Displaying publications 1 - 20 of 24 in total

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  1. Mat Afandi MA, Maarof M, Chowdhury SR, Bt Hj Idrus R
    Tissue Eng Regen Med, 2020 12;17(6):835-845.
    PMID: 32767029 DOI: 10.1007/s13770-020-00283-3
    BACKGROUND: One of the long-standing problems of myoblasts in vitro expansion is slow cell migration and this causes fibroblast population to exceed myoblasts. In this study, we investigated the synergistic effect of laminin and epidermal growth factor (EGF) on co-cultured myoblasts and fibroblasts for cell attachment, proliferation and migration.

    METHODS: Skeletal human muscle cells were cultured in four different conditions; control, EGF, laminin (Lam) and laminin EGF (Lam + EGF). Using live imaging system, their cellular properties; attachment, migration and growth were exposed to Rho kinase inhibitor, Y-27632, and EGF-receptor (EGF-R) inhibitor, gefitinib were measured.

    RESULTS: Myoblast migration and proliferation was enhanced significantly by synergistic stimulation of laminin and EGF (0.61 ± 0.14 µm/min, 0.008 ± 0.001 h-1) compare to that by EGF alone (0.26 ± 0.13 µm/min, 0.004 ± 0.0009 h-1). However, no changes in proliferation and migration were observed for fibroblasts among the culture conditions. Inhibition of Rho kinase resulted in the increase of the myoblast migration on the laminin-coated surface with EGF condition (0.64 ± 0.18 µm/min). Compared to the untreated conditions, myoblasts cultured on the laminin-coated surface and EGF demonstrated elongated morphology, and average cell length increase significantly. In contrast, inhibition of EGF-R resulted in the decrease of myoblast migration on the laminin coated surface with EGF supplemented condition (0.43 ± 0.05 µm/min) in comparison to the untreated control (0.53 ± 0.05 µm/min).

    CONCLUSION: Laminin and EGF preferentially enhance the proliferation and migration of myoblasts, and Rho kinase and EGF-R play a role in this synergistic effect. These results will be beneficial for the propagation of skeletal muscle cells for clinical applications.

  2. Barekatain B, Khezrimotlagh D, Aizaini Maarof M, Ghaeini HR, Salleh S, Quintana AA, et al.
    PLoS One, 2013;8(8):e69844.
    PMID: 23940530 DOI: 10.1371/journal.pone.0069844
    In recent years, Random Network Coding (RNC) has emerged as a promising solution for efficient Peer-to-Peer (P2P) video multicasting over the Internet. This probably refers to this fact that RNC noticeably increases the error resiliency and throughput of the network. However, high transmission overhead arising from sending large coefficients vector as header has been the most important challenge of the RNC. Moreover, due to employing the Gauss-Jordan elimination method, considerable computational complexity can be imposed on peers in decoding the encoded blocks and checking linear dependency among the coefficients vectors. In order to address these challenges, this study introduces MATIN which is a random network coding based framework for efficient P2P video streaming. The MATIN includes a novel coefficients matrix generation method so that there is no linear dependency in the generated coefficients matrix. Using the proposed framework, each peer encapsulates one instead of n coefficients entries into the generated encoded packet which results in very low transmission overhead. It is also possible to obtain the inverted coefficients matrix using a bit number of simple arithmetic operations. In this regard, peers sustain very low computational complexities. As a result, the MATIN permits random network coding to be more efficient in P2P video streaming systems. The results obtained from simulation using OMNET++ show that it substantially outperforms the RNC which uses the Gauss-Jordan elimination method by providing better video quality on peers in terms of the four important performance metrics including video distortion, dependency distortion, End-to-End delay and Initial Startup delay.
  3. Maarof M, Mh Busra MF, Lokanathan Y, Bt Hj Idrus R, Rajab NF, Chowdhury SR
    Drug Deliv Transl Res, 2019 02;9(1):144-161.
