This study investigates the feasibility of processed human amnion (HAM) as a substrate for chondrogenic differentiation of mesenchymal stem cells (MSCs). HAM preparations processed by air drying (AD) and freeze drying (FD) underwent histological examination and MSC seeding in chondrogenic medium for 15 days. Monolayer cultures were used as control for chondrogenic differentiation and HAMs without cell seeding were used as negative control. Qualitative observations were made using scanning electron microscopy analysis and quantitative analyses were based on the sulfated glycosaminoglycans (GAG) assays performed on day 1 and day 15. Histological examination of HAM substrates before seeding revealed a smooth surface in AD substrates, while the FD substrates exhibited a porous surface. Cell attachment to AD and FD substrates on day 15 was qualitatively comparable. GAG were significantly highly expressed in cells seeded on FD HAM substrates. This study indicates that processed HAM is a potentially valuable material as a cell-carrier for MSC differentiation.
As the cellular prion protein (PrP(C)) has been implicated in carcinogenesis, we aimed to investigate the effects of cancer cell-specific PrP(C) overexpression from the invasion, metastasis, and apoptosis aspects, by performing cell motility assays, cell proliferation assays under anchorage-dependent and anchorage-independent conditions, and apoptosis evasion when subjected to multiple anti-cancer drugs. Overexpression of PrP(C) in LS 174T was achieved by stable transfection. PrP(C) overexpression was shown to increase cell proliferation in anchorage-dependent and anchorage-independent manners, as shown by more viable cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, more colonies formed in soft agar assay and increased resistance to anoikis in poly-2-hydroxyethyl methacrylate-coated surface. PrP(C) overexpression also increased cell motility and invasiveness of LS 174T. Cell adhesion to extracellular matrix using collagen- and fibronectin-coated surfaces revealed increased cell attachment in LS 174T cells overexpressing PrP(C). Analysis of apoptotic and necrotic cells by propidium iodide/annexin V-fluorescein isothiocyanate microscopy and 7-amino-actinomycin D/annexin V-phycoerythrin flow cytometry revealed that PrP(C) overexpression attenuated doxorubicin-induced apoptosis. Human apoptosis antibody array with 35 apoptosis-related proteins revealed that three inhibitor of apoptosis proteins (IAPs)-survivin, X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein-1 (cIAP-1)-were upregulated in LS 174T cells overexpressing PrP(C) in doxorubicin-induced apoptosis. In conclusion, the overexpression of PrP(C) could enhance the invasiveness and survival of LS 174T colorectal cancer cells, indicating that PrP(C) plays a role in colorectal cancer biology.
Calcium silicate (CaSiO3, CS) ceramics are promising bioactive materials for bone tissue engineering, particularly for bone repair. However, the low toughness of CS limits its application in load-bearing conditions. Recent findings indicating the promising biocompatibility of graphene imply that graphene can be used as an additive to improve the mechanical properties of composites. Here, we report a simple method for the synthesis of calcium silicate/reduced graphene oxide (CS/rGO) composites using a hydrothermal approach followed by hot isostatic pressing (HIP). Adding rGO to pure CS increased the hardness of the material by ∼40%, the elastic modulus by ∼52%, and the fracture toughness by ∼123%. Different toughening mechanisms were observed including crack bridging, crack branching, crack deflection, and rGO pull-out, thus increasing the resistance to crack propagation and leading to a considerable improvement in the fracture toughness of the composites. The formation of bone-like apatite on a range of CS/rGO composites with rGO weight percentages ranging from 0 to 1.5 has been investigated in simulated body fluid (SBF). The presence of a bone-like apatite layer on the composite surface after soaking in SBF was demonstrated by X-ray diffraction (XRD) and field emission scanning electron microscopy (FESEM). The biocompatibility of the CS/rGO composites was characterized using methyl thiazole tetrazolium (MTT) assays in vitro. The cell adhesion results showed that human osteoblast cells (hFOB) can adhere to and develop on the CS/rGO composites. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of cells on the CS/rGO composites were improved compared with the pure CS ceramics. These results suggest that calcium silicate/reduced graphene oxide composites are promising materials for biomedical applications.
