The Combined Use Of PDLSCs And PRP/PRF On The Periodontal Healing Of Avulsed Teeth Reimplantation

Tooth avulsion, defined as the complete displacement of the tooth from its originalalveolar socket, is one of the most severe types of dental trauma. The treatment consists ofreplantation, immediately if possible. Unfortunately, in most, if not all, cases, knocked-outteeth have been either out of the alveolar socket for a prolonged period of time or keptunder non-physiological conditions, which increases the risk of periodontal limgment(PDL) cell necrosis and, hence, results in ankylosis and replacement resorption of thetooth root post-reimplantation. Of note, periodontal healing cannot be achieved for a verylarge majority of knocked-out teeth, resulting in rapid root resorption or, more frequently,dental ankylosis with subsequent bone substitution and, finally, tooth loss over time.Knowing that the major reason for tooth reimplantation failure is due to the damage of thestem cells remaining on the avulsed tooth, it is logical to postulate that the adjuvant use of outside cultured stem cells may improve the long-term prognosis and survival of theseteeth. Hence, some studies have performed to identify suitable cell population toward thegoal of regeneration of periodontal tissues, and there is mounting evidence thatperiodontal ligament stem cells (PLDSCs) are the best cell choice. Although usingPLDSCs in reimplantation of avulsed teeth more or less promoted the periondontalhealing, the effect was still unsatisfied enough. The possible explanation for thisobservation was that PLDSCs achieve their effects through two ways: one was that thetransplanted stem cells directly participated the repair of PDL; the other was that theresidual stem cells in the surrounding tissues were activated and stem cells homing to theinjury sites were facilitated. In either way, the differentiation of the PDLSCs involvedtissues regeneration was influenced by their environments. In vivo, the proliferation anddifferentiation of stem cells and tissue regeneration were regulated by the themicroenviroment composed by signal molecules, including growth factors (GFs) andcytokines, while this effect could be imitated by ectogenic GFs in vitro. Nevertheless, theapplication of GFs in tissue engenieering still presents major limitations such as high cost,hard to purify, short half-life and so on, so it is necessary to find a way to make sure thatthe GFs could maintain a certain level to educe their special biological effects.Unfortunately, there are still some technical issues restricting the composition andcontrolled release of GFs. In our opinion, all of these problems are solved perfectly byplatelet concentrates, which contains several types of GFs in the same ratio withphysiological condition. These GFs not only play important roles in cells’ proliferation,differentiation and even apoptosis, but also compose a regulation system and interact witheach other. Platelet concentrates have been used in repair of bone, cartilage and plasticsurgery, but seldom in periodontal tissues, while the limited reports were focused onsingle cases. Whereas, by for now, the studies on the relationship and its mechanismbetween platelet concentrates and PDLSCs have not been reported, and the role of plateletconcentrates in periodontal healing of avulsed tooth was not clear yet.In this article, we hypothesized that a cell transplant consisting of cell sheet fragmentsof periodontal ligament stem cells (PDLSCs) and platelet-rich fibrin (PRF) granules might be able to regenerate PDL tissues around the reimplanted teeth. For application in animalmodels of tooth reimplantation, dog PDLSCs were conducted into cell sheets to enhancethe density of the final seed cells, and autologous PRF membrane was used as a growthfactor-rich scaffold that may facilitate the immobilization of the cells on the surface of theroot surface. This cell transplant method, based on tissue-engineering concepts, may openup new opportunities for the clinical management of periodontally involved hopelessavulsed teeth. The content of this item includes two parts summarized as following.Part1: Effect of the PRP/PRF on the reimplantation of avulsed tooth1. The ideal centrifugal parameters for PRP preparation was selected by evaluatingthe quality and quantity of platelets in PRP prepared with different methods. PRF wasprepared according to the standard method of400g/min. HE staining and scan electronmicroscope were adopted to observe the micro-and ultra-structure of PRP and PRF. Theamount of different kind of growth factors in PRP/PRF was evaluated by enzyme-linkedimmuno sorbent assay (ELISA).2. The effects of the PRP/PRF on the periodontal healing of avulsed teeth wereevaluated using animal models.5dogs were selected and6discontinuously distributedincisors from each dog were carefully extracted to establish the animal model for toothavulsion. According to different adjuvant grafts, the teeth were randomly divided into3groups: adjuvant use of PRP, PRF or nothing. In each one, the teeth