    PMID: 30547385 DOI: 10.1007/s13346-018-00612-z
    Skin substitutes are one of the main treatments for skin loss, and a skin substitute that is readily available would be the best treatment option. However, most cell-based skin substitutes require long production times, and therefore, patients endure long waiting times. The proteins secreted from the cells and tissues play vital roles in promoting wound healing. Thus, we aimed to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts from human skin samples were cultured using serum-free keratinocyte-specific media (KM1 or KM2) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. The acellular 3D skin patch was soft, semi-solid, and translucent. Collagen mixed with DFCM-KM1 and DFCM-KM2 showed higher protein release compared to collagen plus DFCM-FM. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce an immune response. The implantation of the 3D skin patch with or without DFCM on the dorsum of BALB/c mice demonstrated a significantly faster healing rate compared to the no-treatment group 7 days after implantation, and all groups had complete re-epithelialization at day 17. Histological analysis confirmed the structure and integrity of the regenerated skin, with positive expression of cytokeratin 14 and type I collagen in the epidermal and dermal layer, respectively. These findings highlight the possibility of using fibroblast secretory factors together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full-thickness skin loss.
  4. Maarof M, Lokanathan Y, Ruszymah HI, Saim A, Chowdhury SR
    Protein J, 2018 12;37(6):589-607.
    PMID: 30343346 DOI: 10.1007/s10930-018-9800-z
    Growth factors and extracellular matrix (ECM) proteins are involved in wound healing. Human dermal fibroblasts secrete wound-healing mediators in culture medium known as dermal fibroblast conditioned medium (DFCM). However, the composition and concentration of the secreted proteins differ with culture conditions and environmental factors. We cultured human skin fibroblasts in vitro using serum-free keratinocyte-specific media (EpiLife™ Medium [KM1] and defined keratinocyte serum-free medium [KM2]) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. We identified and compared their proteomic profiles using bicinchoninic acid assay (BCA), 1-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1D SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography MS (LC-MS/MS). DFCM-KM1 and DFCM-KM2 had higher protein concentrations than DFCM-FM but not statistically significant. MALDI-TOF/TOF MS identified the presence of fibronectin, serotransferrin, serpin and serum albumin. LC-MS/MS and bioinformatics analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2 and DFCM-FM, respectively. The most significant biological processes identified in gene ontology were cellular process, metabolic process, growth and biological regulation. STRING® analysis showed that most secretory proteins in the DFCMs were associated with biological processes (e.g. wound healing and ECM organisation), molecular function (e.g. ECM binding) and cellular component (e.g. extracellular space). ELISA confirmed the presence of fibronectin and collagen in the DFCMs. In conclusion, DFCM secretory proteins are involved in cell adhesion, attachment, proliferation and migration, which were demonstrated to have potential wound-healing effects by in vitro and in vivo studies.
  5. Riha SM, Maarof M, Fauzi MB
    Polymers (Basel), 2021 May 12;13(10).
    PMID: 34065898 DOI: 10.3390/polym13101546
    Skin tissue engineering has made remarkable progress in wound healing treatment with the advent of newer fabrication strategies using natural/synthetic polymers and stem cells. Stem cell therapy is used to treat a wide range of injuries and degenerative diseases of the skin. Nevertheless, many related studies demonstrated modest improvement in organ functions due to the low survival rate of transplanted cells at the targeted injured area. Thus, incorporating stem cells into biomaterial offer niches to transplanted stem cells, enhancing their delivery and therapeutic effects. Currently, through the skin tissue engineering approach, many attempts have employed biomaterials as a platform to improve the engraftment of implanted cells and facilitate the function of exogenous cells by mimicking the tissue microenvironment. This review aims to identify the limitations of stem cell therapy in wound healing treatment and potentially highlight how the use of various biomaterials can enhance the therapeutic efficiency of stem cells in tissue regeneration post-implantation. Moreover, the review discusses the combined effects of stem cells and biomaterials in in vitro and in vivo settings followed by identifying the key factors contributing to the treatment outcomes. Apart from stem cells and biomaterials, the role of growth factors and other cellular substitutes used in effective wound healing treatment has been mentioned. In conclusion, the synergistic effect of biomaterials and stem cells provided significant effectiveness in therapeutic outcomes mainly in wound healing improvement.
  6. Thambirajoo M, Maarof M, Lokanathan Y, Katas H, Ghazalli NF, Tabata Y, et al.
    Antibiotics (Basel), 2021 Nov 02;10(11).
