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Fulltext In vitro characterization of a chitosan skin regenerating template as a scaffold for cells cultivation

Hilmi AB, Halim AS, Hassan A, Lim CK, Noorsal K, Zainol I

Springerplus, 2013 Dec;2(1):79.
PMID: 23503998 DOI: 10.1186/2193-1801-2-79

Chitosan is a marine-derived product that has been widely used in clinical applications, especially in skin reconstruction. The mammalian scaffolds derived from bovine and porcine material have many limitations, for example, prion transmission and religious concerns. Therefore, we created a chitosan skin regenerating template (SRT) and investigated the behavior of fibroblast cell-scaffold constructs. Primary human dermal fibroblasts (HDF) were isolated and then characterized using vimentin and versican. HDF were seeded into chitosan SRT at a density of 3×10(6) cells/cm(2) for fourteen days. Histological analysis and live cells imaging revealed that the cell-chitosan constructs within interconnected porous chitosan showed significant interaction between the cells as well as between the cells and the chitosan. Scanning electron microscopy (SEM) analysis revealed cells spreading and covering the pores. As the pore sizes of the chitosan SRT range between 40-140 μm, an average porosity is about 93 ± 12.57% and water uptake ratio of chitosan SRT is 536.02 ± 14.29%, it is a supportive template for fibroblast attachment and has potential in applications as a dermal substitute.
In vitro cytotoxicology model of oligo-chitosan and n, o-carboxymethyl chitosan using primary normal human epidermal keratinocyte cultures

Lim CK, Halim AS, Lau HY, Ujang Z, Hazri A

J Appl Biomater Biomech, 2007 May-Aug;5(2):82-7.
PMID: 20799177

Chitosan (beta-1, 4-D-glucosamine) is a deacetylated form of chitin with excellent biological properties in wound management. The natural properties of chitosan have the physical and chemical limitations to be widely used in biomedical fields. The improvement of the physical and chemical properties of chitosan with some additional chemicals will alter its biocompatibility. Therefore, the biological attribute of the modified chitosan must be evaluated. In this study, the cytotoxicity of oligo-chitosan (OC) and N, O- carboxymethyl-chitosan (NO-CMC) derivatives (O-C 1%, O-C 5%, NO-CMC 1% and NO-CMC 5%) was evaluated using primary normal human epidermal keratinocyte (pNHEK) cultures as an in vitro toxicology model at standardized cell passages (fourth passages). 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl tetrazolium bromide (MTT) was used as a cell viability assay. The O-C 1% is one of the most compatible chitosan derivatives because it steadily sustained >70% of viable cells until 72 hr post-treatment. This was followed by O-C 5%, NO-CMC 5% and NO-CMC 1%. Therefore, oligo-chitosan had the ideal properties of a biocompatible material compared to N, O- carboxymethyl-chitosan in this study.
Fulltext In vitro models in biocompatibility assessment for biomedical-grade chitosan derivatives in wound management

Keong LC, Halim AS

Int J Mol Sci, 2009 Mar;10(3):1300-1313.
PMID: 19399250 DOI: 10.3390/ijms10031300

One of the ultimate goals of wound healing research is to find effective healing techniques that utilize the regeneration of similar tissues. This involves the modification of various wound dressing biomaterials for proper wound management. The biopolymer chitosan (beta-1,4-D-glucosamine) has natural biocompatibility and biodegradability that render it suitable for wound management. By definition, a biocompatible biomaterial does not have toxic or injurious effects on biological systems. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability to an uncertain degree. Hence, the modified biomedical-grade of chitosan derivatives should be pre-examined in vitro in order to produce high-quality, biocompatible dressings. In vitro toxicity examinations are more favorable than those performed in vivo, as the results are more reproducible and predictive. In this paper, basic in vitro tools were used to evaluate cellular and molecular responses with regard to the biocompatibility of biomedical-grade chitosan. Three paramount experimental parameters of biocompatibility in vitro namely cytocompatibility, genotoxicity and skin pro-inflammatory cytokine expression, were generally reviewed for biomedical-grade chitosan as wound dressing.
Internal mammary perforators as recipient vessels for deep inferior epigastric perforator and muscle-sparing free transverse rectus abdominis musculocutaneous flap breast reconstruction in an Asian population

Halim AS, Alwi AA

Ann Plast Surg, 2014 Aug;73(2):170-3.
PMID: 23528630 DOI: 10.1097/SAP.0b013e318270704b

