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Fulltext Extraction of mitochondrial DNA from tooth dentin: application of two techniques

Ahmad Azlina, Berahim Zurairah, Sidek Mohamad Ros, Mokhtar Khairani Idah, Samsudin Abdul Rani

Archives of Orofacial Sciences, 2011;6(1):9-14.
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Mitochondrial DNA (mtDNA) is a hereditary material located in mitochondria and is normally maternally inherited. Mutational analysis performed on mtDNA proved that the mutations are closely related with a number of genetic illnesses, besides being exploitable for forensic identification. Those findings imply the importance of mtDNA in the scientific field. MtDNA can be found in abundance in tooth dentin where it is kept protected by the enamel, the hardest outer part of the tooth. In this study, two techniques of mtDNA extraction were compared to determine the efficacy between the two techniques. Teeth used for the study was collected from Dental Clinic, Hospital Universiti Sains Malaysia. After the removal of tooth from the tooth socket of the patient, the tooth was kept at -20C until use. Later, pulp tissue and enamel was excised using dental bur and only the root dentin was utilized for the isolation of mtDNA by crushing it mechanically into powdered form. MtDNA was extracted using the two published methods, Pfeifer and Budowle and then subjected to spectrophotometry DNA quantification and purity, Polymerase chain reaction (PCR) amplification of hypervariable-two region of mtDNA, followed by DNA sequencing to analyze the reliability of the extraction techniques. In conclusion, both techniques proved to be efficient and capable for the extraction of mtDNA from tooth dentin.
Fulltext Evaluation of Osteogenic and Cementogenic Potential of Periodontal Ligament Fibroblast Spheroids Using a Three-Dimensional In Vitro Model of Periodontium

Berahim Z, Moharamzadeh K, Jowett AK, Rawlinson A

Int J Dent, 2015;2015:605813.
PMID: 26633971 DOI: 10.1155/2015/605813

The aim of this study was to develop a three-dimensional in vitro model of periodontium to investigate the osteogenic and cementogenic differentiation potential of the periodontal ligament fibroblast (PDLF) spheroids within a dentin-membrane complex. PDLFs were cultured in both spheroid forms and monolayers and were seeded onto two biological collagen-based and synthetic membranes. Cell-membrane composites were then transferred onto dentin slices with fibroblasts facing the dentin surface and further cultured for 20 days. The composites were then processed for histology and immunohistochemical analyses for osteocalcin, Runx2, periostin, and cementum attachment protein (CAP). Both membranes seeded with PDLF-derived cells adhered to dentin and fibroblasts were present at the dentin interface and spread within both membranes. All membrane-cell-dentine composites showed positive staining for osteocalcin, Runx2, and periostin. However, CAP was not expressed by any of the tissue composites. It can be concluded that PDLFs exhibited some osteogenic potential when cultured in a 3D matrix in the presence of dentin as shown by the expression of osteocalcin. However the interaction of cells and dentin in this study was unable to stimulate cementum formation. The type of membrane did not have a significant effect upon differentiation, but fibroblast seeded-PGA membrane demonstrated better attachment to dentin than the collagen membrane.
Biologic interaction of three-dimensional periodontal fibroblast spheroids with collagen-based and synthetic membranes

Berahim Z, Moharamzadeh K, Rawlinson A, Jowett AK

J. Periodontol., 2011 May;82(5):790-7.
PMID: 21080786 DOI: 10.1902/jop.2010.100533

Cell-based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three-dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen-based membranes that have been used in guided tissue regeneration.
Fulltext Biological Interaction Between Human Gingival Fibroblasts and Vascular Endothelial Cells for Angiogenesis: A Co-culture Perspective

Um Min Allah N, Berahim Z, Ahmad A, Kannan TP

Tissue Eng Regen Med, 2017 Oct;14(5):495-505.
PMID: 30603504 DOI: 10.1007/s13770-017-0065-y

Advancement in cell culture protocols, multidisciplinary research approach, and the need of clinical implication to reconstruct damaged or diseased tissues has led to the establishment of three-dimensional (3D) test systems for regeneration and repair. Regenerative therapies, including dental tissue engineering, have been pursued as a new prospect to repair and rebuild the diseased/lost oral tissues. Interactions between the different cell types, growth factors, and extracellular matrix components involved in angiogenesis are vital in the mechanisms of new vessel formation for tissue regeneration. In vitro pre-vascularization is one of the leading scopes in the tissue-engineering field. Vascularization strategies that are associated with co-culture systems have proved that there is communication between different cell types with mutual beneficial effects in vascularization and tissue regeneration in two-dimensional or 3D cultures. Endothelial cells with different cell populations, including osteoblasts, smooth muscle cells, and fibroblasts in a co-culture have shown their ability to advocate pre-vascularization. In this review, a co-culture perspective of human gingival fibroblasts and vascular endothelial cells is discussed with the main focus on vascularization and future perspective of this model in regeneration and repair.
Properties of Cell Sources in Tissue-Engineered Three-dimensional Oral Mucosa Model: A Review

