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Large-Scale Expansion of Human Mesenchymal Stem Cells

Hassan MNFB, Yazid MD, Yunus MHM, Chowdhury SR, Lokanathan Y, Idrus RBH, et al.

Stem Cells Int, 2020;2020:9529465.
PMID: 32733574 DOI: 10.1155/2020/9529465

Mesenchymal stem cells (MSCs) are multipotent stem cells with strong immunosuppressive property that renders them an attractive source of cells for cell therapy. MSCs have been studied in multiple clinical trials to treat liver diseases, peripheral nerve damage, graft-versus-host disease, autoimmune diseases, diabetes mellitus, and cardiovascular damage. Millions to hundred millions of MSCs are required per patient depending on the disease, route of administration, frequency of administration, and patient body weight. Multiple large-scale cell expansion strategies have been described in the literature to fetch the cell quantity required for the therapy. In this review, bioprocessing strategies for large-scale expansion of MSCs were systematically reviewed and discussed. The literature search in Medline and Scopus databases identified 26 articles that met the inclusion criteria and were included in this review. These articles described the large-scale expansion of 7 different sources of MSCs using 4 different bioprocessing strategies, i.e., bioreactor, spinner flask, roller bottle, and multilayered flask. The bioreactor, spinner flask, and multilayered flask were more commonly used to upscale the MSCs compared to the roller bottle. Generally, a higher expansion ratio was achieved with the bioreactor and multilayered flask. Importantly, regardless of the bioprocessing strategies, the expanded MSCs were able to maintain its phenotype and potency. In summary, the bioreactor, spinner flask, roller bottle, and multilayered flask can be used for large-scale expansion of MSCs without compromising the cell quality.
Fulltext Physicochemical Properties and Biocompatibility of Electrospun Polycaprolactone/Gelatin Nanofibers

Lim WL, Chowdhury SR, Ng MH, Law JX

Int J Environ Res Public Health, 2021 04 29;18(9).
PMID: 33947053 DOI: 10.3390/ijerph18094764

Tissue-engineered substitutes have shown great promise as a potential replacement for current tissue grafts to treat tendon/ligament injury. Herein, we have fabricated aligned polycaprolactone (PCL) and gelatin (GT) nanofibers and further evaluated their physicochemical properties and biocompatibility. PCL and GT were mixed at a ratio of 100:0, 70:30, 50:50, 30:70, 0:100, and electrospun to generate aligned nanofibers. The PCL/GT nanofibers were assessed to determine the diameter, alignment, water contact angle, degradation, and surface chemical analysis. The effects on cells were evaluated through Wharton's jelly-derived mesenchymal stem cell (WJ-MSC) viability, alignment and tenogenic differentiation. The PCL/GT nanofibers were aligned and had a mean fiber diameter within 200-800 nm. Increasing the GT concentration reduced the water contact angle of the nanofibers. GT nanofibers alone degraded fastest, observed only within 2 days. Chemical composition analysis confirmed the presence of PCL and GT in the nanofibers. The WJ-MSCs were aligned and remained viable after 7 days with the PCL/GT nanofibers. Additionally, the PCL/GT nanofibers supported tenogenic differentiation of WJ-MSCs. The fabricated PCL/GT nanofibers have a diameter that closely resembles the native tissue's collagen fibrils and have good biocompatibility. Thus, our study demonstrated the suitability of PCL/GT nanofibers for tendon/ligament tissue engineering applications.
Fulltext Current Progress in Tendon and Ligament Tissue Engineering

Lim WL, Liau LL, Ng MH, Chowdhury SR, Law JX

Tissue Eng Regen Med, 2019 Dec;16(6):549-571.
PMID: 31824819 DOI: 10.1007/s13770-019-00196-w

