Modulation of the differentiation of dental pulp stem cells by different concentrations of β-glycerophosphate

Dentinogenesis is a necessary prerequisite for dental tissue engineering. One of the steps for dentinogenesis is to obtain large quantities of highly purified odontoblasts. Therefore, we have undertaken an experiment applying different concentrations of β-glycerophosphate (β-GP) to induce the differentiation of dental pulp stem cells (DPSCs) in a long-term 28-day culture. In the meanwhile, we have studied the time- and maturation-dependent expression of matrix extracellular phosphoglycoprotein (MEPE) and that of the odontoblast-like marker-dentin sialoprotein (DSP), in order to investigate an optimized mineralized condition. Western blot results revealed that the expression of DSP became lower when accompanied by the increase of the β-GP concentration, and there was also an influence on MEPE expression when different concentrations of β-GP were applied. Meanwhile, the mineralized groups had an inhibitory function on the expression of MEPE as compared with the control group. Above all, all experimental groups successfully generated mineralized nodules by Alizarin Red S and the 5 mM β-GP group formed more mineralized nodules quantitated using the CPC extraction method. In conclusion, there is a significant modulation of the β-GP during the differentiation of the DPSCs. The degree of odontoblast differentiation is β-glycerophosphate concentration dependent. A low concentration of β-GP (5 mM) has been shown to be the optimal concentration for stimulating the maturation of the DPSCs. Moreover, MEPE accompanied with DSP clearly demonstrates the degree of the differentiation.     

Raman Micro-Spectroscopy of Dental Pulp Stem Cells: An Approach to Monitor the Effects of Cone Beam Computed Tomography Low-Dose Ionizing Radiation

The objective of this study was to determine the molecular and biochemical changes in dental pulp stem cells (DPSCs) due to consecutive low-dose ionizing radiation exposures using label-free Raman micro-spectroscopy (RMS). Ionizing radiation produces biological damage leading to health effects of varying severity. The effects and subsequent health implications caused by exposure to low-dose radiation, such as diagnostic exposure, remain ambiguous. We identified Raman biomarkers characteristic to low-dose cone beam computed tomography (CBCT) irradiation of the DPSCs. The biomarkers were monitored inside the cells using the relative intensity distribution of the 785 and 1734?cm^?1 bands. The control cells presented a higher relative intensity of the nucleic acid specific Raman bands, whereas the irradiated cells revealed an increased intensity of the lipid-induced bands. The results obtained in this study demonstrate the capability of RMS for the detection of cell response to diagnostic radiation dose levels. This may indicate the potential of the technique for future applications such as monitoring the radiation responses in pediatric patients suffering repeated radiological exposures.     

Electrospun poly(l‐lactide) nanofibers coated with mineral trioxide aggregate enhance odontogenic differentiation of dental pulp stem cells

A combination of bioceramics and nanofibrous scaffolds holds promising potential for inducing of mineralization in connective tissues. The aim of the present study was to investigate the attachment, proliferation and odontogenic differentiation of dental pulp stem cells (DPSC) on poly(l ‐lactide) (PLLA) nanofibers coated with mineral trioxide aggregate (MTA). Polymeric scaffolds were fabricated via the electrospinning method and their surface was coated with MTA. DPSC were isolated from dental pulp and their biological behavior was evaluated on scaffolds and the control group using MTT assay. Alkaline phosphatase (ALP) activity, biomineralization and the expression of odontogenic genes were analyzed during odontogenic differentiation. Isolated DPSC showed spindle‐shaped morphology with multi‐lineage differentiation potential and were positive for CD73, CD90 and CD105. MTA‐coated PLLA (PLLA/MTA) exhibited nanofibrous structure with average fiber diameter of 756 ± 157 nm and interconnected pores and also suitable mechanical properties. Similar to MTA, these scaffolds were shown to be biocompatible and to support the attachment and proliferation of DPSC. ALP activity transiently peaked on day 14 and was significantly higher in PLLA/MTA scaffolds than in the control groups. In addition, increasing biomineralization was observed in all groups with a higher amount in PLLA/MTA. Odontogenic‐related genes, DSPP and collagen type I showed a higher expression in PLLA/MTA on days 21 and 14, respectively. Taken together, MTA/PLLA electrospun nanofibers enhanced the odontogenic differentiation of DPSC and showed the desired characteristics of a pulp capping material.     

