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  1. Setiawan AM, Kamarudin TA, Abd Ghafar N
    Front Cell Dev Biol, 2022;10:1045103.
    PMID: 36340030 DOI: 10.3389/fcell.2022.1045103
    Bone morphogenetic protein 4 (BMP4) is a member of the transforming growth factor beta (TGF-β) superfamily of cytokines responsible for stem cells' commitment to differentiation, proliferation, and maturation. To date, various studies have utilized BMP4 as a chemical inducer for in vitro differentiation of human mesenchymal stem cells (MSCs) based on its potential. BMP4 drives in vitro differentiation of ADSC via TGF-β signaling pathway by interactions with BMP receptors leading to the activation of smad-dependent and smad-independent pathways. The BMP4 signaling pathways are regulated by intracellular and extracellular BMP4 antagonists. Extracellular BMP4 antagonist prevents interaction between BMP4 ligand to its receptors, while intracellular BMP4 antagonist shutdowns the smad-dependent pathways through multiple mechanisms. BMP4 proved as one of the popular differentiation factors to induce ADSC differentiation into cell from mesodermal origin. However, addition of all-trans retinoic acid is also needed in trans-differentiation of ADSC into ectodermal lineage cells. Suggesting that both BMP4 and RA signaling pathways may be necessary to be activated for in vitro trans-differentiation of ADSC.
  2. Omar N, Abidin FZ, Das S, Abd Ghafar N, Haji Suhaimi F, Abd Latiff A, et al.
    Morphologie, 2010 May;94(305):36-9.
    PMID: 20359930 DOI: 10.1016/j.morpho.2010.03.001
    The latissimus dorsi is a muscle of the back which forms the posterior fold of the axilla and its tendon twists to insert into the floor of the intertubercular sulcus of the humerus. Occasionally, the muscle has a muscular slip which crosses the axilla to insert into the pectoralis major. This muscular slip is often termed as "axillary arch." In the present study, we report bilateral axillary arch detected in a 45-year-old male cadaver. The average vertical length of the axillary arch measured 7 cm. The average maximum width of the uppermost, middle and lower part of the arch measured 2, 3.5 and 3.2 cm, respectively. The presence of the axillary arch is an uncommon finding in humans, considering the fact that it is solely found in the animals who prefer to hang on the trees. A histological study of the axillary arch was also performed and it showed skeletal muscle fibres which was uniformly arranged. The presence of the axillary arch may assist in the adduction of the shoulder. It may also compress the axillary vessels and nerves thereby causing resultant symptoms. Prior anatomical knowledge of the presence of axillary arch may be helpful for surgeons performing radical dissection of the axillary lymph nodes and ligation of axillary vessels, clinicians diagnosing abduction syndromes and interventional radiologists interpreting axillary mass in day to day clinical practice.
  3. Wan Hasan WN, Chin KY, Abd Ghafar N, Soelaiman IN
    Drug Des Devel Ther, 2020;14:969-976.
    PMID: 32184566 DOI: 10.2147/DDDT.S224941
    Purpose: Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. However, the mechanism of action of AnTT in achieving these effects is unclear. This study aims to investigate the mechanism of action of AnTT on MC3T3-E1 pre-osteoblasts via the mevalonate pathway.

    Methods: Murine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 104 cells/mL and treated with 4 concentrations of AnTT (0.001-1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.

    Results: The results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.

    Conclusion: AnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.

