Comparative analysis of three different protocols for cholinergic neuron differentiation in vitro using mesenchymal stem cells from human dental pulp

Abstract Background During the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP). Methods DPSCs were divided into the following six groups: i) Control; ii) DPSCs treated with 50 ng/ml C5a; iii) DPSCs treated with 100 ng/ml C5a; iv) DPSCs treated with 200 ng/ml C5a; v) DPSCs treated with 300 ng/ml C5a; and vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups.Result DPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes and osteoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect.Conclusion In this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.

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Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation

Stem Cell Reviews and Reports ◽

10.1007/s12015-018-9854-5 ◽

2018 ◽

Vol 15 (1) ◽

pp. 67-81 ◽

Cited By ~ 18

Author(s):

Dong Li ◽

Xiao-Ying Zou ◽

Ikbale El-Ayachi ◽

Luis O. Romero ◽

Zongdong Yu ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Action Potential ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Potential Capacity ◽

Human Dental Pulp

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Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

BMC Oral Health ◽

10.1186/s12903-021-01833-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jie Liu ◽

Xiaoling Wei ◽

Junlong Hu ◽

Xiaohan Tan ◽

Xiaocui Kang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Inhibitory Effect ◽

Stem Cell Markers ◽

High Concentration ◽

Differentiation Potential ◽

Human Dental Pulp ◽

Dentin Sialoprotein

Abstract Background During the process of deep decay, when decay approaches the pulp, an immune response is triggered inside the pulp, which activates the complement cascade. The effect of complement component 5a (C5a) on the differentiation of dental pulp mesenchymal stem cells (DPSCs) is related to dentin reparation. The aim of the present study was to stimulate DPSCs with different concentrations of C5a and evaluate the differentiation of odontoblasts using dentin sialoprotein (DSP). Methods DPSCs were divided into the following six groups: (i) Control; (ii) DPSCs treated with 50 ng/ml C5a; (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a. Flow cytometry and multilineage differentiation potential were used to identify DPSCs. Mineralization induction, Real-time PCR and Western blot were conducted to evaluate the differentiation of odontoblast in the 6 groups. Result DPSCs can express mesenchymal stem cell markers, including CD105, CD90, CD73 and, a less common marker, mesenchymal stromal cell antigen-1. In addition, DPSCs can differentiate into adipocytes, neurocytes, chondrocytes and odontoblasts. All six groups formed mineralized nodules after 28 days of culture. Reverse transcription-quantitative PCR and western blotting indicated that the high concentration C5a groups expressed higher DSP levels and promoted DPSC differentiation, whereas the low concentration C5a groups displayed an inhibitory effect. Conclusion In this study, the increasing concentration of C5a, which accompanies the immune process in the dental pulp, has demonstrated an enhancing effect on odontoblast differentiation at higher C5a concentrations in vitro.

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Effect of hydroxyapatite microspheres, amoxicillin-hydroxyapatite and collagen-hydroxyapatite composites on human dental pulp-derived mesenchymal stem cells

10.1101/2021.01.22.427754 ◽

2021 ◽

Author(s):

Yasmine Mendes Pupo ◽

Lidiane Maria Boldrini Leite ◽

Alexandra Cristina Senegaglia ◽

Lisiane Antunes ◽

Jessica Mendes Nadal ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Positive Control ◽

Control Group ◽

Migration Behavior ◽

Migration Assay ◽

Hydroxyapatite Composite ◽

Human Dental Pulp

AbstractThe aim of this study was to evaluate the in vitro behavior of human dental pulp mesenchymal stem cells (hDPSCs) cultured on scaffolds of three hydroxyapatite-based materials: hydroxyapatite microspheres [HAp]; amoxicillin-hydroxyapatite composite [Amx-HAp]; and collagen-hydroxyapatite composite [Col-HAp]. These hydroxyapatites (HAps) were synthesized through three methods: microwave hydrothermal, hydrothermal reactor (teflon pouches), and precipitation, respectively. We performed an in vitro experimental study using dental pulp stem cells obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and were maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. Each experiment was performed in triplicate. Data analysis was performed using Kruskal-Wallis test and Dunn’s post-hoc test. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA, which indicated these genes plays an important role in odontogenesis regardless of induction stimulus. Cytotoxicity assay using hDPSCs demonstrated that Col-HAp group presented a number of non-viable cells statistically lower than the control group (p=0.03). In the migration assay after 24h, biomaterials HAp, Amx-HAp, and Col-HAp revealed the same migration behavior for hDPSCs observed to the positive control. Col-HAp also provided a statistically significant higher migration of hDPSCs than HAp (p=0.02). The migration results in 48h for HAp, Amx-HAp, and Col-HAp was intermediate from those achieved by control groups. There was no statistical difference between positive control and Col-HAp (p>0.05). In general, Col-HAp scaffold showed better features for these dynamic parameters of cell viability and cell migration capacities for hDPSCs, leading to suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data present high clinical importance because Col-HAp can be used in a wide variety of therapeutic areas, including ridge preservation, minor bone augmentation, and periodontal regeneration.

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Cholinergic Nerve Differentiation of Mesenchymal Stem Cells Derived from Long-Term Cryopreserved Human Dental Pulp In Vitro and Analysis of Their Motor Nerve Regeneration Potential In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms19082434 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2434 ◽

Cited By ~ 8

Author(s):

Soomi Jang ◽

Young-Hoon Kang ◽

Imran Ullah ◽

Sharath Shivakumar ◽

Gyu-Jin Rho ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Nerve Regeneration ◽

Dental Pulp ◽

Motor Nerve ◽

Cholinergic Neurons ◽

Human Dental Pulp

The reduction of choline acetyltransferase, caused by the loss of cholinergic neurons, leads to the absence of acetylcholine (Ach), which is related to motor nerve degeneration. The aims of the present study were to evaluate the in vitro cholinergic nerve differentiation potential of mesenchymal stem cells from cryopreserved human dental pulp (hDPSCs-cryo) and to analyze the scale of in vivo motor nerve regeneration. The hDPSCs-cryo were isolated and cultured from cryopreserved dental pulp tissues, and thereafter differentiated into cholinergic neurons using tricyclodecane-9-yl-xanthogenate (D609). Differentiated cholinergic neurons (DF-chN) were transplanted into rats to address sciatic nerve defects, and the scale of in vivo motor nerve regeneration was analyzed. During in vitro differentiation, the cells showed neuron-like morphological changes including axonal fibers and neuron body development, and revealed high expression of cholinergic neuron-specific markers at both the messenger RNA (mRNA) and protein levels. Importantly, DF-chN showed significant Ach secretion ability. At eight weeks after DF-chN transplantation in rats with sciatic nerve defects, notably increased behavioral activities were detected with an open-field test, with enhanced low-affinity nerve growth factor receptor (p75NGFR) expression detected using immunohistochemistry. These results demonstrate that stem cells from cryopreserved dental pulp can successfully differentiate into cholinergic neurons in vitro and enhance motor nerve regeneration when transplanted in vivo. Additionally, this study suggests that long-term preservation of dental pulp tissue is worthwhile for use as an autologous cell resource in the field of nerve regeneration, including cholinergic nerves.

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