Therapeutic Potential of Dental Pulp Stem Cells According to Different Transplant Types

Stem cells are unspecialised cells capable of perpetual self-renewal, proliferation and differentiation into more specialised daughter cells. They are present in many tissues and organs, including the stomatognathic system. Recently, the great interest of scientists in obtaining stem cells from human teeth is due to their easy availability and a non-invasive procedure of collecting the material. Three key components are required for tissue regeneration: stem cells, appropriate scaffold material and growth factors. Depending on the source of the new tissue or organ, there are several types of transplants. In this review, the following division into four transplant types is applied due to genetic differences between the donor and the recipient: xenotransplantation, allotransplantation, autotransplantation and isotransplantation (however, due to the lack of research, type was not included). In vivo studies have shown that Dental Pulp Stem Cells (DPSCs)can form a dentin-pulp complex, nerves, adipose, bone, cartilage, skin, blood vessels and myocardium, which gives hope for their use in various biomedical areas, such as immunotherapy and regenerative therapy. This review presents the current in vivo research and advances to provide new biological insights and therapeutic possibilities of using DPSCs.     

Effect of extracts from safflower seeds on osteoblast differentiation and intracellular calcium ion concentration in MC3T3-E1 cells

Although safflower seeds have long been used in Korea as herbal medicines, very little research has been published on the effects of safflower seed on bone formation or bone density. The study reported here therefore examined bone nodule formation, calcium uptake, alkaline phosphatase activity, and intracellular concentration of calcium ion [Ca(2+)](i) in murine osteoblastic cells of the MC3T3-E1 line that were cultured on modified Eagle's minimal essential medium alone (controls) or with addition of 0.1% crude extract of safflower seed (experimental group I) or 0.1% aqueous fraction of safflower seed (experimental group II). Fluorescence spectrometry measurement of ([Ca(2+)](i)) showed significantly accelerated rates of osteoblast differentiation in experimental group I (3 microL of crude extract in 8 x 10(4) cells) and experimental group II (2 microL of aqueous fraction in 8 x 10(4) cells) compared to the control group.     

ILs and MMPs Levels in Inflamed Human Dental Pulp: A Systematic Review

A wide range of mediators are released from the pulp tissue because of bacterial invasion which causes inflammation. Interleukins (ILs) and matrix metalloproteinases (MMPs) have a leading role in initiating and spreading of inflammation because of their synergic action. Biomarkers such as ILs and MMPs can be identified via several methods, establishing the inflammatory response of the dental pulp. The aim of this systematic review is to evaluate the levels of ILs and/or MMPs in human dental pulp. PubMed, OVID, Cochrane, Scopus, Web of Science and Wiley online library databases were searched for original clinical studies. After applying inclusion and exclusion criteria, a quality assessment of studies was performed based on a modified Newcastle-Ottawa scale. In the review were included articles that evaluated the presence of ILs and/or MMPs in pulp tissue using enzyme-linked immunosorbent assay (ELISA) or western blot or multiplex assay. Six articles were included in the present synthesis. Although various diagnostic methods were used, statistically significant higher levels of ILs and/or MMPs were mostly found in the experimental groups compared to healthy pulp samples. The biomarkers studied can be a promising tool to evaluate pulp tissue health or even in pulpitis treatment.     

Effects of Laser Irradiation on Pulp Cells Exposed to Bleaching Agents

The aim of this study was to evaluate the effect of low‐level laser therapy (LLLT) on odontoblast‐like cells exposed to a bleaching agent. Mouse dental papilla cell‐23 cells were seeded in wells of 24‐well plates. Eight groups were established according to the exposure to the bleaching agent and LLLT (0, 4, 10 and 15 J cm^?2). Enamel–dentin disks were adapted to artificial pulp chambers, which were individually placed in wells containing Dulbecco's modified Eagle's medium (DMEM). A bleaching agent (35% hydrogen peroxide [BA35%HP]) was applied on enamel (15 min) to obtain the extracts (DMEM + BA35%HP components diffused through enamel/dentin disks). The extracts were applied (1 h) to the cells, and then subjected to LLLT. Cell viability (Methyl tetrazolium assay), alkaline phosphatase (ALP) activity, as well as gene expression of ALP, fibronectin (FN) and type I collagen, were evaluated. The bleaching procedures reduced the cell viability, ALP activity and gene expression of dentin proteins. Laser irradiation did not modulate the cell response; except for FN, as LLLT decreased the gene expression of this protein by the cells exposed to the BA35%HP. It can be concluded that BA35%HP decreased the activities of odontoblasts that were not recovered by the irradiation of the damaged cells with low‐level laser parameters tested.     

