Development and Validation of a Miniature Bending Device for Imaging Loaded Bone 1Getsinger, B; 1Riffitts, J; 2Price, C; 1Zhang, H; +2Wang, L

1Rowan University, Glassboro, NJ, +2Department of Mechanical Engineering, University of Delaware, Newark, DE Senior Author: lywang@udel.edu

Introduction: Load-induced fluid flow within the bone lacunar-canalicular system (LCS), through its ability to enhance molecular transport and activate osteocyte’s mechanosensory mechanisms, is critical for the maintenance and adaptation of the skeleton [1] but remains difficult to measure. Recently, our laboratory demonstrated the phenomenon of load-induced fluid flow in intact murine tibia using the fluorescence recovery after photobleaching (FRAP) technique in combination with a uniaxial cyclic loading device [2]. The study provided a direct measurement of solute transport enhancement (~30%) at the LCS level when bone was axially compressed under a relatively small loading magnitude (3N). To further understand the load-dependency of fluid flow, we need to image (performing FRAP on) the bone while the bone is loaded under higher loads. However, within the original design, axial compression with higher load magnitudes tends to result in significant creeping of the specimen and out-of-focus shifting during imaging. In this paper, we describe the development and validation of a major upgrade of the loading device. It is a custom three-point bending apparatus that directly attaches to a standard confocal microscope, providing a much smaller equipment footprint and a greater imaging stability. Together with the FRAP imaging technique, this miniature loading device will be a powerful, easy-to-use tool, for studying bone fluid flow and cellular responses to mechanical stimulation.

Methods: Our custom-bending device is controlled via a LabVIEW interface (National Instruments, TX) and a data acquisition card (Fig. 1). The device consists of a piezoelectric linear actuator (Physik Instrumente, MA), a cantilever beam with four attached strain gauges (forming a full bridge) for measuring the applied load, a water bath, a xyz positioning stage, and a frame for attachment to a standard Zeiss microscope (Fig 1). Loads are applied, in a 3-point bending configuration, through two fingers extending from the cantilever beam and a central support from the base. Under the trigger signals generated by the control software, the loaded bone specimen is imaged via an objective inverter (LSM Technologies, PA) immediately above the central support where specimen deflection and out-of-focus drifting are expected to be negligible.

Similarly to our previous study [3], the ex-vivo/in-situ capabilities of our device were evaluated by loading and optically quantifying surface strains in well-characterized beams of ultra-high molecular weight polyethylene (UHMWPE) (3x3 & 2x2x20mm beams), acrylic (2x2 & 1x1x20mm), cortical bone from bovine femora [~12-mo of age] (1x1x20mm), and whole murine tibia from male C57BL/6J (B6) mice (6 & 18wks of age). This study was approved by our Institutional Animal Care and Use Committee. Engineering strains were quantified using optical landmarks (or patterns) generated via 10µm fluorescent microspheres (FluoSpheres, Invitrogen, CA) applied to the specimen’s surface [3]. In the murine tibiae, strain was recorded at the mid-point of the tibial shaft on the anterior-medial surface. An initial preload of ~0.25N was applied, in displacement control, to the specimens and a high-speed CCD camera (MRc5, 0.5s/frame, Carl Zeiss, NY), installed

on the side port of a Zeiss LSM 510 microscope, was triggered to capture the baseline images. Higher bending loads were applied by incrementally advancing the piezo stage. The load magnitude was monitored through strain gauge readings that had been calibrated against known forces. Ten, 2584x1936-pixel, images were acquired at each load level using the digital camera under DAPI illumination [3]. Images were exported to a semi-automated IMAQ Vision script (NI) for pattern matching and strain analysis [3]. Relationships between flexural stress and strain, or strain and applied load were analyzed using Prism statistical software package (GraphPad, CA).

Results: Using our miniature 3-point bending device we were able to consistently, and reproducibly apply controlled strains in a wide array of materials (Fig 2). In all materials tested, the relationships between flexural stress and surface strain as well as those between surface strain and load were highly linear (Fig 2). For the machined beams, calculated values for the flexural moduli were consistent with previously reported values (Table 1). Ex-vivo studies on murine tibiae demonstrated that the anterior-medial surface of the tibial mid-shaft could be deformed from0to~5000με when the piezo actuator applied forces up to 8N. It was also observed, as expected, that the relationship between surface strain and applied load in these tibia depended on age, with older specimens exhibiting significantly less surface deformation for a given load (με18wk=307*L-78, r2=0.87 vs. με6wk=683 *L-136, r2=0.92; ANCOVA p<0.0001).

Discussion: The ability to directly measure, in-situ and in real-time, load-induced solute transport through the bone LCS has the potential to transform future studies of the cellular and molecular biology of bone adaptation. Here we describe a significant upgrade to the combined loading and imaging system we previously used to directly quantify load-induced solute transport in bone. The good agreement between calculated and reported flexural moduli (for beams), and linear relationship between strains and load (for tibiae) [4] demonstrate the reliability and robustness of our new 3-point bending/imaging system. This new device has demonstrated several distinct advantages, including better integration with the microscope and significantly greater stability of imaging, which will allow us to expand and improve on our previous load-induced transport/fluid flow studies. While the system is currently restricted to displacement feedback control and a load limit of 8-N, these limitations are currently being addressed. Our long-term goal is to use these techniques to explore the biological mechanisms behind mechanosensation and bone adaptation, and provide crucial insights into treatments of bone disease.

Acknowledgments: NIH funding (P200RR016458; RO1AR054385) References: [1] Fritton 2009, Ann Rev Fluid Mech; [2] Price 2010, ORS 56th Ann Meeting; [3] Price 2010, J Ortho Res; [4] Silva 2005, Ana Rec Part A

Fig. 2: Linear relationships among flexural stress and strain in beams of A) UHMWPE, B) acrylic, and C) bovine bone (n=5 each). The relationship between D) strain and applied load in intact B6 mouse tibia depended on specimen age.

Table 1: Experimentally calculated flexural moduli for beams of UHMWPE, acrylic and Bovine bone

Fig.1 A) Schematic of the integrated piezoelectric actuated 3-point bending/imaging device (Note: Microscope base is not shown). B) The flow chart shows the control of the piezo stage, load measurement using the strain gauges, and trigger imaging of loaded specimens.

Poster No. 2238 • ORS 2011 Annual Meeting