  4. Soliman AM, Das S, Abd Ghafar N, Teoh SL
    Front Genet, 2018;9:38.
    PMID: 29491883 DOI: 10.3389/fgene.2018.00038
    Wound healing is a complex biological process that is generally composed of four phases: hemostasis, inflammation, proliferation, and remodeling. The proliferation phase is crucial for effective healing compared to other phases. Many critical events occur during this phase, i.e., migration of fibroblasts, re-epithelialization, angiogenesis and wound contraction. Chronic wounds are common and are considered a major public health problem. Therefore, there is the increasing need to discover new therapeutic strategies. MicroRNA (miRNA) research in the field of wound healing is in its early phase, but the knowledge of the recent discoveries is essential for developing effective therapies for the treatment of chronic wounds. In this review, we focused on recently discovered miRNAs which are involved in the proliferation phase of wound healing in the past few years and their role in wound healing.
  5. Ker-Woon C, Abd Ghafar N, Hui CK, Mohd Yusof YA
    BMC Cell Biol., 2014;15:19.
    PMID: 24885607 DOI: 10.1186/1471-2121-15-19
    Acacia honey is a natural product which has proven to have therapeutic effects on skin wound healing, but its potential healing effects in corneal wound healing have not been studied. This study aimed to explore the effects of Acacia honey (AH) on corneal keratocytes morphology, proliferative capacity, cell cycle, gene and protein analyses. Keratocytes from the corneal stroma of six New Zealand white rabbits were isolated and cultured until passage 1. The optimal dose of AH in the basal medium (FD) and medium containing serum (FDS) for keratocytes proliferation was identified using MTT assay. The morphological changes, gene and protein expressions of aldehyde dehydrogenase (ALDH), marker for quiescent keratocytes and vimentin, marker for fibroblasts were detected using q-RTPCR and immunocytochemistry respectively. Flowcytometry was performed to evaluate the cell cycle analysis of corneal keratocytes.
  6. Zainal Abidin F, Hui CK, Luan NS, Mohd Ramli ES, Hun LT, Abd Ghafar N
    PMID: 21992551 DOI: 10.1186/1472-6882-11-94
    There has been no effective treatment or agent that is available for corneal injury in promoting corneal wound healing. Previous studies on edible bird's nest extract (EBN) had reported the presence of hormone-like substance; avian epidermal growth factor that could stimulate cell division and enhance regeneration. This study aimed to investigate the effects of EBN on corneal keratocytes proliferative capacity and phenotypical changes.
  7. Hlaing KP, Das S, Sulaiman IM, Abd-Latiff A, Abd-Ghafar N, Suhaimi FH, et al.
    Bratisl Lek Listy, 2010;111(5):308-10.
    PMID: 20568426
    The renal artery is known to exhibit variations in its number and position. The present study was performed on 50 cadaveric kidneys to observe the topographical anatomy of the accessory renal arteries (ARA) entering the upper or lower poles of the kidney. Out of 50 kidney cadaveric specimens (irrespective of sex) studied, 2 kidneys (4%) showed the presence of ARA. The presence of ARA was observed on the left and right kidneys, respectively. In one left kidney, we observed in addition to the usual renal artery, an ARA near the lower pole of the kidney which divided into anterior and posterior branches. Another right kidney specimen exhibited the presence of single and double ARA at the upper and the lower poles, respectively. The presence of ARA, both at the upper and lower poles is a rare entity. No medical history of the cadavers was available to corroborate the clinical findings. Additional renal vessels may signify a developmental defect. Anatomical knowledge of the variations in the renal vascular supply may be important for abdominal imaging studies and surgical operations involving renal transplantations. The present study discusses in detail the anatomical features and clinical implications of ARA located at both the upper and lower poles of the kidney (Fig. 2, Ref. 15). Full Text (Free, PDF) www.bmj.sk.
  8. Das S, Haji Suhaimi F, Abd Latiff A, Pa Pa Hlaing K, Abd Ghafar N, Othman F
    Rom J Morphol Embryol, 2009;50(3):509-11.
    PMID: 19690784
