Establishment of 3d oral mucosa model using differentiated stem cells from human exfoliated deciduous teeth

Oral mucosa is a specialized type of tissue that lines the oral cavity. It consists of two main layers: stratified squamous epithelium and lamina propria. The epithelial layer is resided by the epithelial cells, while the lamina propria layer is majorly occupied by fibroblasts. As far as the in vitro oral mucosa is concerned, the construction of an oral mucosa model should be performed in full thickness architecture using both cells mentioned. Therefore, the present study aimed to differentiate stem cells from human exfoliated deciduous teeth (SHED) into fibroblastand epithelial-like cells to be subsequently used in the establishment of a 3D oral mucosa model. The differentiation of SHED was carried out by the involvement of growth factors, namely connective tissue growth factor (CTGF) for fibroblastic differentiation, whereas keratinocyte growth factor (KGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin-like growth factor-2 (IGF-II) were employed in epithelial differentiation, respectively. The characterisation of the induced cells was done by morphological observation, proliferation rate, gene and protein expression analyses using semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR), immunofluorescence staining and flow cytometry. The collagen-glycosaminoglycan-chitosan (CGC) scaffold was constructed by combining collagen/chitosan/chondroitin sulphate/hyaluronic acid (100/12/5/1) thoroughly. The porous scaffold produced was characterized via their structural integrity, porosity, and density. The characterized differentiated cells were then co-cultured on CGC scaffold to generate a 3D oral mucosa model, which was later characterized via histological and immunofluorescence analyses. The results demonstrated the inductive effect of growth factors in both fibroblastic and epithelial differentiation of SHED. SHED derived-fibroblast-like cells are morphologically similar to SHED, while SHED derived-epithelial-like cells resembled native epithelial cells. Statistical analysis using one-way ANOVA of the proliferation assay showed a significant correlation (p<0.05) between the induced cells and growth factors involved. There were significant differences in gene and protein expressions between SHED and both differentiated cells. A white, porous lyophilized CGC scaffold produced was able to maintain its structural integrity and did not degrade throughout the whole experiments. The scaffold also exhibited good porosity and density. The co-culture system showed that the fibroblast- and epithelial-like cells derived from SHED were able to attach and proliferate when being seeded on CGC scaffold. The haematoxylin and eosin (H&E) staining of the established oral mucosa model also exhibited the infiltration and stratification of the fibroblast- and epithelial-like cells in some regions within CGC scaffolds. Also, the production of collagen could be observed via the Masson Trichrome staining. The immunofluorescence staining of the epithelial-like cells grown in the CGC scaffold also supported the presence of those cells. These findings hence provide a new understanding on the potential of SHED in the establishment of oral mucosa model for dental tissue regeneration

Nanotechnology for fabrication of nanoparticles and nanoemulsion

Therapeutic agents, namely drugs (i.e. Active pharmaceutical ingredient, API), biopharmaceutical (e.g. protein/peptide/macromolecules) and natural products (e.g. Nigella sativa oil, alpha mangostin) may have drawback of either highly water-soluble or highly lipophilic. These two extreme properties can mask the actual efficacy of the agents regardless of route of administration due to poor absorption across biological membrane. Nanotechnology appear to provide potential solution to these problems. Here, we had attempted a fabrication of nanoparticles and nanoemulsion for DNA-Nigella sativa oil and Doxycycline-Nigella sativa, respectively. For the nanoparticles, DNA-Nigella sativa was encapsulated into biodegradable poly(lactide-coglycolide) (PLGA: glycolic acid to lactic acid ratio of 50:50; IV 0.1 dl/g) polymer with or without chitosan and with or without sorbitan surfactant, using emulsion-solvent evaporation technique. The chitosan molecular weight (MW) was also varied. The DNA-Nigella sativa (DNN) nanoparticles were characterized for particle size using dynamic light scattering, Nanosizer and zeta potential using a Zetasizer. The results revealed particles of size ~870 to 134 nm showing large variation when immersed in deionized water as compared to alkaline media. Zeta-potential showed negative values for non-chitosan particles and positive values for all chitosan-containing particles. Microimages from Field emission Scanning Electron Microscopy (FESEM) revealed DNN nanoparticles of less than 300 nm. For the nanoemulsion, 3 phases were separately prepared, namely doxycycline/preservatives as the aqueous phase, nigella sativa oil/eugenol as the oil phase and lecithin/surfactant as the nanoemulsifier phase. The three phases were added and homogenized at 3000 rpm using a vacuum homogeneous mixer. This method yielded about 300 nm nanoemulsion droplets, PDI of 0.4 and zeta potential of -69. Further rheological characterization revealed that the nanoemulsion had thixotropic property suitable for its intended use as local antibiotic injected into periodontal gum pocket. In conclusion, with appropriate fabrication method, nanotechnology can be potentially explored to mitigate inherent problems of highly lipophilic or hydrophilic therapeutic agents hence unmask actual efficacy of these agents