One of the major challenges in bone grafting is the lack of sufficient bone vascularization. A rapid and stable bone vascularization at an early stage of implantation is essential for optimal functioning of the bone graft. To address this, the ability of in situ TiO2 nanofibrous surfaces fabricated via thermal oxidation method to enhance the angiogenic potential of human umbilical vein endothelial cells (HUVECs) was investigated. The cellular responses of HUVECs on TiO2 nanofibrous surfaces were studied through cell adhesion, cell proliferation, capillary-like tube formation, growth factors secretion (VEGF and BFGF), and angiogenic-endogenic-associated gene (VEGF, VEGFR2, BFGF, PGF, HGF, Ang-1, VWF, PECAM-1 and ENOS) expression analysis after 2 weeks of cell seeding. Our results show that TiO2 nanofibrous surfaces significantly enhanced adhesion, proliferation, formation of capillary-like tube networks and growth factors secretion of HUVECs, as well as leading to higher expression level of all angiogenic-endogenic-associated genes, in comparison to unmodified control surfaces. These beneficial effects suggest the potential use of such surface nanostructures to be utilized as an advantageous interface for bone grafts as they can promote angiogenesis, which improves bone vascularization.
To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold.
Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.
This study was aimed to improve of the corrosion resistance and mechanical properties of Mg/15TiO2/5HA nanocomposite by silicon and magnesium oxide coatings prepared using a powder metallurgy method. The phase evolution, chemical composition, microstructure and mechanical properties of uncoated and coated samples were characterized. Electrochemical and immersion tests used to investigate the in vitro corrosion behavior of the fabricated samples. The adhesion strength of ~36MPa for MgO and ~32MPa for Si/MgO coatings to substrate was measured by adhesion test. Fabrication a homogenous double layer coating with uniform thicknesses consisting micro-sized particles of Si as outer layer and flake-like particles of MgO as the inner layer on the surface of Mg/15TiO2/5HA nanocomposite caused the corrosion resistance and ductility increased whereas the ultimate compressive stress decreased. However, after immersion in SBF solution, Si/MgO-coated sample indicates the best mechanical properties compared to those of the uncoated and MgO-coated samples. The increase of cell viability percentage of the normal human osteoblast (NHOst) cells indicates the improvement in biocompatibility of Mg/15TiO2/5HA nanocomposite by Si/MgO coating.
The evaluation of the interaction of cells with biomaterials is fundamental to establish the suitability of the biomaterial for a specific application. In this study, the properties of bacterial nanocellulose/acrylic acid (BNC/AA) hydrogels fabricated with varying BNC to AA ratios and electron-beam irradiation doses were determined. The manner these hydrogel properties influence the behavior of human dermal fibroblasts (HDFs) at the cellular and molecular levels was also investigated, relating it to its application both as a cell carrier and wound dressing material. Swelling, hardness, adhesive force (wet), porosity, and hydrophilicity (dry) of the hydrogels were dependent on the degree of cross-linking and the amount of AA incorporated in the hydrogels. However, water vapor transmission rate, pore size, hydrophilicity (semidry), and topography were similar between all formulations, leading to a similar cell attachment and proliferation profile. At the cellular level, the hydrogel demonstrated rapid cell adhesion, maintained HDFs viability and morphology, restricted cellular migration, and facilitated fast transfer of cells. At the molecular level, the hydrogel affected nine wound-healing genes (IL6, IL10, MMP2, CTSK, FGF7, GM-CSF, TGFB1, COX2, and F3). The findings indicate that the BNC/AA hydrogel is a potential biomaterial that can be employed as a wound-dressing material to incorporate HDFs for the acceleration of wound healing.
This study aimed to explore a potential use of fish scale-derived gelatin nanofibrous scaffolds (GNS) in tissue engineering due to their biological and economical merits. Extraction of gelatin was achieved via decalcification, sonication and lyophilization of mixed fish scales. To fabricate nano-scale architecture of scaffolds analogous to natural extracellular matrix, gelatin was rendered into nanofibrous matrices through 6-h electrospinning, resulting in the average diameter of 48 ± 12 nm. In order to improve the water-resistant ability while retaining their biocompatibility, GNS were physically crosslinked with ultraviolet (UV) irradiation for 5 min (UGN5), 10 min (UGN10) and 20 min (UGN20). On average, the diameter of nanofibers increased by 3 folds after crosslinking, however, Fourier transform infrared spectroscopy analysis confirmed that no major alterations occurred in the functional groups of gelatin. A degradation assay showed that UGN5 and UGN10 scaffolds remained in minimum essential medium for 14 days, while UGN20 scaffolds degraded completely after 10 days. All UGN scaffolds promoted adhesion and proliferation of human keratinocytes, HaCaT, without causing an apparent cytotoxicity. UGN5 scaffolds were shown to stimulate a better growth of HaCaT cells compared to other scaffolds upon 1 day of incubation, whereas UGN20 had a long-term effect on cells exhibiting 25% higher cell proliferation than positive control after 7 days. In the wound scratch assay, UGN5 scaffolds induced a rapid cell migration closing up to 79% of an artificial wound within 24 h. The current findings provide a new insight of UGN scaffolds to serve as wound dressings in the future. In the wound scratch assay, UGN5 induced a rapid cell migration closing up to 79% of an artificial wound within 24 h.