    Peroneus tertius (PT) is a muscle of the anterior compartment of the leg. The PT muscle originates from the anterior surface of the fibula and the interosseous membrane and inserts into the medial side of the dorsal region of the fifth metatarsal bone. During routine dissection, we observed the absence of PT on the left lower limb of a cadaver. Usually, the PT is involved in dorsiflexion and eversion of the foot. In many cases, the absence of PT maybe asymptomatic and it may be incidentally detected during cadaveric dissections or autopsies. The existence of PT may help in the swing phase of bipedal walking. The PT may be used for tendon graft surgeries. The pull of the PT may be responsible for causing stress on the fifth metacarpal and account for all stress fractures in any individual. The absence of the PT may puzzle any transplant and foot surgeons performing graft operations. We as anatomists discuss the clinical implications of the absence of PT.
  9. Yusof AM, Abd Ghafar N, Kamarudin TA, Hui CK, Yusof YA
    PMID: 26912349 DOI: 10.1186/s12906-016-1055-7
    This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry.
  10. Ker-Woon C, Abd Ghafar N, Hui CK, Mohd Yusof YA, Wan Ngah WZ
    BMC Cell Biol., 2015;16:2.
    PMID: 25887200 DOI: 10.1186/s12860-015-0053-9
    Acacia honey (AH) has been proven to improve skin wound healing, but its therapeutic effects on corneal epithelium has not been elucidated to date. This study aimed to investigate the effects of AH on cultured corneal epithelial cells (CEC) on in vitro corneal abrasion wound healing model. Six New Zealand white rabbits' CEC were isolated and cultured until passage 1. Circular wound area was created onto a confluent monolayer CEC using a corneal trephine which mimicked corneal abrasion and treated with 0.025% AH supplemented in basal medium (BM) and complete cornea medium (CCM). Wound healing was measured as the percentage of wound closure by the migration of CEC on day 0, day 3 and day 6, post wound creation. The morphological changes of CEC were assessed via phase contrast microscopy. Gene and protein expressions of cytokeratin (CK3), fibronectin and cluster of differentiation 44 (CD44) in AH treated groups and control groups were determined by real-time PCR and immunocytochemistry, respectively.
  11. Azmi MF, Abd Ghafar N, Che Hamzah J, Chua KH, Ng SL
    J Food Biochem, 2021 04;45(4):e13645.
    PMID: 33569805 DOI: 10.1111/jfbc.13645
    This study aimed to investigate the role of Gelam honey (GH) in accelerating reepithelialization of corneal abrasion. Corneal epithelial cells (CEC) isolated from New Zealand white rabbit corneas, were cultured and circular-shaped wounds were created onto them, representing the corneal abrasion model. These wounds were treated with basal (BM) and cornea media (CM) supplemented with GH. The percentage of wound closure was measured on day 0, 3, and 5. Expressions of cytokeratin 3 (CK3), cluster of differentiation 44 (CD 44), and connexin 43 (Cx43) were analyzed via qRT-PCR and immunocytochemistry. The results showed CEC cultured in GH-enriched media reepithelialized faster compared to control. Corneal abrasion treated with CM supplemented with GH closed completely on day 5. CK3, CD44, and Cx43 expressions correspond to the stages of reepithelialization. In conclusion, GH promotes the healing of the ex vivo corneal abrasion model. Further explorations of its potential as adjuvant therapy in treating corneal injuries are needed. PRACTICAL APPLICATIONS: Honey has been reported to have many medicinal properties including antibacterial, anti-inflammatory, and the ability to promote skin wound healing. However, the effects of honey on corneal wound healing have not been fully elucidated. In the present study, we aimed to determine the effects of Gelam honey (GH), well-known local honey obtained from the beehive of Gelam trees (Melaleuca spp.), on the ex vivo corneal abrasion model via cell migration study and analysis of genes and proteins during corneal epithelial wound healing. GH has proven to have accelerated effects on the corneal epithelial cell migration during the closure of the ex vivo corneal abrasion wound model. The expressions of the genes and proteins of the corneal epithelial wound healing markers were in accordance with the stages of healing. Therefore, GH has the potential to be developed as adjuvant therapy in the form of GH-based eye drop in treating corneal injuries.
  12. Abd Ghafar N, Ker-Woon C, Hui CK, Mohd Yusof YA, Wan Ngah WZ
    BMC Complement Altern Med, 2016 Jul 29;16:259.
    PMID: 27473120 DOI: 10.1186/s12906-016-1248-0
    BACKGROUND: The study aimed to evaluate the effects of Acacia honey (AH) on the migration, differentiation and healing properties of the cultured rabbit corneal fibroblasts.