BACKGROUND: Tendon and ligament injuries accounted for 30% of all musculoskeletal consultations with 4 million new incidences worldwide each year and thus imposed a significant burden to the society and the economy. Damaged tendon and ligament can severely affect the normal body movement and might lead to many complications if not treated promptly and adequately. Current conventional treatment through surgical repair and tissue graft are ineffective with a high rate of recurrence.
METHODS: In this review, we first discussed the anatomy, physiology and pathophysiology of tendon and ligament injuries and its current treatment. Secondly, we explored the current role of tendon and ligament tissue engineering, describing its recent advances. After that, we also described stem cell and cell secreted product approaches in tendon and ligament injuries. Lastly, we examined the role of the bioreactor and mechanical loading in in vitro maturation of engineered tendon and ligament.
RESULTS: Tissue engineering offers various alternative ways of treatment from biological tissue constructs to stem cell therapy and cell secreted products. Bioreactor with mechanical stimulation is instrumental in preparing mature engineered tendon and ligament substitutes in vitro.
CONCLUSIONS: Tissue engineering showed great promise in replacing the damaged tendon and ligament. However, more study is needed to develop ideal engineered tendon and ligament.
Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds

Law JX, Chowdhury SR, Saim AB, Idrus RBH

J Tissue Viability, 2017 Aug;26(3):208-215.
PMID: 28615133 DOI: 10.1016/j.jtv.2017.05.003

Advances in tissue engineering led to the development of various tissue-engineered skin substitutes (TESS) for the treatment of skin injuries. The majority of the autologous TESS required lengthy and costly cell expansion process to fabricate. In this study, we determine the possibility of using a low density of human skin cells suspended in platelet-rich plasma (PRP)-enriched medium to promote the healing of full-thickness skin wounds. To achieve this, full-thickness wounds of size 1.767 cm2 were created at the dorsum part of nude mice and treated with keratinocytes (2 × 104 cells/cm2) and fibroblasts (3 × 104 cells/cm2) suspended in 10% PRP-enriched medium. Wound examination was conducted weekly and the animals were euthanized after 2 weeks. Gross examination showed that re-epithelialization was fastest in the PRP+cells group at both day 7 and 14, followed by the PRP group and NT group receiving no treatment. Only the PRP+cells group achieved complete wound closure by 2 weeks. Epidermal layer was presence in the central region of the wound of the PRP+cells and PRP groups but absence in the NT group. Comparison between the PRP+cells and PRP groups showed that the PRP+cells-treated wound was more mature as indicated by the presence of thinner epidermis with single cell layer thick basal keratinocytes and less cellular dermis. In summary, the combination of low cell density and diluted PRP creates a synergistic effect which expedites the healing of full-thickness wounds. This combination has the potential to be developed as a rapid wound therapy via the direct application of freshly harvested skin cells in diluted PRP.
Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing

Xian LJ, Chowdhury SR, Bin Saim A, Idrus RB

Cytotherapy, 2015 Mar;17(3):293-300.
PMID: 25456581 DOI: 10.1016/j.jcyt.2014.10.005

Platelet-rich plasma (PRP) has been found to contain a high concentration of growth factors that are present during the process of healing. Studies conducted found that application of PRP accelerates wound healing. In this study, we characterized the skin cell suspension harvested using the co-isolation technique and evaluated the effects of PRP (10% and 20%, v/v) on co-cultured keratinocytes and fibroblasts in terms of wound healing.
The effects of human serum to the morphology, proliferation and gene expression level of the respiratory epithelium in vitro

Yunus MH, Siang KC, Hashim NI, Zhi NP, Zamani NF, Sabri PP, et al.