    PMID: 34827276 DOI: 10.3390/antibiotics10111338
    Nanotechnology has become an emerging technology in the medical field and is widely applicable for various clinical applications. The potential use of nanoparticles as antimicrobial agents is greatly explored and taken into consideration as alternative methods to overcome the challenges faced by healthcare workers and patients in preventing infections caused by pathogenic microorganisms. Among microorganisms, bacterial infections remain a major hurdle and are responsible for high morbidity and mortality globally, especially involving those with medical conditions and elderly populations. Over time, these groups are more vulnerable to developing resistance to antibiotics, as bacterial biofilms are difficult to destroy or eliminate via antibiotics; thus, treatment becomes unsuccessful or ineffective. Mostly, bacterial biofilms and other microbes can be found on medical devices and wounds where they disperse their contents which cause infections. To inhibit biofilm formations and overcome antibiotic resistance, antimicrobial-loaded nanoparticles alone or combined with other substances could enhance the bactericidal activity of nanomaterials. This includes killing the pathogens effectively without harming other cells or causing any adverse effects to living cells. This review summarises the mechanisms of actions employed by the different types of nanoparticles which counteract infectious agents in reducing biofilm formation and improve antibiotic therapy for clinical usage.
  7. Masri S, Maarof M, Mohd NF, Hiraoka Y, Tabata Y, Fauzi MB
    Biomedicines, 2022 Oct 20;10(10).
    PMID: 36289912 DOI: 10.3390/biomedicines10102651
    The irregular shape and depth of wounds could be the major hurdles in wound healing for the common three-dimensional foam, sheet, or film treatment design. The injectable hydrogel is a splendid alternate technique to enhance healing efficiency post-implantation via injectable or 3D-bioprinting technologies. The authentic combination of natural and synthetic polymers could potentially enhance the injectability and biocompatibility properties. Thus, the purpose of this study was to characterise a hybrid gelatin−PVA hydrogel crosslinked with genipin (GNP; natural crosslinker). In brief, gelatin (GE) and PVA were prepared in various concentrations (w/v): GE, GPVA3 (3% PVA), and GPVA5 (5% PVA), followed by a 0.1% (w/v) genipin (GNP) crosslink, to achieve polymerisation in three minutes. The physicochemical and biocompatibility properties were further evaluated. GPVA3_GNP and GPVA5_GNP with GNP demonstrated excellent physicochemical properties compared to GE_GNP and non-crosslinked hydrogels. GPVA5_GNP significantly displayed the optimum swelling ratio (621.1 ± 93.18%) and excellent hydrophilicity (38.51 ± 2.58°). In addition, GPVA5_GNP showed an optimum biodegradation rate (0.02 ± 0.005 mg/h) and the highest mechanical strength with the highest compression modulus (2.14 ± 0.06 MPa). In addition, the surface and cross-sectional view for scanning electron microscopy (SEM) displayed that all of the GPVA hydrogels have optimum average pore sizes (100−199 μm) with interconnected pores. There were no substantial changes in chemical analysis, including FTIR, XRD, and EDX, after PVA and GNP intervention. Furthermore, GPVA hydrogels influenced the cell biocompatibility, which successfully indicated >85% of cell viability. In conclusion, gelatin−PVA hydrogels crosslinked with GNP were proven to have excellent physicochemical, mechanical, and biocompatibility properties, as required for potential bioinks for chronic wound healing.
  8. Zulkiflee I, Amirrah IN, Fadilah NIM, Wee MFMR, Yusop SM, Maarof M, et al.
    Materials (Basel), 2023 Jan 29;16(3).
    PMID: 36770168 DOI: 10.3390/ma16031162
    A skin wound without immediate treatment could delay wound healing and may lead to death after severe infection (sepsis). Any interruption or inappropriate normal wound healing, mainly in these wounds, commonly resulted in prolonged and excessive skin contraction. Contraction is a common mechanism in wound healing phases and contributes 40-80% of the original wound size post-healing. Even though it is essential to accelerate wound healing, it also simultaneously limits movement, mainly in the joint area. In the worst-case scenario, prolonged contraction could lead to disfigurement and loss of tissue function. This study aimed to fabricate and characterise the elastin-fortified gelatin/polyvinyl alcohol (PVA) film layered on top of a collagen sponge as a bilayer hybrid biomatrix. Briefly, the combination of halal-based gelatin (4% (w/v)) and PVA ((4% (w/v)) was used to fabricate composite film, followed by the integration of poultry elastin (0.25 mg/mL) and 0.1% (w/v) genipin crosslinking. Furthermore, further analysis was conducted on the composite bilayer biomatrix's physicochemical and mechanical strength. The bilayer biomatrix demonstrated a slow biodegradation rate (0.374967 ± 0.031 mg/h), adequate water absorption (1078.734 ± 42.33%), reasonable water vapour transmission rate (WVTR) (724.6467 ± 70.69 g/m2 h) and porous (102.5944 ± 28.21%). The bilayer biomatrix also exhibited an excellent crosslinking degree and was mechanically robust. Besides, the elastin releasing study presented an acceptable rate post-integration with hybrid biomatrix. Therefore, the ready-to-use bilayer biomatrix will benefit therapeutic effects as an alternative treatment for future diabetic skin wound management.
