SionTo the best of our knowledge we are the first group to study the role of 5 mg of HS proteoglycan specifically in a model of DO. Using our well-established mouse DO model [8,12,13,46], we tested the effects of 5 mg of HS [32,48] on bone formation at the GS-7340 web regenerate site. Our hypothesis that HS binding to BMPFigure 6. Frequency of post-operative complications. The frequency of infection and early euthanasia was increased in the HSinjected group compared to controls. For statistical 1655472 analysis, a twotailed un-paired t test was performed between the HS-injected group and controls, in which * indicates p,0.05. doi:10.1371/journal.pone.0056790.gantagonists would result in an increase in endogenous BMPs, and subsequently accelerate bone consolidation within the distraction gap, could not be substantiated. In fact, our results suggested the opposite, showing that 5 mg of HS had a negative effect on bone healing and regeneration. We showed that the Bone-fill scores and biomechanical parameters of the regenerate bone formed in the distracted zone were weaker in HS-injected mice compared to controls. We also observed an increase in postoperative complications such as wound dehiscence and skin infection resulting in an increased early euthanasia rate in the HSinjected mice. This implies that bone and wound healing were both negatively affected in the HS treated group. While mCT analysis showed a decrease in most of the bone morphometric parameters of de novo bone in HS-injected mice, these changes were not statistically significant. Conversely, biomechanical testing parameters and bone-fill scores at 51 days post-osteotomy were significantly lower, in the 5 mg HS group compared to the controls. This discrepancy between mCT and biomechanical testing results may be explained by some limitations of the mCT technique. Although mCT measures bone GLPG0187 regeneration in a quantitative manner it can be challenging to delineate appropriate thresholds and to accurately define the distraction gap in the small tibia of a mouse. Futhermore, mCT assesses the volume of bone in the gap but cannot determine if it is contiguous or uniforme. The bone volume of the samples between our two groups were similar. However, if the regenerate was not contiguous or uniforme in one group, then this would translate into differences in strength between the groups, thereby explaining the discrepancy between the two assessments. Biomechanical testing describes the functional integrity of the regenerate bone as well as its strength and is a better assessment of the 1317923 quality of the regenerate. At 51 days (full consolidation), the Stiffness (K) andHeparan Sulfate and Distraction OsteogenesisFigure 7. Histochemistry images of distracted mouse tibiae. Mouse tibiae immunostained for members of the BMP signaling pathway (BMP2, BMPR1a, BMP-3) at 34 and 51 days. Representative images taken at 4006magnification, scale bar represents 50 mM. Chondrocytes and fibroblastic cells are indicated by the white arrows and letters “C” and “F”, respectively. doi:10.1371/journal.pone.0056790.gUltimate Force (F Ult) scores of the controls were about twice-fold that of the HS group, which were statistically significant (p = 0.0161 and p = 0.0333, respectively). Our immunohistochemistry results further corroborate the evidence that 5 mg of HS has a negative impact on bone regeneration in our model, since the expression of all 10 of the analyzed proteins involved in the osteogenic BMP signalingpathway (ligands.SionTo the best of our knowledge we are the first group to study the role of 5 mg of HS proteoglycan specifically in a model of DO. Using our well-established mouse DO model [8,12,13,46], we tested the effects of 5 mg of HS [32,48] on bone formation at the regenerate site. Our hypothesis that HS binding to BMPFigure 6. Frequency of post-operative complications. The frequency of infection and early euthanasia was increased in the HSinjected group compared to controls. For statistical 1655472 analysis, a twotailed un-paired t test was performed between the HS-injected group and controls, in which * indicates p,0.05. doi:10.1371/journal.pone.0056790.gantagonists would result in an increase in endogenous BMPs, and subsequently accelerate bone consolidation within the distraction gap, could not be substantiated. In fact, our results suggested the opposite, showing that 5 mg of HS had a negative effect on bone healing and regeneration. We showed that the Bone-fill scores and biomechanical parameters of the regenerate bone formed in the distracted zone were weaker in HS-injected mice compared to controls. We also observed an increase in postoperative complications such as wound dehiscence and skin infection resulting in an increased early euthanasia rate in the HSinjected mice. This implies that bone and wound healing were both negatively affected in the HS treated group. While mCT analysis showed a decrease in most of the bone morphometric parameters of de novo bone in HS-injected mice, these changes were not statistically significant. Conversely, biomechanical testing parameters and bone-fill scores at 51 days post-osteotomy were significantly lower, in the 5 mg HS group compared to the controls. This discrepancy between mCT and biomechanical testing results may be explained by some limitations of the mCT technique. Although mCT measures bone regeneration in a quantitative manner it can be challenging to delineate appropriate thresholds and to accurately define the distraction gap in the small tibia of a mouse. Futhermore, mCT assesses the volume of bone in the gap but cannot determine if it is contiguous or uniforme. The bone volume of the samples between our two groups were similar. However, if the regenerate was not contiguous or uniforme in one group, then this would translate into differences in strength between the groups, thereby explaining the discrepancy between the two assessments. Biomechanical testing describes the functional integrity of the regenerate bone as well as its strength and is a better assessment of the 1317923 quality of the regenerate. At 51 days (full consolidation), the Stiffness (K) andHeparan Sulfate and Distraction OsteogenesisFigure 7. Histochemistry images of distracted mouse tibiae. Mouse tibiae immunostained for members of the BMP signaling pathway (BMP2, BMPR1a, BMP-3) at 34 and 51 days. Representative images taken at 4006magnification, scale bar represents 50 mM. Chondrocytes and fibroblastic cells are indicated by the white arrows and letters “C” and “F”, respectively. doi:10.1371/journal.pone.0056790.gUltimate Force (F Ult) scores of the controls were about twice-fold that of the HS group, which were statistically significant (p = 0.0161 and p = 0.0333, respectively). Our immunohistochemistry results further corroborate the evidence that 5 mg of HS has a negative impact on bone regeneration in our model, since the expression of all 10 of the analyzed proteins involved in the osteogenic BMP signalingpathway (ligands.
