In vitro evaluation of the mitogenic effect of platelet-derived growth factor-BB on human periodontal ligament cells cultured with various bone allografts

Background: Several studies have documented the role of growth factors in periodontal regeneration. It has been shown that platelet-derived growth factor (PDGF) is a potent stimulator of human periodontal ligament (PDL) cells. A variety of bone graft materials are used to treat osseous defects caused by periodontal disease. We evaluated the mitogenic effect of PDGF on human PDL cells cultured with different allografts to determine which of the allografts with or without PDGF promoted periodontal regeneration. Methods: Two human demineralized freeze-dried allografts of cortical (DFDBA) and cancellous (DFBA) bone and a non-demineralized freeze-dried allograft (FBA) from cancellous bone were used alone or supplemented with PDGF-BB. Human PDL cultures were derived from the mid-root of 2 maxillary premolars extracted for orthodontic reasons. Cells were grown separately in 24-well dishes with or without 20 mg of each bone allograft. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB. DNA synthesis was estimated by measuring [H-3] thymidine incorporation to determine the effects of the test agents on cell proliferation. Cells were processed and subjected to scintillation counting after 48 hours of incubation. Counts per minute (cpm/well) were determined for each sample. Results: There was no statistically significant difference (P<0.05) on PDL cell proliferation when the allografts were used alone. PDL cells exhibited significantly greater proliferative responses to the 2 demineralized bone allografts, DFDBA and DFBA, when combined with PDGF-BB. A statistically significant difference on DNA synthesis was noticed when PDGF-BB was added to PDL cells cultured with FBA. PDL cells displayed no significant increase in mitogenic activity when cultured with PDGF-BB alone. Conclusions: The findings of this study demonstrate the beneficial role of DFDBA, DFBA, and FBA as synergic agents with PDGF-BB to periodontal regeneration. The significant ability of the 2 decalcified bone allografts, DFDBA and DFBA, combined with PDGF to stimulate PDL cell proliferation might be a useful adjunct in the treatment of periodontal defects

Effect of coating Straumann (R) Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation

Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared not to induce ectopic bone formation, longer-term studies are required to determine whether it promotes the final stages of osteoblast formation and mineralization at gene and protein levels. While used in clinical applications, whether Emdogain and other commercial preparations of EMPs truly possess the capacity to induce the regeneration of bone or other components of the periodontium remains to be established.Krzysztof Marek Mrozik, Stan Gronthos, Danijela Menicanin, Victor Marino, P. Mark Bartol