Comparative assessment of plasmid DNA delivery by encapsulation within or adsorbed on poly (D, L-lactide-co-glycolide) nanoparticles

Purpose: To compare the gene delivery effectiveness of plasmid DNA (pDNA) encapsulated within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with that adsorbed on PLGA nanoparticles. Methods: PLGA nanoparticles were prepared using solvent-evaporation method. To encapsulate pDNA within the particles, it was first complexed with cetyltrimethylammonium bromide (CTAB) and then added to the oil phase during the synthesis. For the adsorption, PLGA nanoparticles were first modified with either CTAB or chitosan and then pDNA was adsorbed on the particle surface by electrostatic interaction. Results: Nanoparticles encapsulating pDNA exhibited better plasmid loading and protection with significantly lower burst release (p < 0.05) compared to that of the nanoparticles with adsorbed plasmid. Cell uptake of chitosan-modified nanoparticles by murine neuroblastoma (N2a) cells was significantly (p < 0.05) higher than that of chitosan-free nanoparticles. Nanoparticles encapsulating pDNA showed higher transfection efficiency (p < 0.05) in N2a cells. Conclusion: Encapsulation of pDNA within PLGA nanoparticles presents a potential strategy for gene delivery that is superior to pDNA adsorbed on the nanoparticle surface. In addition, encapsulation keeps the particle surface free for further modifications such as the addition of targeting ligands

Malaysian Carbon Reduction and Environmental Sustainability Tool (MyCREST) Qualified Professional Training Assessment

In assessing the green building application, CIDB has introduced the Malaysian Carbon Reduction and Environmental Sustainability Tool (MyCREST) which will be fully implemented for government project starting in the year 2018. &nbsp;In order to spread the awareness of MyCREST implementation and train the MyCREST assessor, and TVET workforce, CIDB has conducted series of MyCREST training every year. &nbsp;This research aims to investigate the overall performance of MyCREST training through the training objective assessment method.&nbsp; This research was carried out during the MyCREST in-house training by CIDB involving 33 trainees from all over Malaysia. &nbsp;A questionnaire survey has been developed for this research, and the data has been analyzed using descriptive statistical analysis.&nbsp; Result from this research determined that the majority of the respondents who completed this training have acquired new knowledge on MyCREST. &nbsp;It can be concluded that MyCREST training has achieved the objectives set out by the CIDB. &nbsp;However, in order to improve and to richen the data, observation during the training is suggested to be added in future research

Identification and Characterization of Intraoral and Dermal Fibroblasts Revisited

