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Evaluation of suitable biodegradable scaffolds for engineered bone tissue

Phang MY, Ng MH, Tan KK, Aminuddin BS, Ruszymah BH, Fauziah O

Med J Malaysia, 2004 May;59 Suppl B:198-9.
PMID: 15468886

Tricalcium phosphate/hydroxyapatite (TCP/HA), hydroxyapatite (HA), chitosan and calcium sulphate (CaSO4) were studied and evaluated for possible bone tissue engineered construct acting as good support for osteogenic cells to proliferate, differentiate, and eventually spread and integrate into the scaffold. Surface morphology visualized by SEM showed that scaffold materials with additional fibrin had more cell densities attached than those without, depicting that the presence of fibrin and collagen fibers were truly a favourite choice of cells to attach. In comparison of various biomaterials used incorporated with fibrin, TCP/HA had the most cluster of cells attached.
Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy

Munirah S, Samsudin OC, Aminuddin BS, Ruszymah BH

Tissue Cell, 2010 Oct;42(5):282-92.
PMID: 20810142 DOI: 10.1016/j.tice.2010.07.002

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation.
Formation of tissue engineered composite construct of cartilage and skin using high density polyethylene as inner scaffold in the shape of human helix

Ruszymah BH, Chua KH, Mazlyzam AL, Aminuddin BS

Int J Pediatr Otorhinolaryngol, 2011 Jun;75(6):805-10.
PMID: 21481479 DOI: 10.1016/j.ijporl.2011.03.012

Formation of external ear via tissue engineering has created interest amongst surgeons as an alternative for ear reconstruction in congenital microtia.
Formation of tissue-engineered human auricular cartilage via tissue engineering technique for future use in ear surgery

Saim L, Aminuddin BS, Munirah S, Chua KH, Izuddin Fahmy A, Fuzina NH, et al.

Med J Malaysia, 2004 May;59 Suppl B:192-3.
PMID: 15468883

To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.
Gene expression characteristic in human auricular cartilage tissue engineering

Farah Wahida I, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Isa MR, et al.

Med J Malaysia, 2004 May;59 Suppl B:190-1.
PMID: 15468882

This study was to assess collagen type II and collagen type I gene expression in tissue-engineered human auricular: cartilage formed via tissue engineering technique. Large-scale culture expansions were transformed into 3D in vitro construct and were implanted subcutaneously on the dorsal of athymic mice. After 8 weeks, explanted construct was processed in the same manner of native cartilage to facilitate cells for gene expression analysis. Isolated cells from in vivo construct demonstrated expression of type II collagen gene comparable to native cartilage. This study verified that tissue-engineered auricular cartilage expressed cartilage specific gene, collagen type II after in vivo maturation.
Growth kinetic study on normal and microtic chondrocytes of human auricular cartilage

Ishak MF, Aminuddin BS, Asma A, Lokman BS, Ruszymah BH, Goh BS

Med J Malaysia, 2008 Jul;63 Suppl A:117-8.
PMID: 19025013

Chondrocytes were isolated from normal and microtic human auricular cartilage after ear surgery carried out at Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured and expanded until passage 4. After reached confluence, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and assigned for growth profile analysis. There was no significant difference in cell viability between both normal and microtic samples (p = 0.84). Both samples showed no significant differences for growth profile parameters in terms of growth rate, population doubling time and total number of cell doubling, except in passage 1, where there is significant difference in cell growth rate (p = 0.004). This preliminary data has indicated that chondrocytes from microtic cartilage has the potential to be used in the reconstruction of human pinna in the future.
Fulltext Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging

Ruszymah BH, Izham BA, Heikal MY, Khor SF, Fauzi MB, Aminuddin BS

Med J Malaysia, 2011 Dec;66(5):440-2.
PMID: 22390097 MyJurnal

Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.
Human serum is an advantageous supplement for human dermal fibroblast expansion: clinical implications for tissue engineering of skin

Mazlyzam AL, Aminuddin BS, Saim L, Ruszymah BH

Arch Med Res, 2008 Nov;39(8):743-52.
PMID: 18996287 DOI: 10.1016/j.arcmed.2008.09.001

Standard fibroblast culture medium usually contains fetal bovine serum (FBS). In theory, unknown risks of infection from bovine disease or immune reaction to foreign proteins may occur if standard culture method is used for future human tissue-engineering development. Human serum (HS) theoretically would be another choice in providing a safer approach and autologous clinically reliable cells.
Human serum provided additional values in growth factors supplemented medium for human chondrocytes monolayer expansion and engineered cartilage construction

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:194-5.
PMID: 15468884

We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.
Identification of suitable culture condition for expansion and osteogenic differentiation of human bone marrow stem cells

