Effect of Boron on Osteogenic Differentiation of Human Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) have been well established as an ideal source of cell-based therapy for bone tissue engineering applications. Boron (B) is a notable trace element in humans; so far, the effects of boron on the osteogenic differentiation of BMSCs have not been reported. The aim of this study was to evaluate the effects of boron (0, 1, 10,100, and 1,000?ng/ml) on osteogenic differentiation of human BMSCs. In this study, BMSCs proliferation was analyzed by cell counting kit-8 (CCK8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, Von Kossa staining, and real-time PCR. The results indicated that the proliferation of BMSCs was no different from the control group when added with B at the concentration of 1, 10, and 100?ng/ml respectively (P?>?0.05); in contrast, 1,000?ng/ml B inhibited the proliferation of BMSCs at days?4, 7, and 14 (P?相似文献   

Vitamin D Promotes Odontogenic Differentiation of Human Dental Pulp Cells via ERK Activation

The active metabolite of vitamin D such as 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is a well-known key regulatory factor in bone metabolism. However, little is known about the potential of vitamin D as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro. The purpose of this study was to evaluate the effect of vitamin D₃ metabolite, 1α,25(OH)₂D₃, on odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured with 1α,25(OH)₂D₃ in the absence of differentiation-inducing factors. Treatment of HDPCs with 1α,25(OH)₂D₃ at a concentration of 10 nM or 100 nM significantly upregulated the expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein1 (DMP1), the odontogenesis-related genes. Also, 1α,25(OH)₂D₃ enhanced the alkaline phosphatase (ALP) activity and mineralization in HDPCs. In addition, 1α,25(OH)₂D₃ induced activation of extracellular signal-regulated kinases (ERKs), whereas the ERK inhibitor U0126 ameliorated the upregulation of DSPP and DMP1 and reduced the mineralization enhanced by 1α,25(OH)₂D₃. These results demonstrated that 1α,25(OH)₂D₃ promoted odontoblastic differentiation of HDPCs via modulating ERK activation.     

Effect of Aminated Mesoporous Bioactive Glass Nanoparticles on the Differentiation of Dental Pulp Stem Cells

Mesoporous bioactive nanoparticles (MBNs) have been developed as promising additives to various types of bone or dentin regenerative material. However, biofunctionality of MBNs as dentin regenerative additive to dental materials have rarely been studied. We investigated the uptake efficiency of MBNs-NH₂ with their endocytosis pathway and the role of MBNs-NH₂ in odontogenic differentiation to clarify inherent biofunctionality. MBNs were fabricated by sol-gel synthesis, and 3% APTES was used to aminate these nanoparticles (MBNs-NH₂) to reverse their charge from negative to positive. To characterize the MBNs-NH_2, TEM, XRD, FTIR, zeta(ξ)-potential measurements, and Brunauer–Emmett–Teller analysis were performed. After primary cultured rat dental pulp stem cells (rDPSCs) were incubated with various concentrations of MBNs-NH_2, stem cell viability (24 hours) with or without differentiated media, internalization of MBNs-NH₂ in rDPSCs (~4 hours) via specific endocytosis pathway, intra or extracellular ion concentration and odontoblastic differentiation (~28 days) were investigated. Incubation with up to 50 μg/mL of MBNs-NH₂ had no effect on rDPSCs viability with differentiated media (p>0.05). The internalization of MBNs-NH₂ in rDPSCs was determined about 92% after 4 hours of incubation. Uptake was significantly decreased with ATP depletion and after 1 hour of pre-treatment with the inhibitor of macropinocytosis (p<0.05). There was significant increase of intracellular Ca and Si ion concentration in MBNs-NH₂ treated cells compared to no-treated counterpart (p<0.05). The expression of odontogenic-related genes (BSP, COL1A, DMP-1, DSPP, and OCN) and the capacity for biomineralization (based on alkaline phosphatase activity and alizarin red staining) were significantly upregulated with MBNs-NH₂. These results indicate that MBNs-NH₂ induce odontogenic differentiation of rDPSCs and may serve as a potential dentin regenerative additive to dental material for promoting odontoblast differentiation.     

