Khairani Idah Mokhtar

Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes

Stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs) obtained from the dental pulp of human extracted tooth were cultured and characterized to confirm that these were mesenchymal stem cells. The proliferation rate was assessed using AlamarBlue® cell assay. The differentially expressed genes in SHED and DPSCs were identified using the GeneFishing™ technique. The proliferation rate of SHED (P < 0.05) was significantly higher than DPSCs while SHED had a lower multiplication rate and shorter population doubling time (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h). Two bands were highly expressed in SHED and three bands in DPSCs. Sequencing analysis showed these to be TIMP metallopeptidase inhibitor 1 (TIMP1), and ribosomal protein s8, (RPS8) in SHED and collagen, type I, alpha 1, (COL1A1), follistatin‐like 1 (FSTL1), lectin, galactoside‐binding, soluble, 1, (LGALS1) in DPSCs. TIMP1 is involved in degradation of the extracellular matrix, cell proliferation and anti‐apoptotic function and RPS8 is involved as a rate‐limiting factor in translational regulation; COL1A1 is involved in the resistance and elasticity of the tissues; FSTL1 is an autoantigen associated with rheumatoid arthritis; LGALS1 is involved in cell growth, differentiation, adhesion, RNA processing, apoptosis and malignant transformation. This, along with further protein expression analysis, holds promise in tissue engineering and regenerative medicine.

Cell attachment properties of Portland cement-based endodontic materials: biological and methodological considerations.

INTRODUCTION The attachment and spreading of mammalian cells on endodontic biomaterials are an area of active research. The purpose of this review is to discuss the cell attachment properties of Portland cement (PC)-based materials by using scanning electron microscope (SEM). In addition, methodological aspects and technical challenges are discussed. METHODS A PubMed electronic search was conducted by using appropriate key words to identify the available investigations on the cell attachment properties of PC-based endodontic materials. After retrieving the full text of related articles, the cross citations were also identified. RESULTS A total of 23 articles published between January 1993 and October 2013 were identified. This review summarizes the cell attachment properties of commercial and experimental PC-based materials on different cell cultures by using SEM. Methodological procedures, technical challenges, and relevance of SEM in determining the biological profile of PC-based materials are discussed. CONCLUSIONS SEM observations demonstrate that commercial MTA formulations show favorable cell attachment properties, which is consistent with their successful clinical outcomes. The favorable cell attachment properties of PC and its modified formulations support its potential use as a substitute for mineral trioxide aggregate. However, researchers should carefully select cell types for their SEM investigations that would be in contact with the proposed PC-based combinations in the clinical situation. Despite being a technical challenge, SEM provides useful information on the cell attachment properties of PC-based materials; however, other assays for cell proliferation and viability are essential to come up with an accurate in vitro biological profile of any given PC-based formulation.

Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth.

OBJECTIVE 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. DESIGN Cell proliferation was quantitatively assessed by PrestoBlue, live/dead assay was performed and cell attachment to chitosan scaffold was examined by scanning electron microscopy (SEM). For osteogenic differentiation analysis, alkaline phosphatase activity was quantified, cells were stained with Alizarin Red, and the lineage specific genes/proteins ALP, COL I, BSP, and OCN were analysed by real-time PCR and Western blot. RESULTS SHED remained viable and attached well to the chitosan structure. Moreover, TGFβ1 significantly enhanced the proliferative activity of SHED on the chitosan scaffold. Our data further revealed that chitosan and TGFβ1 enhanced the osteogenic differentiation of SHED, as evidenced by high ALP activity, strong mineral deposition, and the up-regulation of ALP, COL I, BSP, and OCN gene/protein expression. CONCLUSION Together, data from our study indicate that the combination of chitosan scaffolds and TGFβ1 enhanced proliferation and osteogenic differentiation of SHED. These findings suggest that the combined application of chitosan scaffold and TGFβ1 in conjunction with SHED might be beneficial for in vivo bone regeneration.

Transforming Growth Factor-α and Nonsyndromic Cleft Lip With or Without Palate or Cleft Palate Only in Kelantan, Malaysia

Objective: To determine the frequency of the transforming growth factor-alpha (TGFα) Taq1 polymorphism in nonsyndromic cleft lip with or without cleft palate (CL±P) and cleft palate only (CP) in Kelantan, Malaysia. Setting: The study was conducted at the Combined Cleft Clinic and at the Human Genome Centre in Hospital Universiti Sains Malaysia in Kelantan, Malaysia. Design: We examined the C2/Taq1 variant of the TGFα gene in 46 patients with nonsyndromic CL±P or CP only and in 33 controls. The TGFα genotype frequencies in patients were compared with those in controls using the chi-square or Fisher exact test. DNA samples were obtained from peripheral blood. Results: No association was found between TGFαTaq1 polymorphism and CL±P or CP in this case-control study. In addition, no homozygosity for the rare allele C2 was noted in CL±P, CP, or the controls. Conclusion: No evidence of TGFαTaq1 polymorphism was observed in association with CL±P and CP in this study.

Angiogenic potential of extracellular matrix of human amniotic membrane

Combination between tissue engineering and other fields has brought an innovation in the area of regenerative medicine which ultimate aims are to repair, improve, and produce a good tissue construct. The availability of many types of scaffold, both synthetically and naturally have developed into many outstanding end products that have achieved the general objective in tissue engineering. Interestingly, most of this scaffold emulates extracellular matrix (ECM) characteristics. Therefore, ECM component sparks an interest to be explored and manipulated. The ECM featured in human amniotic membrane (HAM) provides a suitable niche for the cells to adhere, grow, proliferate, migrate and differentiate, and could possibly contribute to the production of angiogenic micro-environment indirectly. Previously, HAM scaffold has been widely used to accelerate wound healing, treat bone related and ocular diseases, and involved in cardiovascular repair. Also, it has been used in the angiogenicity study, but with a different technical approach. In addition, both side of HAM could be used in cellularised and decellularised conditions depending on the objectives of a particular research. Therefore, it is of paramount importance to investigate the behavior of ECM components especially on the stromal side of HAM and further explore the angiogenic potential exhibited by this scaffold.

Isolation and Enhancement of a Homogenous in Vitro Human Hertwig’s Epithelial Root Sheath Cell Population

Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.

Immunomodulatory Effect of Cytokines in the Differentiation of Mesenchymal Stem Cells: A Review

Mesenchymal stem cells (MSCs) are stromal origin cells with multilineage differentiation capacity. The immunoregulatory properties of MSCs can be interfered effectively by cytokines. Cytokines, produced by a broad range of cells, act at the systemic level to influence biological phenomena such as inflammation, wound healing, organogenesis and oncogenesis. Cytokines also play vital roles in the differentiation of MSCs into several cell lineages. This review summarizes on how cytokines can affect MSCs differentiation and their relative signaling pathways, which may serve to understand the possible underlying mechanisms. Also, this review reveals the potential clinical use of MSCs as promising therapeutic agents due to their special characteristics such as multipotent differentiation, immunomodulatory properties, and selfrestoration.

Chemical analysis and biological properties of two different formulations of white portland cements.

White Portland cement (WPC) has generated research interests in the field of endodontics. This study compared between the properties of two formulations of white Portland cement (WPC) of different origin (Malaysia [MA] and Egypt [EG]). WPCs with and without calcium chloride dihydrate were prepared. Scanning electron microscope (SEM), energy dispersive X-ray micro-analysis, and X-ray diffraction were used for surface morphology evaluation, elemental, and phase analysis, respectively. After the preparation of optimized serial dilutions, the cytotoxicity was evaluated on human periodontal ligament fibroblasts (HPLFs) and dental pulp stem cells (DPSCs) using methyl-thiazol-diphenyltetrazolium assay after 24 and 72 h. Cell attachment properties were examined under SEM after 24 and 72 h. Results showed that the surface morphology and chemical composition of both formulations demonstrated detectable variations. The cytotoxicity evaluation showed different cellular responses of HPLFs compared to DSPCs. Both formulations favored the viability of HPLFs. However, the fast set formulations demonstrated severe cytotoxicity on DPSCs. Significant differences between EGWPC and MAWPC were identified (p < 0.05). The cell attachment properties were favorable; however, HPLFs attached and spread over the samples better than DPSCs. In conclusion, WPC of different origin may show differences in chemical and biological properties. The addition of CaCl2 ·2H2 O to WPC can affect its properties. Human cell types may react differently towards different formulations of WPCs. SCANNING 38:303-316, 2016.

DPSCs and SHED in Tissue Engineering and Regenerative Medicine

Dental pulp stem cells (DPSCs) from permanent teeth and stem cells from human exfoliated deciduous teeth (SHED) have attracted tremendous interest recently by playing a major role in tissue engineering and regenerative medicine. However, since stem cell technology is still in its infancy, interdisciplinary cooperation between medicine, ba- sic biological research, nanotechnology and materials science is needed to achieve successful clinical applications. Similar to mesenchymal stem cells, DPSCs and SHED can undergo self-renewal and have multipotent differentiation ability, but unlike other sources of stem cells such as embryonic stem cells, which involves the destruction of human embryo, DPSCs and SHED have limited ethical concerns as they are readily and easily accessible, non-invasive and disposed off naturally. Not only DPSCs and SHED can be used for cell based therapies and bio-artificial tissue constructs but also made to differentiate into other cell types. Here, we discuss on definitions, opportunities, advantages and limitations of DPSCs and SHED in tissue engineering and regenerative medicine.

White mineral trioxide aggregate mixed with calcium chloride dihydrate: chemical analysis and biological properties

Objectives This study aimed to evaluate the chemical and biological properties of fast-set white mineral trioxide aggregate (FS WMTA), which was WMTA combined with calcium chloride dihydrate (CaCl2·2H2O), compared to that of WMTA. Materials and Methods Surface morphology, elemental, and phase analysis were examined using scanning electron microscope (SEM), energy dispersive X-ray microanalysis (EDX), and X-ray diffraction (XRD), respectively. The cytotoxicity and cell attachment properties were evaluated on human periodontal ligament fibroblasts (HPLFs) using methyl-thiazol-diphenyltetrazolium (MTT) assay and under SEM after 24 and 72 hours, respectively. Results Results showed that the addition of CaCl2·2H2O to WMTA affected the surface morphology and chemical composition. Although FS WMTA exhibited a non-cytotoxic profile, the cell viability values of this combination were lesser than WMTA, and the difference was significant in 7 out of 10 concentrations at the 2 time intervals (p < 0.05). HPLFs adhered over the surface of WMTA and at the interface, after 24 hours of incubation. After 72 hours, there were increased numbers of HPLFs with prominent cytoplasmic processes. Similar findings were observed with FS WMTA, but the cells were not as confluent as with WMTA. Conclusions The addition of CaCl2·2H2O to WMTA affected its chemical properties. The favorable biological profile of FS WMTA towards HPLFs may have a potential impact on its clinical application for repair of perforation defects.