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Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

ABSTRACT: There is an age-dependent decline of pulp regeneration, due to the decline of migration, proliferation, and cell survival of resident stem cells. Trypsin is a proteolytic enzyme clinically used for tissue repair. Here, we investigated the effects of trypsin pretreatment of pulpectomized teeth prior to cell transplantation on pulp regeneration in aged dogs. The amount of regenerated pulp was significantly higher in trypsin-pretreated teeth compared to untreated teeth. Trypsin pretreatment increased the number of cells attached to the dentinal wall that differentiated into odontoblast-like cells. The trypsin receptor, PAR2, was higher in vitro expression in the periodontal ligament cells (PDLCs) from aged dogs compared to those from young. The direct effects of trypsin on aged PDLCs were increased expression of genes related to immunomodulation, cell survival, and extracellular matrix degradation. To examine the indirect effects on microenvironment, highly extracted proteins from aged cementum were identified by proteomic analyses. Western blotting demonstrated that significantly increased fibronectin was released by the trypsin treatment of aged cementum compared to young cementum. The aged cementum extract (CE) and dentin extract (DE) by trypsin treatment increased angiogenesis, neurite extension and migration activities as elicited by fibronectin. Furthermore, the DE significantly increased the mRNA expression of immunomodulatory factors and pulp markers in the aged DPSCs. These results demonstrated the effects of trypsin on the microenvironment in addition to the resident cells including PDLCs in the aged teeth. In conclusion, the potential utility of trypsin pretreatment to stimulate pulp regeneration in aged teeth and the underlying mechanisms were demonstrated.

SUBMITTER: Iohara K

PROVIDER: S-EPMC7456913 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

Iohara Koichiro K Zayed Mohammed M Takei Yoshifumi Y Watanabe Hideto H Nakashima Misako M

Frontiers in bioengineering and biotechnology 20200814

There is an age-dependent decline of pulp regeneration, due to the decline of migration, proliferation, and cell survival of resident stem cells. Trypsin is a proteolytic enzyme clinically used for tissue repair. Here, we investigated the effects of trypsin pretreatment of pulpectomized teeth prior to cell transplantation on pulp regeneration in aged dogs. The amount of regenerated pulp was significantly higher in trypsin-pretreated teeth compared to untreated teeth. Trypsin pretreatment increas ...[more]

PMID: 32923438

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Project description:BackgroundThe golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.MethodsHuman Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.Results and discussionWe analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.ConclusionOur data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

Project description:BackgroundDental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.MethodsColony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests.ResultshpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation.ConclusionsThese results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Dataset Information

Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

Publications

Treatment of Pulpectomized Teeth With Trypsin Prior to Transplantation of Mobilized Dental Pulp Stem Cells Enhances Pulp Regeneration in Aged Dogs.

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