Mohd Nor NH, Berahim Z, Ahmad A, Kannan TP

Curr Stem Cell Res Ther, 2017;12(1):52-60.
PMID: 27538403

Oral mucosa is a mucous membrane lining the oral cavity. Its main function is to protect the deeper structures against the external factors; thermal, chemical, mechanical and biological stimuli. Apart from that, it also plays a significant role during mastication, deglutition and speech. Some oral diseases or injuries to oral mucosa lead to impairment of the oral functions and aesthetics which eventually result in permanent defect of oral mucosa. In order to overcome this defect, different approaches for the development of reconstructed oral mucosa models have been employed including skin/autologous grafts, guided tissue replacement, vestibuloplasty etc. However, the finding of an acceptable source for the transplantations or autologous grafts seems a bit challenging. To overcome this problem, the development of oral mucosa using tissue engineering approach has been widely studied involving various cell lines from different sources. This paper aims to highlight various cell sources used in the development of tissueengineered oral mucosa models based on articles retrieved from PubMed and MEDLINE databases using the search terms "oral mucosa tissue engineering", regardless of time when published.
Identification of novel fibroblast-like cells from stem cells from human exfoliated deciduous teeth

Mohd Nor NH, Berahim Z, Azlina A, Kannan TP

Clin Oral Investig, 2019 Nov;23(11):3959-3966.
PMID: 30847574 DOI: 10.1007/s00784-019-02827-x

OBJECTIVES: This study aimed to differentiate and characterize fibroblast-like cells from stem cells from human exfoliated deciduous teeth (SHED).
MATERIALS AND METHODS: The differentiation of fibroblast-like cells from SHED was carried out by using specific human recombinant connective tissue growth factor (CTGF). To characterize fibroblastic differentiation, the induced cells were subjected to morphological changes, proliferation rate, gene expression analysis using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and immunofluorescence staining. The commercial primary human gingival fibroblasts served as positive control in this study.
RESULTS: The results from characterization analysis were compared with that of commercial cells to ensure that the cells differentiated from SHED were fibroblast-like cells. The results showed the inductive effect of CTGF for fibroblastic differentiation in SHED. SHED-derived fibroblasts were successfully characterized despite having similar morphological appearance, i.e., (i) significant proliferation rate between fibroblast-like cells and SHED, (ii) high expression of fibroblast-associated markers in qRT-PCR analysis, and (iii) positive staining against collagen type 1, fibroblast-specific protein 1, and human thymic fibroblasts in flow cytometry analysis and immunofluorescence staining. The same expression patterns were found in primary human gingival fibroblasts, respectively. SHED as negative control showed lower expression or no signal, thus confirming the cells differentiated from SHED were fibroblast-like cells.
CONCLUSIONS: Taken together, the protocol adopted in this study suggests CTGF to be an appropriate inducer in the differentiation of SHED into fibroblast-like cells.
CLINICAL RELEVANCE: The fibroblast-like cells differentiated from SHED could be used in future in vitro and in vivo dental tissue regeneration studies as well as in clinical applications where these cells are needed.
Fulltext Application of neurotransmitters and dental stem cells for pulp regeneration: A review

Ramli H, Yusop N, Ramli R, Berahim Z, Peiris R, Ghani N

Saudi Dent J, 2023 Jul;35(5):387-394.
PMID: 37520592 DOI: 10.1016/j.sdentj.2023.05.004

INTRODUCTION: Although there have been many studies on stem cells, few have investigated how neurotransmitters and stem cell proliferation interact to regenerate dental pulp. Dental pulp regeneration is an innovative procedure for reviving dental pulp, if feasible for the entire tooth. Upon tooth injury, activated platelets release serotonin and dopamine in bulk to mobilize dental pulp stem cells to mediate natural dental repair. This has induced research on the role of neurotransmitters in increasing the proliferation rate of stem cells. This review also covers prospective future treatments for dental pulp regeneration.
METHODS: A literature search was performed via PubMed and ScienceDirect from 2001 to 2022, using the keywords "neurotransmitter," "stem cell," "tooth regeneration," "tooth repair," "regenerative dentistry," and "dental pulp." Different inclusion/exclusion criteria were used, and the search was restricted to English articles.
RESULTS: Nine publications reporting neurotransmitter interactions with stem cells for tooth and pulp regeneration were selected.
CONCLUSION: Neurotransmitters were found to interact with dental stem cells. Evidence pointing to neurotransmitters as a factor in the increased proliferation of stem cells was found. This review thus gives hope for tooth pulp regeneration and repair.
Biodegradability of Amniotic Membrane as Potential Scaffold for Periodontal Regeneration

