In-situ nanomechanical testing using X-ray microscopy

Micron-scale X-ray tomography, classically referred to as MicroCT, is a well-established 3D imaging technique and has seen various applications of in situ testing due to flexible sample types, sizes, nondestructive imaging, and lack of need for a vacuum enclosure. Recent advances in lab-based nanoscale X-ray microscopy (XRM) have moved beyond some of the physical constraints of traditional MicroCT by incorporating synchrotron-style optics and detection systems, extending spatial resolution down to 50 nm for samples tens to hundreds of microns in size. These nanoscale X-ray microscopes provide high resolution, nondestructive 3D imaging of interior structures on samples of tens to hundreds of microns in cross-sectional dimension. This new length scale of nondestructive investigation complements existing in situ mechanical testing capabilities by filling an additional portion of the span between the sub-nanometer and bulk experimental regimes, and also provides new opportunities for understanding material deformation behavior at this scale. In a complementary fashion to the variety of imaging tools, in situ mechanical testing also covers a broad range of scales. At the high resolution end of the spectrum, testing is often coupled with techniques such as SEM or TEM for direct observation of the deformation processes. While offering excellent spatial resolution, in situ SEM is limited to observations of the sample surface, and TEM is restricted by very thin samples, which also generates unique material properties due to the small size effects. In an effort to provide new, unique information by merging the worlds of high resolution 3D imaging and nanomechanical testing, a new in situ load stage has been designed and tested in a ZEISS nanoscale X-ray microscope. This work will cover early material investigations performed to uniquely connect bulk material properties with detailed, direct observation of discrete, internal deformation events occurring at the nano- and micron-scale. A case study of the nanoindentation of a sample of elephant dentin will be presented. Dentin is a naturally-occurring nano-composite material (found in teeth) consisting of a collagen matrix, mineralized hydroxyapatite, and anisotropic tubule structures. This natural structure is of interest for biomimetic applications due it’s remarkable mechanical properties. In this study, 3D tomography was performed multiple times at increasing levels of applied load to monitor crack initiation and growth processes, and gain insight into the connections between the novel microstructure, crack shielding mechanisms, and the material’s fracture toughness. As an example of the cracking process, figure 1 provides two orthogonal virtual cross sections of the dentin sample during indentation, revealing the tubule structure as well as the initiation of cracks in the vicinity of the indenter tip

Multiscale biomechanical responses of adapted bone–periodontal ligament–tooth fibrous joints

Reduced functional loads cause adaptations in organs. In this study, temporal adaptations of bone-ligament-tooth fibrous joints to reduced functional loads were mapped using a holistic approach. Systematic studies were performed to evaluate organ-level and tissue-level adaptations in specimens harvested periodically from rats (N=60) given powder food for 6 months over 8,12,16,20, and 24 weeks. Bone-periodontal ligament (PDL)-tooth fibrous joint adaptation was evaluated by comparing changes in joint stiffness with changes in functional space between the tooth and alveolar bony socket. Adaptations in tissues included mapping changes in the PDL and bone architecture as observed from collagen birefringence, bone hardness and volume fraction in rats fed soft foods (soft diet, SD) compared to those fed hard pellets as a routine diet (hard diet, HD). In situ biomechanical testing on harvested fibrous joints revealed increased stiffness in SD groups (SD:239-605 N/mm) (p<0.05) at 8 and 12 weeks. Increased joint stiffness in early development phase was due to decreased functional space (at 8 weeks change in functional space was -33 μm, at 12 weeks change in functional space was -30 μm) and shifts in tissue quality as highlighted by birefringence, architecture and hardness. These physical changes were not observed in joints that were well into function, that is, in rodents older than 12 weeks of age. Significant adaptations in older groups were highlighted by shifts in bone growth (bone volume fraction 24 weeks: Δ-0.06) and bone hardness (8 weeks: Δ-0.04 GPa, 16 weeks: Δ-0.07 GPa, 24 weeks: Δ-0.06 GPa). The response rate (N/s) of joints to mechanical loads decreased in SD groups. Results from the study showed that joint adaptation depended on age. The initial form-related adaptation (observed change in functional space) can challenge strain-adaptive nature of tissues to meet functional demands with increasing age into adulthood. The coupled effect between functional space in the bone-PDL-tooth complex and strain-adaptive nature of tissues is necessary to accommodate functional demands, and is temporally sensitive despite joint malfunction. From an applied science perspective, we propose that adaptations are registered as functional history in tissues and joints

In situ compressive loading and correlative noninvasive imaging of the bone-periodontal ligament-tooth fibrous joint.

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics