E then stained with Goldner-Trichome for comparative histology; and were mounted using Permount (Fisher Scientific, Montreal, QC) for histological analysis. Photomicro?graphs of distracted zones were taken under 506 magnification to detect for mineralized (green-stained) and non-mineralized (redstained) regions.9. Biomechanical TestingBiomechanical testing was performed on samples collected at 34 and 51 days NT 157 web post-surgery and analyzed at the McGill Centre for Bone and Periodontal Research of McGill University (Montreal, Canada). Based on previous studies [46,47], the three-point bending test was chosen over other methods of biomechanical testing and was Gracillin conducted 12926553 using the Mach-1TM Micromechanical Systems device (Bio Syntech Canada, Inc., Laval, QC). The distracted bone was placed on its posterior surface, resting on two supports of a bending apparatus that lie 7 mm apart. 
A bending load was applied downwards on the mid-shaft of the lengthened tibia at a rate of 50 mm/s until failure. Failure loads were analyzed using the Mach-1TM Motion and Analysis software (version 3.0.2, Bio Syntech Canada). A load-displacement curve was generated using this software to measure biomechanical parameters including stiffness (N/mm), ultimate force (N), ultimate displacement (mm), and work to ultimate failure (N*mm).10. Statistical analysisAll statistical tests for this study were performed using GraphPad Prism version 4.0 (GraphPad Software, La Jolla, CA). Statistical analysis was conducted for histomorphometric parameters, biomechanical testing parameters and bone-fill score using unpaired two tailed t-tests to compare the treated and untreated groups at two separate time points (34 and 51 days). The primary outcome was stiffness (N/mm) at 34 and 51 days and the other parameters were secondary outcomes.ResultsTo determine if HS visibly improves local regenerate healing, the distracted bony tissues were qualitatively assessed via imaging technology. As shown in Figure 2, radiological analysis of Faxitron X-ray (top row) and mCT image projection (middle row), 
as well as comparative histology via Goldner-Trichrome staining shows qualitative images of de novo bone formed in the injected distraction site. There were no gross phenotypic differences between the control and 5 mg HS groups.1. mCT static parameters and Bone-Fill scoresmCT results showed little to no significant differences in TV (Tissue Volume), BV (Bone Volume), and BV/TV in the HSFigure 2. Faxitron, microcomputed tomography (mCT) and histology images. Analysis and radiological images of Faxitron X-ray (top row) and mCT projection (middle-row) of distracted mouse tibiae collected at 34 and 51days post-osteotomy. The third and bottom rows show histology images of distracted tibiae after Goldner-Trichrome staining and reveal regions of the distracted zone that are occupied with soft, connective tissue (red-stained) vs. calcified tissue and/or bone matrix (green-stained). Images were taken at the center of the callus area at 1006 magnification (scale bar represents 150 mM). doi:10.1371/journal.pone.0056790.gHeparan Sulfate and Distraction Osteogenesis2. Biomechanical Parameters of HS-injected bones are reduced in HS groupResults of biomechanical testing in Figure 5 show that there were no statistically significant differences in the biomechanical parameters between the groups at 34 days. Stiffness value for the HS-injected group were reduced by about half-fold at 51 days (30.00 N/mm; compared to 6.E then stained with Goldner-Trichome for comparative histology; and were mounted using Permount (Fisher Scientific, Montreal, QC) for histological analysis. Photomicro?graphs of distracted zones were taken under 506 magnification to detect for mineralized (green-stained) and non-mineralized (redstained) regions.9. Biomechanical TestingBiomechanical testing was performed on samples collected at 34 and 51 days post-surgery and analyzed at the McGill Centre for Bone and Periodontal Research of McGill University (Montreal, Canada). Based on previous studies [46,47], the three-point bending test was chosen over other methods of biomechanical testing and was conducted 12926553 using the Mach-1TM Micromechanical Systems device (Bio Syntech Canada, Inc., Laval, QC). The distracted bone was placed on its posterior surface, resting on two supports of a bending apparatus that lie 7 mm apart. A bending load was applied downwards on the mid-shaft of the lengthened tibia at a rate of 50 mm/s until failure. Failure loads were analyzed using the Mach-1TM Motion and Analysis software (version 3.0.2, Bio Syntech Canada). A load-displacement curve was generated using this software to measure biomechanical parameters including stiffness (N/mm), ultimate force (N), ultimate displacement (mm), and work to ultimate failure (N*mm).10. Statistical analysisAll statistical tests for this study were performed using GraphPad Prism version 4.0 (GraphPad Software, La Jolla, CA). Statistical analysis was conducted for histomorphometric parameters, biomechanical testing parameters and bone-fill score using unpaired two tailed t-tests to compare the treated and untreated groups at two separate time points (34 and 51 days). The primary outcome was stiffness (N/mm) at 34 and 51 days and the other parameters were secondary outcomes.ResultsTo determine if HS visibly improves local regenerate healing, the distracted bony tissues were qualitatively assessed via imaging technology. As shown in Figure 2, radiological analysis of Faxitron X-ray (top row) and mCT image projection (middle row), as well as comparative histology via Goldner-Trichrome staining shows qualitative images of de novo bone formed in the injected distraction site. There were no gross phenotypic differences between the control and 5 mg HS groups.1. mCT static parameters and Bone-Fill scoresmCT results showed little to no significant differences in TV (Tissue Volume), BV (Bone Volume), and BV/TV in the HSFigure 2. Faxitron, microcomputed tomography (mCT) and histology images. Analysis and radiological images of Faxitron X-ray (top row) and mCT projection (middle-row) of distracted mouse tibiae collected at 34 and 51days post-osteotomy. The third and bottom rows show histology images of distracted tibiae after Goldner-Trichrome staining and reveal regions of the distracted zone that are occupied with soft, connective tissue (red-stained) vs. calcified tissue and/or bone matrix (green-stained). Images were taken at the center of the callus area at 1006 magnification (scale bar represents 150 mM). doi:10.1371/journal.pone.0056790.gHeparan Sulfate and Distraction Osteogenesis2. Biomechanical Parameters of HS-injected bones are reduced in HS groupResults of biomechanical testing in Figure 5 show that there were no statistically significant differences in the biomechanical parameters between the groups at 34 days. Stiffness value for the HS-injected group were reduced by about half-fold at 51 days (30.00 N/mm; compared to 6.