And antagonists alike) were decreased in the 5 mg HS group at 34 days post-osteotomy. Exactly how HS affects BMP signaling is still unclear. One of the known actions of HS is to bind the antagonists of BMPs, such as Chordin and Noggin [29,49]. Cell-surface HS has been shown to selectively bind and stabilize Noggin and Chordin and to increase their antagonism of BMP signaling [33,49]. Our immunohistochemistry data showed thatTable 2. Summarized results for Immunohistochemistry analysis.Control Protein BMP-2 25033180 Endpoint (days) 34 51 BMP-7 34 51 Smad 1/5/8 34 51 Noggin 34 51 Gremlin 34 51 Chordin 34 51 BMP-3 34 51 BMPR1A 34 51 Chondrocytes ++ + ++ + +++ + ++ + ++ + ++ + ++ + +++ + Fibroblasts 2 + 2 2 2 2 + 2 + 2 + 2 2 2 25 mg HS Chondrocytes + + 2 2 + + + + + + + + + 2 + 2 Fibroblasts 2 2 2 2 2 2 2 2 + 2 + 2 2 2 2Immunohistochemistry results of dissected tibiae at 34 days and 51 days post-osteotomy. doi:10.1371/journal.pone.0056790.tHeparan Sulfate and Distraction Osteogenesis5 mg of exogenous HS resulted in a slight decrease in the endogenous expression of BMP antagonists Noggin, Chordin, Gremlin and BMP3 but also resulted in a slightly decreased expression of endogenous BMP-2 and BMP-7. To account for these findings, we speculate whether HS acts to stabilize the BMP ligand/antagonist interaction rather than ADX48621 manufacturer modulate their protein expression level and thus prolongs the inhibitory effects of the antagonists on bone formation (and wound healing) during DO. In order to confirm the exact role of HS on the mechanism of BMP signaling activity, HS binding assays would be required but that is outside the scope of the present study. Another potential explanation is that HS and BMP antagonists may have different binding sites on the BMP ligand [50,51]. Jackson et al. [31] showed that a single dose local application of 5 mg bone-derived HS had an anabolic effect on rat femoral fracture repair after 2 weeks, potentially by increasing the production of local growth factors (ALP, Runx2, FGF-1, IGF-II, TGF-m1, VEGF). However, similar to our study, HS did not significantly increase BMP-2 or -7 expression. In fact, it has yet to be shown that HS interacts directly with the BMP2 or BMP7/ receptor complex. The delivery method and therapeutic dose of HS that reaches the bone can also influence its effects. A study by Woodruff et al. [32], demonstrated that the use of 5 mg of embryonically derived HS, loaded on a scaffold with a more uniform and prolonged distribution of HS, greatly contributed to improve wound healing and bone healing in a rat critical size cranial defect model at 3 months; whereas no difference was demonstrated at 1 month. In our study, we injected HS diluted into saline directly into the regenerate site, a potentially confined space with a surrounding CHIR-258 lactate web membrane of tissue and as such we may have effectively increased the therapeutic dose of HS over a short period of time. In fact, Jackson et al. [31], demonstrated in their dosing study of a rat fracture repair model, that the therapeutic effects of HS can be dose dependant and that a very elevated therapeutic dose can actually have negative effects on bone healing. Another potential explanation may be related to the pH/ionic microenvironment of the distracted zone, where HS tends to have a lower binding affinity to proteins in acidic milieus [38,39]. In our model of DO, acidosis in the distracted gap resulting from hypoxia [52] likely caused a decrease in cationic presence in the callus. This.And antagonists alike) were decreased in the 5 mg HS group at 34 days post-osteotomy. Exactly how HS affects BMP signaling is still unclear. One of the known actions of HS is to bind the antagonists of BMPs, such as Chordin and Noggin [29,49]. Cell-surface HS has been shown to selectively bind and stabilize Noggin and Chordin and to increase their antagonism of BMP signaling [33,49]. Our immunohistochemistry data showed thatTable 2. Summarized results for Immunohistochemistry analysis.Control Protein BMP-2 25033180 Endpoint (days) 34 51 BMP-7 34 51 Smad 1/5/8 34 51 Noggin 34 51 Gremlin 34 51 Chordin 34 51 BMP-3 34 51 BMPR1A 34 51 Chondrocytes ++ + ++ + +++ + ++ + ++ + ++ + ++ + +++ + Fibroblasts 2 + 2 2 2 2 + 2 + 2 + 2 2 2 25 mg HS Chondrocytes + + 2 2 + + + + + + + + + 2 + 2 Fibroblasts 2 2 2 2 2 2 2 2 + 2 + 2 2 2 2Immunohistochemistry results of dissected tibiae at 34 days and 51 days post-osteotomy. doi:10.1371/journal.pone.0056790.tHeparan Sulfate and Distraction Osteogenesis5 mg of exogenous HS resulted in a slight decrease in the endogenous expression of BMP antagonists Noggin, Chordin, Gremlin and BMP3 but also resulted in a slightly decreased expression of endogenous BMP-2 and BMP-7. To account for these findings, we speculate whether HS acts to stabilize the BMP ligand/antagonist interaction rather than modulate their protein expression level and thus prolongs the inhibitory effects of the antagonists on bone formation (and wound healing) during DO. In order to confirm the exact role of HS on the mechanism of BMP signaling activity, HS binding assays would be required but that is outside the scope of the present study. Another potential explanation is that HS and BMP antagonists may have different binding sites on the BMP ligand [50,51]. Jackson et al. [31] showed that a single dose local application of 5 mg bone-derived HS had an anabolic effect on rat femoral fracture repair after 2 weeks, potentially by increasing the production of local growth factors (ALP, Runx2, FGF-1, IGF-II, TGF-m1, VEGF). However, similar to our study, HS did not significantly increase BMP-2 or -7 expression. In fact, it has yet to be shown that HS interacts directly with the BMP2 or BMP7/ receptor complex. The delivery method and therapeutic dose of HS that reaches the bone can also influence its effects. A study by Woodruff et al. [32], demonstrated that the use of 5 mg of embryonically derived HS, loaded on a scaffold with a more uniform and prolonged distribution of HS, greatly contributed to improve wound healing and bone healing in a rat critical size cranial defect model at 3 months; whereas no difference was demonstrated at 1 month. In our study, we injected HS diluted into saline directly into the regenerate site, a potentially confined space with a surrounding membrane of tissue and as such we may have effectively increased the therapeutic dose of HS over a short period of time. In fact, Jackson et al. [31], demonstrated in their dosing study of a rat fracture repair model, that the therapeutic effects of HS can be dose dependant and that a very elevated therapeutic dose can actually have negative effects on bone healing. Another potential explanation may be related to the pH/ionic microenvironment of the distracted zone, where HS tends to have a lower binding affinity to proteins in acidic milieus [38,39]. In our model of DO, acidosis in the distracted gap resulting from hypoxia [52] likely caused a decrease in cationic presence in the callus. This.