The Experimental And Clinical Researches Of CGF Promoting Pulp Tissue Reconstruction

Teeth are complex organs comprising several mineralized tissues,which enclose a soft tissue termed the dental pulp.A vital pulp is critical for maintenance of tooth homeostasis and nutrition.In cases of absence of functional pulp tissue is not able to support formation of dentine,perception of pain and transmission of sensory stimuli from the pulp dentine complex,immunoresponse as well as formation of dentine as active defence mechanisms against invading toxins and bacteria.The main components of dental pulp tissue are dental pulp cells,which are mesenchymal cells derived from the neural crest.Some of these cells demonstrate high growth potential,self-renewal capacity and possess multiple differentiation properties,and have been designated dental pulp stem cells（DPSCs）.Under certain conditions the DPSCs can differentiate into osteoblasts,which formate reparative dentin.These can provide biological basis for preservation of vital pulp and treatment of dental pulp disease by pulp regeneration.CGF is the third generation of platelet concentrate.CGF was formed by a special centrifuge device with variable centrifugation speed.It had been reported to contain more growth factors and more excellent fibrin tensile strength than previous platelet concentrates.CGF had a three-dimensional fibrin framework.This kind of structure protects growth factors from proteolysis,and causes lower and sustained release of growth factors from the fibrin concentrates,which can promote cell proliferation and migration and differentiation.It has been widely used in bone and other regenerative medicine.However,the effect of CGF on DPSCs proliferation,migration and differentiation had not been investigated.It is of great significance to pulp tissue engineering.Both accidental traumatic pulp exposure and caries caused exposure may bring irreversible damage to dental pulp if not treated properly.Direct pulp capping has been showed to be an effective way for maintaining pulpal vitality and promoting the differentiation of dental pulp cells.The objectives of direct pulp capping are formation of dentin bridges to wall off outside stimulation and preservation of healthy pulp tissue.But long term observation showed that it may lead to dental pulp degeneration and diffuse calcification,even root canal block.The study investigated the possibility of CGF applying to direct pulp capping,so can we improve the success rate by using CGF in clinical.Endodontic treatment of immature permanent teeth with necrotic pulps and open apices is a significant challenge to endodontists.The traditional root canal treatment and apexification procedure cannot stimulate the development of the apical closure and thickening of radicular dentin.So the teeth more prone to fracture,even to be extracted.Regenerative treatment approaches in teeth with incomplete root formation and pulp necrosis have been considered as an alternative to conventional treatment.The research was aimed to identify the effect of CGF on pulp revascularization cases in clinic,which would play the important role in guiding the treatment.TAZ（Transcriptional coactivator with PDZ-binding motif）serves as a transcriptional regulator via its intrinsic transactivation domain.TAZ has been implicated to interact with multiple transcription factors,then located into nuclear,bind to the promoter and activate the target gene.TAZ plays important roles in cell proliferation,migration,and EMT.However,how TAZ regulates the proliferation and migration of DPSCs remains unclear.In conclusion,we evalued the effects of CGF on proliferation and mineralization capacity of hDPSCs,assessed the function of CGF on direct pulp capping in beagle dog.Then we observed the impact of CGF applying to pulp revascularization of permanent tooth.Lastly we analyzed the effects and the molecular mechanism of TAZ on proliferation and migration of hDPSCs.Part one Effects of Concentrated Growth Factor（CGF）on humandental pulp stem cells biological characteristics in vitroObjective:To study the effects of CGF on the proliferation,migration and mineralization of hDPSCs.Methods:hDPSCs were isolated from healthy third molars or premolars of healthy human subjects.Individual dental pulps were extracted and digested by using type I collagenase and dispase.Single-cell suspensions were cultured for primary cells.Cell proliferation was evaluated with cell CCK-8assay.The surface antigen of the 3rd passages hDPSCs were identified by Flow cytometry assay（FCM）.The multipotential differentiation of hDPSCs was detected by Alizarin Red and Oil Red O staining.CGF was obtained from9ml fresh venous blood of healthy volunteers,The CGF clot was pressed onto membranes.CGF membranes were cut into small pieces（3×3 mm²）by using sterilized blade and immersed in saline for next experiment.The cells were divided into 4 groups,included control group and three experiment groups.The experiment groups were treated with different concentration of CGF:1piece of CGF,2 pieces of CGF,4 pieces of CGF.Cell proliferation was evaluated with cell counting kit-8（CCK-8）assay to test the number of viable cells.Cell migration