br using a objective and
using a 20× objective, and four images were randomly selected per an-imal. Leukocytes were analysed on the red channel of RGB images using NIS Elements AR Analysis Spot Finder, on Dark Spot mode, “dark, clus-tered” profile, with a 3.5 μm expected diameter and 7.5 contrast ratio se-lected to differentiate between CORM-3 and morphological features. “Remove bright” was set to 90 to reject erythrocytes. Counting method-ology was validated using a manual count on a small region and was found to be N99% accurate using the above settings. Total leukocyte count was divided by total enumerated region to yield a measurement of leukocytes per unit area.
TRAP staining was performed per manufacturer's protocol using the using the Acid Phosphatase, Leukocyte (TRAP) Kit. Briefly, slides were deparaffinised using histoclear and tissue rehydrated through an ethanol gradient. Tissue was then incubated in TRAP staining solution at 37 °C for 60 min, then counterstained with a 1:4 dilution of Harris' Haematoxylin in Milli-Q-water. The nuclear membranes were blued using Scott's Bluing Reagent. Slides were mounted using Aquamount medium (Fisher Scien-tific) under 0.17 mm cover slips. For analysis, images were cropped to se-lect the growth plate of the tibia. A 12 mm by 12 = mm ROI was positioned to be horizontally centred relative to the growth plate, with the top edge of the ROI aligning with the highest point of the border be-tween the proliferative and hypertrophic zones of the bone. This ROI was then duplicated to the immediate right and left of the initial counting ROI, creating a total of three counting ROI's. Osteoclasts were counted on the basis of TRAP+ staining colouration, cell morphology and presence of multiple nuclei using the Cell Counter ImageJ plugin (NIH).
Imaging was performed using an Eclipse Ti epifluorescent micro-scope (Nikon, Tokyo, JPN). For brightfield imaging, the Nikon DS-Ri2 colour camera was used. For fluorescent imaging, a Lumencor Spectra X light engine was utilised to power LED light sources for excitation. Emission filters for DAPI, FITC, TRITC, Cy5, and Li-Cor 740 dyes were used, and fluorescent emissions were detected using an Andor Zyla 5.5 sCMOS camera. Confocal microscopy was performed using an Olym-pus FV3000RS laser-scanning confocal microscope, utilising OBIS LS/LX laser modules (Coherent, Santa Clara, CA, USA) and FV3000 Spectral De-tector and High-sensitivity Spectral Detector units (Olympus, Center Valley, PA, USA). Final image processing was performed using NIS Ele-ments Advanced Research package (Nikon) and ImageJ software (NIH).
2.21. Statistical analysis
All experiments were performed independently at least twice, and each condition within an experiment was done in duplicates or tripli-cates. When sample size was b30, or normal distribution and variance equality were not confirmed, non-parametric tests were used. To com-pare two groups, a Splicing Mann-Whitney test was performed (non-parametric t-test), and when comparing more than two groups a Kruskal-Wallis test was applied (non-parametric variance analysis). Both tests were followed by a multiple comparison test (adjusted p values for multiple comparison). For the flow-chamber assay, a two-way ANOVA test and Dunnett's multiple comparisons test was done, and each column was compared to native MSC. For the survival analysis, a log-rank (Mantel-Cox) test was performed to compare treated groups to the control group. *p b .05, ** p b .01, ***p b .001 and ****p b .0001. For animal exper-iments, the value for each animal and the median of the group were plot-ted. A power analysis was done from the first animal studies performed in order to determine the minimum animal number to be used for following studies. The first study's goal was to evaluate the effect of three mRNA engineered MSC therapies (CD, OPG and CD/OPG BM-MSC) compared to PBS and Mock MSC. In our pilot data, the mean and standard deviation of the log-transferred before and after tumour growth ratio was 1.71 (1.03) in PBS group and − 0.91 (1.90) in CD/OPG group respectively. For the ex-vivo bone analysis, the mean (SD) of bone loss was 0.18