  9. Fadilah NIM, Riha SM, Mazlan Z, Wen APY, Hao LQ, Joseph B, et al.
    Front Bioeng Biotechnol, 2023;11:1160577.
    PMID: 37292094 DOI: 10.3389/fbioe.2023.1160577
    Skin tissue engineering possesses great promise in providing successful wound injury and tissue loss treatments that current methods cannot treat or achieve a satisfactory clinical outcome. A major field direction is exploring bioscaffolds with multifunctional properties to enhance biological performance and expedite complex skin tissue regeneration. Multifunctional bioscaffolds are three-dimensional (3D) constructs manufactured from natural and synthetic biomaterials using cutting-edge tissue fabrication techniques incorporated with cells, growth factors, secretomes, antibacterial compounds, and bioactive molecules. It offers a physical, chemical, and biological environment with a biomimetic framework to direct cells toward higher-order tissue regeneration during wound healing. Multifunctional bioscaffolds are a promising possibility for skin regeneration because of the variety of structures they provide and the capacity to customise the chemistry of their surfaces, which allows for the regulated distribution of bioactive chemicals or cells. Meanwhile, the current gap is through advanced fabrication techniques such as computational designing, electrospinning, and 3D bioprinting to fabricate multifunctional scaffolds with long-term safety. This review stipulates the wound healing processes used by commercially available engineered skin replacements (ESS), highlighting the demand for a multifunctional, and next-generation ESS replacement as the goals and significance study in tissue engineering and regenerative medicine (TERM). This work also scrutinise the use of multifunctional bioscaffolds in wound healing applications, demonstrating successful biological performance in the in vitro and in vivo animal models. Further, we also provided a comprehensive review in requiring new viewpoints and technological innovations for the clinical application of multifunctional bioscaffolds for wound healing that have been found in the literature in the last 5 years.
  10. Fadilah NIM, Ahmat N, Hao LQ, Maarof M, Rajab NF, Idrus RBH, et al.
    Polymers (Basel), 2023 May 24;15(11).
    PMID: 37299233 DOI: 10.3390/polym15112436
    Wound care management is incredibly challenging for chronic injuries, despite the availability of various types of wound care products in the market. However, most current wound-healing products do not attempt to mimic the extracellular matrix (ECM) and simply provide a barrier function or wound covering. Collagen is a natural polymer that involves a major constituent of the ECM protein, thus making it attractive to be used in skin tissue regeneration during wound healing. This study aimed to validate the biological safety assessments of ovine tendon collagen type-I (OTC-I) in the accredited laboratory under ISO and GLP settings. It is important to ensure that the biomatrix will not stimulate the immune system to produce any adverse reaction. Therefore, we successfully extracted collagen type-I from the ovine tendon (OTC- I) using a method of low-concentration acetic acid. The three-dimensional (3D) skin patch of spongy OTC-I was a soft and white colour, being tested for safety and biocompatibility evaluations based on ISO 10993-5, ISO 10993-10, ISO 10993-11, ISO 10993-23, USP 40 <151>, and OECD 471. For the dermal sensitisation and acute irritation test, none of the tested animals displayed any erythema or oedema effects (p > 0.005). In addition, there were no abnormalities detected in the organ of the mice after being exposed to OTC-I; additionally, no morbidity and mortality were observed in the acute systemic test under the guideline of ISO 10993-11:2017. The grade 0 (non-reactive) based on ISO 10993-5:2009 was graded for the OTC-I at 100% concentration and the mean number of the revertant colonies did not exceed 2-fold of the 0.9% w/v sodium chloride compared to the tester strains of S. typhimurium (TA100, TA1535, TA98, TA1537), and E. coli (WP2 trp uvrA). Our study revealed that OTC-I biomatrix does not present any adverse effects or abnormalities in the present study's condition of induced skin sensitization effect, mutagenic and cytotoxic towards cells and animals. This biocompatibility assessment demonstrated a good agreement between in vitro and in vivo results regarding the absence of skin irritation and sensitization potential. Therefore, OTC-I biomatrix is a potential medical device candidate for future clinical trials focusing on wound care management.
