26 features. Due to the particular characteristics of the skeletal tissues (i.e., the existence of mineralized 27 extracellular matrices), a large number of staining methods applicable to either decalcified or 28 undecalcified tissues are available. However, it is usually the case that several staining methods need 29 to be sequentially applied in order to achieve the different endpoints required to fully assess skeletal 30 tissue structure and dynamics, and to allow morphometric quantification. We describe herein a novel 31 staining method, the RGB trichrome, amenable for application to decalcified, paraffin embedded 32 human musculoskeletal tissues. The acronym RGB corresponds to the three primary dyes used: 33 picrosirius Red, fast Green, and alcian Blue. Although these individual pigments are commonly used 34 either isolated, in binary combinations, or as part of more complex polychrome staining methods, when 35 merged in the RGB trichrome staining produce high-quality/high-contrast images, permitting not only 36 clear identification of different tissues (i.e., the different types of cartilage, bone and fibrous connective 37 tissue), but also discrimination between calcified and uncalcified bone and cartilage, as well as an 38 unexpected diversity of shades of color, while displaying singular properties among polychrome 39 staining methods, such as the unveiling of the bone osteocyte dendritic/canalicular network. Hence, 40we propose the RGB trichrome as simple but highly-reliable tool for the preclinical and clinical study of 41 the musculoskeletal system. 42 43 45 endocrine and metabolic roles 1. It is composed of bone, cartilage and skeletal muscle, together with 46 connecting structures, such as tendons and ligaments. The sustained increase in life expectancy 47 witnessed in the last decades in low-, mid-, and high-income countries has resulted in an increase in 48 the number and proportion of elderly people, and in the worldwide prevalence of age-related diseases 49 2. Aging of the musculoskeletal system is a leading cause of disability, and some of its age-related 50 alterations, such as osteoporosis and osteoarthritis, are among the major epidemics of the 21st 51 century, being, together with sarcopenia, highly-prevalent causes of mobility disability, and risk factors 52 for falls and fractures in the elderly population 3-7. 53Histology and histomorphometry are powerful tools for the study of skeleta...