SHED Proliferation Research Articles

Fibroblast growth factor-6 enhances CDK2 and MATK expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real-time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P<0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MATK, (P<0.002 and P<0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MATK genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.

Synergistic effects of Rho kinase inhibitor Y-27632 and Noggin overexpression on the proliferation and neuron-like cell differentiation of stem cells derived from human exfoliated deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHEDs) are highly proliferative, clonogenic, and multipotent stem cells with a neural crest cell origin. This property could be a desirable option for potential therapeutic applications. In this study, we focus on the effects of Rho kinase inhibitors Y-27632 and Noggin on the proliferation of SHEDs and their differentiation into neuron-like cells. SHEDs were extracted from 10 samples of deciduous teeth obtained from healthy children aged from 5 to 10. The passaged SHEDs were transfected with Noggin, Y-27632, or their combination. By means of MTT and colony formation assays, the effects of Y-27632 and Noggin on cell viability and colony formation were detected. Cellular morphology and neurosphere formation were observed under a microscope. Y-27632 transfection in SHEDs showed enhanced cell viability, colony formation, and neurosphere formation indicating that Y-27632 could promote cell proliferation of SHEDs. Furthermore, we observed that the SHEDs treated with Noggin in combination with Y-27632 displayed typical neuron-like cell morphology and reticular processes. Noggin or Y-27632 alone or in combination induced obviously increased NSE, Nestin, and GFAP levels, which were highest in SHEDs treated with the combination of Noggin and Y-27632. These findings suggest that Y-27632 promotes the proliferation of SHEDs, and Y-27632 and Noggin in combination have a synergistic effect on promoting differentiation of SHEDs into neuron-like cells.

Does photobiomodulation change the synthesis and secretion of angiogenic proteins by different pulp cell lineages?

This study aimed to compare the synthesis and secretion of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, and FGF-2 between pulp fibroblasts from human primary teeth (HPF) and stem cell from human deciduous teeth (SHED) before and after photobiomodulation. HPF were obtained from explant technique and characterized by immunohistochemistry, while SHED were obtained from digestion technique and characterized by flow cytometry. HPF (control group) and SHED were plated, let to adhere, and put on serum starvation to synchronize the cell cycles prior to photobiomodulation. Then, both cell lineages were irradiated with 660-nm laser according to the following groups: 2.5 and 3.7 J/cm2. MTT and crystal violet assays respectively verified viability and proliferation. ELISA Multiplex Assay assessed the following proteins: VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, FGF-2, at 6, 12, and 24 h after photobiomodulation, in supernatant and lysate. Two-way ANOVA/Tukey test evaluated cell viability and proliferation, while angiogenic production and secretion values were analyzed by one-way ANOVA (P < .05). Statistically similar HPF and SHED viability and proliferation patterns occurred before and after photobiomodulation (P > .05). HPF exhibited statistically greater values of all angiogenic proteins than did SHED, at all study periods, except for FGF-2 (supernatant; 12 h); VEGFR1 (lysate; non-irradiated; 12 h); and VEGFR1 (lysate; non-irradiated; 24 h). Photobiomodulation changed the synthesis and secretion of angiogenic proteins by HPF. HPF produced and secreted greater values of all tested angiogenic proteins than did SHED before and after irradiation with both energy densities of 2.5 and 3.7 J/cm2.

Long-term effect of low-level diode laser irradiation on proliferation of stem cells from human exfoliated deciduous teeth after cryopreservation protocol

Low-level laser irradiation (LLLI) could be useful for the stimulation of stem-cell proliferation. LLLI has important physiological properties which increase the mitochondrial activity of cells. This study aimed to evaluate the biological effect of LLLI on cryopreserved stem cell from human exfoliated deciduous teeth (SHEDs). Cryopreserved and characterised SHEDs from a previous study were used for this study. The SHEDs were exposed to GaAlAs diode laser with energy densities of 1.2 (Group 2) and 2 J cm−2 (Group 3). For the control group (Group 1), SHEDs were cultured in culture medium including Dulbecco’s modified Eagle’s Medium supplemented with 15% foetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, 1% gentamycin, and amphotericin-B. The cells in the experimental groups were irradiated as previously indicated, and after the irradiation were incubated for 0, 24, 48, and 72 h at 37 °C in 5% CO2. Proliferation of SHEDs was analysed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay at incubation periods of 0, 24, 48, and 72 h after the single-laser irradiation. The effects of laser irradiation on apoptosis of SHEDs at the last incubation period (72 h) were detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method. Group 2 showed increased TUNEL-positive cells compared to Groups 1 and 3, but no significant differences between the three groups were observed (p > 0.05). Comparing Groups 2 and 3 to Group 1, the proliferation of SHEDs significantly increased after 24, 48, and 72 h (p < 0.05), but no significant differences were observed between the groups at 0 h (p > 0.05). In addition to this, at 72 h, Group 3 showed significantly higher proliferative effect on SHEDs compared to Group 2 (p > 0.05). In terms of clinical usage of long-term cryopreserved SHEDs, we can recommend to clinicians and researchers that prior to their use, LLLI may be used to increase the proliferation rate.