    METHODS: Stromal derived corneal fibroblasts from New Zealand White rabbit (n = 6) were isolated and cultured until passage 1. In vitro corneal ulcer was created using a 4 mm corneal trephine onto confluent cultures and treated with basal medium (FD), medium containing serum (FDS), with and without 0.025 % AH. Wound areas were recorded at day 0, 3 and 6 post wound creation. Genes and proteins associated with wound healing and differentiation such as aldehyde dehydrogenase (ALDH), vimentin, alpha-smooth muscle actin (α-SMA), collagen type I, lumican and matrix metalloproteinase 12 (MMP12) were evaluated using qRT-PCR and immunocytochemistry respectively.

    RESULTS: Cells cultured with AH-enriched FDS media achieved complete wound closure at day 6 post wound creation. The cells cultured in AH-enriched FDS media increased the expression of vimentin, collagen type I and lumican genes and decreased the ALDH, α-SMA and MMP12 gene expressions. Protein expression of ALDH, vimentin and α-SMA were in accordance with the gene expression analyses.

    CONCLUSION: These results demonstrated AH accelerate corneal fibroblasts migration and differentiation of the in vitro corneal ulcer model while increasing the genes and proteins associated with stromal wound healing.

  13. Ng SL, Nordin A, Abd Ghafar N, Suboh Y, Ab Rahim N, Chua KH
    Parasit Vectors, 2017 12 28;10(1):625.
    PMID: 29282148 DOI: 10.1186/s13071-017-2547-0
    BACKGROUND: In recent years, the concern of Acanthamoeba keratitis has increased since the infection is often associated with contact lens use. Partial 18S rRNA genotypic identification of Acanthamoeba isolates is important to correlate with pathophysiological properties in order to evaluate the degree of virulence. This is the first report of genotypic identification for clinical isolates of Acanthamoeba from corneal scrapings of keratitis in Malaysia. This study is also the first to correlate the mRNA expression of MBP and AhLBP as virulent markers for axenic strains of Acanthamoeba.

    RESULTS: In this study, ten clinical isolates were obtained from corneal scrapings. Rns genotype and intra-genotypic variation at the DF3 region of the isolates were identified. Results revealed that all clinical isolates belonged to the T4 genotype, with T4/6 (4 isolates), T4/2 (3 isolates), T4/16 (2 isolates) and one new genotype T4 sequence (T4/36), being determined. The axenic clinical isolates were cytopathogenic to rabbit corneal fibroblasts. MBP and AhLBP mRNA expression are directly correlated to Acanthamoeba cytopathic effect.

    CONCLUSIONS: All ten Malaysian clinical isolates were identified as genotype T4 which is predominantly associated with AK. Measuring the mRNA expression of Acanthamoeba virulent markers could be useful in the understanding of the pathogenesis of Acanthamoeba keratitis.

  14. Wan Hasan WN, Abd Ghafar N, Chin KY, Ima-Nirwana S
    Drug Des Devel Ther, 2018;12:1715-1726.
    PMID: 29942115 DOI: 10.2147/DDDT.S168935
    PURPOSE: Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis. However, detailed studies of the effects of AnTT on preosteoblastic cells were limited. This study was conducted to investigate the osteogenic effect of AnTT on preosteoblast MC3T3-E1 cells in a time-dependent manner.

    MATERIALS AND METHODS: Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001-1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively.

    RESULTS: The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner (P<0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups (P<0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 (P<0.05).

    CONCLUSION: Our results suggest that AnTT enhances the osteogenic activity by promoting the bone formation-related genes and proteins in a temporal and sequential manner.

