F gene expression [34,35]. The pH/ionic microenvironment has also been shown to affect the binding affinity of HS [22,36]. HS tends to have a higher affinity to proteins in the presence of cations (e.g. zinc and copper) [37], whereas its binding affinity decreases in a low cationic presence [38,39]. In light of this controversy, the purpose of this study was to investigate the effects of exogenous, locally-applied kidney-derived HS in a wild-type mouse model of DO; by examining the effects on (a) bone formation through radiology, microCT and biomechanical testing; and (b) at the molecular level the 22948146 effect on expression of specific BMP proteins by means of immunohistochemistry.Materials and Methods 1. EthicsThe McGill University Animal Care Committee approved all experimental procedures (protocol #5162). Throughout surgery, mice were anesthetized using inhaled isoflurane and subcutaneously injected with 0.1 ml of buprenorphine (1 mg/kg-Sigma) for pain management. Animals were monitored once daily immediately after surgery and then 3? times per week. During the study, humane endpoints were used in accordance with McGill’s standard operating 
protocol. In case of infection at the surgical site, wound dehiscence, weight loss (.20 ) or if the animal became cachectic, had difficulty eating, drinking or moving around freely, or had a Body order RE-640 Condition Score (BCS) less than 2, the animal was euthanized. The mice were euthanized by CO2 asphyxia under general anesthesia at the time of sacrifice. This method is 1662274 consistent with AVMA (American Veterinary Medical Association) euthanasia guidelines on the use of CO2 as a euthanizing agent.2. AnimalsMice were all adult male wild-type C57B16/J mice (Charles River, Montreal, QC), 2? months of age with an average weight ?of 22.0 g (n = 115 for the entire study). Of the 115 mice, 97 mice survived and were processed for analysis. A total of 18 mice were euthanized due to surgical complications: 7 intra-operatively due to fracture and 11 in the post-operative period due to either skin dehiscence, infection or foot necrosis. The samples were sacrificed at two time points (mid-consolidation and full consolidation) and allocated to four groups: faxitron, mCT, 370-86-5 immunohistochemistry and biomechanical testing with an objective of having at least 6 samples per group per time point. Due to surgical complications and early euthanizia some groups were left with 5 samples per group. Faxitron was performed on all samples other than the samples allocated for immunohistochemistry (refer to Figure 1 for sample distribution).3. Distraction osteogenesis (DO) procedureMurine tibial DO was performed using a miniature Ilizarov fixator (Paolo Alto, CA), as previously described by Isefuku et al. [5] and our group [12,40]. Two 0.25-mm pins (Austerlitz, Marlborough, MA) were drilled 90u apart into the proximal and distal metaphysis of the right tibia and secured into position using 2 rings and 8 hexagonal nuts. Three threaded rods were used to connect the two parallel rings. A transverse osteotomy was performed along the middle diaphysis of the right tibia, between the proximal and distal pins, using a no. 11 surgical scalpel (Fisher Scientific, Osaka, Japan). The fibula was then broken using the back end of the scalpel. Distraction began at a rate of 0.4 mm every 24 hours for 12 days after a 5-day latency period. On post-operative day 11 (middistraction), 5 mg of kidney-derived heparan sulfate (HS) (Sigma) diluted into 20 ul of saline,.F gene expression [34,35]. The pH/ionic microenvironment has also been shown to affect the binding affinity of HS [22,36]. HS tends to have a higher affinity to proteins in the presence of cations (e.g. zinc and copper) [37], whereas its binding affinity decreases in a low cationic presence [38,39]. In light of this controversy, the purpose of this study was to investigate the effects of exogenous, locally-applied kidney-derived HS in a wild-type mouse model of DO; by examining the effects on (a) bone formation through radiology, microCT and biomechanical testing; and (b) at the molecular level the 22948146 effect on expression of specific BMP proteins by means of immunohistochemistry.Materials and Methods 1. EthicsThe McGill University Animal Care Committee approved all experimental procedures (protocol #5162). Throughout surgery, mice were anesthetized using inhaled isoflurane and subcutaneously injected with 0.1 ml of buprenorphine (1 mg/kg-Sigma) for pain management. Animals were monitored once daily immediately after surgery and then 3? times per week. During the study, humane endpoints were used in accordance with McGill’s standard operating protocol. In case of infection at the surgical site, wound dehiscence, weight loss (.20 ) or if the animal became cachectic, had difficulty eating, drinking or moving around freely, or had a Body Condition Score (BCS) less than 2, the animal was euthanized. The mice were euthanized by CO2 asphyxia under general anesthesia at the time of sacrifice. This method is 1662274 consistent with AVMA (American Veterinary Medical Association) euthanasia guidelines on the use of CO2 as a euthanizing agent.2. AnimalsMice were all adult male wild-type C57B16/J mice (Charles River, Montreal, QC), 2? months 
of age with an average weight ?of 22.0 g (n = 115 for the entire study). Of the 115 mice, 97 mice survived and were processed for analysis. A total of 18 mice were euthanized due to surgical complications: 7 intra-operatively due to fracture and 11 in the post-operative period due to either skin dehiscence, infection or foot necrosis. The samples were sacrificed at two time points (mid-consolidation and full consolidation) and allocated to four groups: faxitron, mCT, immunohistochemistry and biomechanical testing with an objective of having at least 6 samples per group per time point. Due to surgical complications and early euthanizia some groups were left with 5 samples per group. Faxitron was performed on all samples other than the samples allocated for immunohistochemistry (refer to Figure 1 for sample distribution).3. Distraction osteogenesis (DO) procedureMurine tibial DO was performed using a miniature Ilizarov fixator (Paolo Alto, CA), as previously described by Isefuku et al. [5] and our group [12,40]. Two 0.25-mm pins (Austerlitz, Marlborough, MA) were drilled 90u apart into the proximal and distal metaphysis of the right tibia and secured into position using 2 rings and 8 hexagonal nuts. Three threaded rods were used to connect the two parallel rings. A transverse osteotomy was performed along the middle diaphysis of the right tibia, between the proximal and distal pins, using a no. 11 surgical scalpel (Fisher Scientific, Osaka, Japan). The fibula was then broken using the back end of the scalpel. Distraction began at a rate of 0.4 mm every 24 hours for 12 days after a 5-day latency period. On post-operative day 11 (middistraction), 5 mg of kidney-derived heparan sulfate (HS) (Sigma) diluted into 20 ul of saline,.
