Effects Of Diseased Cementum In Combination With EMD On The Cementum Regeneration

Backgroud and ObjectiveThe ultimate goal of periodontitis treatment is to reconstruct the periodontal tissues on the basis of inflammation control. Nowadays there are several stragies applied in periodontal regeneration, such as guided tissue regeneration (GTR), grafts of bone materials, application of growth factors as well as tissue engineer, which is a hot spot in the field of regeneration. However, these stragies could only result in partial reconstruction of periodontal attachment, the complete regeneration of periodontal tissue is unavailable. The regeneration of cementum seems critically difficult as it is still unable to predict the amount and quality of regenerated cementum.Cementum, a thin mineralized connective tissue, constitutes the integrity of periodontium with its major function as the site of attachment for principal collagen fibers. Besides the majority of collagens, the extracellular matrix (ECM) of cementum is a rich pool of various growth factors and non-collagen proteins, such as bone sialoprotein (BSP)、osteopontin (OPN)、fibronectin (FN)、bone morphogenetic protein(BMP-2,-3,-4)、 platelet-derived growth factor (PDGF-a and β)、transforming growth factor (TGF-β). These components form the unique microenviroment of cementum. There is accumulating evidence that the preservation of root cementum may favor periodontal regeneration, especially formation of new cementum. The former study in our research group also found that the EDTA conditioned pre-existing healthy cementum may promote hPDLCs differentiate towards cemtoblasts, thus play a role in cementum regeneration. But how about the role of periodontitis affected cementum in cementum regeneration? Is there any growth factor that can be combined to promote its function?In periodontitis, the structure and composition are affected when the chronic inflammatory process destroys gingival collagen fibers and extends to the root surface, this is so called diseased cementum. Although there are results supporting that the diseased cementum can be periodontal healthy after simple polishing on the basis of removal of calculus and bacterial plaque, the matrix composition of diseased cementum was changed, several non-collagen proteins were affected, such as bone sialoprotein (BSP), osteoprotein (OPN) and fibronectin (FN). The changes in the matrix could result in the alteration of the cementum microenviroment inevitably, the effect of diseased cementum on cementum regeneration is obscure.Enamel matrix derivative (EMD), which is widely used in regenerative periodontal therapy, is believed to induce the formation of cementum and promote new attachment formation. EMD is an extracellular matrix that contains proteins similar to that derived from Hertwig epithelial root sheath (HERS) and the application of EMD in periodontal defects may mimic the events that take place during the development of root. It was reported that EMD could induce the formation of acellular extrinsic fiber cementum (AEFC) in animal studies, application of EMD could yield more clinical attachment compared with GTR in clinical trials. A number of recent studies explored the effects of EMD on the biological characters of several types of cells in vitro, the results implied that EMD could induce the differentiation of some types of cells, such as periodontal ligament cell and bone marrow stromal cell towards osteoblast/cementoblast lineage. However, there were no reports regarding the comparision of regenerative effects induced by EMD respectively on cementum surface and dentin surface.Thus, this study aimed to explore the effect of diseased cementum on cementum regeneration, and observe if this effect can be amplified in the presence with EMD.Methods 1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.1.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro.48extracted teeth due to periodontitis were collected, and notches were made at teeth on the coronal and apical line of the pockets by a pencil immediately after extraction. Root debridement was performed by EMS ultrasonic sealer to completely remove the calculus and plaques. Symmetrical root slices were made using diamond bur and one of the symmetrical slices of each root was further scaled to remove root cementum and distributed into the D (dentin) group, the other symmetrical slice was divided into the C (cementum) group in which the cementum was preserved in situ. All slices were conditioned with24%ethylene diamine tetraacetie acid (EDTA) gel for2min and supplemented with10%penicillin and streptomycin for24hours at4℃for sterilization. hPDLCs were isolated according to a modification of the method of Somerman.24root slices from each group were placed into48-well plates, with one chip in each well. The second passage hPDLCs were harvested and suspended at a concentration of1x10/ml and cell suspension was sparsely seeded onto each root slice. After a7-day co-culture,4slices from each group were selected to undergo SEM observation, and the remained20slices in each group were used for real-time PCR to detect the mRNA expression of cementum attachment protein (CAP) and cementum protein-23(CP-23).1.