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Biomaterials

Biomaterials - Poster Session B

Poster C6 - Enhancing Microindentation Accuracy in Soft Biomaterials

Thursday, October 24, 2024
2:45 PM - 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Accurately determining the mechanical properties of soft tissues, such as cartilage, is crucial for enhancing the precision of current medical technologies. Nanoindentation and microindentation, which involve using a small tip to probe local mechanical properties, are valuable tools in this context. However, traditional indentation methods, developed for hard materials, encounter significant challenges when applied to compliant, hydrated biomaterials due to adhesion and substrate effects. Adhesion between the indenter tip and the sample can lead to overestimations of the modulus, while substrate effects occur when thin samples cause the underlying stiffer material to skew measurements. The rule of thumb for substrate effect is that indents should not exceed 1/10th of the sample thickness; this guideline, however, has been primarily investigated in the context of stiff materials, and critical thicknesses for indentation of soft materials remain unexplored.

The primary objective of this study was to observe and quantify the substrate effect during microindentation of soft biomaterials in the presence and absence of adhesion. This study investigates the microindentation of soft materials in three environments: air, water, and surfactant solution. Experiments conducted in air and water were expected to exhibit considerable adhesion, resulting in unreliable modulus measurements. Conversely, the surfactant solution was expected to eliminate adhesion, allowing for accurate data acquisition with traditional analysis methods. By comparing the data obtained from these different environments, the study aims to identify whether the 1/10th rule of thumb is appropriate for soft material microindentation, and whether it is dependent on the presence or absence of adhesion.

Materials and

Methods:  Eighteen samples of EcoFlex 10 silicone, with thicknesses ranging from 0.254 mm to 10 mm, were prepared in custom well plates to investigate the substrate effect and adhesion during testing.

Microindentation tests were performed using the Activelife Biodent reference probe microindenter (Santa Barbara, CA) equipped with cylindrical tips of two diameters: 1.15 mm (Tip A) and 1.56 mm (Tip B). Five indents were conducted on each sample. Each indent applied 10 cycles of sinusoidal displacement with an amplitude of 300 microns and a frequency of 1 Hz.

Loading and unloading reduced moduli were calculated by dividing the sample stiffness (measured by the loading slope and the unloading slope) by the tip diameter. To assess the influence of environmental conditions, indentations were performed in each sample under three different hydration conditions: air, water, and a surfactant solution. This approach aimed to elucidate the role of surfactants in minimizing adhesion and improving measurement accuracy.

Data analysis involved plotting modulus values against sample thickness to identify the critical thicknesses beyond which the substrate effect becomes negligible. To determine critical thickness, the Solver tool in Excel was first used to fit an exponential model of the form y = Ae^(Bx) + C to each set of data (Figure 1). Then, to determine the critical thickness below which the substrate effect caused substantial error in the modulus measurement, the thickness at which the best fit equation for modulus was 5% higher that the equilibrium modulus value reached in thick samples was identified.

Results, Conclusions, and Discussions: When investigating the substrate effect, it was found that modulus values stabilized for thicker samples, with higher values unloading curve moduli (180-200 kPa), and lower values for loading curve moduli (120-150 kPa). However, as sample thickness decreased, the measured modulus increased exponentially, indicating a significant substrate effect (Figure 1). Critical thickness values, the minimum thickness beyond which moduli seemed minimally affected by substrate effect, ranged from 1.93 - 2.61 mm (mean = 2.18 mm), with no trend with hydration condition or tip diameter. These critical thicknesses are less than 10x the indent depth (3 mm). Hence, the rule of thumb of not indenting more than 1/10 the sample thickness seems to apply for soft biomaterials, but may be more conservative than needed.

When investigating the effects of adhesion on equilibrium modulus values (moduli measured in the thickest samples) it was observed that, for both tips, both the loading and unloading modulus values were typically lower in the surfactant environment compared to air and water. The exception was for the unloading modulus measured using tip B, where moduli in surfactant and air were comparable (~130 kPa), but moduli in water were higher (~150 kPa). This trend indicates a reduction in adhesion when using surfactant leading to more accurate measurements during microindentation, as has previously been observed in nanoindentation.

Figure 1 shows that the measured modulus data was noisy for thinner samples. This was observed for all datasets, and may be due to an increased sensitivity to minor variations in thickness or the parallel loading of the tip with the sample when the underlying stiff substrate is impacting modulus measurements. Despite the noisy data, since all critical thickness values were less than 3 mm, it seems reasonable to assume that following the rule of thumb of not indenting more than 1/10 of the sample thickness will still avoid the substrate effect. However, future work should investigate a wider range of tip diameters, indent depths, and sample moduli to establish a rule of thumb for critical thickness that would be applicable across a wide range of soft material testing.

Acknowledgements (Optional):