Tissue Engineering Initiatives in Malaysia

( Ruszymah Bt. Hj. Idrus ) 한국조직공학과 재생의학회 2006 조직공학과 재생의학 Vol.3 No.1

Mesenchymal Stromal Cells from the Maternal Segment of Human Umbilical Cord is Ideal for Bone Regeneration in Allogenic Setting

Jezamine Lim,Zainul Rashid Mohamad Razi,Jia Xian Law,Azmawati Mohammed Nawi,Ruszymah Binti Haji Idrus,Tan Geok Chin,Muaatamarulain Mustangin,Min Hwei Ng 한국조직공학과 재생의학회 2018 조직공학과 재생의학 Vol.15 No.1

Umbilical cord (UC) is a discarded product from the operating theatre and a ready source of mesenchymal stromal cells (MSCs). MSCs from UC express both embryonic and adult mesenchymal stem cell markers and are known to be hypoimmunogenic and non-tumorigenic and thus suitable for allogeneic cell transplantation. Our study aimed to determine the degree of immunotolerance and bone-forming capacity of osteodifferentiated human Wharton’s jelly-derived mesenchymal stromal cells (hWJ-MSCs) from different segments of UC in an allogenic setting. UCs were obtained from healthy donors delivering a full-term infant by elective Caesarean section. hWJ-MSCs were isolated from 3 cm length segment from the maternal and foetal ends of UCs. Three-dimensional fibrin constructs were formed and implanted intramuscularly into immunocompetent mice. The mice were implanted with 1) fibrin construct with maternal hWJ-MSCs, 2) fibrin construct with foetal hWJ-MSCs, or 3) fibrin without cells; the control group received sham surgery. After 1 month, the lymphoid organs were analysed to determine the degree of immune rejection and bone constructs were analysed to determine the amount of bone formed. A pronounced immune reaction was noted in the fibrin group. The maternal segment constructs demonstrated greater osteogenesis than the foetal segment constructs. Both maternal and foetal segment constructs caused minimal immune reaction and thus appear to be safe for allogeneic bone transplant. The suppression of inflammation may be a result of increased anti-inflammatory cytokine production mediated by the hWJMSC. In summary, this study demonstrates the feasibility of using bone constructs derived from hWJ-MSCs in an allogenic setting.

Electrospun Collagen Nanofibers and Their Applications in Skin Tissue Engineering

Jia Xian Law,Ling Ling Liau,Aminuddin Saim,Ying Yang,Ruszymah Idrus 한국조직공학과 재생의학회 2017 조직공학과 재생의학 Vol.14 No.6

Electrospinning is a simple and versatile technique to fabricate continuous fibers with diameter ranging from micrometers to a few nanometers. To date, the number of polymers that have been electrospun has exceeded 200. In recent years, electrospinning has become one of the most popular scaffold fabrication techniques to prepare nanofiber mesh for tissue engineering applications. Collagen, the most abundant extracellular matrix protein in the human body, has been electrospun to fabricate biomimetic scaffolds that imitate the architecture of native human tissues. As collagen nanofibers are mechanically weak in nature, it is commonly cross-linked or blended with synthetic polymers to improve the mechanical strength without compromising the biological activity. Electrospun collagen nanofiber mesh has high surface area to volume ratio, tunable diameter and porosity, and excellent biological activity to regulate cell function and tissue formation. Due to these advantages, collagen nanofibers have been tested for the regeneration of a myriad of tissues and organs. In this review, we gave an overview of electrospinning, encompassing the history, the instrument settings, the spinning process and the parameters that affect fiber formation, with emphasis given to collagen nanofibers’ fabrication and application, especially the use of collagen nanofibers in skin tissue engineering.

Synergistic Effect of Laminin and Epidermal Growth Factor on Biological and Morphological Properties of Co-Cultured Myoblasts and Fibroblasts

Mat Afandi Mohd Asyraf,Maarof Manira,Chowdhury S. R.,Bt. Hj. Idrus Ruszymah 한국조직공학과 재생의학회 2020 조직공학과 재생의학 Vol.17 No.6

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.

