Angiogenic effect of platelet-rich concentrates on dental pulp stem cells in inflamed microenvironment.

OBJECTIVE: In this study, we aimed to determine the suitable concentrations of human platelet lysate (HPL) and platelet-rich plasma (PRP) for maintaining the in vitro proliferative and angiogenic potential of inflamed dental pulp stem cells. MATERIALS AND METHODS: Lipopolysaccharide (LPS)-induced inflamed dental pulp-derived stem cells (iDPSCs) were treated with different concentrations of HPL and PRP (10% and 20%) followed by determination of viability using Alamar Blue assay. Expression of angiogenesis-, adhesion-, and inflammation-regulating genes was also analyzed using RT-qPCR array. Furthermore, expression of growth factors at protein level in the cell culture microenvironment was measured using multiplex assay. RESULTS: Viability of iDPSCs was significantly (p < 0.05) higher in 20% HPL-supplemented media compared to iDPSCs. Expression of 10 out of 12 selected angiogenic genes, four out of seven adhesion molecules, and seven out of nine cytokine-producing genes were significantly (p < 0.05) higher in cells maintained in 20% HPL-supplemented media compared to that in FBS-supplemented media. Furthermore, expression of all the selected growth factors was significantly higher (p < 0.05) in the supernatants from 20% HPL media at 12 and 24 h post-incubation. CONCLUSION: This study suggests that 20% HPL could be optimum to stimulate angiogenesis-related factors in iDPSCs while maintaining their viability. CLINICAL RELEVANCE: This data may suggest the potential use of 20% HPL for expanding DPSCs scheduled for clinical trials for regenerative therapies including dental pulp regeneration.

Immune responses of human dental pulp stem cells in lipopolysaccharide-induced microenvironment.

This study aimed to investigate the effect of inflammatory stimuli on dental pulp stem cells (DPSCs) by assessing their proliferation and expression of genes as well as proteins in lipopolysaccharide (LPS)-induced microenvironment (iDPSCs). DPSCs were first characterized for their mesenchymal properties prior to challenging them with a series of LPS concentrations from 12 to 72 h. Following to this, their proliferation and inflammatory based genes as well as protein expression were assessed. iDPSCs had demonstrated significant expression of mesenchymal markers. Upon exposure to LPS, the viability dropped distinctly with increasing concentration, as compared to control (P < 0.05). The expression of pro-inflammatory genes such as interleukin 6, interleukin 8 were augmented with exposure to LPS (P < 0.05). Similarly, cytokines like tumour necrosis factor (TNF) α and interleukin 1α had increased in dose dependant manner upon LPS exposure (P < 0.05). Our results suggest that LPS concentration between 1 and 2 µg/mL demonstrated inflammation induction in DPSCs that may simulate inflamed microenvironment of dental pulp in clinical scenario. Thus, optimizing iDPSCs secretome profile could be a promising approach to test various regenerative protocols in inflamed microenvironment.

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells.

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Expression patterns of immune genes in long-term cultured dental stem cells.

BACKGROUND AND OBJECTIVES: Long-term culture system is used to prevent the impediment of insufficient cells and is good for low starting materials such as dental pulp or periodontal ligament. In general, although cell viability and functionality are the most common aspects taken into consideration in culturing cells for a long term, they may not truly represent the biological state of the cells. Hence, we explored the behaviour of another important aspect which is the immune properties in long-term cultured cells. METHODS: Dental pulp stem cells from deciduous (SHED; n = 3) and permanent (DPSCs; n = 3) teeth as well as periodontal ligament stem cells (PDLSCs; n = 3) were cultured under identical culture condition. The immune properties of each cell lines were profiled at passage 2 [P2] and passage 9 [P9] as early and late passages, respectively. This was further validated at the protein level using the Luminex platform. RESULTS: A major shift of genes was noticed at P9 with SHED being the highest. SHED cultured at P9 displayed many genes representing pathogen recognition (P < 0.001), immune signalling (P < 0.001, pro-inflammatory (P < 0.001), anti-inflammatory (P < 0.001) and immune-related growth and stimulation factor (P < 0.001) as compared to DPSCs and PDLSCs. Surprisingly, SHED also expressed many cytotoxicity genes (P < 0.001). CONCLUSIONS: Communally, instabilities of immune genes from our findings suggest that long-term cultured cells may not be feasible for transplantation purposes. CLINICAL RELEVANCE: A complete biological characterization covering all major aspects including immune properties should be made as prerequisite criteria prior to the use of long-term cultured stem cells in clinical settings.

