| Pulp tissues play a critical role in dentin formation and nutrition. When suffered fromtrauma or infection, the undifferentiated mesenchymal stem cells (MSCs) in pulp tissuewill migrate to injured site, proliferate and differentiate into odontoblast-like cells, whichwill secrete dentinal matrix and form reparative dentin. Pulp tissue possesses this kind ofcharacteristics of self-reparation, which is also the biological foundation of vital pulptherapy. The traditional way of vital pulp therapy plays its role by bioactive molecule, butthese molecules were easily to be absorbed in vivo, which limits its application in clinicalfor large dosage. However, the pulpectomy and root canal therapy could not make asatisfied result as well for the enhancement of friability and the reduction of function ofteeth. Therefore, the tissue engineering technology was adopted to repair and evenregenerate pulp tissues by using DPSCs as seed cells. Tissue engineering is a method to form new functional tissues and organs by mimicking the microenvironments of growthfactors and transplanting the construct of cells, biological scaffolds and growth factors toinjured sites, while the biological scaffolds degradation could occur together with theformation of new tissues. Nevertheless, this application of pulp tissue engineering was stilllimited in clinical for many issues, such as cross-infection and immunological rejectioncaused by biological materials, the dosage, half-life period, species and vehicles of growthfactors and so on.Platelet-rich fibrin (PRF) belongs to the second-generation platelet concentratesgeared to simplified preparation without biochemical blood handling, so that the ethicalcontroversy, immunological rejection and cross-infection can be avoided. It containsquantities of key growth factors and has been proved to be able to promote theproliferation and differentiation of MSCs, which offers a possibility for the reparation andregeneration of dental pulp-dentin complex. However, the mechanism of that is stillunknown. The present study initially isolated, cultivated and identified the canine DPSCs,and then explored the possibility and biological effect of the reparation and regenerationof dental pulp-dentin complex using PRF both in vitro and in vivo. This study mayprovide experimental foundation and theoretical basis for application of PRF in clinical.1. Isolation, cultivation and identification of canine DPSCsPulp tissue was extracted from the permanent maxillary first molar of canine anddental pulp cells were successfully obtained by using the enzymes digesting method.Putative stem cells were obtained by using a limiting dilution technique, and theirmesenchymal stem cell (MSC) properties were characterized by colony forming unit assay,cell surface marker characterization, the proliferative capability, and theirosteogenic/dentinogenesis and adipogenic differentiation potential.2. Platelet-rich fibrin preparationThe venous blood is collected without any anticoagulant in10-mL tubes bydisposable syringe from canine jugular vein which was then oppressed by gauze forhemostasising, then immediately centrifuged at about3000r/min for12minutes, which balanced with the same volume of water. Three layers are naturally formed in the tube: abase of red blood cells at the bottom, acellular plasma on the surface, and finally a PRFclot between the two. The PRF clot was gently compressed into a membrane, which waslongitudinally cut and divided the red end evenly. Dose-dependent effect (using1/8,2/8,3/8PRF membranes) was assessed and added to the cultures on the first day of theexperiment to analyze the biologic effects of PRF on the proliferation andodonto/osteogenic differentiation potential of DPSCs from the dog in vitro. Theobservations lasted for7days.3. In vitro effects of PRF on proliferation and mineralization of the canine DPSCs in vitroDifferent concentrations of PRF were added into normal and osteo/odontogenicmedium to study the effect of PRF on the ability of proliferation and odonto/osteogenicdifferentiation potential of DPSCs in vitro. The results showed thatthe adjunction of PRFstimulated the proliferation of canine DPSCs, with time dependence, while the promotioneffects had no relationship with the concentration of PRF. Different concerntration of PRFupregulated the expression of the early odonto/osteoblastic markers (ALP, DSPP) and latemarker (DMP1) of DPSCs, which presents simultaneously time-andconcentration-dependent.4. In vivo effects of PRF on the dental-pulp regeneration by DPSCsin dogsBased on the results above, we structure DPSCs/PRF compound membranes andtransplant them subcutaneously into the dorsal surface of5-week-old nude mices. Afterthe observation period of4and8weeks, the specimens were stained with HE and Massonstaining in a routine way. The histological observation showed that the group ofDPSCs/PRF compound membranes have stronger ability of regenerating dentin-pulp-likestructure after transplantation in vivo either in nude mices or dogs, which were largelysuperior to the DPSCs membrane simply. The results of Masson staining, DSPP and BrdUlabeling immunohistochemistry in dog specimens showed that there was the formation ofblood vessels, odontoblast-like cells and dentin-like matrix obviously in the marrow cavity,and the main cytological basis forming tissue were canine DPSCs, and significantly higherthan the experimental group and blank control group at different time points (p<0.05). In summary, There is a good biocompatibility between autologous PRF and canineDPSCs. PRF of different concentrations has enhanced the proliferation and differentiationof DPSCs in dogs in vivo, take advantage of which is expected to provide new opportunityfor tissue-engineered dentin-pulp complex. We have successfully combined DPSCs withautologous PRF and realized pulp/dentin regeneration ectopicly and orthotopicly throughthe DPSCs/PRF compound membranes structured by our research group, which isexpected to provide new opportunities for the construction and clinical application oftissue-engineered dentin-pulp complex. |
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