Effect of non-enzymatically altered microstructure of trabecular bone on microhardness

Abstract

Bone is a composite material consisting of organic and mineral phases, alterations of the constituents will modify its biomechanical properties. Aging bone undergoes various biochemical changes that might cause the declined mechanical performance. Most of the organic phase of the bone is composed of type I collagen which is subjected to posttranslational modifications, including non-enzymatic glycation (NEG). NEG causes the formation of advanced glycation end-products (AGEs) that leads to formation of intra- and inter-fibrillar cross-links between collagen fibers. The presence of a stiffer collagen network in bone might be related to age-related increased fragility and various diseases, such as diabetes, where there is a high incidence of bone fracture. Microindentation is an investigational tool used to determine hardness of bone. The aim of this study is to study explore the effect of NEG on microindentation the hardness results of bovine bone with respect to the variation with applied mass, dwell time, wet/dry state. Specimens from 1.5-year-old bovine femur bone cut perpendicular to the long axis were polished until surface roughness became less than 0.05 μm. The circular pieces cut into four and divided into 2 groups. One group was rested in a solution containing 0.6 M ribose, 25 mM e-amino caproic acid, 5 mM benzamidin, 10 mM N-ethyl maleimide, 30 mM HEPES and Hank's buffer for the non-enzymatic glycation reaction for four weeks. Control group was incubated in the same solution except for the ribose. 3 samples were collected weekly from each group. Effect of indentation load were studied with 0.098, 0.245, 0.490, 0.980 vs 1.96 N forces applied for 10 s. Duration of indentation was studied with 0.980 N force applied for 5, 10, 20 and 30 s. Microindentation measurements were repeated 5 times on each sample in the wet and dry state. ANOVA is used for comparison of load or duration effect, paired sample t test is used for comparison of wet and dry state with SPSS 17.0 statistical program. p NEG bovine cortical bones were found to be significantly different with 0.245, 0.490, 0.980 vs 1.96 N micro-indentation loads compared to control group in samples collected from 1 to 4 weeks ( p p Special nano-structure of mineralized collagen is the most important factor determining its strength and ability to resist large deformations consistent with its physiological function. Microhardness values of bone is found to be could be altered by wet and dry state of the bone apart from the amount of applied load of indentation (>0.098 N) but not by duration of indentation. Microhardness of bone was shown to be effected by non-enzymatic glycation causing a stiffer collagen network. Altered microstructure and thus mechanical properties of bone could explain increased risk of fracture with increasing age.