Stem cell-derived exosomes for dentin-pulp complex regeneration: a mini-review.

This mini-review was conducted to present an overview of the use of exosomes in regenerating the dentin-pulp complex (DPC). The PubMed and Scopus databases were searched for relevant articles published between January 1, 2013 and January 1, 2023. The findings of basic in vitro studies indicated that exosomes enhance the proliferation and migration of mesenchymal cells, as human dental pulp stem cells, via mitogen-activated protein kinases and Wingless-Int signaling pathways. In addition, they possess proangiogenic potential and contribute to neovascularization and capillary tube formation by promoting endothelial cell proliferation and migration of human umbilical vein endothelial cells. Likewise, they regulate the migration and differentiation of Schwann cells, facilitate the conversion of M1 pro-inflammatory macrophages to M2 anti-inflammatory phenotypes, and mediate immune suppression as they promote regulatory T cell conversion. Basic in vivo studies have indicated that exosomes triggered the regeneration of dentin-pulp-like tissue, and exosomes isolated under odontogenic circumstances are particularly strong inducers of tissue regeneration and stem cell differentiation. Exosomes are a promising regenerative tool for DPC in cases of small pulp exposure or for whole-pulp tissue regeneration.

Enhanced Osteogenic Differentiation of Pluripotent Stem Cells via γ-Secretase Inhibition.

Bone healing is a complex, well-organized process. Multiple factors regulate this process, including growth factors, hormones, cytokines, mechanical stimulation, and aging. One of the most important signaling pathways that affect bone healing is the Notch signaling pathway. It has a significant role in controlling the differentiation of bone mesenchymal stem cells and forming new bone. Interventions to enhance the healing of critical-sized bone defects are of great importance, and stem cell transplantations are eminent candidates for treating such defects. Understanding how Notch signaling impacts pluripotent stem cell differentiation can significantly enhance osteogenesis and improve the overall healing process upon transplantation. In Rancourt's lab, mouse embryonic stem cells (ESC) have been successfully differentiated to the osteogenic cell lineage. This study investigates the role of Notch signaling inhibition in the osteogenic differentiation of mouse embryonic and induced pluripotent stem cells (iPS). Our data showed that Notch inhibition greatly enhanced the differentiation of both mouse embryonic and induced pluripotent stem cells.

Dental pulp stem cells stimulate neuronal differentiation of PC12 cells.

Dental pulp stem cells (DPSCs) secrete neurotrophic factors which may play an important therapeutic role in neural development, maintenance and repair. To test this hypothesis, DPSCs-conditioned medium (DPSCs-CM) was collected from 72 hours serum-free DPSCs cultures. The impact of DPSCs-derived factors on PC12 survival, growth, migration and differentiation was investigated. PC12 cells were treated with nerve growth factor (NGF), DPSCs-CM or co-cultured with DPSCs using Transwell inserts for 8 days. The number of surviving cells with neurite outgrowths and the length of neurites were measured by image analysis. Immunocytochemical staining was used to evaluate the expression of neuronal markers NeuN, microtubule associated protein 2 (MAP-2) and cytoskeletal marker ßIII-tubulin. Gene expression levels of axonal growth-associated protein 43 and synaptic protein Synapsin-I, NeuN, MAP-2 and ßIII-tubulin were analysed by quantitative polymerase chain reaction (qRT-PCR). DPSCs-CM was analysed for the neurotrophic factors (NGF, brain-derived neurotrophic factor [BDNF], neurotrophin-3, and glial cell-derived neurotrophic factor [GDNF]) by specific ELISAs. Specific neutralizing antibodies against the detected neurotrophic factors were used to study their exact role on PC12 neuronal survival and neurite outgrowth extension. DPSCs-CM significantly promoted cell survival and induced the neurite outgrowth confirmed by NeuN, MAP-2 and ßIII-tubulin immunostaining. Furthermore, DPSCs-CM was significantly more effective in stimulating PC12 neurite outgrowths than live DPSCs/PC12 co-cultures over the time studied. The morphology of induced PC12 cells in DPSCs-CM was similar to NGF positive controls; however, DPSCs-CM stimulation of cell survival was significantly higher than what was seen in NGF-treated cultures. The number of surviving PC12 cells treated with DPSCs-CM was markedly reduced by the addition of anti-GDNF, whilst PC12 neurite outgrowth was significantly attenuated by anti-NGF, anti-GDNF and anti-BDNF antibodies. These findings demonstrated that DPSCs were able to promote PC12 survival and differentiation. DPSCs-derived NGF, BDNF and GDNF were involved in the stimulatory action on neurite outgrowth, whereas GDNF also had a significant role in promoting PC12 survival. DPSCs-derived factors may be harnessed as a cell-free therapy for peripheral nerve repair. All experiments were conducted on dead animals that were not sacrificed for the purpose of the study. All the methods were carried out in accordance with Birmingham University guidelines and regulations and the ethical approval is not needed.

