Dolph Houben

82 CHAPTER 4 Cyclic Reference Point Indentation To evaluate bone properties on a micro scale, we performed cyclic reference point indentation (RPI). The Total Indentation Distance (TID) can directly assess the ability of the bone matrix to resist crack initiation and propagation. Normalized Indentation Distance Increase (NIDI) is inversely correlated with toughness and fracture [27] . After calibration, cyclic reference point indentation (RPI) testing was performed on a Biodent HFC system (ActiveLife Scientific, Inc., Santa Barbara, CA) using a cone-spherical test probe tip (BP2). Bone sections were irrigated with phosphate buffered saline (PBS) solution to maintain hydration. Twenty cycles of loading alternating between 0 N to 6 N at a frequency of 2 Hz were applied. Before testing each specimen, a polymethyl methacrylate block was indented to provide an internal reference standard. Since the tibia cross- section is roughly triangular, sites on each of the three sides of each specimen were selected for indentation. Each side was oriented such that the indenter made contact perpendicular to the surface. Axial measurements of the cortex were obtained by placing the specimen on a thin rubber sheet that prevented sliding during testing. One indentation at each site was performed. Results from testing were averaged for each cross-section to obtain a single value for each specimen. Quantitative Histomorphometry A 5 mm segment of the allotransplant was reserved for histological analyses, fixed in 10% buffered formalin for 48 hours, embedded in methyl methacrylate, sectioned using a diamond band saw, thereafter ground to 15um-thick sections (Exakt Technologies Inc., Oklahoma City, OK). The unstained sections were analyzed with fluoroscopy (10X, Olympus BX51) since dual fluorescent labels were given prior to sacrifice 10 days apart. Both the endosteal and periosteal surfaces (six random fields on each surface) were analyzed to quantify label uptake: labeled surface (LS), single labeled surface (sLS) and double-labeled surface (dLS) per bone surface (BS), mineralizing bone surface (MS/BS). Based on these remodeling parameters we were able to calculate the bone formation ratio (BFR/BS (mcm 3 /mcm 2/ d)). Quantifications and calculations were done with the semi-automatic bone image analysis software (Osteomeasure; Osteometrics, Atlanta, GA). Statistical Analysis Since the collected data was collected from a low sample size (N=14) and not normally distributed, a non-parametrical test (Wilcoxon rank sum test) was used to detect a difference between the two groups (allotransplants with patent AV-bundle versus allotransplants with ligated AV-bundle). For the same reasons, a non-parametric (Wilcoxon signed-rank test) test was used to detect a difference between the operated and contra-lateral tibia. A Fisher’s exact test was used to detect a difference in union at the time of sacrifice. All statistical tests were two-sided and differences were considered significant for p-values of < 0.05. Statistical analyses were performed using the statistical program JMP Pro 13.0.0 (SAS Institute Inc.). Statistical analysis was supported by the Center for Translational Sciences Activities (CTSA) at Mayo Clinic.