Comparative evaluation of osteogenic differentiation potential of stem cells derived from dental pulp and exfoliated deciduous teeth cultured over granular hydroxyapatite based scaffold

Abstract Background Mesenchymal stem cells isolated from the dental pulp of primary and permanent teeth can be differentiated into different cell types including osteoblasts. This study was conducted to compare the morphology and osteogenic potential of stem cells from exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSC) in granular hydroxyapatite scaffold (gHA). Preosteoblast cells (MC3T3-E1) were used as a control group. Methodology The expression of stemness markers for DPSC and SHED was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR). Alkaline phosphatase assay was used to compare the osteoblastic differentiation of these cells (2D culture). Then, cells were seeded on the scaffold and incubated for 21 days. Morphology assessment using field emission scanning electron microscopy (FESEM) was done while osteogenic differentiation was detected using ALP assay (3D culture). Results The morphology of cells was mononucleated, fibroblast-like shaped cells with extended cytoplasmic projection. In RT-PCR study, DPSC and SHED expressed GAPDH, CD73, CD105, and CD146 while negatively expressed CD11b, CD34 and CD45. FESEM results showed that by day 21, dental stem cells have a round like morphology which is the morphology of osteoblast as compared to day 7. The osteogenic potential using ALP assay was significantly increased (p < 0.01) in SHED as compared to DPSC and MC3T3-E1 in 2D and 3D cultures. Conclusion gHA scaffold is an optimal scaffold as it induced osteogenesis in vitro. Besides, SHED had the highest osteogenic potential making them a preferred candidate for tissue engineering in comparison with DPSC.

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Insight into the Role of Dental Pulp Stem Cells in Regenerative Therapy

Biology ◽

10.3390/biology9070160 ◽

2020 ◽

Vol 9 (7) ◽

pp. 160 ◽

Cited By ~ 5

Author(s):

Shinichiro Yoshida ◽

Atsushi Tomokiyo ◽

Daigaku Hasegawa ◽

Sayuri Hamano ◽

Hideki Sugii ◽

...

Keyword(s):

Stem Cells ◽

Tissue Regeneration ◽

Dental Pulp ◽

High Growth ◽

Dental Pulp Stem Cells ◽

Permanent Teeth ◽

Deciduous Teeth ◽

Pulp Tissue ◽

Differentiation Potential ◽

Cell Based Therapy

Mesenchymal stem cells (MSCs) have the capacity for self-renewal and multilineage differentiation potential, and are considered a promising cell population for cell-based therapy and tissue regeneration. MSCs are isolated from various organs including dental pulp, which originates from cranial neural crest-derived ectomesenchyme. Recently, dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) have been isolated from dental pulp tissue of adult permanent teeth and deciduous teeth, respectively. Because of their MSC-like characteristics such as high growth capacity, multipotency, expression of MSC-related markers, and immunomodulatory effects, they are suggested to be an important cell source for tissue regeneration. Here, we review the features of these cells, their potential to regenerate damaged tissues, and the recently acquired understanding of their potential for clinical application in regenerative medicine.

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CD146 controls the quality of clinical grade mesenchymal stem cells from human dental pulp

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02559-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lan Ma ◽

Zhiqing Huang ◽

Di Wu ◽

Xiaoxing Kou ◽

Xueli Mao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Culture Conditions ◽