assays were performed by using Transwell assay.For odontogenesis,hDPSCs were stained with Alizarin Red.The mineralized nodules were observed to test the mineralization capacities of CGF on hDPSCs.Alkaline phosphatase（ALP）enzyme activity was detected to analyze the effect of CGF on hDPSCs mineralization.To confirm the effect of CGF on hDPSCs mineralization,mRNA expression levels of typical odontogenic genes（DSPP,DMP1,OCN,BSP）in hDPSCs were evaluate by real-time PCR.Western blot analysis was used to assay the protein expression levels of DSPP,DMP-1 and ALP in hDPSCs.Results:Primary h DPSCs presented spindle,long fusiform,abundant cytoplasm under the microscope.Flow Cytometry analysis showed that hDPSCs were positive for CD44 and CD29,but negative for CD34 and CD45.After different induction of hDPSCs exhibited multi-directional differentiation ability,formed Alizarin-Red-positive mineral deposits and Oil-Red-O-positive lipid droplet.The CCK-8 analysis revealed that CGF could promote the proliferation of hDPSCs.Transwell migration assay showed that the cell numbers in CGF treated group were significantly higher than those in the control group.Real-time PCR results demonstrated that CGF increased mRNA levels of DSPP,DMP-1,BSP and ALP in hDPSCs at day 7 and day 14compared with control group.Simultaneously,4 pieces of CGF conversely repressed the mRNA levels at day14.Western blot analysis results showed the protein expression levels of DSPP,DMP-1 and ALP were significantly up-regulated compared with the control group,while 4 pieces of CGF decreased protein expression levels of ALP,DSPP,DMP-1 at day 14compared with other groups.The results were consistent with real-time PCR results.Conclusion:CGF could facilitate h DPSCs proliferation and migration in vitro.CGF could promote hDPSCs mineralization,and up-regulate expression levels of mineralization related genes,DSPP,DMP-1,ALP,BSP.Part Two Animal experiment study of CGF on directly pulp cappingObjective:To evaluate the effects of Ca（OH）₂,MTA,CGF on directly pulp capping in vivo of Beagle dog.To investigate the feasibility of CGF appling to directly pulp capping.Methods:CGF was obtained from 9ml venous blood of Beagle dog small saphenous vein on hind limb,The CGF clot was pressed onto membranes.CGF membranes were cut into small pieces（2×2 mm²）by using sterilized blade and immersed in saline for next experiment.A total of 48second and third premolars were selected for this study.All experimental teeth were randomly divided into 4 groups（n=12 per group）,including group CGF,group MTA,group Dycal and negative control group.During the experiment,the dogs were induced general anesthesia by the intramuscular injection with0.07⁰.08 mg/kg of Su-Mian-XinⅡ.Subsequently Sodium Pentobarbital was injected by 0.3 ml/kg for anesthesia maintenance.One percent Lidocaine with1:10⁵ of Epinephrine was injected for local anesthesia.The operative area was disinfected with 0.2%Chlorhexidine.Rubber dam isolation was placed for each experimental tooth.The teeth and surrounding rubber dam were coated with Iodine Tincture.The pulp chamber was mechanically exposed and an access cavity was prepared from the occlusal surface with a high speed round carbide bur.The access cavity was flush with sterile saline solution,and a small moist cotton pellet was placed into the access cavity to stop the bleeding.After hemostasis had been achieved,different materials were placed directly over the exposed surface of pulp.The access cavity was then sealed with glass ionomer cement.Radiographs were taken for all experimental teeth after the treatment.Three months after treatment the teeth were extracted and fixed in10%buffered formalin.Results:Preoperative and 3-month postoperative radiographs were taken.The results revealed that dentin bridging with Ca（OH）₂ was thick,pulp chamber was narrowed,the dentin of root canal was thickened.Dentin bridging with MTA was formation,pulp chamber was narrowed,the dentin of root canal was thickened,but the degree was diminished compared with MTA.CGF specimens had the less dentin bridging,narrowed pulp chamber and the thickened dentin of root canal were not obvious compared with Ca（OH）₂ and MTA.Hematoxylin-eosin–stained histopathologic evaluation showing comp-lete calcific barrier formation was observed in Ca（OH）₂ specimens.Fiber hyperplasia and odontoblasts disappear of pulp were found in Ca（OH）₂specimens.MTA specimens had the moderate calcific barrier formation,and the pulp showed fiber hyperplasia,congestion and odontoblasts disappear.CGF specimens had the most thin calcific barrier formation among all experiments.CGF specimens showed that odontoblasts regular arranged and predentin formed after pulp capping with CGF for 3 months.Conclusion:Mineral trioxide aggregate and Ca（OH）₂ are traditional pulping capping agent,could be used in clinical for direct pulp capping.CGF could enhance the healing of exposed pulp by directly pulp capping in vivo,promote the recovery of pulp,and maintain the normal physiological function of pulp tissue.Part Three Clinic research of CGF on revascularization of immaturepermanent teethObjective:To explore the capacity of CGF to regenerate dental pulp in permanent teeth