Advancing stem cell therapy from bench to bedside: lessons from drug therapies.

The inadequacy of existing therapeutic tools together with the paucity of organ donors have always led medical researchers to innovate the current treatment methods or to discover new ways to cure disease. Emergence of cell-based therapies has provided a new framework through which it has given the human world a new hope. Though relatively a new concept, the pace of advancement clearly reveals the significant role that stem cells will ultimately play in the near future. However, there are numerous uncertainties that are prevailing against the present setting of clinical trials related to stem cells: like the best route of cell administration, appropriate dosage, duration and several other applications. A better knowledge of these factors can substantially improve the effectiveness of disease cure or organ repair using this latest therapeutic tool. From a certain perspective, it could be argued that by considering certain proven clinical concepts and experience from synthetic drug system, we could improve the overall efficacy of cell-based therapies. In the past, studies on synthetic drug therapies and their clinical trials have shown that all the aforementioned factors have critical ascendancy over its therapeutic outcomes. Therefore, based on the knowledge gained from synthetic drug delivery systems, we hypothesize that by employing many of the clinical approaches from synthetic drug therapies to this new regenerative therapeutic tool, the efficacy of stem cell-based therapies can also be improved.

Different isolation methods alter the gene expression profiling of adipose derived stem cells.

Human adipose stem cells (ASCs) has been in the limelight since its discovery as a suitable source of mesenchymal stem cells (MSCs) in regenerative medicine. Currently, two major techniques are used to isolate ASCs, namely liposuction and tissue biopsy. These two methods are relatively risk-free but the question as to which method could give a more efficient output remains unclear. Thus, this study was carried out to compare and contrast the output generated in regards to growth kinetics, differentiation capabilities in vitro, and gene expression profiling. It was found that ASCs from both isolation methods were comparable in terms of growth kinetics and tri-lineage differentiation. Furthermore, ASCs from both populations were reported as CD44(+), CD73(+), CD90(+), CD166(+), CD34(-), CD45(-) and HLA-DR(-). However, in regards to gene expression, a group of overlapping genes as well as distinct genes were observed. Distinct gene expressions indicated that ASCs (liposuction) has endoderm lineage propensity whereas ASCs (biopsy) has a tendency towards mesoderm/ectoderm lineage. This information suggests involvement in different functional activity in accordance to isolation method. In conclusion, future studies to better understand these gene functions should be carried out in order to contribute in the applicability of each respective cells in regenerative therapy.

Evaluation of the marginal fit of a CAD/CAM zirconia-based ceramic crown system.

PURPOSE: To evaluate the marginal fit of zirconia (Zi) CAD/CAM crowns in terms of gap and overhang compared to lithium disilicate (LDS) computer-aided design crowns, as well as the effect of finish line design on marginal accuracy. MATERIALS AND METHODS: Stone dies were acquired from two master metal dies (n = 20 each) with two different finish lines and were scanned to produce digital models. Ceramic crowns (ZS-Ronde Zi, KaVo and IPS e.max CAD LDS, Ivoclar Vivadent) were designed and milled on the resulting 40 dies: 10 Zi-shoulder, 10 Zi-chamfer, 10 LDSshoulder, and 10 LDS-chamfer. Marginal gap and overhang were evaluated at six designated margin locations. The data were obtained, and the influence of material and finish line on the marginal fit of crowns was assessed using two-way analysis of variance and Bonferroni multiple comparisons test (α = .05). RESULTS: Mean marginal gap and overhang on Zi crowns were 30 ± 14 µm and 79 ± 27 µm for the shoulder, respectively, and were 68 ± 34 µm and 104 ± 34 µm for the chamfer. The corresponding values for LDS crowns were 57 ± 22 µm and 74 ± 29 µm for the shoulder, and 62 ± 12 µm and 59 ± 27 µm for the chamfer. ANOVA revealed that the differences in marginal gap between the two materials were not significant (P > .05), but that the finish line effect and interaction were significant (P < .05). With regard to marginal overhang, significant differences were found between Zi and LDS crowns (P < .05), although the finish line geometries did not show any significant differences (P > .05). LDS crowns showed no differences between shoulder and chamfer margins for gap or overhang (P > .05), whereas significant differences were found in marginal gap between the Zi shoulder and chamfer margins (P lt; .005). CONCLUSION: In terms of marginal accuracy, shoulder margins produced smaller marginal gaps compared to chamfers for Zi CAD/CAM crowns.

Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications.

BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

Correction to: MicroRNAomic Transcriptomic Analysis Reveal Deregulation of Clustered Cellular Functions in Human Mesenchymal Stem Cells During in Vitro Passaging.

The original version of this article unfortunately contained a mistake.

Amenable epigenetic traits of dental pulp stem cells underlie high capability of xeno-free episomal reprogramming.

BACKGROUND: While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming. METHODS: Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13-18 days). RESULTS: The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation. CONCLUSION: In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.

Autologous serum supplement favours in vitro regenerative paracrine factors synthesis.

OBJECTIVES: Foetal bovine serum (FBS) is often the serum supplement of choice for in vitro human cell culture. This study compares the effect of FBS and autologous human serum (AuHS) supplement in human peripheral blood mononuclear cell (PBMC) culture to prepare secretome. MATERIALS AND METHODS: The PBMC (n = 7) were cultured either in RPMI-1640 containing L-glutamine and 50 units/ml Penicillin-Streptomycin (BM) or in BM with either AuHS or FBS. Viability, proliferation and differentiation of PBMC were evaluated. Paracrine factors present in the secretomes (n = 6) were analysed using ProcartaPlex Human Cytokine panel (17 plex). Ingenuity Pathway Analysis (IPA) was performed to predict activation or inhibition of biological functions related to tissue regeneration. RESULTS: The viability of PBMC that were cultured with FBS supplement was significantly reduced at 96 h compared to those at 0 and 24 h (P < .05). While the reduction of the viability of PBMC that were cultured with AuHS supplement was not significantly different compared to those at 0 and 24 h. The FBS secretomes prepared at 24 h was found to contain significantly higher amount of EGF (P < .05) compared to that in AuHS or BM secretome. The AuHS secretomes contained significantly higher amount of HGF at 24 (P < .05) and 96 h (P < .01), and VEGF-A at 24 h (P < .05) compared to those in the FBS secretomes. SDF-1 was not detected in the FBS secretomes prepared at either 24 or 96 hours. Double immunocytochemical staining revealed a marked increase in co-localization of SDF-1 and its receptor in PBMC that were cultured with AuHS supplement compared to that cultured with FBS supplement. CONCLUSION: In secretome preparation, AuHS supplement favours synthesis of paracrine factors that are needed for regenerative therapy.

Neuro-fuzzy method for predicting the viability of stem cells treated at different time-concentration conditions.

Dental stem cells isolated for human dental pulp are an excellent source for regenerative medicine and dentistry. Simulation of clinical scenario is one of the crucial challenges for evaluation of the efficacy of DPSCs in various regenerative therapies. In this study we evaluated the viability of DPSCs after treatment with artificial bacterial lipopolysaccharides (LPS) as the main component responsible for inducing inflammatory response in majority of the inflammatory conditions in clinical scenario. Although a number of studies have previously treated stem cells with LPS from bacteria, however the accuracy level of the outcome was not established. Here we have analyzed the outcome using adaptive neuro-fuzzy inferences system (ANFIS) to predict the viability of human DPSCs after treatment with bacterial LPS.

Nanomechanical properties, wear resistance and in-vitro characterization of Ta2O5 nanotubes coating on biomedical grade Ti-6Al-4V.

Tantalum pentoxide nanotubes (Ta2O5 NTs) can dramatically raise the biological functions of different kinds of cells, thus have promising applications in biomedical fields. In this study, Ta2O5 NTs were prepared on biomedical grade Ti-6Al-4V alloy (Ti64) via physical vapor deposition (PVD) and a successive two-step anodization in H2SO4: HF (99:1)+5% EG electrolyte at a constant potential of 15V. To improve the adhesion of nanotubular array coating on Ti64, heat treatment was carried out at 450°C for 1h under atmospheric pressure with a heating/cooling rate of 1°Cmin-1. The surface topography and composition of the nanostructured coatings were examined by atomic force microscopy (AFM) and X-ray electron spectroscopy (XPS), to gather information about the corrosion behavior, wear resistance and bioactivity in simulated body fluids (SBF). From the nanoindentation experiments, the Young's modulus and hardness of the 5min anodized sample were ~ 135 and 6GPa, but increased to ~ 160 and 7.5GPa, respectively, after annealing at 450°C. It was shown that the corrosion resistance of Ti64 plates with nanotubular surface modification was higher than that of the bare substrate, where the 450°C annealed specimen revealed the highest corrosion protection efficiency (99%). Results from the SBF tests showed that a bone-like apatite layer was formed on nanotubular array coating, as early as the first day of immersion in simulated body fluid (SBF), indicating the importance of nanotubular configuration on the in-vitro bioactivity.