Comparison of Odontogenic Differentiation of Human Dental Follicle Cells and Human Dental Papilla Cells

Classical tooth development theory suggests that dental papilla cells (DPCs) are the precursor cells of odontoblasts, which are responsible for dentin development. However, our previous studies have indicated that dental follicle cells (DFCs) can differentiate into odontoblasts. To further our understanding of tooth development, and the differences in dentinogenesis between DFCs and DPCs, the odontogenic differentiation of DFCs and DPCs was characterized in vitro and in vivo. DFCs and DPCs were individually combined with treated dentin matrix (TDM) before they were subcutaneously implanted into the dorsum of mice for 8 weeks. Results showed that 12 proteins were significantly differential, and phosphoserine aminotransferase 1 (PSAT1), Isoform 2 of hypoxia-inducible factor 1-alpha (HIF1A) and Isoform 1 of annexin A2 (ANXA2), were the most significantly differential proteins. These proteins are related to regulation of bone balance, angiogenesis and cell survival in an anoxic environment. Both DFCs and DPCs express odontogenic, neurogenic and peridontogenic markers. Histological examination of the harvested grafts showed that both DFCs and DPCs form pulp-dentin/cementum-periodentium-like tissues in vivo. Hence, DFCs and DPCs have similar odontogenic differentiation potential in the presence of TDM. However, differences in glucose and amino acid metabolism signal transduction and protein synthesis were observed for the two cell types. This study expands our understanding on tooth development, and provides direct evidence for the use of alternative cell sources in tooth regeneration.     

Adhesion,Vitality and Osteogenic Differentiation Capacity of Adipose Derived Stem Cells Seeded on Nitinol Nanoparticle Coatings

Autologous cells can be used for a bioactivation of osteoimplants to enhance osseointegration. In this regard, adipose derived stem cells (ASCs) offer interesting perspectives in implantology because they are fast and easy to isolate. However, not all materials licensed for bone implants are equally suited for cell adhesion. Surface modifications are under investigation to promote cytocompatibility and cell growth. The presented study focused on influences of a Nitinol-nanoparticle coating on ASCs. Possible toxic effects as well as influences on the osteogenic differentiation potential of ASCs were evaluated by viability assays, scanning electron microscopy, immunofluorescence and alizarin red staining. It was previously shown that Nitinol-nanoparticles exert no cell toxic effects to ASCs either in soluble form or as surface coating. Here we could demonstrate that a Nitinol-nanoparticle surface coating enhances cell adherence and growth on Nitinol-surfaces. No negative influence on the osteogenic differentiation was observed. Nitinol-nanoparticle coatings offer new possibilities in implantology research regarding bioactivation by autologous ASCs, respectively enhancement of surface attraction to cells.     

Angiogenic Properties of Human Dental Pulp Stem Cells

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.     

Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways

Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, β1 and β3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.     

Morphology-Based Prediction of Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells (hBMSCs) are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP) activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions). The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient''s own cell images to predict a new patient''s cellular potential. The prediction accuracy was found to be greatly enhanced by incorporation of patients'' own cell features in the modeling, indicating the practical strategy for clinical usage. Consequently, our results provide strong evidence for the feasibility of using a quantitative time series of phase-contrast cellular morphology for non-invasive cell quality prediction in regenerative medicine.     

Radiation-Induced Alterations of Osteogenic and Chondrogenic Differentiation of Human Mesenchymal Stem Cells

While human mesenchymal stem cells (hMSCs), either in the bone marrow or in tumour microenvironment could be targeted by radiotherapy, their response is poorly understood. The oxic effects on radiosensitivity, cell cycle progression are largely unknown, and the radiation effects on hMSCs differentiation capacities remained unexplored. Here we analysed hMSCs viability and cell cycle progression in 21% O₂ and 3% O₂ conditions after medical X-rays irradiation. Differentiation towards osteogenesis and chondrogenesis after irradiation was evaluated through an analysis of differentiation specific genes. Finally, a 3D culture model in hypoxia was used to evaluate chondrogenesis in conditions mimicking the natural hMSCs microenvironment. The hMSCs radiosensitivity was not affected by O₂ tension. A decreased number of cells in S phase and an increase in G2/M were observed in both O₂ tensions after 16 hours but hMSCs released from the G2/M arrest and proliferated at day 7. Osteogenesis was increased after irradiation with an enhancement of mRNA expression of specific osteogenic genes (alkaline phosphatase, osteopontin). Osteoblastic differentiation was altered since matrix deposition was impaired with a decreased expression of collagen I, probably through an increase of its degradation by MMP-3. After induction in monolayers, chondrogenesis was altered after irradiation with an increase in COL1A1 and a decrease in both SOX9 and ACAN mRNA expression. After induction in a 3D culture in hypoxia, chondrogenesis was altered after irradiation with a decrease in COL2A1, ACAN and SOX9 mRNA amounts associated with a RUNX2 increase. Together with collagens I and II proteins decrease, associated to a MMP-13 expression increase, these data show a radiation-induced impairment of chondrogenesis. Finally, a radiation-induced impairment of both osteogenesis and chondrogenesis was characterised by a matrix composition alteration, through inhibition of synthesis and/or increased degradation. Alteration of osteogenesis and chondrogenesis in hMSCs could potentially explain bone/joints defects observed after radiotherapy.     

