Ruszymah Bh

The potential of intra-articular injection of chondrogenic-induced bone marrow stem cells to retard the progression of osteoarthritis in a sheep model.

In recent years, the use of bone marrow mesenchymal stem cell (BMSC) implantation has provided an alternative treatment for osteoarthritis. The objective of this study is to determine whether or not an intra-articular injection of a single dose of autologous chondrogenic induced BMSC could retard the progressive destruction of cartilage in a surgically induced osteoarthritis in sheep. Sheep BMSCs were isolated and divided into two groups. One group was cultured in chondrogenic media containing (Hams F12:DMEM, 1:1) FD+1% FBS+5 ng/ml TGFβ3+50 ng/ml IGF-1 (CM), and the other group was cultured in the basal media, FD+10% FBS (BM). The procedure for surgically induced osteoarthritis was performed on the donor sheep 6 weeks prior to intra-articular injection into the knee joint of a single dose of BMSC from either group, suspended in 5 ml FD at density of 2 million cells/ml. The control groups were injected with basal media, without cells. Six weeks after injection, gross evidence of retardation of cartilage destruction was seen in the osteoarthritic knee joints treated with CM as well as BM. No significant ICRS (International Cartilage Repair Society) scoring was detected between the two groups with cells. However macroscopically, meniscus repair was observed in the knee joint treated with CM. Severe osteoarthritis and meniscal injury was observed in the control group. Interestingly, histologically the CM group demonstrated good cartilage histoarchitecture, thickness and quality, comparable to normal knee joint cartilage. As a conclusion, intra-articular injection of a single dose of BMSC either chondrogenically induced or not, could retard the progression of osteoarthritis (OA) in a sheep model, but the induced cells indicated better results especially in meniscus regeneration.

Prevalence of otitis media with effusion amongst pre-school children in Malaysia

A cross-sectional screening test was done to determine the prevalence of otitis media with effusion amongst, preschool children in two district in Malaysia, namely Kuala Lumpur an urban district and Kuala Selangor a rural district. It involved 1097 preschool children aged between 5 and 6 years old. Presence of otitis media effusion (OME) is based on abnormal otoscopic finding, Type B tympanogram and absence of ipsilateral acoustical reflex. The overall prevalence rate of OME was 13.8%. The prevalence in Kuala Lumpur was 17.9%, while in Kuala Selangor it was 9.48%. Bottle feeding during infancy and high socioeconomic status of the parents was statistically associated with higher incidence of OME. Other factors such as race, premature delivery, passive smoking, allergy, asthma and family size, had no influence on the prevalence of otitis media with effusion.

Shelf-life evaluation of bilayered human skin equivalent, MyDerm™.

Skin plays an important role in defense against infection and other harmful biological agents. Due to its fragile structure, skin can be easily damaged by heat, chemicals, traumatic injuries and diseases. An autologous bilayered human skin equivalent, MyDerm™, was engineered to provide a living skin substitute to treat critical skin loss. However, one of the disadvantages of living skin substitute is its short shelf-life, hence limiting its distribution worldwide. The aim of this study was to evaluate the shelf-life of MyDerm™ through assessment of cell morphology, cell viability, population doubling time and functional gene expression levels before transplantation. Skin samples were digested with 0.6% Collagenase Type I followed by epithelial cells dissociation with TrypLE Select. Dermal fibroblasts and keratinocytes were culture-expanded to obtain sufficient cells for MyDerm™ construction. MyDerm™ was constructed with plasma-fibrin as temporary biomaterial and evaluated at 0, 24, 48 and 72 hours after storage at 4°C for its shelf-life determination. The morphology of skin cells derived from MyDerm™ remained unchanged across storage times. Cells harvested from MyDerm™ after storage appeared in good viability (90.5%±2.7% to 94.9%±1.6%) and had short population doubling time (58.4±8.7 to 76.9±19 hours). The modest drop in cell viability and increased in population doubling time at longer storage duration did not demonstrate a significant difference. Gene expression for CK10, CK14 and COL III were also comparable between different storage times. In conclusion, MyDerm™ can be stored in basal medium at 4°C for at least 72 hours before transplantation without compromising its functionality.