The Effect of Calcium-Silicate Cements on Reparative Dentinogenesis Following Direct Pulp Capping on Animal Models

Dental pulp vitality is a desideratum for preserving the health and functionality of the tooth. In certain clinical situations that lead to pulp exposure, bioactive agents are used in direct pulp-capping procedures to stimulate the dentin-pulp complex and activate reparative dentinogenesis. Hydraulic calcium-silicate cements, derived from Portland cement, can induce the formation of a new dentin bridge at the interface between the biomaterial and the dental pulp. Odontoblasts are molecularly activated, and, if necessary, undifferentiated stem cells in the dental pulp can differentiate into odontoblasts. An extensive review of literature was conducted on MedLine/PubMed database to evaluate the histological outcomes of direct pulp capping with hydraulic calcium-silicate cements performed on animal models. Overall, irrespective of their physico-chemical properties and the molecular mechanisms involved in pulp healing, the effects of cements on tertiary dentin formation and pulp vitality preservation were positive. Histological examinations showed different degrees of dental pulp inflammatory response and complete/incomplete dentin bridge formation during the pulp healing process at different follow-up periods. Calcium silicate materials have the ability to induce reparative dentinogenesis when applied over exposed pulps, with different behaviors, as related to the animal model used, pulpal inflammatory responses, and quality of dentin bridges.     

Chemical constituents from the leaves of Manglietia phuthoensis and their effects on osteoblastic MC3T3-E1 cells

Two new phenyl glycosides, mangliesides A and B (1, 2), a new ionol glycoside, manglieside C (3), two new lignan glycosides, mangliesides D and E (4, 5), were isolated from the leaves of Manglietia phuthoensis, along with two known lignans, 3-methoxymagnolol (6) and obovatol (7). Their structures were established by means of 1D and 2D NMR, electrospray ionization (ESI)-MS and HR-ESI-MS experiments. Among them, compounds 2 and 5 significantly (p<0.05) increased the growth and differentiation of osteoblastic MC3T3-E1 cells in vitro.     

The pharmacological effects of morroniside and loganin isolated from Liuweidihuang Wan, on MC3T3-E1 cells

Liuweidihuang wan (LW), initially a well-known formula for curing "wu chi wu ruan", is commonly used nowadays for clinical treatment of postmenopausal osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP), and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.     

Laponite–Gelatin Nanofibrous Microsphere Promoting Human Dental Follicle Stem Cells Attachment and Osteogenic Differentiation for Noninvasive Stem Cell Transplantation

Nanofibrous microspheres (NFM) are emerging as prominent next-generation biomimetic injectable scaffold system for stem cell delivery and different tissue regeneration where nanofibrous topography facilitates ECM-like stem cells niches. Addition of osteogenic bioactive nanosilicate platelets within NFM can provide osteoconductive cues to facilitate matrix mediated osteogenic differentiation of stem cells and enhance the efficiency of bone tissue regeneration. In this study, gelatin nanofibrous microspheres are prepared containing fluoride-doped laponite XL21 (LP) using the emulsion mediated thermal induce phase separation (TIPS) technique. Systematic studies are performed to understand the effect of physicochemical properties of biomimicking NFM alone and with different concentrations of LP on human dental follicle stem cells (hDFSCs), their cellular attachment, proliferation, and osteogenic differentiation. The study highlights the effect of LP nanosilicate with biomimicking nanofibrous injectable scaffold system aiding in enhancing stem cell differentiation under normal physiological conditions compared to NFM without LP. The laponite–NFM shows suitability as excellent injectable biomaterials system for stem cell attachment, proliferation and osteogenic differentiation for stem cell transplantation and bone tissue regeneration.     