Neurotrophic effects of dental pulp stem cells on trigeminal neuronal cells.

Evidence indicates that dental pulp stem cells (DPSC) secrete neurotrophic factors which play an important role in neurogenesis, neural maintenance and repair. In this study we investigated the trophic potential of DPSC-derived conditioned medium (CM) to protect and regenerate isolated primary trigeminal ganglion neuronal cells (TGNC). DPSC and TGNC were harvested by enzymatic digestion from Wister-Hann rats. CM was collected from 72 h serum-free DPSC cultures and neurotrophic factors; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were analysed by specific enzyme-linked immunosorbent assays (ELISAs). Primary co-cultures of DPSC and TGNC were established to evaluate the paracrine effects of DPSC. In comparison, NGF was used to evaluate its neurotrophic and neuritogenic effect on TGNC. Immunocytochemistry was performed to detect the neuronal-markers; neuronal nuclei (NeuN), microtubule-associated protein-2 (MAP-2) and ßIII-tubulin. Quantitative real time polymerase chain reaction (qRT-PCR) was used to analyse neuronal-associated gene expression of NeuN, MAP-2, ßIII-tubulin in addition to growth-associated protein-43 (GAP-43), Synapsin-I and thermo-sensitive transient receptor potential vanilloid channel-1 (TRPV1). DPSC-CM contained significant levels of NGF, BDNF, NT-3 and GDNF. DPSC and DPSC-CM significantly enhanced TGNC survival with extensive neurite outgrowth and branching as evaluated by immunocytochemistry of neuronal markers. DPSC-CM was more effective in stimulating TGNC survival than co-cultures or NGF treated culture. In comparison to controls, DPSC-CM significantly upregulated gene expression of several neuronal markers as well as TRPV1. This study demonstrated that DPSC-derived factors promoted survival and regeneration of isolated TGNC and may be considered as cell-free therapy for TG nerve repair.

The Effect of Different Routes of Injection of Bone Marrow Mesenchymal Stem Cells on Parotid Glands of Rats Receiving Cisplatin: A Comparative Study.

BACKGROUND AND OBJECTIVES: Cisplatin is a powerful antitumor chemotherapeutic agent that is widely used in the treatment of many cancers but it has many side effects on many organs including salivary glands. Bone marrow is considered to be a rich environment that comprises many types of stem cells of which BMSCs (Bone marrow mesenchymal stem cells) are the most studied with potentiality to differentiate into many cell types. This study was conducted to evaluate the effect of different routes of injection of BMSCs on parotid glands of rats receiving cisplatin. METHODS AND RESULTS: Sprague-Dawley rats were divided into 3 groups: a negative control group receiving phosphate buffered saline, a positive control group receiving cisplatin, and an experimental group where rats received cisplatin and then received iron oxide-labeled BMSCs by either intravenous or intraparotid routes or both. Animals were sacrificed at periods of 3,6,10 and 15 days after cisplatin injection, then histological, ultrastructural and immunohistochemical studies were done. The experimental stem cell treated group showed better histological features and increased PCNA proliferation index when compared to the control. The systemic and combination groups showed better results than the local group. Iron oxide-labeled cells were detected with Prussian blue stain. CONCLUSIONS: This study proved that BMSCs can improve cisplatin induced cytotoxicity in parotid glands. Systemic administration showed to have a better effect than local intraparotid administration and comparable effect to combined administration.

Efficacy of Human Platelet Rich Fibrin Exudate vs Fetal Bovine Serum on Proliferation and Differentiation of Dental Pulp Stem Cells.