Human Amniotic Membrane as a Matrix for Endothelial Differentiation of VEGF-Treated Dental Stem Cells

Endothelial cells cover the surface of the capillary wall and literature review has cemented its angiogenic roles in wound healing and tissue regeneration. However, the angiogenic in vitro models available are inadequate to understand the endothelial differentiation process. A construct was made using human amniotic membrane (HAM) as a matrix to assist the dental stem cells to differentiate into endothelial-like cells. This study aimed to assess the biological interaction between stem cells from human exfoliated deciduous teeth (SHED) and the stromal side (SS) of the glycerol-preserved HAM in angiogenic-induced environment media using VEGF. The changes were evaluated through cell morphology, migration, as well as gene expression level. There were morphological changes observed in SHED in angiogenic-induced media. SHED appeared to be differentiated from fibroblast-like cells to a new structure, mimicking endothelial-like structure through microscopy analysis. Besides, the cross-section of the construct revealed that the cells seeded on the matrix were able to maintain its monolayer structure at day 1, 7 and 14 but infiltrated into the HAM at day 21, suggesting cell migration. The cells were also able to maintain its stemness (Nestin, Nanog and CD29) and at the same time express the angiogenic markers (IL-8, VEGF and MMP-2). HAM promotes SHED proliferation, migration and has the potential as a differentiating matrix for endothelial-like cells.

Fibroblast growth factor-6 enhances CDK2 and MATK expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real-time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P&lt;0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MATK, (P&lt;0.002 and P&lt;0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MATK genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.

Effect of single and multiple doses of low-level laser therapy on viability and proliferation of stem cells from human exfoliated deciduous teeth (SHED).

The present study aimed to evaluate in vitro whether the low-level laser (LLL) delivering fractionated total energy (multiple irradiation) or single irradiation stimulates regeneration-associated events (viability and proliferation) in stem cells from human exfoliated deciduous teeth (SHED). Cells received LLL irradiation (InGaAlP-660nm), according to the following experimental groups: G1 (single irradiation 2.5J/cm2, 10mW, 10s, 0.10J), G2 (single irradiation 5.0J/cm2, 10mW, 20s, 0.20J), G3 (single irradiation 7.5J/cm2, 10mW, 30s, 0.30J), G4 (twoirradiations 2.5J/cm2, 10mW, 10s; total energy 0.20J), G5 (threeirradiations 2.5J/cm2, 10mW, 10s; total energy 0.30J), and G6 (non-irradiated). Cell viability was assessed by MTT and trypan blue exclusion (TBE) methods, while cell proliferation was evaluated by crystal violet (CV) and sulforhodamine B (SRB) assays after 24, 48, and 72h after the first irradiation. By MTT, there was no difference between groups at 24 and 72h. At 48h, the groups subjected to multiple irradiation (G4 and G5) presented higher cell viability rates. The average percentages of viable cells for all groups by TBE method were 91.04%, 96.63%, and 97.48% at 24, 48, and 72h, respectively. By CV, there was no significant difference between groups at 24 and 48h; at 72h, G2, G3, and G4 presented higher cell proliferation. By SRB, G1 and G4 presented lower proliferation rates in all the periods. When the groups presenting the same total energy were compared, G2 (0.20J) presented lower cell viability rates and higher cell proliferation rates in comparison with G4; G3 (0.30J) presented similar results to those of G5, with higher cell viability and proliferation. The application of laser delivering fractionated total energy (two or three applications of 2.5J/cm2) induced higher cell viability at 48h, while the single irradiation with 2.5J/cm2 did not stimulate metabolic activity in such period and the proliferation over time. The 5.0 and 7.5J/cm2 single doses and the threeapplications of 2.5J/cm2 maintained cell viability and stimulated proliferation of SHED at 72h.