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.24root slices from C and D group were placed into48-well plate, and hPDLCs with concentration of1x106/ml were seeded onto root slices. Root slices in this part were divided into four groups, cementum slices(group C) and dentin slices (group D) incubated in the DMEM culture medium respectively, as well as cementum slices (group E+C) and dentin slices (group E+D) incubated in DMEM culture medium containing EMD respectively, each group contained12root slices. After a7-day co-culture,12root slices of each group were rinsed with PBS and wrapped with sterilized expanded polytetrafluoroethylene (ePTFE), and then were inserted beneath the dorsal skin of nude mice. Each mouse received four slices, with one from each group. The mice were sacrificed3weeks after surgery, all the specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was carried out. The newly-formed tissue in four groups were observed and BSP staining was carried out in specimens with newly formed cementum.2. The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects.8male beagle dogs were chosen, and4teeth (the right and left third and fourth premolars in the mandible) from each dog were used for this experiment. Four weeks before the experiment day, class III furcation defects were surgically created (height:5mm) in experiment teeth and cotton balls saturated with anaerobic bacteria were placed in all defects to initiate inflammation. The cotton ball was removed from the defects2weeks before the day of experiment, and then plaque control was achieved by subgingival scaling and oral hygiene with0.2%chlorhexidine gluconate irriation. On the day of experiment, no severe inflammation and gingival recession were observed, four experimental teeth of each dog received one of the following treatments randomly. C:preservation of root cementum in situ+24%EDTA conditioning D:removal of root cementum+24%EDTA conditioning E+C:preservation of root cementum in situ+24%EDTA conditioning+EMD E+D:removal of root cementum+24%EDTA conditioning+EMD All the buccal mucoperiosteal flaps were elevated, and the furactions were treated according to the design, then collagenous membranes were placed on buccal side of all the defects. Flaps on the lingual side were not elevated in consideration of the objective of the study and convenience in operation. All the dogs were sacrificed by4%paraformaldehyde perfusion. All the specimens were harvested and2specimens from each group were embedded with resin and the slices were stained with toluidine blue for histological observation. The remained6Specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was used for histological observation and histometric study.Results1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.2.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro..The cells began to grow out from the periodontal ligament explants after7day to10day. Then they proliferated quickly and the cells were digested when they reached80%confluence. Immunohistochemical staining results showed that the second passage hPDLCs were positive for vimentin and negative for keratin, certificating that they were originated from ectomesenchyme. SEM results showed that the cells seeded on the cementum slices and dentin slices grew well, cells on the dentin surface exhibited a spindle shape while cells on cementum were much larger without typical spindle shape. Both CAP and CP-23expression were significantly higher on the cementum surface than on the dentin surface.(p=0.001for CAP and p=0.003for CP-23respectively).2.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.All the mice survived without inflammation throughout the experiment period,1root slice in group C fell off as the sutures loose, other11specimens in this group were obtained.12specimens of other three groups were obtained. Results of HE staining showed that neither group D nor group E+D had newly formed cementum-like tissue (NFC) formation,7specimens in group C showed NFC formation while10specimens in group E+C showed NFC formation, the results between group C and group E+C were statistically different.2.The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects. We artificially created32inflamed Class III furcation defects in beagle dogs. Within a healing period of8weeks, healing of the wounds in all groups occurred uneventfully, with no infection or suppuration. Histomorphometric results showed that the percentage of regenerated cementum (PRP) and the percentage of regenerated bone (PRB) in C group were statistically higher than those in D group, PRP and PRB in E+D and E+C groups were statistically higher than those in groups without EMD, but there were no differences between E+D group and E+C group. D group:The newly formed periodontal ligament was loosely arranged without principle fiber bundles, and favorable new attachment was not established. C group:Newly formed cementum was laid on the top of old cementum, newly formed periodontal ligament was in orderly arranged, with principle fiber inserting into new bone and new cementum, and favorable new attachment was established. The quality of new attachment in group E+D and E+C was almost good, except for the artifacts between newly formed cementum and dentin surface observed in group E+D. Results from resin slices were similar, but artifacts were not seen.Conclusion1. Preservation of diseased cementum promoted the differentiation of inoculated hPDLCs towards cementoblasts, and the effect was amplified by the presence of EMD.2. Preservation of diseased cementum favored the cementum regeneration as well as formation of new attachment, and the application of EMD could strengthen the effect.