Ion within the distracted site of rabbit and mouse models of DO [14,15]. In humans, large supraphysiological doses of exogenous BMPs have to be administered in order to significantly improve bone growth. These doses can have harmful effects, such as ectopic bone formation and potential for malignancies, notwithstanding the extremely elevated costs related with the use of recombinant BMPs [16,17,18,19]. An alternative strategy to administeringHeparan Sulfate and Distraction Osteogenesisexogenous BMPs is to manipulate endogenous BMPs by neutralizing or counteracting the activities of their antagonists, such as Noggin or BMP3. Several methods have been shown to inhibit BMP antagonists, including the use of antibodies, RNA interference or naturally-occurring substances such as the extracellular proteoglycan heparan sulfate, or HS [20,21]. HS is a membrane-bound proteoglycan [22] that has been previously reported to interact with BMP antagonists as well as BMP ligands to modulate protein activity. HS is an endogenous, commercially available, cost effective and clinically feasible alternative to antibody-mediated or RNAi-mediated gene silencing modulation techniques. Structurally, HS is composed of a core protein and highly sulfated glycosaminoglycan side chains of Dglucuronic acid-N-acetyl-D-glucosamine 
repeats [23]. These negatively charged side chains of HS have been shown to bind a myriad of proteins [24,25], including soluble BMP ligands (e.g. BMP2, BMP4, BMP7) [26,27,28] and BMP antagonists (e.g. 1662274 Noggin) [29], which can have anti- and pro-osteogenic effects on bone, respectively. Previous in vitro studies have proposed two different models for the mechanism of action of how HS can bind BMPs and their secreted antagonists. In the first model, HS is proposed to transport BMPs from cell to cell through restricted diffusion; whereas in the second model, HS was shown to retain BMP antagonists such as Noggin to establish an inverse gradient of BMP activity [20]. In vivo studies have also demonstrated that the interaction of HS with BMP antagonists can block the activity of these inhibitors thereby potentiating BMP activity during bone Rubusoside healing [20,22,23,30]. One previous animal study demonstrated that in a rat fracture repair model there was 20 increased bone formation when injected with 5 mg of bone derived HS [31]. Another animal study showed that in a critical size rat cranial defect, 5 mg of embryonically derived HS played an 1516647 important role in accelerating bone healing by 3 months [32]. Therefore, based on its previously reported therapeutic potential in in vitro and in vivo studies, we postulated that exogenous application of the naturallyoccurring HS, particularly at a dose of 5 mg, may maximize the bioavailability of endogenous BMPs during DO; by inhibiting the action of BMP antagonists, and thus improve bone regeneration in a murine model of DO. It is important to note, that while some studies have attested to the positive effects of HS on bone regeneration, other reports have showed that HS showed no significant effect on bone [20,33]. The conflicting data on the role of HS on bone formation and BMP signaling can be explained by a number of reasons, including variations in the sulfation patterns, the PS 1145 manufacturer microenvironment, and pH/ionic presence of the target tissue. For example, the sulfation pattern of proteoglycans, including HS, can drastically affect their binding affinity to different ligands, resulting in stimulation or inhibition o.Ion within the distracted site of rabbit and mouse models of DO [14,15]. In humans, large supraphysiological doses of exogenous BMPs have to be administered in order to significantly improve bone growth. These doses can have harmful effects, such as ectopic bone formation and potential for malignancies, notwithstanding the extremely elevated costs related with the use of recombinant BMPs [16,17,18,19]. An alternative strategy to administeringHeparan Sulfate and Distraction Osteogenesisexogenous BMPs is to manipulate endogenous BMPs by neutralizing or counteracting the activities of their antagonists, such as Noggin or BMP3. Several methods have been shown to inhibit BMP antagonists, including the use of antibodies, RNA interference or naturally-occurring substances such as the extracellular proteoglycan heparan sulfate, or HS [20,21]. HS is a membrane-bound proteoglycan [22] that has been previously reported to interact with BMP antagonists as well as BMP ligands to modulate protein activity. HS is an endogenous, commercially available, cost effective and clinically feasible alternative to antibody-mediated or RNAi-mediated gene silencing modulation techniques. Structurally, HS is composed of a core protein and highly sulfated glycosaminoglycan side chains of Dglucuronic 
acid-N-acetyl-D-glucosamine repeats [23]. These negatively charged side chains of HS have been shown to bind a myriad of proteins [24,25], including soluble BMP ligands (e.g. BMP2, BMP4, BMP7) [26,27,28] and BMP antagonists (e.g. 1662274 Noggin) [29], which can have anti- and pro-osteogenic effects on bone, respectively. Previous in vitro studies have proposed two different models for the mechanism of action of how HS can bind BMPs and their secreted antagonists. In the first model, HS is proposed to transport BMPs from cell to cell through restricted diffusion; whereas in the second model, HS was shown to retain BMP antagonists such as Noggin to establish an inverse gradient of BMP activity [20]. In vivo studies have also demonstrated that the interaction of HS with BMP antagonists can block the activity of these inhibitors thereby potentiating BMP activity during bone healing [20,22,23,30]. One previous animal study demonstrated that in a rat fracture repair model there was 20 increased bone formation when injected with 5 mg of bone derived HS [31]. Another animal study showed that in a critical size rat cranial defect, 5 mg of embryonically derived HS played an 1516647 important role in accelerating bone healing by 3 months [32]. Therefore, based on its previously reported therapeutic potential in in vitro and in vivo studies, we postulated that exogenous application of the naturallyoccurring HS, particularly at a dose of 5 mg, may maximize the bioavailability of endogenous BMPs during DO; by inhibiting the action of BMP antagonists, and thus improve bone regeneration in a murine model of DO. It is important to note, that while some studies have attested to the positive effects of HS on bone regeneration, other reports have showed that HS showed no significant effect on bone [20,33]. The conflicting data on the role of HS on bone formation and BMP signaling can be explained by a number of reasons, including variations in the sulfation patterns, the microenvironment, and pH/ionic presence of the target tissue. For example, the sulfation pattern of proteoglycans, including HS, can drastically affect their binding affinity to different ligands, resulting in stimulation or inhibition o.