The use of internal mammary perforators (IMPs) as recipient vessels in autologous free flap breast reconstruction has many additional benefits compared with the internal mammary or thoracodorsal vessels. Our goals were to analyze the characteristics of these vessels and to evaluate the reliability of using them in an Asian population.
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.
Keloid pathogenesis via Drosophila similar to mothers against decapentaplegic (SMAD) signaling in a primary epithelial-mesenchymal in vitro model treated with biomedical-grade chitosan porous skin regenerating template

Lim CK, Halim AS, Yaacob NS, Zainol I, Noorsal K

J Biosci Bioeng, 2013 Apr;115(4):453-8.
PMID: 23177217 DOI: 10.1016/j.jbiosc.2012.10.010

The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-β1 (TGF-β1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-β1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-β1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-β1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-β1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.
Fulltext Keloid scarring: understanding the genetic basis, advances, and prospects

Halim AS, Emami A, Salahshourifar I, Kannan TP

Arch Plast Surg, 2012 May;39(3):184-9.
PMID: 22783524 DOI: 10.5999/aps.2012.39.3.184

Keloid disease is a fibroproliferative dermal tumor with an unknown etiology that occurs after a skin injury in genetically susceptible individuals. Increased familial aggregation, a higher prevalence in certain races, parallelism in identical twins, and alteration in gene expression all favor a remarkable genetic contribution to keloid pathology. It seems that the environment triggers the disease in genetically susceptible individuals. Several genes have been implicated in the etiology of keloid disease, but no single gene mutation has thus far been found to be responsible. Therefore, a combination of methods such as association, gene-gene interaction, epigenetics, linkage, gene expression, and protein analysis should be applied to determine keloid etiology.
Laminated pedicled latissimus dorsi myocutaneous flap for single-stage reconstruction of a composite anterior neck and tracheal defect

Wu LY, Halim AS, Sain AH

J Otolaryngol, 2005 Feb;34(1):69-72.
PMID: 15966482
Fulltext Long-Term Nasolabial Appearance Post-Unilateral Cleft Lip Repair in a Single Center: A Descriptive Study

Al-Chalabi MMM, Wan Sulaiman WA, Halim AS

Cureus, 2023 Jul;15(7):e41683.
PMID: 37575768 DOI: 10.7759/cureus.41683

Introduction Modern treatments still aim to keep the impact of surgical intervention low and the outcome of surgeries as good as a surgeon can. Assessing the long-term nasolabial appearance of patients who underwent cleft lip (CL) repair surgery is one of the methods of evaluating the outcomes of cleft surgery. Methods This is a retrospective cross-sectional descriptive study of data records of unilateral CL patients. The data records of all patients who underwent unilateral CL repair by the reconstructive science unit at Hospital Universiti Sains Malaysia (HUSM) within the first two years of their lives and whose current age is 14 years or above were accessed and analyzed. Results The data records of 50 patients were analyzed, including 13 (26%) males and 37 (74%) females. The surgeons opined that 28% of the patients had an acceptable nasolabial appearance, while there were 10 (20%) patients whose nasolabial appearance was considered unacceptable by the reviewing surgeons. Fifteen (30%) patients were described as having an acceptable lip appearance with secondary nasal deformity, and 11 (22%) patients had an acceptable nasal appearance with secondary lip deformity. There were no surgical modifications or postoperative complications among the patients. None of our variables reported a significant association with long-term nasolabial appearance. Conclusion The long-term evaluation of the nasolabial appearance in individuals with CL following surgical correction significantly improves the service and care provided to patients to achieve optimum results. Although our results showed no relationship between gender, age at operation, type or diagnosis of cleft, and family history and long-term nasolabial appearance, frequent assessments will enhance surgical results.
Long-term effect on foot and ankle donor site following vascularized fibular graft resection in children

Sulaiman AR, Wan Z, Awang S, Che Ahmad A, Halim AS, Ahmad Mohd Zain R

J Pediatr Orthop B, 2015 Sep;24(5):450-5.
PMID: 26049965 DOI: 10.1097/BPB.0000000000000197