Proliferation Rate ◽

Permanent Teeth ◽

Deciduous Teeth ◽

Clinical Grade ◽

Surface Molecule ◽

Differentiation Potential

Abstract Background Human mesenchymal stem cells from dental pulp (hMSC-DP), including dental pulp stem cells from permanent teeth and exfoliated deciduous teeth, possess unique MSC characteristics such as expression of specific surface molecules and a high proliferation rate. Since hMSC-DP have been applied in numerous clinical studies, it is necessary to establish criteria to evaluate their potency for cell-based therapies. Methods We compared stem cell properties of hMSC-DP at passages 5, 10 and 20 under serum (SE) and serum-free (SF) culture conditions. Cell morphology, proliferation capacity, chromosomal stability, surface phenotypic profiles, differentiation and immunoregulation ability were evaluated. In addition, we assessed surface molecule that regulates hMSC-DP proliferation and immunomodulation. Results hMSC-DP exhibited a decrease in proliferation rate and differentiation potential, as well as a reduced expression of CD146 when cultured under continuous passage conditions. SF culture conditions failed to alter surface marker expression, chromosome stability or proliferation rate when compared to SE culture. SF-cultured hMSC-DP were able to differentiate into osteogenic, adipogenic and neural cells, and displayed the capacity to regulate immune responses. Notably, the expression level of CD146 showed a positive correlation with proliferation, differentiation, and immunomodulation, suggesting that CD146 can serve as a surface molecule to evaluate the potency of hMSC-DP. Mechanistically, we found that CD146 regulates proliferation and immunomodulation of hMSC-DP through the ERK/p-ERK pathway. Conclusion This study indicates that SF-cultured hMSC-DP are appropriate for producing clinical-grade cells. CD146 is a functional surface molecule to assess the potency of hMSC-DP.

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Association of Dental Pulp Stem Cells and Ricinus Bone Compound in a Model of Bone Defect

JOURNAL OF BIOENGINEERING AND TECHNOLOGY APPLIED TO HEALTH ◽

10.34178/jbth.v3i3.129 ◽

2020 ◽

Vol 3 (3) ◽

pp. 267-278

Author(s):

Alan Jesus ◽

Adriano Jesus ◽

Flávia Lima ◽

Luiz Freitas ◽

Cássio Meira ◽

...

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Bone Defect ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Autogenous Bone ◽

Control Group

Autogenous bone grafting is needed in some bone tissue defects; however, it causes secondary surgical wounds and morbidity. Tissue bioengineering may be an alternative approach for bone regeneration. Here we investigated the osteogenic potential of dental pulp stem cells from deciduous teeth (DPSC) in association with a Ricinus bone compound (RBC) in a model of bone defect. The influence of the biomaterial RBC on the proliferation and osteogenic differentiation of DPSC was assessed in vitro by MTT metabolism and alizarin red staining, respectively. The morphologic analysis was performed using the optic and scanning electron (SEM) microscopies. For the in vivo study, 54 Wistar rats submitted to calvarial defects were filled with RBC or RBC+DPSC. A control group had the defects filled only with blood clots. Analyses were performed 15, 30 and 60 days after treatment using digital radiography, optical microscopy, SEM and chemical analysis by electron dispersive spectroscopy. The Ricinus bone compound (RBC) did not inhibit the osteogenic differentiation in vitro. No spontaneous regeneration was observed in the control group. The area of the calvarial defect of the RBC+DPSC group showed greater radiopacity on day 15. The RBC presented no reabsorption, was biocompatible and showed osteointegration, working as a mechanical filling. Only sparse ossification areas were found and those were larger and more developed on the RBC+DPSC group when compared to animals treated only with RBC. RBC in association with DPSC is a promising combination for applications in bone regeneration.

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Multilineage Differentiation Potential of Human Dental Pulp Stem Cells—Impact of 3D and Hypoxic Environment on Osteogenesis In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms21176172 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6172

Author(s):

Anna Labedz-Maslowska ◽

Natalia Bryniarska ◽

Andrzej Kubiak ◽

Tomasz Kaczmarzyk ◽

Malgorzata Sekula-Stryjewska ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Stem Cell Population ◽