Tissue Cell, 2014 Aug;46(4):233-40.
PMID: 24973262 DOI: 10.1016/j.tice.2014.05.003

The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. The present study focused on investigating the effects of human serum (HS) on the qualitative and quantitative properties of the human respiratory epithelium compared to the fetal bovine serum (FBS), as a supplement in culture. Respiratory epithelial (RE) cells derived from human nasal turbinate were co-cultured with fibroblasts, subsequently separated at 80-90% confluency by differential trypsinization. RE cells were then sub-cultured into 2 different plates containing 5% allogenic HS and FBS supplemented media respectively up to passage 1 (P1). Cell morphology, growth rate, cell viability and population doubling time were assessed under light microscope, and levels of gene expression were measured via real time reverse transcriptase-polymerase chain reaction (qRT-PCR). RE cells appeared as polygonal shape and expanded when cultured in HS whereas RE cells in FBS were observed to be easily matured thus limit the RE cells expansion. Proliferation rate of RE cells in HS supplemented media (7673.18 ± 1207.15) was 3 times higher compared to RE in FBS supplemented media (2357.68 ± 186.85). Furthermore, RE cells cultured in HS-supplemented media required fewer days (9.15 ± 1.10) to double in numbers compared to cells cultured in FBS-supplemented media (13.66 ± 0.81). Both the differences were significant (p<0.05). However, there were no significant differences in the viability of RE cells in both groups (p=0.105). qRT-PCR showed comparable expressions of gene Cytokeratin-14 (CK-14), Cytokeratin-18 (CK-18) and Mucin-5 subtype B (MUC5B) in RE cells cultured in both groups (p>0.05). In conclusion, HS is a comparatively better choice of media supplement in accelerating growth kinetics of RE cells in vitro thus producing a better quality of respiratory epithelium for future tracheal reconstruction.
Tissue-Engineered Skin Substitute Enhances Wound Healing after Radiation Therapy

Busra MF, Chowdhury SR, bin Ismail F, bin Saim A, Idrus RB

Adv Skin Wound Care, 2016 Mar;29(3):120-9.
PMID: 26866868 DOI: 10.1097/01.ASW.0000480556.78111.e4

OBJECTIVE: When given in conjunction with surgery for treating cancer, radiation therapy may result in impaired wound healing, which, in turn, could cause skin ulcers. In this study, bilayer and monolayer autologous skin substitutes were used to treat an irradiated wound.
MATERIALS AND METHODS: A single dose of 30 Gy of linear electron beam radiation was applied to the hind limb of nude mice before creating the skin lesion (area of 78.6 mm). Monolayer tissue-engineered skin substitutes (MTESSs) were prepared by entrapping cultured keratinocytes in fibrin matrix, and bilayer tissue-engineered skin substitutes (BTESSs) were prepared by entrapping keratinocytes and fibroblasts in separate layers. Bilayer tissue-engineered skin substitute and MTESS were implanted to the wound area. Gross appearance and wound area were analyzed to evaluate wound healing efficiency. Skin regeneration and morphological appearance were observed via histological and electron microscopy. Protein expressions of transforming growth factor β1 (TGF-β1), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor (VEGF) in skin regeneration were evaluated by immunohistochemistry (IHC).
RESULTS: Macroscopic observation revealed that at day 13, treatments with BTESS completely healed the irradiated wound, whereas wound sizes of 1.1 ± 0.05 and 6.8 ± 0.14 mm were measured in the MTESS-treated and untreated control groups, respectively. Hematoxylin-eosin (H&E) analysis showed formation of compact and organized epidermal and dermal layers in the BTESS-treated group, as compared with MTESS-treated and untreated control groups. Ultrastructural analysis indicates maturation of skin in BTESS-treated wound evidenced by formation of intermediate filament bundles in the dermal layer and low intercellular space in the epidermal layer. Expressions of TGF-β1, PDGF-BB, and VEGF were also higher in BTESS-treated wounds, compared with MTESS-treated wounds.
CONCLUSIONS: These results indicate that BTESS is the preferred treatment for irradiated wound ulcers.
Secretion of wound healing mediators by single and bi-layer skin substitutes

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.
One-Step Purification of Human Skeletal Muscle Myoblasts and Subsequent Expansion Using Laminin-Coated Surface

Chowdhury SR, binti Ismail A, Chee SC, bin Laupa MS, binti Jaffri F, Saberi SE, et al.