Autologous nerve grafts to bridge nerve gaps pose various drawbacks. Nerve tissue engineering to promote nerve regeneration using artificial neural conduits has emerged as a promising alternative.
Numerous preclinical and clinical studies have investigated the regenerative potential and the trophic support of mesenchymal stem cells (MSCs) following their injection into a target organ. Clinicians favor the use of smallest bore needles possible for delivering MSCs into vascular organs like heart, liver and spleen. There has been a concern that small needle bore sizes may be detrimental to the health of these cells and reduce the survival and plasticity of MSCs.
Multipotent stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for tissue regeneration. In the present study we decided to test the inductive effect of chitosan and transforming growth factor-β1 (TGFβ1) as a scaffold/factor combination on SHED proliferation and osteogenic differentiation.
This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.
In dental implant treatment, the long-term prognosis is dependent on the biologic seal formed by the soft tissue around the implant. The in vitro investigation of the implant-soft tissue interface is usually carried out using a monolayer cell-culture model that lacks a polarized-cell phenotype. This study developed a tissue-engineered three-dimensional oral mucosal model (3D OMM) to investigate the implant-soft tissue interface.
The interrelationship between malignant epithelium and the underlying stroma is of fundamental importance in tumour development and progression. In the present study, we used cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), tumours that are characterized by the loss of genes such as TP53 and p16INK4A and with extensive loss of heterozygosity, together with CAFs from their more genetically stable (GS) counterparts that have wild-type TP53 and p16INK4A and minimal loss of heterozygosity (GS-OSCC). Using a systems biology approach to interpret the genome-wide transcriptional profile of the CAFs, we show that transforming growth factor-β (TGF-β) family members not only had biological relevance in silico but also distinguished GU-OSCC-derived CAFs from GS-OSCC CAFs and fibroblasts from normal oral mucosa. In view of the close association between TGF-β family members, we examined the expression of TGF-β1 and TGF-β2 in the different fibroblast subtypes and showed increased levels of active TGF-β1 and TGF-β2 in CAFs from GU-OSCC. CAFs from GU-OSCC, but not GS-OSCC or normal fibroblasts, induced epithelial-mesenchymal transition and down-regulated a broad spectrum of cell adhesion molecules resulting in epithelial dis-cohesion and invasion of target keratinocytes in vitro in a TGF-β-dependent manner. The results demonstrate that the TGF-β family of cytokines secreted by CAFs derived from genotype-specific oral cancer (GU-OSCC) promote, at least in part, the malignant phenotype by weakening intercellular epithelial adhesion.
The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.
Urothelial bladder cancer is a major cause of morbidity and mortality worldwide, causing an estimated 150 000 deaths per year. Whilst non-muscle-invasive bladder tumours can be effectively treated, with high survival rates, many tumours recur, and some will progress to muscle-invasive disease with a much poorer long-term prognosis. Thus, there is a pressing need to understand the molecular transitions occurring within the progression of bladder cancer to an invasive disease. Tumour invasion is often associated with a down-regulation of E-cadherin expression concomitant with a suppression of cell:cell junctions, and decreased levels of E-cadherin expression have been reported in higher grade urothelial bladder tumours. We find that expression of E-cadherin in a panel of bladder cancer cell lines correlated with the presence of cell:cell junctions and the level of PAK5 expression. Interestingly, exogenous PAK5 has recently been described to be associated with cell:cell junctions and we now find that endogenous PAK5 is localised to cell junctions and interacts with an E-cadherin complex. Moreover, depletion of PAK5 expression significantly reduced junctional integrity. These data suggest a role for PAK5 in maintaining junctional stability and we find that, in both our own patient samples and a commercially available dataset, PAK5mRNA levels are reduced in human bladder cancer compared with normal controls. Taken together, the present study proposes that PAK5 expression levels could be used as a novel prognostic marker for bladder cancer progression.