  11. Fadilah NIM, Phang SJ, Kamaruzaman N, Salleh A, Zawani M, Sanyal A, et al.
    Antioxidants (Basel), 2023 Mar 23;12(4).
    PMID: 37107164 DOI: 10.3390/antiox12040787
    Natural-based biomaterials play an important role in developing new products for medical applications, primarily in cutaneous injuries. A large panel of biomaterials with antioxidant properties has revealed an advancement in supporting and expediting tissue regeneration. However, their low bioavailability in preventing cellular oxidative stress through the delivery system limits their therapeutic activity at the injury site. The integration of antioxidant compounds in the implanted biomaterial should be able to maintain their antioxidant activity while facilitating skin tissue recovery. This review summarises the recent literature that reported the role of natural antioxidant-incorporated biomaterials in promoting skin wound healing and tissue regeneration, which is supported by evidence from in vitro, in vivo, and clinical studies. Antioxidant-based therapies for wound healing have shown promising evidence in numerous animal studies, even though clinical studies remain very limited. We also described the underlying mechanism of reactive oxygen species (ROS) generation and provided a comprehensive review of ROS-scavenging biomaterials found in the literature in the last six years.
  12. Tahri S, Maarof M, Masri S, Che Man R, Masmoudi H, Fauzi MB
    Front Bioeng Biotechnol, 2023;11:1200618.
    PMID: 37425369 DOI: 10.3389/fbioe.2023.1200618
    Introduction: Plenty of biomaterials have been studied for their application in skin tissue engineering. Currently, gelatin-hydrogel is used to support three-dimensional (3D) skin in vitro models. However, mimicking the human body conditions and properties remains a challenge and gelatin-hydrogels have low mechanical properties and undergo rapid degradation rendering them not suitable for 3D in vitro cell culture. Nevertheless, changing the concentration of hydrogels could overcome this issue. Thus, we aim to investigate the potential of gelatin hydrogel with different concentrations crosslinked with genipin to promote human epidermal keratinocytes and human dermal fibroblasts culture to develop a 3D-in vitro skin model replacing animal models. Methods: Briefly, the composite gelatin hydrogels were fabricated using different concentrations as follows 3%, 5%, 8%, and 10% crosslinked with 0.1% genipin or non-crosslinked. Both physical and chemical properties were evaluated. Results and discussion: The crosslinked scaffolds showed better properties, including porosity and hydrophilicity, and genipin was found to enhance the physical properties. Furthermore, no alteration was prominent in both formulations of CL_GEL 5% and CL_GEL8% after genipin modification. The biocompatibility assays showed that all groups promoted cell attachment, cell viability, and cell migration except for the CL_GEL10% group. The CL_GEL5% and CL_GEL8% groups were selected to develop a bi-layer 3D-in vitro skin model. The immunohistochemistry (IHC) and hematoxylin and eosin staining (H&E) were performed on day 7, 14, and 21 to evaluate the reepithelization of the skin constructs. However, despite satisfactory biocompatibility properties, neither of the selected formulations, CL_GEL 5% and CL_GEL 8%, proved adequate for creating a bi-layer 3D in-vitro skin model. While this study provides valuable insights into the potential of gelatin hydrogels, further research is needed to address the challenges associated with their use in developing 3D skin models for testing and biomedical applications.
  13. Masri S, Fadilah NIM, Hao LQ, Maarof M, Tabata Y, Hiraoka Y, et al.
    Drug Deliv Transl Res, 2023 Nov 08.