were divided intodifferent subgroups accoding to different extraoral time:30min,1h and2h. Immediatereimplantation was considered to be the negative control, and the total number of groupswas10. The histometric observations were performed to evaluate the periodontal healingof reimplanted teeth.Part2: The combined use of cell sheets of periodontal ligament stem cells andplateletlets concentrates for delayed reimplantation of avulsed tooth1. Periodontal ligament cells were successfully obtained by using the combination oftissue block method and enzymes digesting method. Putative stem cells were obtained by using a limiting dilution technique, and their mesenchymal stem cell (MSC) propertieswere characterized by colony forming unit assay, cell surface marker characterization, andtheir osteogenic/adipogenic differentiation potential.2. The PDLSCs were co-cultured with a serial dose of PRP/PRF to assess the effectof PRP/PRF on PDLSCs. Cell counts and the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay were used to evaluate cellproliferation and viability. The effects of PRP/PRF on cell differentiation were detected byalkaline phosphatase (ALP) activity on days7and14, and the mRNA expression levelsfor bone sialoprotein (BSP), osteocalcin (OCN), collagen I (Col-I), and cementum-derivedprotein (CP23) were determined on days7,14and21.3. To construct the graft of PDLSCs/PRF, the PDLSCs were cultured into the form ofcell sheets and PRF was made into the form of membrane, and then both of them wereconstructed by different ways. The ultrastructures of differnet constructs were observed bySEM to select the best one for its applications in vivo.4. The effects of the PDLSCs/PRF construct on the periodontal healing of delayedreimplantation of avulsed teeth were evaluated using animal models, in comparison withPDLSCs or PRP/PRF.Based on the test in vitro and in vivo, the results showed as following.1. Platelets concentrates, including PRP and PRF, were successfully prepared in thisstudy, and the micro-and ultra-structures observsations demonstrated that both of themwere3-D collagen network. The results of ELISA demonstrated that there were plenty ofGFs in PRP/PRF, such as epidermal growth factor (EGF), insulin-like growth factor(IGF-1), platelet-derived growth factor-AB (PDGF-AB), transforming growth factor(TGF-β) and vascular endothelial growth factor (VEGF). The application of plateletsconcentrates in reimplantation could promote the periondontal healing of avulsed tooth,especially for PRF, showing a negative correlation with the time of extraoral. However,once the knocked-out teeth have been out of the alveolar socket more than2hours, eventhe adjuvant use of PRP/PRF could not advance healing. 2. PDLSCs were successfully obtained by using a limiting dilution technique, whichshowed the similar surface molecule markers with MSCs and had the capacity of CFUsforming and multiple-direction differentiation, including osteogenic and adipogenicdifferentiation. PRP and PRF could stimulate the proliferation and the expression ofPDL-related genes while inhibiting the expression of osteogenesis-related genes, whichmanifested as the decrease of activity of ALP and gene expression of BSP and OCN.3. The PDLSCs/PRF construct was obtained using a combination of cell sheetfragments (approximately0.5mm×0.5mm) and PRF granules (approximately0.5mm×0.5mm×0.5mm) in a certain proportion. The ultra-structure of this construct, whenobserved by SEM, indicated that the cells secreted extracellular matrix, expanded well andextended a large number of cell processes into the pores of the PRF fibrins. The cellsinterlaced with the PRF to form an integral biomaterial. This construct was superior forincreasing the cell density, preserving the extracellular matrix and meeting the threeelements of tissue engineering. The adjuvant use of PDLSCs/PRF could greatly promotethe periodontal healing of delayed reimplantation of avulsed tooth, which was manifestedas organized PDL-like structure and the least pathologic healing.Summary: For the first time, the concept of tissue engineering was applied to thereimplantation of avulsed tooth. Firstly, we prepared different platelet concentrates andthen explored their biological effects on PDLSCs. Secondly, we constructed a cellstransplant combined with cell sheet fragments of PDLSCs and plate-rich granules foravulsed tooth reimplantation. The results revealed that PRP/PRF could promote theperiodontal healing of avulsed tooth by stimulating the proliferation and differentiation ofPDLSCs. However, even the adjuvant use of PRP/PRF could not advance healing of theteeth which have been knock-out for more than2hours. Our data suggest the use of stemcells in the form of cell sheet fragments is effective, and the combination of PRF enrichedwith growth factors may enhance the overall outcomes of tooth reimplantation for teeththat have been extra-oral and dry-stored for2hours. In prospect, the application of thiscell/PRF construct may shed some light on the treatment of avulsed teeth in future dentalpractice.