Tissue Eng Part C Methods, 2015 Nov;21(11):1135-42.
PMID: 26061720 DOI: 10.1089/ten.TEC.2015.0015

Skeletal myoblasts have been extensively used to study muscle growth and differentiation, and were recently tested for their application as cell therapy and as a gene delivery system to treat muscle and nonmuscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10, and 15 min. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity, and yield. It was found that laminin-coated surface favors the attachment of myoblasts. Highest myoblast purity of 78.9% ± 6.8% was achieved by 5 min of preplating only on the laminin-coated surface with a yield of 56.9% ± 3.3%. Primary cells, isolated from skeletal muscle (n = 4), confirm the enhancement of purity through preplating on laminin-coated surface for 5 min. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity of ∼ 98% was achieved when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.
Genotoxicity and cytotoxicity of ovine collagen on human dermal fibroblasts

Busra FM, Chowdhury SR, Saim AB, Idrus RB

Saudi Med J, 2011 Dec;32(12):1311-2.
PMID: 22159390
Fulltext Type II Collagen-Conjugated Mesenchymal Stem Cells Micromass for Articular Tissue Targeting

Sulaiman SB, Chowdhury SR, Busra MFBM, Abdul Rani RB, Mohamad Yahaya NHB, Tabata Y, et al.

Biomedicines, 2021 Jul 23;9(8).
PMID: 34440084 DOI: 10.3390/biomedicines9080880

The tissue engineering approach in osteoarthritic cell therapy often requires the delivery of a substantially high cell number due to the low engraftment efficiency as a result of low affinity binding of implanted cells to the targeted tissue. A modification towards the cell membrane that provides specific epitope for antibody binding to a target tissue may be a plausible solution to increase engraftment. In this study, we intercalated palmitated protein G (PPG) with mesenchymal stem cells (MSCs) and antibody, and evaluated their effects on the properties of MSCs either in monolayer state or in a 3D culture state (gelatin microsphere, GM). Bone marrow MSCs were intercalated with PPG (PPG-MSCs), followed by coating with type II collagen antibody (PPG-MSC-Ab). The effect of PPG and antibody conjugation on the MSC proliferation and multilineage differentiation capabilities both in monolayer and GM cultures was evaluated. PPG did not affect MSC proliferation and differentiation either in monolayer or 3D culture. The PPG-MSCs were successfully conjugated with the type II collagen antibody. Both PPG-MSCs with and without antibody conjugation did not alter MSC proliferation, stemness, and the collagen, aggrecan, and sGAG expression profiles. Assessment of the osteochondral defect explant revealed that the PPG-MSC-Ab micromass was able to attach within 48 h onto the osteochondral surface. Antibody-conjugated MSCs in GM culture is a potential method for targeted delivery of MSCs in future therapy of cartilage defects and osteoarthritis.
Fibroblast-derived matrices-based human skin equivalent as an in vitro psoriatic model for drug testing

Yap WH, Cheah TY, Yong LC, Chowdhury SR, Ng MH, Kwan Z, et al.

J Biosci, 2021;46.
PMID: 34475316

Psoriasis is a chronic skin disease characterized by thickening and disorganization of the skin's protective barrier. Although current models replicate some aspects of the disease, development of therapeutic strategies have been hindered by absence of more relevant models. This study aimed to develop and characterize an in vitro psoriatic human skin equivalent (HSE) using human keratinocytes HaCat cell line grown on fibroblasts-derived matrices (FDM). The constructed HSEs were treated with cytokines (IL-1α, TNF-α, IL-6, and IL22) to allow controlled induction of psoriasis-associated features. Histological stainings showed that FDMHSE composed of a fully differentiated epidermis and fibroblast-populated dermis comparable to native skin and rat tail collagen-HSE. Hyperproliferation (CK16 and Ki67) and inflammatory markers (TNF-α and IL-6) expression were significantly enhanced in the cytokine-induced FDM- and rat tail collagen HSEs compared to non-treated HSE counterparts. The characteristics were in line with those observed in psoriasis punch biopsies. Treatment with all-trans retinoic acid (ATRA) has shown to suppress these effects, where HSE models treated with both ATRA and cytokines exhibit histological characteristics, hyperproliferation and differentiation markers expression like non-treated control HSEs. Cytokine-induced FDM-HSE, constructed entirely from human cell lines, provides an excellent opportunity for psoriasis research and testing new therapeutics.
Development of various composition multicomponent chitosan/fish collagen/glycerin 3D porous scaffolds: Effect on morphology, mechanical strength, biostability and cytocompatibility