    PMID: 37938542 DOI: 10.1007/s13346-023-01447-z
    Chronic wounds are challenging to heal and increase global mortality. The effectiveness of skin graft is limited by rejection, fibrosis, and inadequate donor site. Multifunctionalised-hydrogel skin substitutes promoted higher wound healing by maintaining the moisture microenvironment and permit gas exchange/nourishment in prolong cell viability/activity. The purpose of this study was to evaluate a skin substitute using two strategies; via injectable and 3D bioprinting technique. New hydrogel formulations that composed of gelatin (GE) and polyvinyl-alcohol (PVA) were constructed using a pre-mix crosslinking approach with genipin (GNP) to generate the biodegradable and biocompatible skin substitute with reduced secondary traumatic wound. GPVA5_GNP (6% GE: 5% PVA crosslinked with GNP) was the most stable hydrogel for wound healing application with the longest enzymatic degradation and stable hydrogel for absorption of excess wound exudates. Primary human dermal fibroblasts (HDFs) migrated extensively through 3D bioprinted hydrogels with larger average pore sizes and interconnected pores than injectable hydrogels. Moreover, 3D bioprinted GPVA hydrogels were biocompatible with HDFs and demonstrated > 90% cell viability. HDFs maintained their phenotype and positively expressed collagen type-I, vinculin, short and dense F-actin, alpha-smooth muscle actin, and Ki67. Additionally, the presence of GNP demonstrated antioxidant capacity and high-ability of angiogenesis. The utilisation of the 3D bioprinting (layer-by-layer) approach did not compromise the HDFs' growth capacity and biocompatibility with selected bioinks. In conclusion, it allows the cell encapsulation sustainability in a hydrogel matrix for a longer period, in promoting tissue regeneration and accelerating healing capacity, especially for difficult or chronic wound.
  14. Mohamed Haflah NH, Ng MH, Mohd Yunus MH, Naicker AS, Htwe O, Fahmi M, et al.
    Int J Low Extrem Wounds, 2017 Sep;16(3):212-216.
    PMID: 28862056 DOI: 10.1177/1534734617724974
    Open fracture Gustilo-Anderson grade IIIC is associated with higher risk of infection and problems with soft tissue coverage. Various methods have been used for soft tissue coverage in open fractures with large skin defect. We report a case of a patient who had grade IIIC open fracture of the tibia with posterior tibial artery injury. The patient underwent external fixation and reduction. Because of potential compartment syndrome after vascular repair, fasciotomy of the posterior compartment was performed. This wound, however, became infected and because of further debridement, gave rise to a large skin defect. A tissue engineered skin construct, MyDermTM was employed to cover this large defect. Complete wound closure was achieved 35 days postimplantation. The patient then underwent plating of the tibia for nonunion with no adverse effect to the grafted site. The tibia eventually healed 5 months postplating, and the cosmetic appearance of the newly formed skin was satisfactory.
  15. Maarof M, Law JX, Chowdhury SR, Khairoji KA, Saim AB, Idrus RB
    Cytotechnology, 2016 Oct;68(5):1873-84.
    PMID: 26768914 DOI: 10.1007/s10616-015-9940-3
    Limitations of current treatments for skin loss caused by major injuries leads to the use of skin substitutes. It is assumed that secretion of wound healing mediators by these skin substitutes plays a role in treating skin loss. In our previous study, single layer keratinocytes (SK), single layer fibroblast (SF) and bilayer (BL; containing keratinocytes and fibroblasts layers) skin substitutes were fabricated using fibrin that had shown potential to heal wounds in preclinical studies. This study aimed to quantify the secretion of wound healing mediators, and compare between single and bi-layer skin substitutes. Skin samples were digested to harvest fibroblasts and keratinocytes, and expanded to obtain sufficient cells for the construction of skin substitutes. Acellular fibrin (AF) construct was used as control. Substitutes i.e. AF, SK, SF and BL were cultured for 2 days, and culture supernatant was collected to analyze secretion of wound healing mediators via multiplex ELISA. Among 19 wound healing mediators tested, BL substitute secreted significantly higher amounts of CXCL1 and GCSF compared to SF and AF substitute but this was not significant with respect to SK substitute. The BL substitute also secreted significantly higher amounts of CXCL5 and IL-6 compared to other substitutes. In contrast, the SK substitute secreted significantly higher amounts of VCAM-1 compared to other substitutes. However, all three skin substitutes also secreted CCL2, CCL5, CCL11, GM-CSF, IL8, IL-1α, TNF-α, ICAM-1, FGF-β, TGF-β, HGF, VEGF-α and PDGF-BB factors, but no significant difference was seen. Secretion of these mediators after transplantation may play a significant role in promoting wound healing process for the treatment of skin loss.