  15. Harun MH, Sepian SN, Chua KH, Ropilah AR, Abd Ghafar N, Che-Hamzah J, et al.
    Hum. Cell, 2013 Mar;26(1):35-40.
    PMID: 21748521 DOI: 10.1007/s13577-011-0025-0
    The anterior surface of the eye is covered by several physically contiguous but histologically distinguishable epithelia overlying the cornea, limbus, bulbar conjunctiva, fornix conjunctiva, and palpebral conjunctiva. The self-renewing nature of the conjunctival epithelia makes their long-term survival ultimately dependent on small populations of stem cells. Hence, the objective of this study was to investigate the expression of the stem cell genes Sox2, OCT4, NANOG, Rex1, NES, and ABCG2 in cultured human conjunctival epithelium from different conjunctival zones, namely, the bulbar, palpebral and fornix zones. Three samples were taken from patients with primary pterygium and cataract (age range 56-66 years) who presented to our eye clinic at the UKM Medical Centre. The eye was examined with slit lamp to ensure there was no underlying ocular surface diseases and glaucoma. Conjunctival tissue was taken from patients who underwent a standard cataract or pterygium operation as a primary procedure. Tissues were digested, cultured, and propagated until an adequate number of cells was obtained. Total RNA was extracted and subjected to expression analysis of conjunctival epithelium genes (KRT4, KRT13, KRT19) and stem cell genes (Sox2, OCT4, NANOG, Rex1, NES, ABCG2) by reverse transcriptase-PCR and 2% agarose gel electrophoresis. The expression of Sox2, OCT4, and NANOG genes were detected in the fornical cells, while bulbar cells only expressed Sox2 and palpebral cells only expressed OCT4. Based on these results, the human forniceal region expresses a higher number of stem cell genes than the palpebral and bulbar conjunctiva.
    Study site: Eye clinic, Pusat Perubatan Universiti Kebangsaan Malaysia (PPUKM), Kuala Lumpur, Malaysia
  16. Jusoh AR, Abd Rahman N, Abd Latiff A, Othman F, Das S, Abd Ghafar N, et al.
    Rom J Morphol Embryol, 2010;51(1):163-6.
    PMID: 20191138
    The obturator artery (OA) originates from the internal iliac artery. Variation in the origin of the OA may be asymptomatic in individuals and occasionally be detected during routine cadaveric dissections or autopsies. In the present study, we observed the origin and the branching pattern of the OA on 34 lower limbs (17 right sides and 17 left sides) irrespective of sex. The bifurcation of the common iliac artery into internal and external iliac from the sacral ala varied between 4.3-5.3 cm. The distance of the origin of the anterior division of internal iliac artery from the bifurcation of common iliac artery varied between 1-6 cm. The distance of the origin of the posterior division of the internal iliac artery from the point of bifurcation of the common iliac artery varied between 0-6 cm. Out of 34 lower limbs studied, two specimens (5.8%) showed anomalous origin of the OA originating from the posterior division of the internal iliac artery. Of these two, one limb belonged to the right side while the other was from the left side. The anomalous OA gave off an inferior vesical branch to the prostate in both the specimens. No other associated anomalies regarding the origin or branching pattern of the OA were observed. Prior knowledge of the anatomical variations may be beneficial for vascular surgeons ligating the internal iliac artery or its branches and the radiologists interpreting angiograms of the pelvic region.
  17. Abd Ghafar N, Chua KH, Wan Ngah WZ, Che Hamzah J, Othman F, Abd Rahman R, et al.
    Cell Tissue Bank, 2014 Mar;15(1):25-34.
    PMID: 23292197 DOI: 10.1007/s10561-012-9360-y
    The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.
  18. Md Yusof A, Abd Ghafar N, Kamarudin TA, Chua KH, Azmi MF, Ng SL, et al.
    Cytotechnology, 2019 Dec;71(6):1121-1135.
    PMID: 31606844 DOI: 10.1007/s10616-019-00349-8
    This study evaluated the effects of Gelam honey (GH) on ex vivo corneal fibroblast ulcer model via wound healing assay, gene expression and immunocytochemistry. Corneal fibroblasts from New Zealand white rabbits were culture expanded. The corneal fibroblast wound healing capacity was observed by creating a circular wound onto confluent monolayer cells cultured in basal medium (BM), BM with GH, serum-enriched basal medium (BMS) and BMS with GH respectively. Wound healing assay and phenotypic characterization of the corneal fibroblast were performed at different stages of wound closure. Expression of aldehyde dehydrogenase (ALDH), vimentin, α-smooth muscle actin (α-SMA), lumican, collagen I and matrix metalloproteinase 12 (MMP 12) were measured at day 1, day 3 and complete wound closure day. Corneal fibroblast cultured in BMS with GH demonstrated the fastest wound closure, at day 5 post wounding. The gene expressions of ALDH and vimentin were higher than control groups while α-SMA expression was lower, in GH enriched media. The expressions of lumican, collagen I and MMP 12 were also higher in cells cultured in GH enriched media compared to the control groups. GH was shown to promote in vitro corneal fibroblast wound healing and may be a potential natural adjunct in the treatment of corneal wound.
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