Aqueous extract of Centella asiatica promotes corneal epithelium wound healing in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Centella asiatica is a traditional herbal medicine that has been shown to have pharmacological effect on skin wound healing, and could be potential therapeutic agent for corneal epithelial wound healing. AIM OF THE STUDY This study was done to evaluate the effects of Centella asiatica on the proliferation and migration of rabbit corneal epithelial (RCE) cells in the in vitro wound healing model. MATERIALS AND METHODS RCE cells were cultured with or without supplementation of Centella asiatica aqueous extract. Viability and proliferation of the RCE cells was determined by MTT assay and cell cycle was analyzed by flow cytometry. In vitro re-epithelization was studied by scratch assay and migration rate was evaluated quantitatively by image analyzer. Expression of corneal specific differentiation markers, CK12 and connexin 43, were studied via RT-PCR. RESULTS It was found that supplementation of Centella asiatica did not show any significant effect on the RCE cells proliferation at the concentration up to 500ppm, while at the concentration of 1000ppm significantly inhibited RCE cells proliferation (p<0.05). However, at the concentration up to 62.5ppm, RCE cells shows significant enhancement of migration rate compared to the control group (p<0.05). It was also found that the supplementation of Centella asiatica aqueous extract did not alter the expression of differentiation markers and cell cycle. CONCLUSION In conclusion, supplementation of Centella asiatica aqueous extract at low concentrations could be useful to promote corneal epithelium wound healing.

Safety and Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells Therapy for Retinal Degeneration

Purpose To investigate the safety and efficacy of subretinal injection of human Wharton’s Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats. Methods RCS rats were divided into 2 groups: hWJ-MSCs treated group (n = 8) and placebo control group (n = 8). In the treatment group, hWJ-MSCs from healthy donors were injected into the subretinal space in one eye of each rat at day 21. Control group received saline injection of the same volume. Additional 3 animals were injected with nanogold-labelled stem cells for in vivo tracking of cells localisation using a micro-computed tomography (microCT). Retinal function was assessed by electroretinography (ERG) 3 days before the injection and repeated at days 15, 30 and 70 after the injection. Eyes were collected at day 70 for histology, cellular and molecular studies. Results No retinal tumor formation was detected by histology during the study period. MicroCT scans showed that hWJ-MSCs stayed localised in the eye with no systemic migration. Transmission electron microscopy showed that nanogold-labelled cells were located within the subretinal space. Histology showed preservation of the outer nuclear layer (ONL) in the treated group but not in the control group. However, there were no significant differences in the ERG responses between the groups. Confocal microscopy showed evidence of hWJ-MSCs expressing markers for photoreceptor, Müller cells and bipolar cells. Conclusions Subretinal injection of hWJ-MSCs delay the loss of the ONL in RCS rats. hWJ-MSCs appears to be safe and has potential to differentiate into retinal-like cells. The potential of this cell-based therapy for the treatment of retinal dystrophies warrants further studies.

Formation of tissue engineered composite construct of cartilage and skin using high density polyethylene as inner scaffold in the shape of human helix

BACKGROUND Formation of external ear via tissue engineering has created interest amongst surgeons as an alternative for ear reconstruction in congenital microtia. OBJECTIVE To reconstruct a composite human construct of cartilage and skin in the shape of human ear helix in athymic mice. METHODS Six human nasal cartilages were used and digested with Collagenase II. Chondrocytes were passaged in 175 cm(2) culture flasks at a density of 10,000 cells/cm(2). Frozen human plasma was then mixed with human chondrocytes. Six human skin samples were cut into small pieces trypsinized and resuspended. The keratinocytes were plated in six-well plate culture dishes at a density of 2×105 cells per well. Dermis tissues were digested and the fibroblast cells resuspended in six-well plate at the density of 10,000 cells per well. Fibrin-fibroblast layer and fibrin-keratinocytes were formed by mixing with human plasma to create 6 bilayered human skin equivalent (BSE) constructs. The admixture of fibrin chondrocytes layers was wrapped around high density polyethylene (HDP), and implanted at the dorsum of the athymic mice. The construct was left for 4 weeks and after maturation the mice skin above the implanted construct was removed and replaced by BSE for another 4 weeks. RESULTS Haematoxylin and Eosin showed that the construct consists of fine arrangement and organized tissue structure starting with HDP followed by cartilage, dermis and epidermis. Safranin-O staining was positive for proteoglycan matrix production. Monoclonal mouse antihuman cytokeratin, 34βE12 staining displayed positive result for human keratin protein. CONCLUSIONS The study has shown the possibility to reconstruct ear helix with HDP and tissue engineered human cartilage and skin. This is another step to form a human ear and hopefully will be an alternative in reconstructive ear surgery.

Autologous implantation of bilayered tissue-engineered respiratory epithelium for tracheal mucosal regenesis in a sheep model.