Abstract: Background: Fibroblasts are the common cells used in clinical regenerative medicine and dentistry. These cells are known to appear heterogeneous in vivo. Previous studies have only investigated the biological properties of these cell subpopulations in vitro. Despite sharing similarity in their spindle-shaped appearance, previous literatures revealed that they play distinguished functional and biological activities in the body. Objective: This paper highlights the similarities and differences among these cell subpopulations, particularly between intraoral fibroblasts (human periodontal ligament, gingival and oral mucosa fibroblasts) and dermal fibroblasts based on several factors including their morphology, growth and proliferation rate. Results: It could be suggested that each subpopulation of fibroblasts demonstrate different positionspecified gene signatures and responses towards extracellular signals. These dissimilarities are crucial to be taken into consideration to employ specific methodologies in stimulating these cells in vivo. Conclusion: A comparison of the characteristics of these cell subpopulations is desired for identifying appropriate cellular applications. Keywords: Dermal fibroblast, differences, gingival fibroblast, oral mucosa fibroblast, periodontal ligament fibroblast, similaritie

Surface deformation on thermocapillary convection in a binary fluid with internal heat generation and temperature dependent viscosity

The effects of temperature dependent viscosity and internal heat generation on the onset of steady Bénard-Marangoni convection in a horizontal binary fluid layer heated from below is investigated theoretically. The upper free surface is assumed to be deformable and the lower boundary is considered to be rigid and perfectly insulated to temperature perturbations. The asymptotic solution of the long wavelength is obtained using regular perturbation method with wave number as a perturbation parameter. It is found that the surface deformation of a binary fluid layer enhances the onset of thermocapillary convection while increasing the value of internal heat generation and temperature dependent viscosity will destabilize the binary fluid layer system

Effect of surfactants on plasmid DNA stability and release from Poly (D, L-lactide-co-glycolide) microspheres

Purpose: To evaluate the effect of surfactants on plasmid DNA during preparation and release from polylactic glycolide (PLGA) microspheres. Methods: Various surfactants, both ionic and non-ionic (Span, Tween, Triton X100, cetyltrimethylammonium bromide and sodium dodecyl sulphate), were added during the microsphere preparation and their effect was evaluated. Supercoil index (SCI) was introduced as a harmonised value derived from encapsulation efficiency and supercoil preservation efficiency in order to evaluate the impact of different surfactants on pDNA encapsulation. Results: Polyvinyl alcohol and Span revealed low SCI whereas Tween increased the SCI in a fraction- dependent manner. The Tween blend of hydrophilic-lipophilic balance (HLB) of 16 and Triton X-100 (HLB = 13.5) showed the highest SCI. Span revealed high burst release of pDNA whereas Triton X-100 exhibited low burst release. Following the burst release, diffusion mechanism was found to predominate in DNA release. Conclusion: The microspheres were non-toxic to the neuro-2a cells which suggest they can be potentially used in the gene therapy of neuronal diseases

Onset of Benard-Marangoni instabilities in a double diffusive binary fluid layer with temperature-dependent viscosity

The effect of temperature-dependent viscosity in a horizontal double diffusive binary fluid layer is investigated. When the layer is heated from below, the convection of Benard-Marangoni will start to exists. Linear stability analysis is performed and the eigenvalues from few cases of boundary conditions were obtained. Galerkin method were used to solve the numerical calculation and marginal stability curve is obtained. Results shows that an increase of temperature-dependent viscosity will destabilized the system. The impact of double diffusive coefficients are also revealed. It is found that the effect of Soret parameter exhibits destabilizing reaction on the system while an opposite response is noted with an increase of Dufour parameter

Marangoni convection in a double diffusive binary fluid with temperature dependent viscosity, Coriolis force and internal heat generation

A steady Marangoni convection in a horizontal double diffusive binary fluid is considered. Present study investigated the effects of temperature dependent viscosity, Coriolis force and internal heat generation to the onset of convection. The bottom boundary was set to be insulating or conducting to temperature. A detailed numerical calculation of the marginal stability curves were performed by using the Galerkin method and it is shown that temperature dependent viscosity, internal heat generation and Soret number destabilize the binary fluid layer system while Taylor number and Dufour number act oppositely to the system