| Background:Periodontitis,which progressively destroys tooth-supporting structures,is one of the most widespread infectious diseases and the leading cause of tooth loss in adults.There is mounting evidence that stem cells derived from periodontal ligament(PDL)tissues are able to regrow new alveolar bone,new PDL and new cementum tissues.More importantly,investigations from preclinical trials and small-scale pilot clinical studies suggest that cell-therapy is a promising therapy for the regeneration of lost/damaged periodontal tissue.This study aimed to assesse the safety and feasibility of using autologous PDL-derived stem cells(PDLSCs)as an adjuvant to grafting materials in guided periodontal tissue regeneration(GTR)to treat intrabony defects in human patients.Our data will provide primary clinical evidence for the efficacy of cell transplantation in regenerative dentistry.Following the findings from the trial,the quality of cell sheets yielded from PDLSCs derived from different-aged donors was evaluated.Data from the evaluation will provide us more information to understand the findings from the trial and direct us for the design of new trials to further assesse the efficacy of PDLSC-based treatment in patients with intrabony defects.Methods:1)Preparation and delivery of autologous PDLSC sheets: Selecting unimpacted and non-functional third molars from patients.The PDLSCs were isolated and cultured from the root surfaces of the teeth.Isolated and purified PDLSCs were induced into PDLSC sheets.Then the PDLSCsheets were transported to the clinic for using.2)We conducted a single-center,randomized trial that used autologous PDLSCs in combination with bovine-derived bone mineral materials to treat periodontal intrabony defects.Enrolled patients were randomly assigned to either the Cell group(treatment with GTR and PDLSC sheets in combination with Bio-oss?)or the Control group(treatment with GTR and Bio-oss? without stem cells).This trial was approved by the ethical committees of our Hospital(2011-02)and in 2011,it is registered in the Clinical Trials.gov database(NCT01357785).During a 12-month follow-up study,we evaluated the frequency and extent of adverse events.For the assessment of treatment efficacy,the primary outcome was based on the magnitude of alveolar bone regeneration following the surgical procedure.3)The activity of PDLSCs was first determined based on their colony-forming ability,surface markers,proliferative/differentiative potentials,senescence-associated b-galactosidase(SA-b G)staining,and expression of pluripotency-associated transcription factors.The ability of these cells to form sheets,based on their extracellular matrix(ECM)contents and their functional properties necessary for osteogenic differentiation,was evaluated to predict the age-related changes in the regenerative capacity of the cell sheets in their further application.Results:1)PDLSCs were successfully obtained from 22 of the 24 teeth provided by our clinical researchers involved in this trial.PDLSC sheets were manufactured based on our protocol and safely transported to the clincial center for use.No safety issues were happened during cell isolation,sheet production and delivery.2)A total of 30 periodontitis patients aged 18 to 65 years(48 testing teeth with periodontal intrabony defects)who satisfied our inclusion and exclusion criteria were enrolled in the study and randomly assigned to the Cell group or the Control group.A total of 21 teeth were treated in the Control group and 20 teeth were treated in the Cell group.All patients received surgery and a clinical evaluation.No clinical safety problems that could be attributed to the investigational PDLSCs were identified.Each group showed a significant increase in the alveolar bone height(decrease in the bone-defect depth)over time(p < 0.001).However,no statistically significant differences were detected between the Cell group and the Control group(p > 0.05).3)To investigate the age-related changes in PDLSC sheets,it was found that human PDLSCs could be isolated from the PDL tissue of different-aged subjects.However,the ability of the PDLSCs to proliferate and to undergo osteogenic differentiation and their expression of pluripotency-associated transcription factors displayed age-related decreases.In addition,these cells exhibited an age-related increase in SA-?G expression.Aged cells showed an impaired ability to form functional cell sheets,as determined by morphological observations and Ki-67 immunohistochemistry staining.Based on the production of ECM proteins,such as fibronectin,integrin b1,and collagen type I;alkaline phosphatase(ALP)activity;and the expression of osteogenic genes,such as ALP,Runt-related transcription factor 2,and osteocalcin,cell sheets formed by PDLSCs derived from older donors demonstrated a less potent osteogenic capacity compared to those formed by PDLSCs from younger donors.Conclusions:Stem cell therapy is a promising new therapeutic avenue that may enable the regeneration of lost periodontal tissue,and regenerative dentistry is at the forefront of the transition from basic science research to the clinical reconstructive arena.Although there are many issues that need to be resolved before stem cell therapies become commonplace,clinicians should continue to monitor the progression of these technologies.The data obtained in this trial showed that autologous PDLSC-based treatment for periodontal intrabony defects was safe;however,more rigorous clinical trials are recommended to evaluate the efficacy of this therapy.Future clinical endeavors in cell-based periodontal therapy should identify more suitable scaffolding materials and define safe and effective cell dosing procedures based on well-designed multi-center randomized controlled trials.Collectively,the conclusions are:1)The protocol for PDLSC isolation,sheet preparation and transportation is realiable,safe and practical and hence,can be used in next similar trials.2)This study demonstrates that clinial use of autologous PDLSCs for the treatment of periodontal intrabony defects is safe and does not produce any adverse effects.Alghough no additional benefit was found The efficacy of cell-based periodontal therapy requires further validation by multicenter,randomized controlled studies with an increased sample size.3)Age-associated decline in the matrix contents and osteogenic properties of PDLSC sheets should be taken into account in cell sheet engineering research and the design of next clinical trials. |
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