Ullah S, Zainol I, Chowdhury SR, Fauzi MB

Int J Biol Macromol, 2018 May;111:158-168.
PMID: 29305219 DOI: 10.1016/j.ijbiomac.2017.12.136

The various composition multicomponent chitosan/fish collagen/glycerin 3D porous scaffolds were developed and investigated the effect of various composition chitosan/fish collagen/glycerin on scaffolds morphology, mechanical strength, biostability and cytocompatibility. The scaffolds were fabricated via freeze-drying technique. The effects of various compositions consisting in 3D scaffolds were investigated via FT-IR analysis, porosity, swelling and mechanical tests, and effect on the morphology of scaffolds investigated microscopically. The biostability and cytocompatibility tests were used to explore the ability of scaffolds to use for tissue engineering application. The average pore sizes of scaffolds were in range of 100.73±27.62-116.01±52.06, porosity 71.72±3.46-91.17±2.42%, tensile modulus in dry environment 1.47±0.08-0.17±0.03MPa, tensile modulus in wet environment 0.32±0.03-0.14±0.04MPa and biodegradation rate (at day 30) 60.38±0.70-83.48±0.28%. In vitro culture of human fibroblasts and keratinocytes showed that the various composition multicomponent 3D scaffolds were good cytocompatibility however, the scaffolds contained high amount of fish collagen excellently facilitated cell proliferation and adhesion. It was found that the high amount fish collagen and glycerin scaffolds have high porosity, enough mechanical strength and biostability, and excellent cytocompatibility.
Safety and efficacy of dermal fibroblast conditioned medium (DFCM) fortified collagen hydrogel as acellular 3D skin patch

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.
Proteomic Analysis of Human Dermal Fibroblast Conditioned Medium (DFCM)

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.
Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications

Fauzi MB, Lokanathan Y, Aminuddin BS, Ruszymah BHI, Chowdhury SR

Mater Sci Eng C Mater Biol Appl, 2016 Nov 01;68:163-171.
PMID: 27524008 DOI: 10.1016/j.msec.2016.05.109

Collagen is the most abundant extracellular matrix (ECM) protein in the human body, thus widely used in tissue engineering and subsequent clinical applications. This study aimed to extract collagen from ovine (Ovis aries) Achilles tendon (OTC), and to evaluate its physicochemical properties and its potential to fabricate thin film with collagen fibrils in a random or aligned orientation. Acid-solubilized protein was extracted from ovine Achilles tendon using 0.35M acetic acid, and 80% of extracted protein was measured as collagen. SDS-PAGE and mass spectrometry analysis revealed the presence of alpha 1 and alpha 2 chain of collagen type I (col I). Further analysis with Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDS) confirms the presence of triple helix structure of col I, similar to commercially available rat tail col I. Drying the OTC solution at 37°C resulted in formation of a thin film with randomly orientated collagen fibrils (random collagen film; RCF). Introduction of unidirectional mechanical intervention using a platform rocker prior to drying facilitated the fabrication of a film with aligned orientation of collagen fibril (aligned collagen film; ACF). It was shown that both RCF and ACF significantly enhanced human dermal fibroblast (HDF) attachment and proliferation than that on plastic surface. Moreover, cells were distributed randomly on RCF, but aligned with the direction of mechanical intervention on ACF. In conclusion, ovine tendon could be an alternative source of col I to fabricate scaffold for tissue engineering applications.
Human Amniotic Membrane with Aligned Electrospun Fiber as Scaffold for Aligned Tissue Regeneration

