Preliminary Study Of Platelet-rich Plasma Scaffolds Induce Dental Pulp Regeneration In Vivo

Objective: To explore the feasibility of pulp regeneration using differenttypes of platelet-rich plasma(PRP）scaffolds and PRP scaffolds combined withdental pulp cells(DPCs) in vivo respectively. To evaluate the capacity ofrecruiting endogenous cells with different types of PRP scaffolds, and provideexperimental foundation and theoretical basis for clinical treatment.Methods:Part1: Leucocyte-rich platelet-rich plasma (L-PRP) scaffold was madeusing modified Curasn’s method. The deciduous root segments of minipigs wereprepared by chemical methods. Four groups were randomly established (6samples in each group):(1)the whole blood group;(2)100%L-PRP;(3)50%L-PRP：100%L-PRP were diluted into50%L-PRP using DMEMmedium (v/v);(4)blank group: root fragments with empty canal space. The rootfragments with different contents were subcutaneously implanted into severecombined immunodeficient (SCID) mice. Five weeks after transplantation, thesamples were harvested and processed for histological examination. Part2: Leucocyte-poor platelet-rich plasma(P-PRP)scaffold was madeusing two-step method. The human dental plus cells(hDPCs) were isolated andcultured with tissue block culture method and indentified. The human rootswere segmented and the root canals were pretreated by mechanical-chemicalmethods. The roots fragments were randomly divided into three groups (8samples in each group):①control group, P-PRP scaffold;②experimental group,the P-PRP scaffolds were combined with hDPCs;③blank group, empty rootcanals. The root fragments were implanted subcutaneously in SCID mice. Eightweeks after transplantation, the samples were subjected to histologicalexamination.Results1.The levels of platelet and leukocyte in L-PRP was6-fold and4-foldcompared with those in the whole blood respectively. Two SCID mice in100%L-PRP group suffered skin damage and inflammation.2. Newly formed connective tissue was found in the empty space of theroot fragments inserted50%L-PRP scaffolds but no vasculogenesis. The rootfragments filled with a large number of inflammatory cells,and a small amountof scattered spindle cells were observed in100%L-PRP scaffolds group.3. The forth generation of hDPCs showed a positive reaction for alkalinephosphatese, then observed a few calcium nodule after stained with alizarin redand von kossa. The levels of platelet in P-PRP was1.55-fold compared to that inthe whole blood, and the leukocyte count was much lower than those in thewhole blood. Irregular formed tissue was observed in the root fragments whichhad been solely transplanted with P-PRP scaffolds.4. The root fragments with P-PRP and hDPCs has revealed that the canalwas filled with regenerated vascularized pulp-like tissue, but with no dentin-like tissue. A majority of the cells in regenerated pulp-like tissue stained positivelyfor the anti-human mitochondria antibodies, indicating their human origin. But aminority of the cells strained negatively which were gathered near the root canalopening area, confirming there are host cells involved in tissue regeneration.Conclusion1.It is feasible to induce the pulp-like tissue regeneration via transplantingP-PRP scaffold combined with human dental pull cells(hDPCs) in vivo.2.Endogenous cells can be recruited by transplanting L-PRP or P-PRPscaffolds alone and got involved in tissue regeneration, but the type and thenumber of the recruited cells were limited.