This study was carried out to evaluate the long-term effect on the donor side of the foot and ankle following vascularized fibular graft resection in children. Eight patients underwent resection of the fibula for the purpose of a vascularized fibular graft by a surgical team who practiced leaving at least 6 cm residual distal fibula. The age of these children at the time of surgery was between 3 and 12 years. They were reviewed between 3 and 12 years after surgery. Two patients who underwent resection of the middle shaft of the fibula at 3 and 5 years of age developed abnormal growth of the distal tibia, leading to ankle valgus. They were treated with growth modulation of the distal tibial physis and supramalleolar osteotomy with tibiofibular synostosis. Another patient who underwent the entire proximal fibula resection at the age of 6 years had developed hindfoot valgus because of weakness of the tibialis posterior muscle. He required talonavicular fusion and flexor hallucis to tibialis posterior muscle transfer. Patients operated at the age of older than 8 years neither had ankle nor hindfoot deformity. We concluded that resection of the middle shaft of the fibula for the purpose of a vascularized fibula graft, leaving a 6 cm distal fibular stump in children younger than 6 years old, may give rise to abnormal growth of the distal tibial physis, leading to valgus ankle. The entire proximal fibular resection for the similar purpose in a 6-year-old child may give rise to weakness of tibialis posterior and hindfoot valgus.
Long-term outcome of free fibula osteocutaneous flap and massive allograft in the reconstruction of long bone defect

Halim AS, Chai SC, Wan Ismail WF, Wan Azman WS, Mat Saad AZ, Wan Z

J Plast Reconstr Aesthet Surg, 2015 Dec;68(12):1755-62.
PMID: 26420474 DOI: 10.1016/j.bjps.2015.08.013

Reconstruction of massive bone defects in bone tumors with allografts has been shown to have significant complications including infection, delayed or nonunion of allograft, and allograft fracture. Resection compounded with soft tissue defects requires skin coverage. A composite osteocutaneous free fibula offers an optimal solution where the allografts can be augmented mechanically and achieve biological incorporation. Following resection, the cutaneous component of the free osteocutaneous fibula flaps covers the massive soft tissue defect. In this retrospective study, the long-term outcome of 12 patients, who underwent single-stage limb reconstruction with massive allograft and free fibula osteocutaneous flaps instead of free fibula osteal flaps only, was evaluated. This study included 12 consecutive patients who had primary bone tumors and had follow-up for a minimum of 24 months. The mean age at the time of surgery was 19.8 years. A total of eight patients had primary malignant bone tumors (five osteosarcomas, two chondrosarcomas and one synovial sarcoma), and four patients had benign bone tumors (two giant-cell tumors, one aneurysmal bone cyst, and one neurofibromatosis). The mean follow-up for the 12 patients was 63 months (range 24-124 months). Out of the 10 patients, nine underwent lower-limb reconstruction and ambulated with partial weight bearing and full weight bearing at an average of 4.2 months and 8.2 months, respectively. In conclusion, augmentation of a massive allograft with free fibula osteocutaneous flap is an excellent alternative for reducing the long-term complication of massive allograft and concurrently addresses the soft tissue coverage.
Maternal uniparental heterodisomy of chromosome 6 in a boy with an isolated cleft lip and palate

Salahshourifar I, Halim AS, Sulaiman WA, Zilfalil BA

Am J Med Genet A, 2010 Jul;152A(7):1818-21.
PMID: 20583164 DOI: 10.1002/ajmg.a.33526

We describe a chromosome 6 uniparental disomy (UPD6) in a boy, discovered during a screening for the genetic cause of cleft lip and palate. In the medical literature, almost all documented cases of UPD6 are paternal in origin, and only four were maternal. We present here a report of complete maternal chromosome 6 uniparental heterodisomy. Haplotype analysis was performed using highly polymorphic short tandem repeat (STR) markers that span both arms of chromosome 6. Analysis of these markers revealed the presence of two maternal alleles but no paternal allele, indicating an instance of maternal uniparental heterodisomy. Chromosome analysis of peripheral blood lymphocytes confirmed a normal male karyotype. Advanced maternal age at the time of the infant's birth and heterodisomy of markers around the centromere favors a meiosis-I error. No specific phenotype has been reported for maternal UPD6. Therefore, the cleft lip and palate in the present case probably occurred due to other risk factors. This report provides further evidence that maternal UPD6 has no specific clinical consequences and adds to the collective knowledge of this rare chromosomal finding.
Mechanism Action of Platelets and Crucial Blood Coagulation Pathways in Hemostasis