Priming Dental Pulp Stem Cells from Human Exfoliated Deciduous Teeth with Fibroblast Growth Factor-2 Enhances Mineralization Within Tissue-Engineered Constructs Implanted in Craniofacial Bone Defects.

The craniofacial area is prone to trauma or pathologies often resulting in large bone damages. One potential treatment option is the grafting of a tissue‐engineered construct seeded with adult mesenchymal stem cells (MSCs). The dental pulp appears as a relevant source of MSCs, as dental pulp stem cells display strong osteogenic properties and are efficient at bone formation and repair. Fibroblast growth factor‐2 (FGF‐2) and/or hypoxia primings were shown to boost the angiogenesis potential of dental pulp stem cells from human exfoliated deciduous teeth (SHED). Based on these findings, we hypothesized here that these primings would also improve bone formation in the context of craniofacial bone repair. We found that both hypoxic and FGF‐2 primings enhanced SHED proliferation and osteogenic differentiation into plastically compressed collagen hydrogels, with a much stronger effect observed with the FGF‐2 priming. After implantation in immunodeficient mice, the tissue‐engineered constructs seeded with FGF‐2 primed SHED mediated faster intramembranous bone formation into critical size calvarial defects than the other groups (no priming and hypoxia priming). The results of this study highlight the interest of FGF‐2 priming in tissue engineering for craniofacial bone repair. stem cells translational medicine 2019;8:844&857

Effects of graphene oxide and graphene oxide quantum dots on the osteogenic differentiation of stem cells from human exfoliated deciduous teeth

Graphene and its derivatives, graphene oxide (GO) and graphene oxide quantum dots (GOQDs), have recently attracted much attention as bioactive factors in differentiating stem cells towards osteoblastic lineage. The stem cells from human exfoliated deciduous teeth (SHEDs) possess the properties of self-renewal, extensive proliferation, and multiple differentiation potential, and have gradually become one of the most promising mesenchymal stem cells (MSCs) in bone tissue engineering. The purpose of this study was to explore the effects of GO and GOQDs on the osteogenic differentiation of SHEDs. In this study, GO and GOQDs facilitated SHED proliferation up to 7 days in vitro at the concentration of 1 μg/ml. Because of their excellent fluorescent properties, GOQD uptake by SHEDs was confirmed and distributed in the SHED cytoplasm. Calcium nodules formation, alkaline phosphatase (ALP) activity, and RNA and protein expression increased significantly in SHEDs treated with osteogenic induction medium containing GOQDs but decreased with osteogenic induction medium containing GO. Interestingly, the Wnt/β-catenin signaling pathway appeared to be involved in osteogenic differentiation of SHEDs induced with GOQDs. In summary, GO and GOQDs at the concentration of 1 μg/ml promoted SHED proliferation. GOQDs induced the osteogenic differentiation of SHEDs, whilst GO slightly inhibited it.

Fibroblast growth factor-6 enhances CDK2 and MATK expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real-time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P<0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MATK, (P<0.002 and P<0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MATK genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.

Interleukin-17A promotes osteogenic differentiation by increasing OPG/RANKL ratio in stem cells from human exfoliated deciduous teeth (SHED).

Stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for bone tissue regeneration. This study evaluated the effects of interleukin-17A (IL-17A) on the osteogenic differentiation of SHED. SHED were cultured in complete alpha minimum essential medium supplemented with osteoinducing reagents and treated with recombinant IL-17A. The cells were quantitatively analysed for proliferative activity by MTS assay, cell markers expression, and apoptotic activity by flow cytometry. For osteogenic differentiation, alkaline phosphatase (ALP) activity was quantified; mineralization assays were carried out using von Kossa and Alizarin red, and expression of osteogenic markers were analysed by real-time polymerase chain reaction and Western blot. The results showed that treatment with IL-17A increased proliferative activity in a dose-dependent manner, but reduced the expression of stem cell markers (c-Myc and Nanog) as the days progressed. IL-17A induced osteogenic differentiation in SHED as evidenced by high ALP activity, increased matrix mineralization, and upregulation of the mRNA expression of the osteogenic markers ALP, alpha 1 type 1 collagen (Col1A1), runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG) but downregulation of receptor activator of nuclear factor κB ligand (RANKL) as well as altering the OPG/RANKL ratio. Findings from our study indicate that IL-17A enhances proliferation and osteogenic differentiation of SHED by regulating OPG/RANKL mechanism thus suggests therapeutic potential of IL-17A in bone regeneration.

Fibroblast growth factor-6 enhances CDK2 and MAKT expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P&lt;0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MAKT, (P&lt;0.002 and P&lt;0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MAKT genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.

Fibroblast growth factor-6 enhances CDK2 and MAKT expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth

Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P&lt;0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MAKT, (P&lt;0.002 and P&lt;0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MAKT genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.

Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm−2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P < 0.05). At 24 h, SHED under nutritional deficit showed lower viability and proliferation after 1.2 J cm−2 irradiation. All of the irradiated groups revealed significantly higher viability and proliferation in SHED maintained under nutritional deficit than in regular nutritional conditions, except in the 3.7 and 6.2 J cm−2 groups by MTT assay. In the crystal violet assay, SHED irradiated with 1.2 J cm−2 showed no difference between the different nutritional conditions. Decrease of FBS concentration in the culture medium seems to enhance the sensitivity of SHED to the effects of photobiomodulation therapy. Nutritional stress conditions improved cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm−2.

Stem Cells from Human Extracted Deciduous Teeth Expanded in Foetal Bovine and Human Sera Express Different Paracrine Factors After Exposure to Freshly Prepared Human Serum.

The response of stem cells to paracrine factors within the host's body plays an important role in the regeneration process after transplantation. The aim of this study was to determine the viability and paracrine factor profile of stem cells from human extracted deciduous teeth (SHED) pre-cultivated in media supplemented with either foetal bovine serum (FBS) or pooled human serum (pHS) in the presence of individual human sera (iHS). SHED (n=3) from passage 4 were expanded in FBS (FBS-SHED) or pHS (pHS-SHED) supplemented media until passage 7. During expansion, the proliferation of SHED was determined. Cells at passage 7 were further expanded in human serum from four individual donors (iHS) for 120h followed by assessment of cell viability and profiling of the secreted paracrine factors. Proliferation of SHED was significantly higher (p<0.05) in pHS supplemented media compared to FBS supplemented media. pHS-SHED also maintained their higher proliferation rate compared to FBS-SHED in the presence of iHS. In iHS supplemented media, FBS-SHED expressed significantly higher levels of SDF-1A (p<0.05) after 24h compared to pHS-SHED. Similar results were found for HGF (p<0.01), LIF (p<0.05), PDGF-BB (p<0.05), SDF-1A (p<0.01), and IL-10 (p<0.05) when cell culture supernatants from FBS-SHED were profiled 120h post-incubation. SHED expanded in pHS instead of FBS have higher proliferative capacity and show an altered secretion profile. Further studies are needed to determine whether these differences could result in better engraftment and regeneration following transplantation.

Characterization of the biological effect of BiodentineTM on primary dental pulp stem cells

Biodentine™ is relatively a new tricalcium silicate cement that has gained great attention of the researchers due to its biological potential in comparison with other materials. The aim of this study was to investigate the optimum concentrations of Biodentine in relation to its stimulatory or inhibitory effect on proliferation, migration and adhesion of stem cells of human exfoliated deciduous teeth (SHED). The cell cultures of SHED were treated with Biodentine™ extract at four different concentrations; 20mg/ml, 2mg/ml, 0.2mg/ml and 0.02mg/ml. Cells cultured without Biodentine™ were kept as a blank control. The proliferation potential of SHED cells was evaluated by MTT viability analysis for 6 days. Migration potential was investigated by wound healing and transwell migration assays. The growth, survival and communication potential of these cells was determined by Adhesion assay. A significant increase was observed in the proliferation and migration of SHED at (2mg/ml, 0.2mg/ml and 0.02mg/ml) while higher concentration of Biodentine™ (20mg/ml) exhibited cytotoxic effect on the cells. However, three tested Biodentine™ concentrations were similar in effect (non-significant) to adhesion ability of cells when compared with blank control. Our findings suggest that lower concentrations of Biodentine™ can be considered as the optimum concentrations to enhance the stimulatory effect of Biodentine on SHED.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660nm, 30mW, continuous action mode) using two different energy densities (0.5J/cm2-16s; 1.0J/cm2-33s). Irradiation was performed at 0 and 48h, with the laser probe fixed at a distance of 0.5cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p<0.05). The results showed that an energy density of 1.0J/cm2 promoted an increase in cell proliferation at 48 and 72h compared to the control and 0.5J/cm2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660nm, 30mW, 1.0J/cm2) promote the proliferation of SHEDs and the maintenance of cell viability.

Biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs).

The aim is to investigate in vitro biological effects of silk fibroin 3D scaffolds on stem cells from human exfoliated deciduous teeth (SHEDs) in terms of proliferation, morphological appearance, cell viability, and expression of mesenchymal stem cell markers. Silk fibroin 3D scaffolding materials may represent promising suitable scaffolds for their application in regenerative endodontic therapy approaches. SHEDs were cultured in silk fibroin 3D scaffolds. Then, cell numbers were counted and the Alamar blue colorimetric assay was used to analyse cell proliferation after 24, 48, 72, and 168h of culture. The morphological features of SHEDs cultured on silk fibroin scaffolds were evaluated by scanning electron microscopy (SEM). Finally, cell viability and the expression of mesenchymal stem cell markers were analysed by flow cytometry. One-way analysis of variance (ANOVA) followed by a Bonferroni post-test was performed (P<0.05). At 24 and 48h of culture, SHED proliferation on scaffolds was modest compared to the control although still significant (p<0.05). However, cell proliferation progressively increased from 72 to 168h compared with the control (p<0.001; p<0.01). In addition, flow cytometry analysis showed that the culture of SHEDs on silk fibroin scaffolds did not significantly alter the level of expression of the mesenchymal markers CD73, CD90, or CD105 up to 168h; in addition, cell viability in silk fibroin was similar to than obtained in plastic. Moreover, SEM studies revealed a suitable degree of proliferation, cell spreading, and attachment, especially after 168h of culture. The findings from the current study suggest that silk fibroin 3D scaffolds had a favourable effect on the biological responses of SHEDs. Further in vivo investigations are required to confirm these results.

Biocompatibility of biological material polylactic acid with stem cells from human exfoliated deciduous teeth.

To investigate the biocompatibility of the biomaterial, polylactic acid (PLA) with stem cells from human exfoliated deciduous teeth (SHED) and its induction of mineralization as a type of scaffold material. To determine the impacts of biomaterial PLA on proliferation and mineralization of SHED, the expression of surface molecules of SHED isolated and cultured in vitro was detected by flow cytometry. In addition, cell proliferation was measured using MTT and Edu assays, and the evaluation of mineralized differentiation was performed using Alizarin Red S staining. In addition, the expression levels of osteogenic marker genes were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. SHED were successfully isolated and identified. The MTT and Edu results indicated that the proliferation of SHED cultured in PLA and normal medium was not significantly different. The Alizarin Red S staining demonstrated that the mineralization capability was significantly higher in the SHED that were cultured in PLA medium. Furthermore, RT-qPCR and western blot analyses indicated that the expression levels of osteogenic marker genes were higher in the SHED cultured in PLA medium. These results suggested that PLA possesses good biocompatibility with SHED and may effectively induce the mineralization of SHED and serve as a scaffold material.

Pooled Human Serum Increases Regenerative Potential of In Vitro Expanded Stem Cells from Human Extracted Deciduous Teeth.

In regenerative therapy, in vitro expansion of stem cells is critical to obtain a significantly higher number of cells for successful engraftment after transplantation. However, stem cells lose its regenerative potential and enter senescence during in vitro expansion. In this study, the influence of foetal bovine serum (FBS) and pooled human serum (pHS) on the proliferation, morphology and migration of stem cells from human extracted deciduous teeth (SHED) was compared. SHED (n=3) was expanded in KnockOut DMEM supplemented with either pHS (pHS-SM) or FBS (FBS-SM). pHS was prepared using peripheral blood serum of six healthy male adults, aged between 21 and 35years old. The number of live SHED was significantly higher, from passage 5 to 7, when cultured in pHS-SM compared to those cultured in FBS-SM (p<0.05). Number of cells having flattened morphology, characteristics of partially differentiated and senescent cells, was significantly lower (p<0.05) in pHS-SM (3%) compared to those in FBS-SM (7%). Furthermore, migration of SHED in pHS-SM was found to be more directional. The presence of selected ten paracrine factors known for their proliferation and migration potential was detected in all six individual human sera, used to produce pHS, none of which were detected in FBS. Ingenuity Pathway Analysis showed the possible involvement of the 'ephrin receptor signalling pathway' to regulate the proliferation and migration of SHED in pHS-SM. In conclusion, pHS-SM showed significantly higher proliferation rate and could maintain significantly lower number of senescent cells and support directional migration of cells.