Shelf Life Evaluation of Clinical Grade Chondrogenic Induced Aged Adult Stem Cells for Cartilage Regeneration.

The study objectives include, enhancing the proliferations of aged bone marrow stem cells (BMSCs) and adipose stem cells (ADSCs); and evaluating the shelf lives of clinical grade chondrogenically induced cells from both samples. ADSCs and BMSCs from 56 patients (76 ± 8 yrs) were proliferated using basal medium (FD) and at (5, 10, 15, 20 and 25) ng/ml of basal fibroblast growth factor (bFGF). They were induced to chondrogenic lineage and stored for more than 120 hrs in FD, serum, Dulbecco's phosphate buffered saline (DPBS) and saline at 4 °C. In FD, cells stagnated and BMSCs' population doubling time (PDT) was 137 ± 30 hrs, while ADSCs' was 129.7 ± 40 hrs. bFGF caused PDT's decrease to 24.5 ± 5.8 hrs in BMSCs and 22.0 ± 6.5 hrs in ADSCs (p = 0.0001). Both cells were positive to stem cell markers before inductions and thereafter, expressed significantly high chondrogenic genes (p = 0.0001). On shelf life, both cells maintained viabilities and counts above 70% in FD and serum after 120 hrs. BMSCs' viabilities in DPBS fell below 70% after 96 hrs and saline after 72 hrs. ADSCs' viability fell below 70% in DPBS after 24 hrs and saline within 24 hrs. Concentrations between 20 ng/ml bFGF is ideal for aged adult cells' proliferation and delivery time of induced BMSCs and ADSCs can be 120 hrs in 4 °C serum.

Long-term evaluation of osteoarthritis sheep knee, treated with TGF-ß3 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-ß3 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.

The evaluation of cartilage differentiations using transforming growth factor beta3 alone and with combination of bone morphogenetic protein-6 on adult stem cells.

In our quest to standardize our formula for a clinical trial, transforming growth factor-beta3 (TGF-ß3) alone and in combination with bone morphogenetic protein-6 (BMP-6) were evaluated for their effectiveness in cartilage differentiation. Bone Marrow Stem Cells (BMSCs) and Adipose Derived Stem Cells (ADSCs) were induced to chondrogenic lineage using two different media. Native chondrocytes served as positive control. ADSCs and BMSCs proved multipotency by tri-lineage differentiations. ADSC has significantly higher growth kinetics compare to Chondrocyte only p ≤ 0.05. Using TGF-ß3 alone, BMSC revealed higher expressions for hyaline cartilage genes compare to ADSCs. Chondrocyte has significantly higher early chondrogenic markers expression to ADSCs and BMSCs, while BMSCs was only higher to ADSC at chondroadherin, p ≤ 0.0001. On mature chondrogenic markers, chondrocytes were significantly higher to ADSCs and BMSCs for aggrecan, collagen IX, sry (sex determining region y)-box9, collagen II and fibromodullin; and only to ADSC for collagen XI. BMSC was higher to ADSC for aggrecan and collagen IX, p ≤ 0.0001. The combination of TGF-ß3 + BMP-6 revealed increased gene expressions on both BMSCs and ADSCs for early and mature chondrogenic markers, but no significance difference. For dedifferentiation markers, ADSC was significantly higher to chondrocyte for collagen I. Glycosaminoglycan evaluations with both formulas revealed that chondrocytes were significantly higher to ADSCs and BMSCs, but none was significant to each other, p ≤ 0.0001. Combination of 10 ng TGF-ß3 with 10 ng of BMP-6 enhanced chondrogenic potentials of BMSCs and ADSCs compare to TGF-ß3 alone. This could be the ideal cocktail for either cell's chondrogenic induction.

Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications.

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.

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 × 10(4) ± 2.77 × 10(4) cells/cm(2)) 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.

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 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.

Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment.

In the present study in vitro expansion of human keratinocytes by supplementing dermal fibroblasts conditioned medium (DFCM) has been reported. Effect of two different DFCM acquired by culturing fibroblasts in keratinocyte-specific medium (defined keratinocytes serum free medium, DFCM-DKSFM) and fibroblast-specific serum free medium (F12: DMEM nutrient mix, DFCM-FD) have been compared. Growth kinetics of keratinocytes in terms of efficiency of cell attachment, expansion index, apparent specific growth rate and growth potential at the end of culture was evaluated in culture supplemented with DFCM-DKSFM and DFCM-FD in comparison with control i.e. DKSFM only. Results indicated that supplementation of DFCM caused significant increase in keratinocyte attachment. Efficiency of keratinocyte attachment in culture supplemented with bFCM-DKSFM was significantly higher compared to those cultured in DFCM-FD and DKSFM. In addition, the expansion index of keratinocytes in cultures supplemented with DFCM-DKSFM and DFCM-FD were 3.7 and 2.2 times higher than that of control condition even though the apparent growth rate and proliferative potential was found significantly lower. These results suggested that supplementation of DFCM enhanced expansion of keratinocyte by increasing efficiency of cell attachment, and DFCM-DKSFM provided suitable condition for in vitro expansion of keratinocytes compared to DFCM-FD and control condition.

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.

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, 34ßE12 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.

Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

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.

Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy.

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-ß2), 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.

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.

Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes.

Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9-69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3-12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p<0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p<0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of keratinocytes but not fibroblasts. Telomerase activity is an important criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression.

Derivation of neurospheres from bone marrow stromal cells.

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.

Differential gene expression of human adipose-derived stem cells in osteogenic induction.

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.

Usage of allogeneic single layered tissue engineered skin enhance wound treatment in sheep.

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.

A scanning electron microscopic study of in vivo tissue engineered respiratory epithelium in sheep.

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.

Living bilayered human skin equivalent: promising potentials for wound healing.

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.

Measurement of sulphated glycosaminoglycans production after autologous 'chondrocytes-fibrin' constructs implantation in sheep knee joint.

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.