Analysis of growth kinetics and colony-forming unit efficiency of human dental pulp stem cells cultured in different media and supplements

Dental tissues are considered as ideal autologous sources of multipotent stem cells. Presently, human dental pulp stem cells (DPSCs) are largely being isolated and expanded in media containing fetal bovine serum (FBS). However, the use of FBS has limitations due to its animal origin. Therefore, the present study evaluated the morphology, proliferation rate, population doubling time (PDT) and colony-forming unit fibroblast (CFU-F) efficiency of DPSCs cultured in animal serum-containing medium (SCM) and serumfree medium (SFM) in addition to serum-free culture conditions by supplementing human blood-derivatives such as platelet lysate (PL), fresh frozen plasma (FFP) and umbilical cord blood serum (UCS) at 2.5%, 5% and 7.5% concentrations. Established DPSCs had spindle-shape during primary culture but acquired characteristic fibroblast-like features when cultured in PL, FFP and UCS. DPSCs in SCM, SFM and PL had significantly (P<0.05) higher proliferative potential than those in UCS and FFP and these observations were supported by PDT values. The CFU efficiency of DPSCs was confirmed in all culture conditions with a slightly varied clonogenic potential in blood-derived components. Based on the growth kinetics and CFU ability, it is concluded that PL could be considered as a suitable alternative to FBS for the ex vivo expansion of DPSCs.

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Cellular Properties and Expression of Pluripotent Markers in Human Dental Pulp Stem Cells Cultured in Serum-Free Medium

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48893.14864 ◽

2021 ◽

Author(s):

Chethan Kumar ◽

Basan Gowda Sharanappa Kurkalli ◽

Shishir Shetty ◽

Akshay Bairapura Manjappa ◽

Veena Shetty ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Population Doubling ◽

Animal Origin ◽

Serum Free Medium ◽

Free Medium ◽

Serum Free ◽

Alp Activity ◽

Human Dental Pulp

Introduction: The standard isolation and expansion of human Dental Pulp Stem Cells (DPSCs) under invitro conditions normally involve the usage of Fetal Bovine Serum (FBS). However, its animal-origin poses possible concerns for clinically relevant procedures. This critical issue compels the use of Xenogeneic-Free (XF) or human-origin alternatives to FBS for culture expansion and differentiation of DPSCs to determine the usefulness for translating into therapeutic clinical applications. Aim: To evaluate the cellular characteristics and expression of pluripotent markers in DPSCs cultured using Serum-Containing Medium (SCM-DPSCs) and Serum-Free Medium (SFM-DPSCs). Materials and Methods: This in-vitro descriptive study was conducted at NITTE (Deemed to be University), Mangaluru, Karnataka, India, from June 2019 to August 2020. DPSCs were isolated from impacted third molars. The culture expanded DPSCs in serum-containing and serum-free media were analysed on their morphology, viability, proliferation rate, Population Doubling Time (PDT), Alkaline Phosphatase (ALP) activity, cell surface markers expression, osteogenic and adipogenic potential, and the relative expression of selected pluripotent genes. Results: The primary culture of DPSCs established in SCM and SFM showed spindle shaped fibroblastic morphology with >80% viability from passage 1 (P1) to P4. A significant (p-value<0.05) difference in the proliferation rates in terms of cell numbers between SCM-DPSCs and SFM-DPSCs was observed (day 6: 3×105 vs 0.8×105; day 9: 5.8×105 vs 1.27×105; day 12: 7.8×105vs 1.56×105, respectively). The average PDT values recorded in SCM- and SFM-DPSCs were 44.33 hours and 58.41 hours, respectively. A slightly higher expression of ALP activity was observed in SCM-DPSCs than in SFM-DPSCs. Flow cytometry analysis showed that both DPSCs were positive for CD29, CD73, CD90, and negative for CD34 and CD45. The expression of OCT4 and NANOG was relatively higher in SCM-DPSCs compared to SFM-DPSCs. Further, SCM-DPSCs showed the higher levels of SOX2 and SSEA4, but did not exhibit any significant differences in their expression levels. Conclusion: The results showed that DPSCs in FBS displayed better growth kinetics and stemness markers expression along with more propensities towards lineage differentiation. SFM can be used to establish and expand DPSCs with characteristics of multipotent stem cells, but needs further research for its optimisation.

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Neurogenic Differentiation of Human Dental Pulp Stem Cells on Graphene-Polycaprolactone Hybrid Nanofibers

Nanomaterials ◽

10.3390/nano8070554 ◽

2018 ◽

Vol 8 (7) ◽

pp. 554 ◽

Cited By ~ 8

Author(s):

Hoon Seonwoo ◽

Kyung-Je Jang ◽

Dohyeon Lee ◽

Sunho Park ◽

Myungchul Lee ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Morphological Characteristics ◽

Electrospun Nanofibers ◽

Dental Pulp Stem Cells ◽

Neurogenic Differentiation ◽

Dental Tissues ◽

Differentiated Cells ◽

Human Dental Pulp

Stem cells derived from dental tissues—dental stem cells—are favored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages, such as high proliferation and a highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here, reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties, such as influence on the cellular alignments and cell viability of DPSCs, were also analyzed. The influences of NFs on DPSCs neurogenesis were also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated an aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.