Differentiation Potential ◽

Human Dental Pulp

Human dental pulp harbours unique stem cell population exhibiting mesenchymal stem/stromal cell (MSC) characteristics. This study aimed to analyse the differentiation potential and other essential functional and morphological features of dental pulp stem cells (DPSCs) in comparison with Wharton’s jelly-derived MSCs from the umbilical cord (UC-MSCs), and to evaluate the osteogenic differentiation of DPSCs in 3D culture with a hypoxic microenvironment resembling the stem cell niche. Human DPSCs as well as UC-MSCs were isolated from primary human tissues and were subjected to a series of experiments. We established a multiantigenic profile of DPSCs with CD45−/CD14−/CD34−/CD29+/CD44+/CD73+/CD90+/CD105+/Stro-1+/HLA-DR− (using flow cytometry) and confirmed their tri-lineage osteogenic, chondrogenic, and adipogenic differentiation potential (using qRT-PCR and histochemical staining) in comparison with the UC-MSCs. The results also demonstrated the potency of DPSCs to differentiate into osteoblasts in vitro. Moreover, we showed that the DPSCs exhibit limited cardiomyogenic and endothelial differentiation potential. Decreased proliferation and metabolic activity as well as increased osteogenic differentiation of DPSCs in vitro, attributed to 3D cell encapsulation and low oxygen concentration, were also observed. DPSCs exhibiting elevated osteogenic potential may serve as potential candidates for a cell-based product for advanced therapy, particularly for bone repair. Novel tissue engineering approaches combining DPSCs, 3D biomaterial scaffolds, and other stimulating chemical factors may represent innovative strategies for pro-regenerative therapies.

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Comparison of Pluripotency, Differentiation, and Mitochondrial Metabolism Capacity in Three-Dimensional Spheroid Formation of Dental Pulp-Derived Mesenchymal Stem Cells

BioMed Research International ◽

10.1155/2021/5540877 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Young-Bum Son ◽

Dinesh Bharti ◽

Saet-Byul Kim ◽

Chan-Hee Jo ◽

Eun-Yeong Bok ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Dental Pulp ◽

Time Course ◽

Three Dimensional ◽

3D Culture ◽

Mitochondrial Metabolism ◽

Osteogenic Potential ◽

Spheroid Formation ◽

Differentiation Potential

Mesenchymal stem cells (MSCs) are valuable candidates in tissue engineering and stem cell-based therapy. Traditionally, MSCs derived from various tissues have been successfully expanded in vitro using adherent culture plates commonly called as monolayer two-dimensional (2D) cultures. Recently, many studies demonstrated that stemness and multilineage differentiation potential could be enhanced to greater extent when MSCs are cultured as suspended aggregates by means of three-dimensional (3D) culturing techniques. However, there are limited reports on changed mitochondrial metabolism on 3D spheroid formation of MSCs. Therefore, the present study was aimed at investigating the stemness, differentiation potential, and mitochondrial metabolism capacity of 3D dental pulp-derived MSC (DPSC) spheroids in comparison to monolayer cultured DPSCs. We isolated dental pulp-derived MSCs (DPSCs) and successfully developed a 3D culture system which facilitated the formation of MSC spheroids. The cell aggregation was observed after 2 hours, and spheroids were formed after 24 hours and remained in shape for 72 hours. After spheroid formation, the levels of pluripotent markers increased along with enhancement in adipogenic and osteogenic potential compared to 2D cultured control cells. However, decreased proliferative capacity, cell cycle arrest, and elevated apoptosis rate were observed with the time course of the 3D culture except for the initial 24-hour aggregation. Furthermore, oxygen consumption rates of living cells decreased with the time course of the aggregation except for the initial 24 hours. Overall, our study indicated that the short-term 3D culture of MSCs could be a suitable alternative to culture the cells.

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Association of Dental Pulp Stem Cells and Ricinus Bone Compound in a Model of Bone Defect

JOURNAL OF BIOENGINEERING AND TECHNOLOGY APPLIED TO HEALTH ◽

10.34178/jbth.v3i3.145 ◽

2020 ◽

Vol 3 (3) ◽

pp. 267-278

Author(s):

Alan Araújo de Jesus ◽

Adriano Araújo de Jesus ◽

Flávia Oliveira de Lima ◽

Luiz Antônio Rodrigues de Freitas ◽

Cássio Santana Meira ◽

...

