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 sheep's 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.
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.
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.
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.
A potential cure for hearing loss would be to regenerate hair cells by stimulating cells of the damaged inner ear sensory epithelia to proliferate and differentiate into hair cells. Here, we investigated the possibility to isolate, culture-expand and characterize the cells from the cochlea membrane of adult mice. Our results showed that the cultured cells isolated from mouse cochlea membrane were heterogenous in nature. Morphologically there were epithelial like cells, hair cell like, nerve cell like and fibroblastic cells observed in the culture. The cultured cells were immunopositive for specific hair cell markers including Myosin 7a, Calretinin and Espin.
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.
This study aimed to compare the effects of three different media on the in vivo chondrogenesis of sheep bone marrow stem cells (BMSC). Sheep BMSC were cultured in F12:DMEM + 10% FBS, chondrogenic medium containing 5ng/ml TGF,3 + 50ng/ml IGF-1 and UKM-MECC for three weeks. The cultured cells were then harvested for construct formation with fibrin. Constructed tissues were implanted subcutaneously into nude mice for in vivo development. Cell aggregates were formed in both chondrogenic medium and UKM-MECC demonstrated the early chondrogenesis process. After five weeks of in vivo development, both chondrogenic medium and UKM-MECC promoted cartilage matrix synthesis confirmed by Safranin O staining.
The present work was to determine the development and re-epithelization of bilayered corneal construct (BCC) in vitro and in vivo using scanning electron microscopy (SEM). The in vitro BCC was transplanted to the rabbit's eye and after 90 days the BCC was harvested and analyzed. The corneas were processed for morphology studies. The result indicates that the BICC that was transplanted for 90 days showed good development and re-epithelization of epithelial layer similar to the normal cornea.
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.
Nerve stem cells have a unique characteristic in that they form spherical aggregates, also termed neurospheres, in vitro. The study demonstrated the successful derivation of these neurospheres from bone marrow culture. Their plasticity as nerve stem cells was confirmed. The findings further strengthens the pluripotency of cell populations within the bone marrow.
Human adipose-derived stem cells (HADSC) have demonstrated the capacity of differentiating into bone depending on the specific induction stimuli and growth factors. However, investigation on stem cell characteristic after osteogenic differentiation is still lacking. The goal of this study was to investigate the differential expression of sternness and osteogenic genes in non-induced HADSC compared with HADSC after osteogenic induction using quantitative Real Time RT-PCR. Our results showed that OCT-4, REX-1, FZD9, OSC, RUNX, and ALP were up regulated after osteogenic induction. This may indicated that HADSCs after osteogenic induction still possessed some stemness properties.
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.
Normal tracheal mucociliary clearance is the key to maintaining the health and defense of respiratory airway. Therefore the present of cilia and mucous blanket are important for tracheal epithelium to function effectively. In the present study, we prepared a tissue engineered respiratory epithelium construct (TEREC) made of autologous respiratory epithelium cells, fibroblast and fibrin from sheep owns blood which replaced a created tracheal mucosal defect. Scanning electron microscopy (SEM) showed encouraging result where immature cilia were present on the surface of TEREC. This result indicates that engineered respiratory epithelium was able to function as normal tissue.
This study was to determine if autologous bone marrow mesenchymal stem cells (BMSCs) cultured in chondrogenic medium could repair surgically induced osteoarthritis. Sheep BMSCs were cultured in medium containing 5ng/ml TGFbeta3 + 50ng/ml IGF-1 for three weeks. The cultured cells were then suspended at density of 2x10(6) cell/ml and injected intraarticularly into the osteoarthritic knee joint. After six weeks, the distal head of the femur and the proximal tibial plateau were removed and stained with H&E. The results indicated that knee joints treated with autologous BMSCs cultured in chondrogenic medium showed clear evidence of articular cartilage regeneration in comparison with other groups.
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.
A major factor limiting survival following extensive thermal injury is insufficient availability of donor sites to provide enough skin for the required grafting procedures. Limitation of autologous grafting promotes the usage of allograft skin substitutes to promote wound healing. Here, we investigated the wound healing potential of allograft single layered tissue engineered skin which comprises of either keratinocytes (SLTES-K) or fibroblast (SLTES-F) with fibrin as the delivery system. Results from gross and microscopic evaluation showed our single layered tissue engineered skin constructed with keratinocytes or fibroblast after gamma radiation with the dosage of 2Gy could serve as allograft for the treatment of skin loss.
The emergence of tissue engineering and stem cell research has created a tremendous response amongst scientist in Malaysia. However, despite the enthusiastic to embark on the research we have to carefully divert the research towards our needs. This is due to our responsibility to address the mounting problem of communicable diseases here and a very limited funding. As commercialization is a key objective the combination of products towards treating or diagnosing communicable and non-communicable diseases in the developing country is another important factor. The discussion here is mainly on the evolution of tissue engineering in Malaysia and taking a model of tissue engineering in otolaryngology.
Spinal fusion using autologous bone graft is performed in an increasing rate for many spinal disorders. However, graft harvesting procedure is associated with prolonged operation time and potential donor site morbidity. We produced an engineered 'bone graft' substitute by using porous hydroxyapatite (HA) scaffold seeded with autologous bone marrow osteoprogenitor cells (OPCs) and fibrin. This obviates bone graft harvesting, thus eliminates donor site morbidity and shortens the operation time. The aim of this study is to evaluate Hydroxyapatite (HA) ceramics as scaffold for autologous tissue engineered bone construct for spinal fusion in a sheep model. The sheep's marrow was aspirated from iliac crest. The bone marrow mesenchymal stem cells (BMMSCs) were cultured for several passages in the presence of growth and differentiation factors to increase the number of OPCs. After the cultures reached confluence, they were trypsinized and seeded on Hydroxyapatite scaffold (HA). Approximately 5 million cells were generated after 3 weeks of culture. Microscopically, very tight Colony Forming Units (CFU-Fs) were seen on monolayer culture. The Von Kossa and Alizarin Red staining of monolayer culture showed positive mineralization areas; indicating the presence of OPCs. Sheep underwent a posterolateral spinal fusion in which scaffolds with or without OPCs seeded were implanted on both sides of the lumbar spine (L1-L2). Intended fusion segments were immobilized using wires. At the end of third month, the fusion constructs were harvested for histological examination. Fibrous tissue infiltration found in the inter-connecting pores of plain HA ceramics indicates inefficient new bone regeneration. New bone was found surrounding the HA ceramics seeded with autologous cells. The new bone is probably formed by the sheep BMMSCs that were initially encapsulating HA while it remained intact. The new bone is naturally fused with the vertebrae. In conclusion, the incorporation of autologous bone marrow cells improved the effectiveness of HA ceramics as 'bone graft' substitute for spinal fusion.
Twenty percent of all childhood deafness is due to mutations in the GJB2 gene (Connexin 26). The aim of our study was to determine the prevalence and spectrum of GJB2 mutations in childhood deafness in Malaysia. We analyzed the GJB2 gene in 51 deaf students from Sekolah Pendidikan Khas Alor Setar, Kedah. Bidirectional sequencing indicates that 25% of our childhood deafness has mutation in their GJB2 gene. Sixty two percent of these children demonstrate V37I missense mutation. Interestingly, V37I mutation in the GJB2 gene have been reported as polymorphism in Western countries, however in our country it behaved as a potentially disease-causing missense mutation, causing childhood deafness as it was not found in the normal control.
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.