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Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Journal of Applied Oral Science ◽

10.1590/1678-7757-2021-0296 ◽

2021 ◽

Vol 29 ◽

Author(s):

Thanasup GONMANEE ◽

Tawepong ARAYAPISIT ◽

Kutkao VONGSAVAN ◽

Chareerut PHRUKSANIYOM ◽

Hathaitip SRITANAUDOMCHAI

Keyword(s):

Stem Cells ◽

Neuronal Differentiation ◽

Dental Pulp ◽

Culture Conditions ◽

Dental Pulp Stem Cells ◽

Human Dental Pulp

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Bioprinting of three-dimensional dentin–pulp complex with local differentiation of human dental pulp stem cells

Journal of Tissue Engineering ◽

10.1177/2041731419845849 ◽

2019 ◽

Vol 10 ◽

pp. 204173141984584 ◽

Cited By ~ 14

Author(s):

Jonghyeuk Han ◽

Da Sol Kim ◽

Ho Jang ◽

Hyung-Ryong Kim ◽

Hyun-Wook Kang

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Dental Pulp ◽

Three Dimensional ◽

Dental Pulp Stem Cells ◽

Patient Specific ◽

Fibrinogen Concentration ◽

Dental Tissues ◽

Human Dental Pulp ◽

Dental Pulp Stem Cell

Numerous approaches have been introduced to regenerate artificial dental tissues. However, conventional approaches are limited when producing a construct with three-dimensional patient-specific shapes and compositions of heterogeneous dental tissue. In this research, bioprinting technology was applied to produce a three-dimensional dentin–pulp complex with patient-specific shapes by inducing localized differentiation of human dental pulp stem cells within a single structure. A fibrin-based bio-ink was designed for bioprinting with the human dental pulp stem cells. The effects of fibrinogen concentration within the bio-ink were investigated in terms of printability, human dental pulp stem cell compatibility, and differentiation. The results show that micro-patterns with human dental pulp stem cells could be achieved with more than 88% viability. Its odontogenic differentiation was also regulated according to the fibrinogen concentration. Based on these results, a dentin–pulp complex having patient-specific shape was produced by co-printing the human dental pulp stem cell–laden bio-inks with polycaprolactone, which is a bio-thermoplastic used for producing the overall shape. After culturing with differentiation medium for 15 days, localized differentiation of human dental pulp stem cells in the outer region of the three-dimensional cellular construct was successfully achieved with localized mineralization. This result demonstrates the possibility to produce patient-specific composite tissues for tooth tissue engineering using three-dimensional bioprinting technology.

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Neurogenic Differentiation of Human Dental Pulp Stem Cells on Graphene-Polycaprolactone Hybrid Nanofibers

10.20944/preprints201806.0452.v1 ◽

2018 ◽

Author(s):

Hoon Seonwoo ◽

Kyung-Je Jang ◽

Dohyeon Lee ◽

Sunho Park ◽

Myungchul Lee ◽

...

Keyword(s):

Stem Cells ◽

Dental Pulp ◽

Morphological Characteristics ◽

Electrospun Nanofibers ◽

Dental Pulp Stem Cells ◽

Neurogenic Differentiation ◽

Dental Tissues ◽

Differentiated Cells ◽

Human Dental Pulp

Stem cells derived from dental tissues—dental stem cells—are flavored due to their easy acquisition. Among them, dental pulp stem cells (DPSCs) extracted from the dental pulp have many advantages such as high proliferation and highly purified population. Although their ability for neurogenic differentiation has been highlighted and neurogenic differentiation using electrospun nanofibers (NFs) has been performed, graphene-incorporated NFs have never been applied for DPSC neurogenic differentiation. Here reduced graphene oxide (RGO)-polycaprolactone (PCL) hybrid electrospun NFs were developed and applied for enhanced neurogenesis of DPSCs. First, RGO-PCL NFs were fabricated by electrospinning with incorporation of RGO and alignments, and their chemical and morphological characteristics were evaluated. Furthermore, in vitro NF properties such as influence on the cellular alignments and cell viability of DPSCs were also analyzed. The influences of NFs on DPSCs neurogenesis was also analyzed. The results confirmed that an appropriate concentration of RGO promoted better DPSC neurogenesis. Furthermore, the use of random NFs facilitated contiguous junctions of differentiated cells, whereas the use of aligned NFs facilitated aligned junction of differentiated cells along the direction of NF alignments. Our findings showed that RGO-PCL NFs can be a useful tool for DPSC neurogenesis, which will help regeneration in neurodegenerative and neurodefective diseases.

Download Full-text