Aminuddin Bs

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.

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.

Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry

Transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are known to stimulate the rate of chondrocyte proliferation. The theoretical risk of malignant transformation associated with growth factor stimulation of chondrocytes should be addressed; aneuploidy has been found to occur in human cartilaginous tumors. In this study, chondrocytes were obtained from six human auricles and cultured in vitro for 6 weeks in the presence or absence of TGF-beta and bFGF. Cells were analyzed for DNA at 3-, 4-, 5-, and 6-week intervals by flow cytometry (FACScan), which demonstrated no evidence of aneuploidy. A persistent increase in S-phase was noted in cells cultured only with TGF-beta. Cells were implanted in athymic mice, and after 8 weeks of implantation, the cartilage constructs formed were examined histologically. The tissue-engineered cartilage cultured originally in bFGF most resembled normal, native cartilage. Specimens cultured in TGF-beta produced suboptimal cartilage morphology. Flow cytometry shows no evidence of aneuploidy, with chondrocytes maintaining their normal diploid state. Further studies incorporating additional methods of analysis need to be done.

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.

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheeps bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1-L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1-L2, (b) TCP/HA constructs group/L2-L3, and (c) autogenous bone graft group/L5-L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.

Identification of suitable culture condition for expansion and osteogenic differentiation of human bone marrow stem cells

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.

Long-term evaluation of osteoarthritis sheep knee, treated with TGF-β3 and BMP-6 induced multipotent stem cells

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‐&bgr;3 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. HighlightsADSCs and BMSCs had increased chondrogenic gene expressions using TGF‐&bgr;3 and BMP‐6.Each treated groups had significantly improved cartilage scores compare to controls.PKH26 dye can be used for an elongated in vivo trackingThe EMG results revealed improved joint recovery of the treated groups.