Chowdhury SR, Ng MH, Hassan NS, Aminuddin BS, Ruszymah BH

Hum. Cell, 2012 Sep;25(3):69-77.
PMID: 22968953

This study was undertaken in order to identify the best culture strategy to expand and osteogenic differentiation of human bone marrow stem cells (hBMSCs) for subsequent bone tissue engineering. In this regard, the experiment was designed to evaluate whether it is feasible to bypass the expansion phase during hBMSCs differentiation towards osteogenic lineages by early induction, if not identification of suitable culture media for enhancement of hBMSCs expansion and osteogenic differentiation. It was found that introduction of osteogenic factors in alpha-minimum essential medium (αMEM) during expansion phase resulted in significant reduction of hBMSCs growth rate and osteogenic gene expressions. In an approach to identify suitable culture media, the growth and differentiation potential of hBMSCs were evaluated in αMEM, F12:DMEM (1:1; FD), and FD with growth factors. It was found that αMEM favors the expansion and osteogenic differentiation of hBMSCs compared to that in FD. However, supplementation of growth factors in FD, only during expansion phase, enhances the hBMSCs growth rate and significantly up-regulates the expression of CBFA-1 (the early markers of osteogenic differentiation) during expansion, and, other osteogenic genes at the end of induction compared to the cells in αMEM and FD. These results suggested that the expansion and differentiation phase of the hBMSCs should be separately and carefully timed. For bone tissue engineering, supplementation of growth factors in FD only during the expansion phase was sufficient to promote hBMSCs expansion and differentiation, and preferably the most efficient culture condition.
In vitro development of autologous tissue engineered human articular neocartilage for orthopaedic surgery

Samsudin OC, Aminuddin BS, Munirah S, Chua KH, Fuzina NH, Isa MR, et al.

Med J Malaysia, 2004 May;59 Suppl B:15-6.
PMID: 15468796

Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.
Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Eur Cell Mater, 2005 Jun 17;9:58-67; discussion 67.
PMID: 15962238

This study was to investigate the effects of insulin-transferrin-selenium (ITS) on the proliferation and quantitative gene expression of adult human nasal septum chondrocytes in monolayer culture expansion and the formation of tissue engineered hyaline cartilage. Effects of ITS on human nasal septum chondrocytes monolayer culture expansion and gene expression were evaluated in various culture media either added with 2% fetal bovine serum (FBS) or 1 ng/mL basic fibroblast growth factor plus 1 ng/mL transforming growth factor or both serum and growth factors supplementation in comparison with medium added with 10%FBS. Chondrocytes cultured in medium added with 2% fetal bovine serum and growth factors either supplemented with or without ITS were then mixed with pluronic F-127 hydrogel for in vivo tissue engineered cartilage formation in nude mice model. Engineered tissues were removed after 8 weeks of implantation and evaluated with histological staining, immunohistochemistry, transmission electron microscopy and quantitative gene expression analysis. ITS promoted human chondrocytes proliferation and reduced chondrocytes dedifferentiation in media supplemented with serum and growth factors. ITS with 2% FBS and growth factors provided 15-fold increased in chondrocytes number by the end of the culture period compared to the standard culture medium used in chondrocytes culture (medium added with 10% FBS). Engineered tissue resulted from ITS supplementation demonstrated higher quality of cartilage formation. In conclusion, our study has demonstrated the benefits of ITS supplementation in human chondrocytes monolayer culture and tissue engineering cartilage formation.
Interaction between insulin-like growth factor-1 with other growth factors in serum depleted culture medium for human cartilage engineering

Chua KH, Aminuddin BS, Fuzina NH, Ruszymah BH

Med J Malaysia, 2004 May;59 Suppl B:7-8.
PMID: 15468792

The regulation roles of insulin-like growth factor-1 (IGF-1) with basic fibroblast growth factor (bFGF) and transforming growth factor beta 2 (TGFbeta2) in human nasal septum chondrocytes monolayer culture and cartilage engineering was investigated in this study. The role of IGF-1 with bFGF and TGFbeta2 was investigated by measuring chondrocyte growth kinetic and collagen genes expression. IGF-1 together with bFGF and TGFbeta2 promote cartilage tissue engineering, increase type II collagen expression and enhance the histological features of engineered cartilage.
Isolation techniques of murine bone marrow progenitor cells and their adipogenic, neurogenic and osteogenic differentiation capacity

Ng AM, Kojima K, Kodoma S, Ruszymah BH, Aminuddin BS, Vacanti AC

Med J Malaysia, 2008 Jul;63 Suppl A:121-2.
PMID: 19025015

Bone marrow derived progenitor cells have been widely studied for its multipotent property and have proofed to be an important resource in regenerative medicine. However, the propagation of murine bone marrow appeared to be a great challenge as compared to other mammalian species. In this study, various isolation techniques and the plasticity of the isolated cells were evaluated. Our result shows that magnetic sorting technique yielded the most viable cells and displayed wider differentiation capacity.
Fulltext Linkage and association studies in a Malaysian family with autosomal recessive non-syndromic hearing loss