Ling KE, Roslan SM, Taib H, Berahim Z

Cureus, 2023 Sep;15(9):e45394.
PMID: 37854737 DOI: 10.7759/cureus.45394

Background In the periodontal regenerative procedure, the membrane used should possess good mechanical stability with suitable resorption time to allow restoration of the lost periodontium. Amniotic membrane (AM) has regenerative potential as a scaffold or barrier membrane due to its various beneficial properties. However, its degradation rate is not clearly reported. Methodology This study aimed to evaluate the resorption capacity of AM and its surface architecture after being subjected to hydrolytic degradation analysis in phosphate buffer solution (PBS). AM was cut into sizes of 10 × 10 mm2 for three replicates. The membranes were weighed before and at different time intervals (days 7, 14, 21, and 28) after immersion in PBS. The degradation rate was determined by the percentage of mean weight loss from the initial weight at different time intervals. The AM surface profile was observed under scanning electron microscopy (SEM) before and after 28 days of immersion. Results The result shows a 92% loss of weight over 28 days with the highest attained in the first seven days (67%), followed by 7%, 17%, and 1% after days 14, 21, and 28, respectively. SEM of the AM surface before the degradation test showed a polygonal shape forming a well-arranged mosaic pattern covered with microvilli. At day 28, the remaining AM appears as porous surface architecture, irregularly arranged fibers, and no microvilli seen. Conclusions This study demonstrated that over four weeks of degradation analysis, AM was not entirely degraded but had lost some of the microstructure. The biodegradability of AM should be further evaluated to elucidate its stability within adequate time parallel with the tissue healing process in periodontal tissue regeneration.
Synergistic effects of chitosan scaffold and TGFβ1 on the proliferation and osteogenic differentiation of dental pulp stem cells derived from human exfoliated deciduous teeth

Farea M, Husein A, Halim AS, Abdullah NA, Mokhtar KI, Lim CK, et al.

Arch Oral Biol, 2014 Dec;59(12):1400-11.
PMID: 25222336 DOI: 10.1016/j.archoralbio.2014.08.015

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.
Cementoblastic lineage formation in the cross-talk between stem cells of human exfoliated deciduous teeth and epithelial rests of Malassez cells

Farea M, Husein A, Halim AS, Berahim Z, Nurul AA, Mokhtar KI, et al.

Clin Oral Investig, 2016 Jul;20(6):1181-91.
PMID: 26392396 DOI: 10.1007/s00784-015-1601-6

The purpose of this study was to evaluate the synergistic effect of epithelial rests of Malassez cells (ERM) and transforming growth factor-β1 (TGF-β1) on proliferation, cementogenic and osteogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED).
Identification and Characterization of Intraoral and Dermal Fibroblasts Revisited

Mohd Nor NH, Berahim Z, Azlina A, Mokhtar KI, Kannan TP

Curr Stem Cell Res Ther, 2017;12(8):675-681.
PMID: 28969579 DOI: 10.2174/1574888X12666170929124621

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.
Fulltext Phytochemical Compounds of Raw Versus Methanol-Extracted Kelulut, Tualang, and Manuka Honeys

Chu LI, Berahim Z, Mohamad S, Shahidan WNS, Yhaya MF, Zainuddin SLA

Cureus, 2023 Apr;15(4):e38297.
PMID: 37255896 DOI: 10.7759/cureus.38297

Honey has been widely used for medicinal purposes since ancient times. It is produced by stinging bees or stingless bees by processing the collected nectar or plant sap in their bodies into raw honey. Extraction of honey will result in the pooling of crude volatile bioactive materials that could enhance its benefits. This work aims to compare the phytochemical characteristics of raw and methanol-extracted honeys in the Kelulut, Tualang and Manuka honeys. All types of raw honey samples were extracted by using the methanol extraction method and both groups were analysed using gas chromatography/mass spectrometry (GC/MS) at the National Poison Centre, Universiti Sains Malaysia, Malaysia. The findings showed that 23 compounds were identified in raw Kelulut honey and 18 compounds in methanol-extracted Kelulut honey; 28 compounds were identified in raw Tualang honey and 29 compounds in methanol-extracted Tualang honey; 19 compounds in raw Manuka honey and 22 compounds in methanol-extracted Manuka honey. There were differences in the phytochemical substances detected in raw and methanol-extracted honeys. The major compounds in raw honeys were mostly from the ketone, alcohol, and ester groups, whereas the ketone group was dominant in methanol-extracted honeys. Most bioactive substances identified in the methanol-extracted variant of honeys were more concentrated than the raw variant. A majority of these substances have antimicrobial characteristics.
Fulltext Isolation and enhancement of a homogenous in vitro human Hertwig's epithelial root sheath cell population

Farea M, Halim AS, Abdullah NA, Lim CK, Mokhtar KI, Berahim Z, et al.

Int J Mol Sci, 2013;14(6):11157-70.
PMID: 23712356 DOI: 10.3390/ijms140611157

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.

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