Emodin-8-O-β-D-glucoside from Polygonum amplexicaule D. Don var. sinense Forb. promotes proliferation and differentiation of osteoblastic MC3T3-E1 cells

Polygonum amplexicaule D. Don var. sinense Forb. (Polygonaceae) (PAF) is a famous traditional herb used to treat fractures, rheumatoid arthritis, muscle injury and pain. The present study was designed to investigate a PAF derived-chemical compound emodin-8-O-β-D-glucoside (EG) on the proliferation and differentiation of osteoblastic MC3T3-E1 cell in vitro. A compound was isolated from PAF extract by HPLC and identified as emodin-8-O-β-D-glucoside (EG) by spectroscopic methods. EG significantly promoted cell proliferation at 0.1-100 ng/mL, and increased the cell proportion in S-phase from 16.34% to 32.16%. Moreover, EG increased alkaline phosphatase (ALP) expression in MC3T3-E1 cells at the concentration from 0.1 to 100 ng/mL and inhibited PGE(2 )production induced by TNF-α in osteoblasts at the concentrations ranging from 10-100 ng/mL, suggesting that cell differentiation was induced in MC3T3-E1 osteoblasts. Taken together, these results indicated compound EG directly stimulated cell proliferation and differentiation of osteoblasts, therefore this study preliminarily explored the pharmacological mechanism of PAF to promote the healing of bone rheumatism and various fractures.     

Chemical characterization and ultrastructure study of pulp fibers

Understanding the ultrastructure and chemical characterization of pulp fibers is highly important in utilizing wood as a raw material in a wide scope of applications, such as forest biomass-based biorefineries and low-cost renewable materials. The observation of the ultrastructure is not possible without advanced microscopy and spectroscopy techniques. Therefore, this study focuses on exploring the ultrastructure of pulp fibers with helium ion microscopy (HIM) and scanning electron microscopy (SEM). For the analysis of chemical characterization in the pulp fibers, Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS) were performed. For these studies, the pulp fiber samples were obtained mainly from three different wood species, i.e. spruce, birch and eucalyptus. They were received in the never dried state and dried with a critical point drier (CPD) to minimize pore collapse. The spectroscopy results showed a strong signal from crystalline cellulose and confirmed the absence of lignin after Kraft pulping and bleaching. However, with XPS about 2% of lignin was detected in eucalyptus pulp. The results obtained with the microscopy techniques are compared and indicating the nanofibril size, shape, surface roughness as well as their orientation in pulp fibers. To our knowledge, this is the first time that HIM is applied to study the ultrastructure of pulp fibers and compared against more conventional microscopy and spectroscopy techniques. The main differences between HIM and SEM were found to be related to the focusing and magnification. The individual nano- and microfibrils as well as their bundles were more easily visible with HIM than with SEM. Also, with HIM it was possible to get the total area in focus at once which was not the case with SEM. The increased understanding of the ultrastructure and chemical composition of wood pulp enhance the development of novel wood-based products and processes for their manufacture.     

A novel silk/PES hybrid nanofibrous scaffold promotes the in vitro proliferation and differentiation of adipose‐derived mesenchymal stem cells into insulin producing cells

Using stem cells to replace the lost beta cells is a hopeful strategy in the treatment of diabetic patients. Furthermore, during stem cell culture and therapy, it is a need to use a substrate to act as a supportive matrix to mimic 3D in vivo microenvironment. Therefore, in this study, human adipose‐derived stem cells were used to differentiate into insulin‐producing cells (IPCs) on a silk/polyethersulfone (PES) scaffold. After exposing to the differentiation media, 2D and 3D (silk/PES) cultured cells were gradually aggregated and formed spherical shaped clusters. The viability of cells was comparable in both 3D and 2D culture. As the results of gene expression assay in both RNA and protein level showed, the differentiation efficiency was higher in 3D culture. Furthermore, ELISA revealed that the release of C‐peptide and insulin was higher in 3D than 2D culture. It seems that silk/PES nanofibrous hybrid scaffold could provide an appropriate matrix to mimic in vivo microenvironment and therefore increases the IPC differentiation potency of stem cells.     

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm² at a flow rate of 0.2 μL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.