  16. Maarof M, Mohd Nadzir M, Sin Mun L, Fauzi MB, Chowdhury SR, Idrus RBH, et al.
    Polymers (Basel), 2021 Feb 08;13(4).
    PMID: 33567703 DOI: 10.3390/polym13040508
    The current strategy for rapid wound healing treatment involves combining a biomaterial and cell-secreted proteins or biomolecules. This study was aimed at characterizing 3-dimensional (3D) collagen hydrogels fortified with dermal fibroblast-conditioned medium (DFCM) as a readily available acellular skin substitute. Confluent fibroblasts were cultured with serum-free keratinocyte-specific medium (KM1 and KM2) and fibroblast-specific medium (FM) to obtain DFCM. Subsequently, the DFCM was mixed with collagen (Col) hydrogel and chondroitin-4-sulphate (C4S) to fabricate 3D constructs termed Col/C4S/DFCM-KM1, Col/C4S/DFCM-KM2, and Col/C4S/DFCM-FM. The constructs successfully formed soft, semi-solid and translucent hydrogels within 1 h of incubation at 37 °C with strength of <2.5 Newton (N). The Col/C4S/DFCM demonstrated significantly lower turbidity compared to the control groups. The Col/C4S/DFCM also showed a lower percentage of porosity (KM1: 35.15 ± 9.76%; KM2: 6.85 ± 1.60%; FM: 14.14 ± 7.65%) compared to the Col (105.14 ± 11.87%) and Col/C4S (143.44 ± 27.72%) constructs. There were no changes in both swelling and degradation among all constructs. Fourier transform infrared spectrometry showed that all groups consisted of oxygen-hydrogen bonds (O-H) and amide I, II, and III. In conclusion, the Col/C4S/DFCM constructs maintain the characteristics of native collagen and can synergistically deliver essential biomolecules for future use in skin therapeutic applications.
  17. Maarof M, Chowdhury SR, Saim A, Bt Hj Idrus R, Lokanathan Y
    Int J Mol Sci, 2020 Apr 22;21(8).
    PMID: 32331278 DOI: 10.3390/ijms21082929
    Fibroblasts secrete many essential factors that can be collected from fibroblast culture medium, which is termed dermal fibroblast conditioned medium (DFCM). Fibroblasts isolated from human skin samples were cultured in vitro using the serum-free keratinocyte-specific medium (Epilife (KM1), or define keratinocytes serum-free medium, DKSFM (KM2) and serum-free fibroblast-specific medium (FM) to collect DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively). We characterised and evaluated the effects of 100-1600 µg/mL DFCM on keratinocytes based on attachment, proliferation, migration and gene expression. Supplementation with 200-400 µg/mL keratinocyte-specific DFCM-KM1 and DFCM-KM2 enhanced the attachment, proliferation and migration of sub-confluent keratinocytes, whereas 200-1600 µg/mL DFCM-FM significantly increased the healing rate in the wound healing assay, and 400-800 µg/mL DFCM-FM was suitable to enhance keratinocyte attachment and proliferation. A real-time (RT2) profiler polymerase chain reaction (PCR) array showed that 42 genes in the DFCM groups had similar fold regulation compared to the control group and most of the genes were directly involved in wound healing. In conclusion, in vitro keratinocyte re-epithelialisation is supported by the fibroblast-secreted proteins in 200-400 µg/mL DFCM-KM1 and DFCM-KM2, and 400-800 µg/mL DFCM-FM, which could be useful for treating skin injuries.
  18. Imran SAM, Yazid MD, Idrus RBH, Maarof M, Nordin A, Razali RA, et al.
    Int J Mol Sci, 2021 Apr 09;22(8).
