Therapeutic Effect Of Hypoxic Preconditioned Dental Pulp Stem Cells For Treatment Of Apical Periodontitis

Backgrounds and objectives:Apical periodontitis is a bone destructive disease caused by bacterial infection in the dental root canal system. Clinically, root canal treatment can not completely biological repair alveolar bone defects caused by periapical disease. With the extensive study and development of stem cells in bone regeneration, application of stem cells to biologically repair infectious bone defects is the most promising way at present. Human dental pulp stem cells?hDPSCs? have many advantages like abundant resource, convenient obtain, simple cultivation, multiple differentiation potential and little ethical limits. Owing to these advantages, we propose a new strategy of cell therapy for infectious bone defect repair by systemic transplantation of human dental pulp stem cells?hDPSCs?.A large number of studies show that, regenerative potential of MSCs to damaged tissue plays key roles during MSC-based therapy. Researches have indicated that hypoxic preconditioning?HP? is a simple yet effective way of improving MSCs properties. Culturing BM-MSCs under hypoxia was found to enhance cell recruitment and therapeutic benefits after ischemic stroke in mice. Human umbilical cord-derived MSCs showed enhanced angiogenic effects by HP treatment in a mouse model of hind limb ischemia. Nevertheless, the effect of hypoxic preconditioned hDPSCs on bone regenerative potential has not been clearly illuminated so far. Therefore, the investigation of this question will contribute to more effective application of hDPSCs in bone tissue repair and regeneration of infectious bone defect.Herein, the present study aimed to investigate the bone regeneration effect of the damaged alveolar bone through injection of hypoxic preconditioned hDPSCs into apical periodontitis mice model. The results would be constructive for treatment of infection-caused bone destruction and helpful for translation of hDPSCs-based therapy.Materials and methods:Human dental pulp tissue were obtained from premolars or third molars of healthy 8¹4 years old patients who needed extraction for orthodontic treatment in department of oral and maxillofacial surgery, Union Hospital. Isolation and culture of hDPSCs by Collagenase ? digested combined with tissue anchored under sterile conditions.Human DPSCs from passages 3-6 were used for experiments. For normoxic pretreatment, hDPSCs were incubated in standard culture media at 37°C in a humidified atmosphere of 21% O₂, 74% N₂ and 5% CO₂. Hypoxic pretreatment was achieved by a finely controlled Pro Ox-C-chamber system with 3% O₂, 92% N₂ and 5% CO₂.[1] Cell immunofluorescence assay, flow cytometry and cell osteogenic/adipogenic experiments were conducted to identify the characteristics of human dental pulp stem cells.[2] Alizarin red staining and immunofluorescence were used to detect the osteogenic ability of hDPSCs after hypoxic preconditioning.[3] RT-PCR and Western blot assays were used to detect the expression of osteogenic related mRNA and proteins.[4] Through establish a mouse model of apical periodontitis to study the hDPSCsbased therapeutic effect.[5] Using HE staining, Trap staining and immunohistochemistry to observe and compare the periapical inflammatory changes, the activity of osteoclasts and cell ossification between the mice that received normoxic and hypoxic preconditioned hDPSCs.[6] Adopting Micro CT analysis to evaluat e the regeneration effect of the alveolar bone of the mice mentioned above.Results:[1] The characterization of DPSCs by immunocytochemical staining showed positive for vimentin and negative for keratin; flow cytometry displayed the molecular surface antigen markers of hDPSCs are CD29, CD44, CD90, CD105, CD146 and STRO-1, while CD34 and CD45 were little expressed; the Oil-Red O staining and Alizarin red S staining demonstrated these cells have adipogenic and osteogenic differentiation ability.[2] Hypoxic preconditioning promoted the osteogenic differentiation of hDPSCs.[3] Hypoxic preconditioning hDPSCs promoted the expression of Runx2, Sp-7,VEGF and CD45.[4] HE staining showed that the periapical inflammatory lesions in mice aggravated gradually from the acute phase to the chronic phase with the extension of the opening time of the dental pulp. This progression was similar to the clinical periapical disease, indicating that the model was successfully established.[5] Hypoxic hDPSCs injection compared with normoxic one, peri apical inflammation in mice was significantly reduced as well as reduced apical osteoclasts, while more cells in the bone defect region were positive for Runx2, BMP2 and OCN, which are osteogenic related proteins. These results indicated hypoxic pretreatme nt of hDPSCs enhanced the cell osteogenic differentiation capacity in the bone defect area.[6] Micro CT results directly showed that hypoxic preconditioned hDPSCs promoted bone tissue regeneration and repair effect in the site of graft host bone defect.Conclusions:This investigation demonstrates that hypoxic preconditioning promotes osteogenic differentiation of hDPSCs in vitro and significantly enhance repair and regeneration of periapical bone defect. These results displays a promising therapeutic strategy of hDPSCs transplantation against infection-caused bone loss and HP is an effective means of improving cell osteogenesis capability in the lesion for the treatment of infectious bone destruction.