Hasmad H, Yusof MR, Mohd Razi ZR, Hj Idrus RB, Chowdhury SR

Tissue Eng Part C Methods, 2018 06;24(6):368-378.
PMID: 29690856 DOI: 10.1089/ten.TEC.2017.0447

Fabrication of composite scaffolds is one of the strategies proposed to enhance the functionality of tissue-engineered scaffolds for improved tissue regeneration. By combining multiple elements together, unique biomimetic scaffolds with desirable physical and mechanical properties can be tailored for tissue-specific applications. Despite having a highly porous structure, the utility of electrospun fibers (EF) as scaffold is usually hampered by their insufficient mechanical strength. In this study, we attempted to produce a mechanically competent scaffold with cell-guiding ability by fabricating aligned poly lactic-co-glycolic acid (PLGA) fibers on decellularized human amniotic membrane (HAM), known to possess favorable tensile and wound healing properties. Decellularization of HAM in 18.75 μg/mL of thermolysin followed by a brief treatment in 0.25 M sodium hydroxide efficiently removed the amniotic epithelium and preserved the ultrastructure of the underlying extracellular matrix. The electrospinning of 20% (w/v) PLGA 50:50 polymer on HAM yielded beadless fibers with straight morphology. Subsequent physical characterization revealed that EF-HAM scaffold with a 3-min fabrication had the most aligned fibers with the lowest fiber diameter in comparison with EF-HAM 5- and 7-min scaffolds. Hydrated EF-HAM scaffolds with 3-min deposition had a greater tensile strength than the other scaffolds despite having thinner fibers. Nevertheless, wet HAM and EF-HAMs regardless of the fiber thicknesses had a significantly lower Young's modulus, and hence, a higher elasticity compared with dry HAM and EF-HAMs. Biocompatibility analysis showed that the viability and migration rate of skeletal muscle cells on EF-HAMs were similar to control and HAM alone. Skeletal muscle cells seeded on HAM were shown to display random orientation, whereas cells on EF-HAM scaffolds were oriented along the alignment of the electrospun PLGA fibers. In summary, besides having good mechanical strength and elasticity, EF-HAM scaffold design decorated with aligned fiber topography holds a promising potential for use in the development of aligned tissue constructs.
Rapid treatment of full-thickness skin loss using ovine tendon collagen type I scaffold with skin cells

Mh Busra F, Rajab NF, Tabata Y, Saim AB, B H Idrus R, Chowdhury SR

J Tissue Eng Regen Med, 2019 05;13(5):874-891.
PMID: 30811090 DOI: 10.1002/term.2842

The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.
Fulltext Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

Zahari NK, Idrus RBH, Chowdhury SR

Int J Mol Sci, 2017 Oct 30;18(11).
PMID: 29084180 DOI: 10.3390/ijms18112242

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h-1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h-1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.
Fulltext Attachment, Proliferation, and Morphological Properties of Human Dermal Fibroblasts on Ovine Tendon Collagen Scaffolds: A Comparative Study

Busra FM, Lokanathan Y, Nadzir MM, Saim A, Idrus RBH, Chowdhury SR

Malays J Med Sci, 2017 Mar;24(2):33-43.
PMID: 28894402 DOI: 10.21315/mjms2017.24.2.5

INTRODUCTION: Collagen type I is widely used as a biomaterial for tissue-engineered substitutes. This study aimed to fabricate different three-dimensional (3D) scaffolds using ovine tendon collagen type I (OTC-I), and compare the attachment, proliferation and morphological features of human dermal fibroblasts (HDF) on the scaffolds.
METHODS: This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions.
RESULTS: The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions.
CONCLUSION: OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.

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