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OBJECTIVE Prolyl hydroxylases (PHD) are oxygen sensors and therefore pharmacological targets to stimulate periodontal regeneration. Here we evaluate the release profile of the PHD inhibitors dimethyloxaloylglycine and l-mimosine from bone substitutes. MATERIALS Dimethyloxaloylglycine and l-mimosine were lyophilised onto bone substitutes including bovine bone mineral, beta-tricalcium phosphate, and hydroxyapatite. Release kinetic was evaluated by bioassays with gingival and periodontal ligament fibroblasts. We determined the capacity of PHD inhibitors to provoke VEGF expression and to repress metabolic activity and proliferation as assessed by immunoassay, MTT conversion and (3)[H]thymidine incorporation, respectively. RESULTS We found that the PHD inhibitors are released from bovine bone mineral as indicated by the increase of VEGF production in gingival and periodontal ligament fibroblasts. Supernatants obtained after 1h also decreased metabolic activity and proliferation of the fibroblasts. A fibrin matrix prolonged the release of PHD inhibitors up to 192h. A similar cellular response was found when supernatants from PHD inhibitors loaded beta-tricalcium phosphate and hydroxyapatite embedded in fibrin were assessed. CONCLUSIONS In conclusion bone substitutes can serve as carriers for PHD inhibitors that maintain their capacity to provoke a pro-angiogenic response in vitro. These findings provide the basis for preclinical studies to evaluate if this release kinetic can stimulate periodontal regeneration.

Introduction Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. Methods To test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3 [H]thymidine, and 3 [H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). Results We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. Conclusions These results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Objective Chronic inflammation in periodontal disease is associated with increased plasminogen activation and elevated levels of chemokines. It is unknown whether chemokines can regulate the activation of plasminogen via modulation of plasminogen activators (PA) and the corresponding plasminogen activator inhibitors (PAI) in periodontal tissue. Design To establish a link between chemokines and activation of plasminogen, human periodontal ligament fibroblasts (PDL) and gingival fibroblasts (GF) were incubated with IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and platelet factor-4, either alone or in the presence of the inflammatory mediators TGF-β and IL-1. The potential of the cell lysates to activate plasminogen was based on kinetic studies with the substrate casein. Casein zymography was performed to determine the molecular sizes of the PA. Total PAI-1 in the cell-conditioned medium was quantified by immunoassay. Results We report that the chemokines did not affect activation of plasminogen by PDL and GF. Even in the presence of TGF-β which suppressed, and IL-1 which stimulated plasminogen activation, the chemokines had no direct effect. Inhibition of PA and plasmin, but not of matrix metalloproteinases and cysteine proteinases prevented caseinolysis. The plasminogen activation capacity of the cell lysates was represented by a single band with features of uPA. The immunoassay showed that the release of PAI-1 in PDL and GF remained unaffected by the chemokines, also when stimulated with TGF-β. Conclusions These results suggest that plasminogen activation by PDL and GF is not directly affected by the chemokines even in the presence of the inflammatory mediators TGF-β and IL-1.