    PMID: 33918710 DOI: 10.3390/ijms22083888
    Epithelial-Mesenchymal Transition (EMT) was first discovered during the transition of cells from the primitive streak during embryogenesis in chicks. It was later discovered that EMT holds greater potential in areas other than the early development of cells and tissues since it also plays a vital role in wound healing and cancer development. EMT can be classified into three types based on physiological functions. EMT type 3, which involves neoplastic development and metastasis, has been the most thoroughly explored. As EMT is often found in cancer stem cells, most research has focused on its association with other factors involving cancer progression, including telomeres. However, as telomeres are also mainly involved in aging, any possible interaction between the two would be worth noting, especially as telomere dysfunction also contributes to cancer and other age-related diseases. Ascertaining the balance between degeneration and cancer development is crucial in cell biology, in which telomeres function as a key regulator between the two extremes. The essential roles that EMT and telomere protection have in aging reveal a potential mutual interaction that has not yet been explored, and which could be used in disease therapy. In this review, the known functions of EMT and telomeres in aging are discussed and their potential interaction in age-related diseases is highlighted.
  19. Subramaniam T, Shaiful Hadi N, Sulaiman S, Fauzi MB, Hj Idrus RB, Chowdhury SR, et al.
    Burns, 2021 Aug 20.
    PMID: 34893370 DOI: 10.1016/j.burns.2021.08.012
    Skin substitutes are designed dressings intended to promote wound closure. In previous in vitro and in vivo studies on small animal, an acellular skin patch made of collagen hydrogel with dermal fibroblast conditioned medium (Col-DFCM), a collagen sponge scaffold with freshly harvested skin cells (OTC), and a platelet-rich-plasma gel with freshly harvested skin cells (PRP) have been developed and tested for immediate treatment of full-thickness wound. However, to determine the safety and efficacy of these skin patches for clinical applications, further study in a large animal model is needed. The aim of this study is to evaluate the potential of Col-DFCM, OTC and PRP in treating full-thickness wound in an ovine model via histological analysis and immunohistochemistry staining were performed, with the untreated (NT) group serving as the control. Gross examination was conducted on day 7, 14 and 21 to determine the wound closure rate. The findings of percentage of wound size reduction showed that the wound healed fastest in the presence of Col-DFCM (91.34 ± 23.35%) followed by OTC (84.49 ± 23.13%), PRP (77.73 ± 20.9%) and NT group (73.94 ± 23.71%). Histological evaluation with Hematoxylin & Eosin (H & E) and Masson's trichrome staining was used to study the structure of the wound area. The results showed that OTC treated wound was more mature as indicated by the presence of a thinner epidermis followed by the Col-DFCM, PRP and NT group. Immunohistochemistry analysis also confirmed the integrity and maturity of the regenerated skin, with positive expression of cytokeratin 10 (CK10) and involucrin in the epidermal layer. In conclusion, Col-DFCM, OTC and PRP treatments promote healing of full-thickness wound and have the potential to be used clinically for rapid treatment of full-thickness wound.
  20. Abdul Ghani N', Razali RA, Chowdhury SR, Fauzi MB, Bin Saim A, Ruszymah BHI, et al.
    Biomedicines, 2022 Dec 09;10(12).
    PMID: 36551960 DOI: 10.3390/biomedicines10123203
    A key event in wound healing is re-epithelialisation, which is mainly regulated via paracrine signalling of cytokines, chemokines, and growth factors secreted by fibroblasts. Fibroblast-secreted factors can be collected from the used culture medium, known as dermal fibroblast conditioned medium (DFCM). The goal of this study was to optimise the culture condition to acquire DFCM and evaluate its effect on keratinocyte attachment, proliferation, migration, and differentiation. Confluent fibroblasts were cultured with serum-free keratinocyte-specific (DFCM-KM) and fibroblast-specific (DFCM-FM) medium at different incubation times (Days 1, 2, and 3). DFCM collected after 3 days of incubation (DFCM-KM-3 and DFCM-FM-3) contained a higher protein concentration compared to other days. Supplementation of DFCM-KM-3 enhanced keratinocyte attachment, while DFCM-FM-3 significantly increased the keratinocyte wound-healing rate, with an increment of keratinocyte area and collective cell migration, which was distinctly different from DFCM-KM-3 or control medium. Further analysis confirmed that the presence of calcium at higher concentrations in DFCM-FM facilitated the changes. The confluent dermal fibroblasts after 3 days of incubation with serum-free culture medium produced higher proteins in DFCM, resulting in enhanced in vitro re-epithelialisation. These results suggest that the delivery of DFCM could be a potential treatment strategy for wound healing.
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