Overcoming compromised bone regeneration in diabetes

In oral surgery critical size bony defects are challenging in particular when healing is compromised. Thus, in diabetes representing a situation of compromised tissue regeneration, there is a demand to enhance bone formation. Our strategy to enhance bone formation in diabetes is to supplement bone substitutes with pro-angiogenic molecules. This strategy is based on two arguments: (i) angiogenesis is the fundament of bone formation and (ii) angiogenesis is impaired in diabetic patients. (1, 2) Strategies based on recombinant growth factors are expensive and the molecules are sensitive to degradation upon prolonged storage. Therefore we follow a strategy that utilizes small molecules: Prolyl hydroxylase (PHD) inhibitors can stimulate angiogenesis and thereby support bone regeneration. (3, 4) PHD inhibitors can even overcome situations of compromised wound healing in diabetic animals. (5) However, repeated application of the PHD inhibitors which is performed in these models is not appropriate in oral surgery. We therefore propose an one-step approach were PHD inhibitors are applied together with bone substitutes. We will combine bone substitutes and PHD inhibitors and optimize this combination with regard to the capacity to induce VEGF in vitro and the capacity to induce orthotopic bone formation in vivo specifically considering the compromised healing situation of diabetic rats. To reach this goal, we propose two specific aims: Specific aim 1: To select the best performing combination of PHD inhibitors with inorganic bone mineral to increase VEGF in vitro. The PHD inhibitors dimethyloxaloylglycine, desferrioxamine, L-mimosine, and cobalt chloride will be lyophilized at various concentrations onto inorganic bone mineral. The best performing PHD inhibitor is slowly released from inorganic bone mineral and causes a maximal increase of VEGF in periodontal fibroblasts. This part of the project will be performed in Ann Arbor, MI, USA The best performing PHD inhibitors will then be tested in a bone regeneration model. In addition there will be the opportunity to participate in the ongoing hard tissue regeneration projects that are performed in Ann Arbor, MI, USA. This will be of benefit for the upcoming in vivo study for aim 2 in Vienna, Austria. Specific aim 2: To assess the candidate PHD inhibitor to induce bone formation in vivo. The PHD inhibitors lyophilized onto inorganic bone mineral will be tested for the effect on bone regeneration in a calvaria defect model in diabetic rats. Bone regeneration will be assessed by micro computer tomography, histology, and histomorphometry. This part of the project will be performed in Vienna, Austria. Overall our study will help to overcome compromised healing in diabetes by developing a smart biomaterial able to enhance bone formation under diabetic conditions. We believe that this proof-of concept study has implications for future patient therapy.

In oral surgery critical size bony defects are challenging in particular when healing is compromised. Thus, in diabetes representing a situation of compromised tissue regeneration, there is a demand to enhance bone formation. We proposed a strategy to enhance bone formation in diabetes by supplementing bone substitutes with pro-angiogenic molecules. This strategy is based on two arguments: (i) angiogenesis is the fundament of bone formation and (ii) angiogenesis is impaired in diabetic patients. Strategies based on recombinant growth factors are expensive and the molecules are sensitive to degradation upon prolonged storage. Therefore we follow a strategy that utilizes small molecules: Prolyl hydroxylase inhibitors can stimulate angiogenesis and thereby support bone regeneration. Prolyl hydroxylase inhibitors can also help to overcome situations of compromised wound healing in diabetic animals. However, repeated application of prolyl hydroxylase inhibitors which was used in these models is not appropriate in oral surgery. We therefore proposed a one-step approach where prolyl hydroxylase inhibitors are applied together with biomaterials. We combined bone substitutes and collagen barrier membranes with prolyl hydroxylase inhibitors and optimized this combination with regard to their capacity to induce vascular endothelial growth factor (VEGF) in vitro and the capacity to induce bone formation in vivo specifically considering the compromised healing situation of diabetic rats. We found that the prolyl hydroxylase inhibitors released from inorganic bone mineral and collagen barrier membranes maintain their capacity to increase VEGF production in our in vitro bioassays. Overall a faster release from collagen barrier membranes was observed than from inorganic bone mineral. Mimicking in vitro the in vivo situation where bone substitutes are embedded in the fibrin matrix of the blood clot further prolonged the release of prolyl hydroxylases. In addition we found that prolyl hydroxylase inhibitors reduce the katabolic activity of oral cells and decrease the formation and activity of bone-resorbing osteoclasts. In the course of this study we also established new ex vivo 3-D culture models. Based on the results, prolyl hydroxylase inhibitors lyophilized onto inorganic bone mineral were tested for the effect on bone regeneration in a calvaria defect model in diabetic rats. We found that the prolyl hydroxylase inhibitor dimethyloxaloylglycine increased the vessel diameter but did not increase bone formation. Desferrioxamine did not show a significant effect in this model.Overall our results contribute to the development of new smart biomaterial which stimulate bone formation under diabetic conditions. These results will support the development of new personalized approaches to stimulate oral tissue regeneration in diabetic patients.