Dolph Houben

100 CHAPTER 5 Histology The selected bone segment was fixed in 10% buffered formalin for 48 hours, embedded in methyl methacrylate, sectioned using a diamond band saw and ground to 15µm thick sections (Exact technologies Inc., Oklahoma City, OK). The SRBS stained slides were used to analyze transplant viability by quantifying osteocytes, osteoblasts, and empty lacunae using light microscopy (20X, Olympus BX51) on the endosteal surface and periosteal surface (six random fields on each surface). Quantifications and calculations were done with the semi-automatic bone image analysis software (Osteomeasure; Osteometrics, Atlanta, GA). Bone viability was measured by calculating the percentage of either vacant or osteocyte-occupied lacunae using the Osteomeasure system. The percentage of empty lacunae was calculated by dividing the number of empty lacunae by the total (occupied and empty) lacunae X 100. Micro CT angiography The 20mm mid-VCA segment was fixed in 10% buffered formalin (48 hours) and decalcified over a 7 week period in Richard-Allan Scientific™ Decalcifying Solution (Thermofisher, Waltham, MA). Micro-CT scanning was performed using an Inveon PET CT scanner (Siemens Medical Solutions USA, Inc., Malvern, PA) using settings of 80 kV and 500uA and imaging software (PMOD Technologies, Zurich, Switzerland) at a medium-high magnification resolution. We used the BMA application of AnalyzePro sofware (Analyze, Mayo Clinics, Rochester, MN) to measure total transplant volume, cortex volume and medullary canal volumes. The segmentation portion of the application were used to separate the contrast-filled cortical and medullary vessels from surrounding bone. Capillary density within the allotransplants and normal tibiae was then calculated, reported as a percentage of total bone, medullary space and cortical bone volumes occupied by vessels. RNA extraction, cDNA synthesis, and real-time quantitative PCR (RT-qPCR) Another 2mm section of the allotransplant was cut with a cooled sterile oscillating bone saw. The section was cleared of excess soft tissue, snap frozen in liquid nitrogen and stored at -80 ⁰C for later analysis. Bone sections were then individually pulverized in liquid nitrogen using the A11 basic analytical mill (IKA-Werke GmbH & Co. KG, Germany). RNA was extracted from the pulverized bone with PureLink RNA mini kit, TRIzol reagent and an on-column Pure link DNase treatment (Thermo Fisher Scientific, Cat no. 12813018A, 12034977, 12185-010, Carlsbad, CA). Quantification and determination of RNA purity were performed with a Nano-drop Spectrometer (ThermoScientific Nano-drop Technologies, Wilmington, DE) and absence of RNA degradation was confirmed by gel electrophoreses of the total RNA before conversion to cDNA. An iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA) was used for the reverse transcription reaction. Total RNA (200 ng) was mixed with nuclease-free water, iScript reverse transcriptase, and 5X reverse transcription reaction mix and converted to cDNA following the iScript protocol. RT-qPCR was performed to quantify the expression of target genes with iQ SYBR green Supermix using the CFX384 Real-Time detection system (Bio-Rad Hercules, CA). Transcript quantity measurements were normalized to GAPDH , and gene expression levels were