Tissue Engineered Endodontic Reconstruction Based On DLP Printing Technology And Biological Performance Evaluation

BackgroundDental pulp develops from the ectodermal mesenchyme,mainly plays the functions of nutrition,feeling,formation of dentin,defense and repair.It contains several components such as nerves,blood vessels,dental pulp stem cells and odontoblasts.Due to the existence of hard dentin around the dental pulp,as well as the small root apex and insufficiency effective collateral circulation,it is difficult to recover the dental pulp once inflammation occurs or other stimuli and injury happen.Currently,root canal therapy is a kind of conventional therapy for irreversible pulp lesions or periapical lesions.However,without the pulp's supporting,the fragility of tooth increased and the tooth is easy to stain.The methods of vital pulpotomy,apexification and endodontic revascularization cannot solve this problem perfectly,thus,it is urgent to find a new therapy.AimThis article aims to provide a new treatment strategy for clinical pulp lesions and periapical lesions by constructing functional dental pulp in situ by tissue engineering method.Methods and resultsFirstly,we isolate hDPSC from human dental pulp through enzymatic digestion.hDPSC has strong proliferation ability,meanwhile,it highly expresses mesenchymal stem cell markers such as CD105,CD73 and CD90,and hardly expresses the endothelial marker CD144.Therefore,hDPSC is a kind of mesenchymal stem cell with strong ability of proliferation and stemness.When cultured in vitro,hDPSC can differentiate to the directions of osteogenesis,adipogenesis,cartilage formation,nerve formation,and angiogenesis.Nuclear magnetic resonance spectroscopy indicates that we have successfully synthesized Gel MA.Similar to gelatin,Gel MA has excellent biocompatibility,meanwhile,the mechanical property improves and it could be photo-crosslinked because part of the amino group on the gelatin is replaced by MA.Lyophilized Gel MA has typical porous structure.Subsequently,we use DLP printer layer-by-layer construct hDPSC-loaded Gel MA microspheres with a diameter of 400 ?m.Cells inside the microspheres can proliferate and spread when culture in basal medium,indicating Gel MA hydrogel has good biocompatibility,moreover,it can slowly degrade in body.By comparing the biological behaviors of hDPSC in Gel MA microspheres,on2D-Gel MA or on 2D-PS substrates,including cell viability,spreading,proliferation,stem maintenance and differentiation ability,we learn that,hDPSC significantly increases in both 3D and 2D environments,the ones on 2D-PS substrates proliferate most rapidly.The cells cultured in 3D environment spread slower and have smaller size comparing to the ones on 2D-PS group,but they have stronger ability to maintain stemness.In addition,soft substrate help maintains stemness rather than hard substrate.Comparing with the 2D culture environment(Gel MA substrate or PS substrate),3D cultured hDPSC has the ability to differentiate into the neuroblastic and angioblasticdirections.As for the direction of dentin forming potential,there is no significant difference between the groups.Moreover,Gel MA degradation does not cause significant inflammation.Subsequently,we perform in situ animal experiment and ectopic animal experiment to construct tissue engineered dental pulp.Firstly,we prepare partially demineralized dentin fragments with exposed dentin tubule,as outer scaffolds for constructing dental pulp-dentin complexes.After implanting hDPSC-loaded microspheres,hDPSC aggregation and hDPSC division into the root canal for 4 or 8weeks,pink and soft tissue forms,we name it tissue engineered dental pulp.Large numbers of cells in the tissue engineered pulp are in proliferative phase.The tissue engineered pulp forming from the microsphere group and cell aggregation group has microvessel inside,moreover,neural signals and odontogenetic signals are detected.The expression levels of these markers in microsphere group are consistent with the expression of human normal pulp,which are relatively higher than other groups,and the indicators of proliferation,stemness and inflammation are also similar with the normal pulp.In situ dental pulp regeneration experiment in small pigs shows that hDPSC-loaded Gel MA microsphere and cell aggregation can form tissue engineered dental pulp in the root canal.Dense tissues are formed in the apical region,highly vascularized dental pulp is visible in the middle,with some uniformly arranged odontoblast-like cells around.Comparing to the cell aggregation group,the tissue in upper region of the pulp cavity forming in microsphere group is closer to the original pulp structure.There is no complete tissue engineered dental pulp in cell division group.The tissue forms mainly in the apical region(1/3)of the root canal.It is considered that the cell division is partially lost after sedimentation.In addition,hDPSC-loaded microsphere can repair spinal cord injury both in histological and functional levels,suggesting that nerve function may also exist in tissue engineered tooth pulp.We further study the neurodifferentiation ability of hDPSC-loaded microsphere,results show that hDPSC-loaded microspheres can repair spinal cord injury models bothin histological and functional levels,suggesting that tissue engineering pulps may also have neurodifferentiation capabilities.ConclusionIn this study,DLP-based 3D printing technology is used to construct DPSC-loaded Gel MA microspheres,and the microspheres are injected to the root canal for regenerated dental pulp.The main conclusions of this study are as follows,firstly,hDPSC can be isolated from human dental pulp by enzyme digestion.hDPSC is a kind of mesenchymal stem cell with strong proliferation ability and stemness.Secondly,the hDPSC-loaded Gel MA microspheres constructed with DLP printing technology have a3 D soft environment for cell growth,which is conducive for hDPSC to maintain the stemness and retain multi-directional differentiation ability.Thirdly,the hDPSC on the microspheres has the ability to maintain stemness and proliferate,form blood vessels and nerves,as well as form odontoblast.This method can be used to construct tissue engineering pulp,which is expected to be applied as a tissue engineering therapy for pulpal lesions and periapical lesions in the future.