BACKGROUND AND OBJECTIVES: The imperative role of dental pulp stem cells (DPSCs) in regenerative therapy demands an in-vitro expansion which must deal with the safety and ethical problems associated with fetal bovine serum (FBS). The primary aim of this study was to compare the effects of human platelet rich fibrin (hPRF) exudate Vs FBS on proliferation and osteodifferentiation of human dental pulp stem cells (hDPSCs). The secondary one was to determine the optimum concentration of hPRF exudate inducing hDPSCs proliferation and osteodifferentiation. METHODS: The direct method was used to prepare hPRF exudate. hDPSCs were isolated from impacted mandibular third molars of twelve donors by the outgrowth method. For cell viability and proliferation rate testing, 96 well plates were used and the assay was done in duplicate and the trial repeated four times under the same conditions. Six wells were used to contain 10% FBS, serum free media, 1%, 5%, 10% and 20% concentrations of hPRF exudates, respectively. The proliferation assay was carried out by MTS tetrazolium cell proliferation assay kit and Elisa reader. The study design for osteodifferentiation protocol was exactly as the proliferation one and instead the assay was carried out by alizarin red with Elisa reader. RESULTS: Compared to 10% FBS, 10% hPRF exudate was the optimum concentration for hDPSCs proliferation, while 1% hPRF exudate was the optimum concentration for osteodifferentiation of hDPSCs. Conclusions: Avoiding the risk of zoonosis which may be occurred with FBS, it is recommended to use 10% hPRF exudate for proliferation and 1% for osteodifferentiation.

Can mandibular lingual canals be used as a forensic fingerprint?

OBJECTIVES: This study aimed to identify whether the lingual canals of the mandible can be used as a unique fingerprint when dealing with forensic victim identification. MATERIALS AND METHODS: The study consisted of two parts; an observational part and an objective image analysis part. In the observational part a total of 100 in vivo high resolution CBCT datasets of human mandibles were included in the process of simulated matching of ante-mortem (AM) and post-mortem (PM) data. For the objective image analysis part 10 dry human mandibles were scanned with 2 different Cone Beam Computed tomography (CBCT) machines. In the observational part of the study trained observers attempted to correctly identify matching pairs of images taken from the same mandible out of a series of 100 mandibles. The aim was to simulate matching of the neurovascular structures on AM and PM mandibular midline images and determine the percentage of mandibles identified correctly. In the objective image analysis part, simulated matching was carried out using a specific CBCT dataset acquired to mimic a PM dataset and 10 datasets acquired from a different CBCT device which served as the source of potential AM cases. Comparison between AM and PM datasets resulted in the matching of the AM data and PM data obtained from the same mandible, leading to an assumed correct identification. RESULTS: The observational part of the study showed an average 95% correct identification of the mandibular midline neurovascular structures. Registration of mandibles resulted in perfect overlap of the same mandible from 2 different CBCT machine with an error distance equalling zero, while the registration of different mandibles deviated on average error distance 0.13 mm to 0.18 mm. CONCLUSION: The percentage of fit for the simulated AM and PM data of the same mandible was 100%. This finding together with the significant deviations noted for the non-matching cases, may have a potential role in forensic identification in the same way that fingerprints are recognised as being a unique identifying feature.

Inhibition of 4-NQO-induced F433 rat tongue carcinogenesis by oleuropein-rich extract.

Olive leaf extract provides nutritional support for detoxification at the cellular level, when the body is under stress. The present study aimed to evaluate the chemopreventive effect of oleuropein-rich extract (ORE) on 4-NQO-induced rat tongue carcinogenesis. Eighty male F344 rats, 6 weeks age were divided into 5 groups (10 animals each for groups 1 and 2 and 20 each for groups 3, 4, and 5). Group 1 served as an untreated control. Group 2 was given ORE-containing diet alone. Rats of groups 3, 4, and 5 were given daily 20 ppm 4-NQO in drinking water for 8 weeks. Group 4 was fed diets containing ORE, concomitantly with the time of carcinogen exposure and continued 1 week after its stoppage. Group 5 was fed diets mixed with ORE starting 1 week after cessation of 4-NQO treatment. The experiment was terminated when the rats aged 37 weeks, and all animals were euthanized. The tongues were carefully inspected for pathological lesions, excised, and were processed for c-Met and Ki-67 immunohistochemical examination. The gross inspection, histopathological and immunohistochemical results of the present study showed a beneficial regression effect of ORE on tumor progression, especially when it was administered concomitantly with 4-NQO rather than when given after the stoppage of the carcinogenic material. In conclusion, ORE has a chemopreventive role in tongue squamous cell carcinoma, and further studies are needed to explore the molecular mechanisms of its tumor suppressive effect at this level.