Periayah MH, Halim AS, Mat Saad AZ

Int J Hematol Oncol Stem Cell Res, 2017 Oct 01;11(4):319-327.
PMID: 29340130

Blood is considered to be precious because it is the basic necessity for health; our body needs a steady provision of oxygen, supplied via blood, to reach billions of tissues and cells. Hematopoiesis is the process that generates blood cells of all lineages. However, platelets are the smallest blood component produced from the very large bone marrow cells called megakaryocytes and they play a fundamental role in thrombosis and hemostasis. Platelets contribute their hemostatic capacity via adhesion, activation and aggregation, which are triggered upon tissue injury, and these actions stimulate the coagulation factors and other mediators to achieve hemostasis. In addition, these coordinated series of events are the vital biological processes for wound healing phases. The aim of this review is to summarize and highlight the important pathways involved in achieving hemostasis that are ruled by platelets. In addition, this review also describes the mechanism action of platelets, including adhesion, activation, aggregation, and coagulation, as well as the factors that aid in hemostasis and wound healing.
Method optimization on the use of postocclusive hyperemia model to assess microvascular function

Yvonne-Tee GB, Rasool AH, Halim AS, Wong AR, Rahman AR

Clin. Hemorheol. Microcirc., 2008;38(2):119-33.
PMID: 18198413

Recent development had allowed non-invasive assessment of microvascular function in vivo; however, the method has not been fully optimized and standardized. In this study, we aimed to characterize the "effective" occlusion duration needed to elicit sufficient postocclusive hyperemia (PORH) responses in forearm skin using laser Doppler fluximetry (LDF), in subjects with differing age, gender and menstrual phases.
Fulltext Modified Five-Flap Z-plasty for Surgical Correction of Webbed Neck Deformity in Turner Syndrome

Mohd Said MT, Halim AS, Mat Zain MA, Koh KL

Cureus, 2023 May;15(5):e39312.
PMID: 37346202 DOI: 10.7759/cureus.39312

Webbed neck deformity is a congenital anomaly that exists in several syndromes. Various techniques for surgically correcting the webbed neck deformity have been described in the literature, each comes with its own advantages and disadvantages. The aim of surgery is to achieve normal neck contour and symmetrical hairline, avoid excessive scarring over the anterior and lateral neck, and limit recurrence. In this report, we described our experience in managing a case of Turner syndrome with bilateral webbed neck deformity using the modified five-flap Z-plasty technique.
Fulltext Modular endoprosthetic replacement after total femur resection for malignant bone tumor

Faisham WI, Zulmi W, Halim AS

Med J Malaysia, 2005 Jul;60 Suppl C:45-8.
PMID: 16381283

Total femur endoprothesis is an alternative replacement for massive malignant bone tumor with intramedullary extension or skip lesion. Four patients underwent total femoral resection and replacement with megaprosthesis: three had primary malignant bone tumor and one had salvage procedure for aseptic loosening of the distal femoral replacement. Tumor-free margins were achieved in all patients with two patients required vascularized latissimus dorsi free flap cover for reconstruction of soft tissue defects. The average follow-up was 24 months (range 16 - 60 months). All four patients were still alive with three of them being disease-free and one survived even with the presence of lung metastasis. The functional results obtained were either excellent or good in all patients in accordance to the Musculoskeletal Tumors Society grading system.
Mutation screening of IRF6 among families with non-syndromic oral clefts and identification of two novel variants: review of the literature

Salahshourifar I, Wan Sulaiman WA, Halim AS, Zilfalil BA

Eur J Med Genet, 2012 Jun;55(6-7):389-93.
PMID: 22440537 DOI: 10.1016/j.ejmg.2012.02.006

Non-syndromic oral clefts share the main clinical features of Van der Woude Syndrome (VWS), with the exception of the lower lip pit. Thus, about 15% of VWS cases are indistinguishable from cases with non-syndromic oral clefts. IRF6 mutations are the major cause of VWS; however, variants in this gene show strong association with non-syndromic oral clefts, with a higher increased risk among cases with cleft lip only (CLO). A total of 39 individuals, including 16 patients with CLO and 23 patients with a family history of cleft, were examined for IRF6 mutations in the present study. Seven variants, including five known (c.-75-4 A>; G, c.-73T>; C, c.459G>; T 5, c.820G>; A, and c.1060 + 37C>; T) and two novel (c.-75-23G>; C and c.1380G>; T), were found. Both novel variants were inherited from non-affected parents and we did not find also in the 120 control chromosomes. In silico analysis revealed that both c.1380G>; T and c.-75-23G>; C variants may disrupts a putative exonic splicing enhancer and intronic splicing binding site for SC35, respectively. Taken together, the presence of deleterious IRF6 variants in patients with non-syndromic oral clefts could be most likely an evidence for VWS. While, IRF6 variants could, at best, contribute to clefting as part of a complex inheritance pattern, with both additional genes and environmental factors having a role.
Noninvasive assessment of cutaneous vascular function in vivo using capillaroscopy, plethysmography and laser-Doppler instruments: its strengths and weaknesses