Self-assembly programming of DNA polyominoes.

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously.

Optimization of pre-transplantation conditions to enhance the efficacy of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are considered a potential tool for cell based regenerative therapy due to their immunomodulatory property, differentiation potentials, trophic activity as well as large donor pool. Poor engraftment and short term survival of transplanted MSCs are recognized as major limitations which were linked to early cellular ageing, loss of chemokine markers during ex vivo expansion, and hyper-immunogenicity to xeno-contaminated MSCs. These problems can be minimized by ex vivo expansion of MSCs in hypoxic culture condition using well defined or xeno-free media i.e., media supplemented with growth factors, human serum or platelet lysate. In addition to ex vivo expansion in hypoxic culture condition using well defined media, this review article describes the potentials of transient adaptation of expanded MSCs in autologous serum supplemented medium prior to transplantation for long term regenerative benefits. Such transient adaptation in autologous serum supplemented medium may help to increase chemokine receptor expression and tissue specific differentiation of ex vivo expanded MSCs, thus would provide long term regenerative benefits.

Triggering Mechanisms of Thermosensitive Nanoparticles Under Hyperthermia Condition.

Nanoparticle-based hyperthermia is an effective therapeutic approach that allows time- and site-specific treatment with minimized off-site effects. The recent advances in materials science have led to design a diversity of thermosensitive nanostructures that exhibit different mechanisms of thermal response to the external stimuli. This article aims to provide an extensive review of the various triggering mechanisms in the nanostructures used as adjuvants to hyperthermia modalities. Understanding the differences between various mechanisms of thermal response in these nanostructures could help researchers in the selection of appropriate materials for each experimental and clinical condition as well as to address the current shortcomings of these mechanisms with improved material design.

Global search for right cell type as a treatment modality for cardiovascular disease.

INTRODUCTION: Acute myocardial infarction is the primary cause of heart disease-related death in the world. Reperfusion therapy is currently the backbone of treatment for acute myocardial infarction albeit with many limitations. With the emergence of stem cells as potential therapeutic agents, attempts in using them to enhance cardiac function have increased exponentially. However, it has its own disadvantages, and we postulate that the primary drawback is choosing the right cell type and solving this may significantly contribute to ambitious goal of using stem cells in the regeneration medicine. AREAS COVERED: This article discusses several types of stem cells that have been proven to be likely candidates for treating cardiovascular diseases and their uses up to date, focusing on their biological characterization and potential usage in preclinical and clinical work. EXPERT OPINION: The research on cell therapies for cardiovascular disease is promising, but there is still much uncertainty surrounding the efficacy of these cells in clinical settings. With a wide range of cells available as potential treatment for cardiovascular diseases, one should avoid from being overzealous and extrapolating when reporting their data. Future studies should focus more in understanding the biological functions of the available cells lines.

Finite element analysis of zygomatic implants in intrasinus and extramaxillary approaches for prosthetic rehabilitation in severely atrophic maxillae.

PURPOSE: To compare the extramaxillary approach with the widely used intrasinus approach via finite element method. MATERIALS AND METHODS: A unilateral three-dimensional model of the craniofacial area surrounding the region of interest was developed using computed tomography image datasets. The zygomatic implants were modeled using three-dimensional computer-aided design software and virtually placed according to the described techniques together with one conventional implant and a prosthesis. The bone was assumed to be linear isotropic with a stiffness of 13.4 GPa, while the implants were of titanium alloy with a stiffness of 110 GPa. Masseter forces were applied at the zygomatic arch, and occlusal loads were applied to the surface of the prosthesis. The stresses and displacements generated on the surrounding bone and within the implant due to the simulated loading configuration were analyzed. RESULTS: The bone-implant interface and zygomatic implant body for the intrasinus approach produced 1.41- and 4.27-fold higher stress, respectively, compared with the extramaxillary approach under vertical loading. However, under lateral loading, the extramaxillary approach generated 2.48-fold higher stress than the intrasinus at the bone-implant interface. The zygomatic implant in the extramaxillary approach had twofold higher micromotion than those with intrasinus approach under lateral loading. CONCLUSIONS: No one technique was found to be superior; however, if lateral loading is used, the intrasinus approach is the most favorable for the rehabilitation of severely atrophic maxillae.