Centella asiatica (L.)-Neurodifferentiated Mesenchymal Stem Cells Promote the Regeneration of Peripheral Nerve

Hanita Mohd Hussin,Mahazura Mat Lawi,Nor Hazla Mohamed Haflah,Abdul Yazid Mohd Kassim,Ruszymah Bt Hj Idrus,Yogeswaran Lokanathan 한국조직공학과 재생의학회 2020 조직공학과 재생의학 Vol.17 No.2

BACKGROUND: Centella asiatica (L.) is a plant with neuroprotective and neuroregenerative properties; however, its effects on the neurodifferentiation of mesenchymal stem cells (MSCs) and on peripheral nerve injury are poorly explored. This study aimed to investigate the effects of C. asiatica (L.)-neurodifferentiated MSCs on the regeneration of peripheral nerve in a critical-size defect animal model. METHODS: Nerve conduit was developed using decellularised artery seeded with C. asiatica-neurodifferentiated MSCs (ndMSCs). A 1.5 cm sciatic nerve injury in Sprague–Dawley rat was bridged with reversed autograft (RA) (n = 3, the gold standard treatment), MSC-seeded conduit (MC) (n = 4) or ndMSC-seeded conduit (NC) (n = 4). Pinch test and nerve conduction study were performed every 2 weeks for a total of 12 weeks. At the 12th week, the conduits were examined by histology and transmission electron microscopy. RESULTS: NC implantation improved the rats’ sensory sensitivity in a similar manner to RA. At the 12th week, nerve conduction velocity was the highest in NC compared with that of RA and MC. Axonal regeneration was enhanced in NC and RA as shown by the expression of myelin basic protein (MBP). The average number of myelinated axons was significantly higher in NC than in MC but significantly lower than in RA. The myelin sheath thickness was higher in NC than in MC but lower than in RA. CONCLUSION: NC showed promising effects on nerve regeneration and functional restoration similar to those of RA. These findings revealed the neuroregenerative properties of C. asiatica and its potential as an alternative strategy for the treatment of critical size nerve defect.

Sox-9 Transient Transfection Enhances Chondrogenic Expression of Osteoarthritic Human Articular Chondrocytes In Vitro: Preliminary Analysis

( Munirah Sha` Ban ),( Samsudin Osman Cassim ),( Nor Hamdan Mohd Yahya ),( Aminuddin Bin Saim ),( Ruszymah Bt Hj Idrus ) 한국조직공학·재생의학회 2011 조직공학과 재생의학 Vol.8 No.1

In this study, we are taking step to actively manage osteoarthritis that may help gain control over osteoar-thritic pain and delay the degenerative changes in articular cartilage in future. We transiently over expressed cartilage transcriptional factor, human sox-9 gene in chondrocytes derived from consented osteoarthritic patients after joint surgery. The expression vector carrying human sox-9 gene, pAdTrack-sox9 was transformed into One Shot® TOP10 Chemically Competent E. coli according to the manufacturer protocol. Plasmid purification was performed in accordance with QIAGEN® plasmid purification kit procedure. We compared the efficiency between two transfection techniques i.e. lipofection using Lipofectamine™ 2000 kit from Invitrogen, USA and nucleofection using Human Chondrocytes Nucleofector® kit from Amaxa Biosystem, Germany. Chondrocytes were cultured and transfected with sox-9 gene at passage 1 according to the manufacturers’ protocols. Transfected chondrocytes were expanded until passage 3. Expression of chondrogenic markers namely collagen type II, aggrecan core protein and sox-9 were evaluated by quantitative RT-PCR method using iScriptTM One Step RT-PCR Kit with SYBR® Green, BIO-RAD. Chondrogenic dedifferentiation marker, collagen type I was also analyzed using the quantitative RT-PCR method. Expression level of each targeted gene was normalized to the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Overall efficiency ranging from 50% to 60% could be achieved using both transfection techniques. Transiently transfecting cells demonstrated remarkable competency sustaining specific chondrogenic genes namely collagen type II, aggrecan core protein and sox-9, significantly better than in the non-transfected cells. It is believed that this preliminary finding has to be extended to develop its full potential since sox-9 transcription factor is essential for chondrocyte differentiation and cartilage formation. Sox9 gene therapy would delay the degenerative changes in articular cartilage which is consistent to the up-regulation of cartilage-specific markers especially collagen type II synthesis in vivo.