Yvonne-Tee GB, Rasool AH, Halim AS, Rahman AR

Clin. Hemorheol. Microcirc., 2006;34(4):457-73.
PMID: 16687786

Given that functional abnormalities of the microcirculation are one of the primary abnormalities in cardiovascular disease pathogenesis, various noninvasive clinical tools have been developed recently to assess the microvascular function, particularly at the skin. The common techniques used to assess cutaneous microvascular function in vivo include capillaroscopy, venous occlusion plethysmography, and laser-Doppler instruments (laser-Doppler fluximetry and laser-Doppler imaging). These noninvasive techniques can be used as an early measure of functional abnormalities within the microvascular tree, predominantly in population at high risk for cardiovascular events. This review discusses some underlying application principle of these techniques, including its clinical significance, method reproducibility and limitations.
Fulltext Osteosarcoma: the outcome of limb salvage surgery

Faisham WI, Zulmi W, Halim AS, Biswal BM, Mutum SS

Med J Malaysia, 2004 Dec;59 Suppl F:24-34.
PMID: 15941157

We reviewed the surgical and oncological management 23 consecutive patients with osteosarcoma of the long bones to determine the outcome of limb salvage technique performed in our centre. All patients received neoadjuvant chemotherapy. There were 15 males and 8 females with a mean age at diagnosis of 19 years (9 to 36). The median follow-up was 30 months (10 to 60). Fifteen had lesion around the knee joint followed by three in the proximal humerus, two in distal humerus, two in the pelvis, and one in the distal tibia. Six patients presented with lung metastases at diagnosis. We performed limb salvage surgery to control local disease in 16 patients and amputation in 7. The resection margins of the primary lesion were adequate and free of tumour cells in all patients. Local recurrence developed in 1 patient of limb salvage group. The overall median survival was 22 months and actuarial survival was 52% at 3 years. Eleven patients died of pulmonary metastases within 2 years of follow-up. Median survival of the limb salvage surgery group was 30 months compared to 6 months in the amputation group. As per our experience, limb salvage technique is a feasible option in extremity osteosarcoma without compromising survival.
Outcome of the Modified Meek Technique in the Management of Major Pediatric Burns

Lee SZ, Halim AS, Wan Sulaiman WA, Mat Saad AZ

Ann Plast Surg, 2018 09;81(3):295-301.
PMID: 29994880 DOI: 10.1097/SAP.0000000000001565

INTRODUCTION: The modified Meek micrografting technique has been used in the treatment of severely burned patients and a number of articles have examined the use of the modified Meek technique in adults and in mixed-age groups. However, there is a paucity of research pertaining to the outcome in the pediatric age group. The aim of this study is to present our favorable outcome in pediatric major burns using the modified Meek technique.
METHODS: A retrospective review of burn cases in Hospital Universiti Sains Malaysia from 2010 to 2015 was conducted. Cases of major burns among pediatric patients grafted using the Meek technique were examined.
RESULTS: Twelve patients were grafted using the Meek technique. Ten (91.7%) patients were male, whereas 2 (8.3%) were female. The average age of patients was 6 years (range, 2-11 years). The average total body surface area was 35.4% (range, 15%-75%). Most burn mechanisms were due to flame injury (66.7%) as compared with scalds injury (16.7%) and chemical injury (16.7%). There was no mortality. All patients were completely grafted with a good donor site scar. The average graft take rate was 82.3%, although 8 cases had positive tissue cultures from the Meek-grafted areas. The average follow-up duration was 3.6 years (range, 1.1-6.7 years). Only 1 case developed contracture over minor joint.
CONCLUSIONS: The Meek technique is useful when there is a paucity of donor site in the pediatric group. The graft take is good, contracture formation is low, and this technique is cost-effective.

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