Farah WI, Aminuddin BS, Ruszymah BH

Malays J Pathol, 2006 Jun;28(1):23-33.
PMID: 17694956 MyJurnal

Hearing loss is a common sensory deficit in humans. The hearing loss may be conductive, sensorineural, or mixed, syndromic or nonsyndromic, prelingual or postlingual. Due to the complexity of the hearing mechanism, it is not surprising that several hundred genes might be involved in causing hereditary hearing loss. There are at least 82 chromosomal loci that have been identified so far which are associated with the most common type of deafness--non-syndromic deafness. However, there are still many more which remained to be discovered. Here, we report the mapping of a locus for autosomal recessive, non-syndromic deafness in a family in Malaysia. The investigated family (AC) consists of three generations--parents who are deceased, nine affected and seven unaffected children and grandchildren. The deafness was deduced to be inherited in an autosomal recessive manner with 70% penetrance. Recombination frequencies were assumed to be equal for both males and females. Using two-point lod score analysis (MLINK), a maximum lod score of 2.48 at 0% recombinant (Z = 2.48, theta = 0%) was obtained for the interval D14S63-D14S74. The haplotype analysis defined a 14.38 centiMorgan critical region around marker D14S258 on chromosome 14q23.2-q24.3. There are 16 candidate genes identified with positive expression in human cochlear and each has great potential of being the deaf gene responsible in causing non-syndromic hereditary hearing loss in this particular family. Hopefully, by understanding the role of genetics in deafness, early interventional strategies can be undertaken to improve the life of the deaf community.
Living bilayered human skin equivalent: promising potentials for wound healing

Mazlyzam AL, Aminuddin BS, Saim L, Ruszymah BH

Med J Malaysia, 2008 Jul;63 Suppl A:32-3.
PMID: 19024969

The angiogenic potential of native skin (NS), keratinocytes single skin equivalent (SSE-K), fibroblasts single skin equivalent (SSE-F) and bilayered skin equivalent secreting angiogenic growth factors such as transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) in the in vitro systems at 24, 48, 72 hours and 7 days was compared using Enzyme-Linked Immunosorbent Assay (ELISA). Bilayered skin equivalent exhibit highest release of growth factors within 24 hours to 7 days of culture compared to NS, SSE-K and SSE-F. This proved the potential of bilayered skin equivalent in producing and sustaining growth factors release to enhance angiogenesis, fibroblasts proliferation, matrix deposition, migration and growth of keratinocytes.
Long-term evaluation of osteoarthritis sheep knee, treated with TGF-β3 and BMP-6 induced multipotent stem cells

Ude CC, Shamsul BS, Ng MH, Chen HC, Ohnmar H, Amaramalar SN, et al.

Exp Gerontol, 2018 04;104:43-51.
PMID: 29421350 DOI: 10.1016/j.exger.2018.01.020

BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery.
METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.
RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.
CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-β3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.
Measurement of sulphated glycosaminoglycans production after autologous 'chondrocytes-fibrin' constructs implantation in sheep knee joint

Munirah S, Samsudin OC, Chen HC, Salmah SH, Aminuddin BS, Ruszymah BH

Med J Malaysia, 2008 Jul;63 Suppl A:35-6.
PMID: 19024971

Chondrocytes were isolated from articular cartilage biopsy and were cultivated in vitro. Approximately 30 million of cultured chondrocytes per ml were incorporated with autologous plasma-derived fibrin to form three-dimensional construct. Full-thickness punch hole defects were created in lateral and medial femoral condyles. The defects were implanted either with the autologous 'chondrocytes-fibrin' construct (ACFC), autologous chondrocytes (ACI) or fibrin blank (AF). Sheep were euthanized after 12 weeks. The gross morphology of all defects treated with ACFC implantation, ACI and AF exhibited median scores which correspond to a nearly normal appearance according to the International Cartilage Repair Society (ICRS) classification. ACFC significantly enhanced cartilage repair compared to ACI and AF in accordance with the modified O'Driscoll histological scoring scale. The relative sulphated glycosaminoglycans content (%) was significantly higher (p < 0.05) in ACFC when compared to control groups; ACI vs. fibrin only vs. untreated (blank). Results showed that ACFC implantation exhibited superior cartilage-like tissue regeneration compared to ACI. If the result is applicable to the human, it possibly will improve the existing treatment approaches for cartilage restoration in orthopaedic surgery.
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.
Pediatric auricular chondrocytes gene expression analysis in monolayer culture and engineered elastic cartilage

Ruszymah BH, Lokman BS, Asma A, Munirah S, Chua K, Mazlyzam AL, et al.

Int J Pediatr Otorhinolaryngol, 2007 Aug;71(8):1225-34.
PMID: 17531328

This study was aimed at regenerating autologous elastic cartilage for future use in pediatric ear reconstruction surgery. Specific attentions were to characterize pediatric auricular chondrocyte growth in a combination culture medium and to assess the possibility of elastic cartilage regeneration using human fibrin.

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