The Re-Expression of Collagen Type 2, Aggrecan and Sox 9 in Tissue-Engineered Human Articular Cartilage

( Munirah Sha`ban ),( Aminuddin Bin Saim ),( Samsudin Osman Cassim ),( Chua Kien Hui ),( Fuzina Nor Hussein ),( Ruszymah Bt Hj Idrus ) 한국조직공학과 재생의학회 2005 조직공학과 재생의학 Vol.2 No.4

This study was designed to verify the optimal · basic culture media that promote chondrocytes proliferation in vitro in order to facilitate adequate amount of chondrocytes for cartilage reconstructionas well as maintaining cartilage specific phenotype. Human articular chondrocytes were cultured in three types of basic culture media Ham`s F12, DMEM and the equivalent mixture of F12:DMEM. Cultured chondrocytes were trypsinized as they reached confluency. The viability and total number of cell were recorded at every passage. Large-scale culture expansion was used to reconstruct tissue-engineered cartilage. Quantitative RT-PCR analysis was used to evaluate the expression of collagen Type II, collagen Type I, aggrecan and Sox 9 gene, both in monolayer culture and in the engineered cartilage. The mixture of F12:DMEM promotes significantly greater (p<0.05) chondrocytes proliferation at every passage compared to the individual medium. Monolayer cultured chondrocytes exhibited down-regulation expression pattern of collagen Type II gene, aggrecan and Sox 9, whilst the expression of collagen Type I is up-regulated. Tissue-engineered cartilage morphologically and histologically resembled normal hyaline cartilage. Moreover, tissue-engineered cartilage re-expressed the specific chondrogenesis markers; collagen Type II, aggrecan and Sox 9. In conclusion, the mixture of F12:DMEM enhanced human articular chondrocytes proliferation thus provided adequate amount of chondrocytes for cartilage reconstruction. The new cartilage formed phenotypically resembles native cartilage. This results hold promise for the use of tissue-engineered cartilage implant for future orthopaedic reconstructive surgery.

Incorporation of Smooth Muscle Cells Derived from Human Adipose Stem Cells on Poly(Lactic-co-Glycolic Acid) Scaffold for the Reconstruction of Subtotally Resected Urinary Bladder in Athymic Rats

Salem Salah Abood,Rashidbenam Zahra,Jasman Mohd Hafidzul,Ho Christopher Chee Kong,Sagap Ismail,Singh Rajesh,Yusof Mohd Reusmaazran,Md. Zainuddin Zulkifli,Haji Idrus Ruszymah Bt,Ng Min Hwei 한국조직공학과 재생의학회 2020 조직공학과 재생의학 Vol.17 No.4

Background: The urinary tract can be affected by both congenital abnormalities as well as acquired disorders, such as cancer, trauma, infection, inflammation, and iatrogenic injuries, all of which may lead to organ damage requiring eventual reconstruction. As a gold standard, gastrointestinal segment is used for urinary bladder reconstruction. However, one major problem is that while bladder tissue prevents reabsorption of specific solutes, gastrointestinal tissue actually absorbs them. Therefore, tissue engineering approach had been attempted to provide an alternative tissue graft for urinary bladder reconstruction. Methods: Human adipose-derived stem cells isolated from fat tissues were differentiated into smooth muscle cells and then seeded onto a triple-layered PLGA sheet to form a bladder construct. Adult athymic rats underwent subtotal urinary bladder resection and were divided into three treatment groups (n = 3): Group 1 (“sham”) underwent anastomosis of the remaining basal region, Group 2 underwent reconstruction with the cell-free scaffold, and Group 3 underwent reconstruction with the tissue-engineered bladder construct. Animals were monitored on a daily basis and euthanisation was performed whenever a decline in animal health was detected. Results: All animals in Groups 1, 2 and 3 survived for at least 7 days and were followed up to a maximum of 12 weeks post-operation. It was found that by Day 14, substantial ingrowth of smooth muscle and urothelial cells had occurred in Group 2 and 3. In the long-term follow up of group 3 (tissue-engineered bladder construct group), it was found that the urinary bladder wall was completely regenerated and bladder function was fully restored. Urodynamic and radiological evaluations of the reconstructed bladder showed a return to normal bladder volume and function.Histological analysis revealed the presence of three muscular layers and a urothelium similar to that of a normal bladder. Immunohistochemical staining using human-specific myocyte markers (myosin heavy chain and smoothelin) confirmed the incorporation of the seeded cells in the newly regenerated muscular layers. Conclusion: Implantation of PLGA construct seeded with smooth muscle cells derived from human adipose stem cells can lead to regeneration of the muscular layers and urothelial ingrowth, leading to formation of a completely functional urinary bladder.