Determination of the Depth- and Time- Dependent Mechanical Behavior of Mouse Articular Cartilage Using Cyclic Reference Point Indentation

Mouse models of osteoarthritis and cartilage degeneration are important and powerful tools for investigating the molecular mechanisms of the disease pathology. Because of the vast number of genetically modified mouse models that are available for research, the ability to use these models is particularly attractive for the mechanobiologic interactions in the pathogenesis of osteoarthritis. However, the very small scale of mouse articular cartilage, where the healthy tissue is only 80 µm in thickness, poses challenges in quantifying mechanical characteristics of the tissue. We introduce here a novel approach that combines experimental and analytical methods to quantify the nuanced mechanical changes during cartilage degeneration at this scale. Cyclic reference point indentation is used to directly test the murine articular cartilage to obtain the force-deformation and the phase-shift characteristics of the tissue. The cartilage zonal thicknesses are confirmed from histology. These data are then fitted to a parallel spring model to determine the depth-dependent tissue stiffness and modulus. Using this approach, we investigated the effects of trypsin degradation on the zonal mechanical behavior of mouse articular cartilage. We observe a decline of the superficial zone stiffness coupled with the loss of the superficial layer. Subsequent degradation by trypsin allowed the identification of middle- and deep- zone properties. Taken together, this approach can be a useful tool for understanding the disease mechanisms of cartilage homeostasis and degeneration, and for monitoring of therapies for osteoarthritis.

Determination of Elastic Modulus in Mouse Bones Using a Nondestructive Micro-Indentation Technique Using Reference Point Indentation

The determination of the elastic modulus of bone is important in studying the response of bone to loading and is determined using a destructive three-point bending method. Reference point indentation (RPI), with one cycle of indentation, offers a nondestructive alternative to determine the elastic modulus. While the elastic modulus could be determined using a nondestructive procedure for ex vivo experiments, for in vivo testing, the three-point bending technique may not be practical and hence RPI is viewed as a potential alternative and explored in this study. Using the RPI measurements, total indentation distance (TID), creep indentation distance, indentation force, and the unloading slope, we have developed a numerical analysis procedure using the Oliver–Pharr (O/P) method to estimate the indentation elastic modulus. Two methods were used to determine the area function: (1) Oliver–Pharr (O/P—based on a numerical procedure) and (2) geometric (based on the calculation of the projected area of indentation). The indentation moduli of polymethyl methacrylate (PMMA) calculated by the O/P (3.49–3.68 GPa) and geometric (3.33–3.49 GPa) methods were similar to values in literature (3.5–4 GPa). In a study using femurs from C57Bl/6 mice of different ages and genders, the three-point bending modulus was lower than the indentation modulus. In femurs from 4 to 5 months old TOPGAL mice, we found that the indentation modulus from the geometric (5.61 ± 1.25 GPa) and O/P (5.53 ± 1.27 GPa) methods was higher than the three-point bending modulus (5.28 ± 0.34 GPa). In females, the indentation modulus from the geometric (7.45 ± 0.86 GPa) and O/P (7.46 ± 0.92 GPa) methods was also higher than the three-point bending modulus (7.33 ± 1.13 GPa). We can conclude from this study that the RPI determined values are relatively close to three-point bending values.

The effect of aminoguanidine (AG) and pyridoxamine (PM) on ageing human cortical bone

Objectives Advanced glycation end-products (AGEs) are a post-translational modification of collagen that form spontaneously in the skeletal matrix due to the presence of reducing sugars, such as glucose. The accumulation of AGEs leads to collagen cross-linking, which adversely affects bone quality and has been shown to play a major role in fracture risk. Thus, intervening in the formation and accumulation of AGEs may be a viable means of protecting bone quality. Methods An in vitro model was used to examine the efficacy of two AGE-inhibitors, aminoguanidine (AG) and pyridoxamine (PM), on ageing human cortical bone. Mid-diaphyseal tibial cortical bone segments were obtained from female cadavers (n = 20, age range: 57 years to 97 years) and randomly subjected to one of four treatments: control; glucose only; glucose and AG; or glucose and PM. Following treatment, each specimen underwent mechanical testing under physiological conditions via reference point indentation, and AGEs were quantified by fluorescence. Results Treatment with AG and PM showed a significant decrease in AGE content versus control groups, as well as a significant decrease in the change in indentation distance, a reliable parameter for analyzing bone strength, via two-way analysis of variance (ANOVA) (p < 0.05). Conclusions The data suggest that AG and PM prevent AGE formation and subsequent biomechanical degradation in vitro. Modulation of AGEs may help to identify novel therapeutic targets to mitigate bone quality deterioration, especially deterioration due to ageing and in AGE-susceptible populations (e.g. diabetics). Cite this article: O. Abar, S. Dharmar, S. Y. Tang. The effect of aminoguanidine (AG) and pyridoxamine (PM) on ageing human cortical bone. Bone Joint Res 2018;7:105–110. DOI: 10.1302/2046-3758.71.BJR-2017-0135.R1.