The objective of this study was to regenerate the tracheal epithelium using autologous nasal respiratory epithelial cells in a sheep model. Respiratory epithelium and fibroblast cells were harvested from nasal turbinates and cultured for 1 week. After confluence, respiratory epithelium and fibroblast cells were suspended in autologous fibrin polymerized separately to form a tissue-engineered respiratory epithelial construct (TEREC). A 3 × 2 cm2 tracheal mucosal defect was created, and implanted with TEREC and titanium mesh as a temporary scaffold. The control groups were divided into 2 groups: polymerized autologous fibrin devoid of cells (group 1), and no construct implanted (group 2). All sheep were euthanized at 4 weeks of implantation. Gross observation of the trachea showed minimal luminal stenosis formation in the experimental group compared to the control groups. Macroscopic evaluation revealed significant mucosal fibrosis in control group 1 (71.8%) as compared to the experimental group (7%). Hematoxylin and eosin staining revealed the presence of minimal overgrowth of fibrous connective tissue covered by respiratory epithelium. A positive red fluorescence staining of PKH26 on engineered tissue 4 weeks after implantation confirmed the presence of cultured nasal respiratory epithelial cells intercalated with native tracheal epithelial cells. Scanning electron microscopy showed the presence of short microvilli representing immature cilia on the surface of the epithelium. Our study showed that TEREC was a good replacement for a tracheal mucosal defect and was able to promote natural regenesis of the tracheal epithelium with minimal fibrosis. This study highlighted a new technique in the treatment of tracheal stenosis.

Bone marrow and adipose stem cells can be tracked with PKH26 until post staining passage 6 in in vitro and in vivo

Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.

Stem cell genes are poorly expressed in chondrocytes from microtic cartilage

OBJECTIVES This study was aimed to see the difference between chondrocytes from normal cartilage compared to chondrocytes from microtic cartilage. Specific attentions were to characterize the growth of chondrocytes in terms of cell morphology, growth profile and RT-PCR analysis. STUDY DESIGN Laboratory experiment using auricular chondrocytes. METHODS Chondrocytes were isolated from normal and microtic human auricular cartilage after ear reconstructive surgeries carried out at the Universiti Kebangsaan Malaysia Medical Centre. Chondrocytes were cultured in vitro and subcultured until passage 4. Upon confluency, cultured chondrocytes at each passage (P1, P2, P3 and P4) were harvested and subjected to growth profile and gene expression analyses. Comparison was made between the microtic and normal chondrocytes. RESULTS For growth profile analysis cell viability did not show significant differences between both samples. There are no significance differences between both samples in terms of its growth rate, except in passage 1 where microtic chondrocytes were significant lower in their growth rate. Population doubling time and total number of cell doubling of all samples also did not show any significant differences. Gene expression is measured using Real Time-Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). There is no significant differences in the expression of collagen type I, collagen type II, collagen type X, aggrecan core protein, elastin and sox9 genes in both samples. There are significant lower in the expression of sox2, nestin, BST-1 and OCT-4 gene in microtic chondrocytes compared to the normal chondrocytes. Stem cells markers are included in this study as stemness in cells may imply a greater proliferative potential and plasticity in vitro. CONCLUSION Chondrocytes from microtic samples have the same properties as chondrocytes from normal samples and hold promises to be used as a starting material in the reconstruction of the external ear in future clinical application. The reduction in sox2, nestin, BST-1 and OCT-4 gene expression in microtic samples could be the possible cause of the arrested development of the external ear.

Surface modification of electrospun poly(methyl methacrylate) (PMMA) nanofibers for the development of in vitro respiratory epithelium model.

Scaffold design is an important aspect of in vitro model development. In this study, nanoscaffold surface modification, namely UV radiation and genipin cross-linking to immobilize collagen on the surface of electrospun poly (methyl methacrylate) (PMMA) nanofiber sheet was investigated. Samples were divided into four groups; PMMA nanofibers (PMMA), collagen-coated PMMA nanofibers (PMMACOL), genipin cross-linked collagen-coated PMMA nanofibers (PMMAGEN), and UV-irradiated collagen-coated PMMA nanofibers (PMMAUV). 6 h of UV radiation significantly reduced the hydrophobicity of PMMA nanofibers from (131.88° ± 1.33°) to (110.04° ± 0.27°) (p < 0.05). The amount of collagen immobilized was significantly higher in PMMAGEN group (239.36 ± 16.63 μg collagen/mg nanofibers) (p < 0.05) compared to the other groups. RECs on all scaffold expressed epithelial cell-specific markers (CK18 and CK14), mucin-producing cell marker (MUC5Ac) and were actively proliferating, based on the positive expression of Ki67. Total number of attached cells was significantly the highest in PMMAUV group on day 9 (6.44 × 104 ± 2.77 × 104 cells/cm2) and it has the highest proliferation rate from day 4 to 9 (0.005 ± 0.003 h−1) compared to the other groups. Even though PMMAGEN group showed the highest collagen adsorption, in terms of cells attachment and proliferation, PMMAUV group showed a better outcome compared to the other groups. Thus, PMMAUV scaffold is more suitable to be used in the construction of in vitro respiratory epithelial model.