with long-term follow-up,and to assess the function of CGF on revascularization of the immature,nonvital permanent tooth.Methods:CGF was obtained from 9ml fresh venous blood of the patient who needed to be treated,CGF membranes were cut into small pieces（2×2mm²）and immersed in saline for next treatment.Twelve immature permanent tooth with pulp necrosis from nine patients were recruited,the study included5 incisors and 7 premolars with or without signs and（or）symptoms of periapical pathology.Intraoral periapical radiographics were taken pre-operative.After the teeth were isolated with rubber dam,an access cavity was prepared by using a high speed handpiece with water spray,removed the corruption necrosis pulp.Each tooth received copious irrigation with of 1%NaOCl（sodium hypochlorite）,and then the access cavities were sealed by temporary sealer with application of a triple antibiotic paste of Metronidazole,Ciprofloxacin and Cefaclor in the canal.In the next appointment,the temporary seal was removed.The canals were flushed with NaOCl,and physiological saline,and dried with sterile paper points.A size#30 K-file was inserted over the apical area of the canals,evoked apical tissues bleeding into canals.CGF membranes were put into the canal to reach a level 1 mm below the cementoenamel junction by vertical comdensation plugger,the access cavities were sealed by 3 mm MTA and temporary sealer.At 1 week recall,the tooth was no any symptom,the coronal portions of the root canals were sealed by composite.Patient would be recalled at 1,3,6,12,18,24 months.Preoperative and postoperative radiographs were taken,and the pulp vitality was detected.Results:Fourteen teeth from 12 patients were enrolled in this study,1patient returned with apical periodontitis after revascularization treatment,and then performed apexification.Four cases showed pulp vitality,evidence of increase in root length,increased dentinal wall thickness and root development.Seven cases exhibited apical periodontitis was eliminated,and there was radiographic evidence of continuing thickness of dentinal walls,apical closure,or increased root length.Two cases showed apical periodontitis had be eliminated,the root development was ceased,not apical closure.Conclusion:Revascularization could be performed as an effective method for increasing root length,thickening of the root wall and apical closure.CGF could be used as root canal sealer of revascularization,increasing the success rate.Part Four Effects of TAZ on human dental pulp stem cell proliferationand migrationObjective:The present study was aimed to observe the expression level of TAZ（The transcriptional co-activator with PDZ-binding motif）in human dental pulp stem cells（h DPSCs）,and to observe the effect of siTAZ on hDPSCs proliferation and migration,as well as the molecular mechanisms underlying its actions.Methods:Cell immune fluorescence stain was performed to analyze the expression level of TAZ.After TAZ was silenced,following hDPSC transfection with TAZ-specific small interfering si RNA（siTAZ）,Real-time PCR and Western blot were performed to detect the efficiency of TAZ silence.MTT and BrdU were performed to analyze the effect of TAZ silenced on hDPSCs proliferation.Wound-healing assay and Transwell was used to test the effect of TAZ silencing on hDPSCs migration.Real-time PCR and Western blot were performed to verify the function of TGF-βsignal pathway on TAZ regulating hDPSCs proliferation and migration.Results:hDPSCs were processed for immunofluorescence.TAZ was revealed to be high expressed green fluorescence in hDPSCs.Successful transfection with siTAZ in hDPSCs was confirmed by using qPCR and Western blot analysis.MTT and Brd U showed that siTAZ inhibits hDPSC proliferation in vitro.Wound-healing assay and Transwell assay revealed that the migration of hDPSCs was inhibited by transfection with siTAZ.Treatment of hDPSCs with siTAZ significantly decreased the mRNA and protein expression levels of CTGF and Cyr61.Western blot analysis demonstrated Smad3/4 protein that involved in the regulation of CTGF and Cyr61expression via a TGF-βmediated pathway were significantly down-regulated in TAZ-depleted cells.Conclusion:TAZ is high expression on hDPSCs.TAZ silence could suppress hDPSCs proliferation and migration in vitro.TAZ may be implicated in TGF-βsignaling pathways that may modulate the expression of downstream gene Smad3,Smad4,CTGF and Cyr61,which involved in hDPSCs proliferation and migration.Summary:1.CGF could facilitate hDPSCs proliferation and migration in vitro.2.CGF could promote hDPSCs mineralization,and up-regulate expression levels of mineralization related genes,DSPP,DMP-1,ALP,BSP.3.CGF could enhance the healing of exposed pulp by direct pulp capping in vivo,promote the recovery of pulp,and the normal physiological function of pulp.4.CGF could be used as root canal sealer of revascularization,increasing the success rate.5.TAZ is high expression on h DPSCs.6.TAZ silence could suppress h DPSCs proliferation and migration in vitro.7.TAZ may be implicated in TGF-βsignaling pathways that may modulate the expression of downstream gene Smad3,Smad4,CTGF and Cyr61,which involved in hDPSCs proliferation and migration.