Expression, Purification, and Characterization of a Dentin Phosphoprotein Produced by Escherichia coli, and Its Odontoblastic Differentiation Effects on Human Dental Pulp Cells

To investigate the functions of recombinant human dentin phosphoprotein (rhDPP), we examined cell adhesion, viability and the odontoblastic differentiation activity of human dental pulp cells (hDPCs). Firstly, rhDPP was constructed using pBAD-HisA plasmid in Escherichia coli. Cell adhesion and viability of hDPCs by rhDPP was examined using a crystal violet assay and a MTT assay, ALP, mineralization activity and odontoblastic differentiation-related mRNA levels of hDPCs were measured to elucidate the odontoblastic differentiation effect of rhDPP on hDPCs. Initially, rhDPP significantly and dose-dependently increased hDPCs adhesion versus the untreated control (p?<?0.05). Cell viability was also significantly increased by rhDPP at 5?days (p?<?0.001). Furthermore, the odontoblastic differentiation effect of rhDPP was verified by measuring ALP activity, mineralization activity and the mRNA levels of odontoblastic differentiation markers. Taken together, rhDPP is expected to play an important role on hDPCs, thereby suggesting its potential use for tooth repair and regeneration.     

Osteogenic Differentiation of Human Mesenchymal Stem Cells in Mineralized Alginate Matrices

Mineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.     

Effects of Nerve Growth Factor and Basic Fibroblast Growth Factor Promote Human Dental Pulp Stem Cells to Neural Differentiation

Dental pulp stem cells (DPSCs) were the most widely used seed cells in the field of neural regeneration and bone tissue engineering, due to their easily isolation, lack of ethical controversy, low immunogenicity and low rates of transplantation rejection. The purpose of this study was to investigate the role of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on neural differentiation of DPSCs in vitro. DPSCs were cultured in neural differentiation medium containing NGF and bFGF alone or combination for 7 days. Then neural genes and protein markers were analyzed using western blot and RT-PCR. Our study revealed that bFGF and NGF increased neural differentiation of DPSCs synergistically, compared with bFGF and NGF alone. The levels of Nestin, MAP-2, βIII-tubulin and GFAP were the most highest in the DPSCs?+?bFGF?+?NGF group. Our results suggested that bFGF and NGF signifiantly up-regulated the levels of Sirt1. After treatment with Sirt1 inhibitor, western blot, RT-PCR and immunofluorescence staining showed that neural genes and protein markers had markedly decreased. Additionally, the ERK and AKT signaling pathway played a key role in the neural differentiation of DPSCs stimulated with bFGF?+?NGF. These results suggested that manipulation of the ERK and AKT signaling pathway may be associated with the differentiation of bFGF and NGF treated DPSCs. Our date provided theoretical basis for DPSCs to treat neurological diseases and repair neuronal damage.     

Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable. Periosteum-derived cells were isolated from the tibiae of adult Wistar rats (n = 12). The osteogenic potential with regard to alkaline phosphatase activity, morphology, nodule formation and mineralization was studied by culturing them in an osteogenic medium for up to 4 months. Seventy-five percent of the cultures (n = 9) did not show any increase in alkaline phosphatase activity nor nodule formation during long-term culture for up to 4 months. Nevertheless, in 25% of the cultures, alkaline phosphatase activity started from negligible (<5 mM pNP/mg protein) and increased towards approximately 50 mM pNP/mg protein. Three-dimensional nodule formation was observed at passages 3–5. In further passages (P5–P7), nodule formation capacity decreased and a diffuse mineralization pattern was observed. Suitable cultures with osteogenic capacity, can be selected at early passages based on the presence of cuboidal cells. These cells have the advantage of retaining their osteogenic potential even after prolonged cultivation (6–7 passages) before final differentiation occurs. Although periosteal cells are suitable for long term in vitro evaluation of biomaterials, the isolation and selection is time consuming. Hence, a more appropriate source to study cell/biomaterial interactions should be more convenient.     

Transcriptional Regulation of Zinc Transporters in Human Osteogenic Sarcoma (Saos-2) Cells to Zinc Supplementation and Zinc Depletion

Biological Trace Element Research - Bone is a passive storage organ for zinc, which contains about 30% of the total body zinc. However, during extreme zinc deficiency, only a small fraction of zinc...