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Bone Defect ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Autogenous Bone ◽

Control Group

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Human Dental Pulp Stem Cells Exhibit Osteogenic Differentiation Potential

Open Life Sciences ◽

10.1515/biol-2020-0023 ◽

2020 ◽

Vol 15 (1) ◽

pp. 229-236

Author(s):

Sadia Awais ◽

Samira Shabbir Balouch ◽

Nabeela Riaz ◽

Mahmood S Choudhery

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Histological Analysis ◽

Morphological Changes ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Proliferative Potential ◽

Differentiation Potential ◽

Human Dental Pulp

AbstractBone regeneration after trauma, pathologic and surgical procedures is considered a major medical challenge. Due to limitations in using conventional approaches, cell based regenerative strategies may provide an alternative option to address such issues. In the current study, we sought to determine the osteogenic potential of dental pulp stem cells (DPSCs) isolated from impacted 3rd molars. DPSCs were isolated from human dental pulp tissue (n=6) using explant culture. Growth characteristics of DPSCs were determined using plating efficiency, and the number and time of population doublings. After characterization, DPSCs were induced to differentiate into osteoblasts and were assessed using polymerase chain reactions (PCR) and histological analysis. Results indicated that DPSCs can be isolated from impacted human third molars, and that DPSCs exhibited typical fibroblastic morphology and excellent proliferative potential. In addition, morphological changes, histological analysis and expression of lineage specific genes confirmed osteogenic differentiation of DPSCs. In conclusion, DPSCs isolated from impacted 3rd molars have high proliferative potential and ability to differentiate into osteoblasts.

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Stem Cells Derived from Human Exfoliated Deciduous teeth [shed] in Neuronal Disorders: A Review

Current Stem Cell Research & Therapy ◽

10.2174/1574888x16666201221151512 ◽

2020 ◽

Vol 16 ◽

Author(s):

Minu Anoop ◽

Indrani Datta

Keyword(s):

Stem Cells ◽

Neurodegenerative Diseases ◽

Dental Pulp ◽

Therapeutic Potential ◽

Permanent Teeth ◽

Deciduous Teeth ◽

Proliferative Potential ◽

Pulp Tissue ◽

Human Exfoliated Deciduous Teeth

: Most conventional treatments for neurodegenerative diseases fail due to their focus on neuroprotection rather than neurorestoration. Stem cell‐based therapies are becoming a potential treatment option for neurodegenerative diseases as they can home in, engraft, differentiate and produce factors for CNS recovery. Stem cells derived from human dental pulp tissue differ from other sources of mesenchymal stem cells due to their embryonic neural crest origin and neurotrophic property. These include both dental pulp stem cells [DPSCs] from dental pulp tissues of human permanent teeth and stem cells from human exfoliated deciduous teeth [SHED]. SHED offer many advantages over other types of MSCs such as good proliferative potential, minimal invasive procurement, neuronal differentiation and neurotrophic capacity, and negligible ethical concerns. The therapeutic potential of SHED is attributed to the paracrine action of extracellularly released secreted factors, specifically the secretome, of which exosomes is a key component. SHED and its conditioned media can be effective in neurodegeneration through multiple mechanisms, including cell replacement, paracrine effects, angiogenesis, synaptogenesis, immunomodulation, and apoptosis inhibition, and SHED exosomes offer an ideal refined bed-to-bench formulation in neurodegenerative disorders. However, in spite of these advantages, there are still some limitations of SHED exosome therapy, such as the effectiveness of long-term storage of SHED and their exosomes, the development of a robust GMP-grade manufacturing protocol, optimization of the route of administration, and evaluation of the efficacy and safety in humans. In this review, we have addressed the isolation, collection and properties of SHED along with its therapeutic potential on in vitro and in vivo neuronal disorder models as evident from the published literature.

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Evaluation of immunophenotyping, proliferation and osteogenic differentiation potential of SSEA-4 positive stem cells derived from pulp of deciduous teeth

Archives of Oral Biology ◽

10.1016/j.archoralbio.2018.09.014 ◽

2018 ◽

Vol 96 ◽

pp. 201-207

Author(s):

Farzaneh Aghajani ◽

Somaieh Kazemnejad ◽

Tabassom Hooshmand ◽

Zahra Ghaempanah ◽

Amir-Hassan Zarnani

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Deciduous Teeth ◽

Differentiation Potential

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A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms21072280 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2280 ◽

Cited By ~ 3

Author(s):

Terezia Okajcekova ◽

Jan Strnadel ◽

Michal Pokusa ◽

Romana Zahumenska ◽

Maria Janickova ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Surface Markers ◽

Osteogenic Medium ◽

Differentiated Cells ◽

Mineralization Potential ◽

Gene And Protein Expression

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification—OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.

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