Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) are widely used in the clinic because they involve fewer ethical issues and safety concerns compared to other stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). MSCs derived from umbilical cord Wharton's jelly (WJ-MSCs) have excellent proliferative potential and a faster growth rate and can retain their multipotency for more passages in vitro compared to adult MSCs from bone marrow or adipose tissue. WJ-MSCs are used clinically for repairing tissue injuries of the spinal cord, liver and heart with the aim of regenerating tissue. On the other hand, WJ-MSCs are also used clinically to ameliorate immune-mediated diseases based on their ability to modulate immune responses. In the field of tissue engineering, WJ-MSCs capable of differentiating into multiple cell lineages have been used to produce a variety of engineered tissues in vitro that can then be transplanted in vivo. This review discusses the characteristics of WJ-MSCs, the differences between WJ-MSCs and adult MSCs, clinical studies involving WJ-MSCs and future perspectives of WJ-MSC research and clinical applications. To summarize, WJ-MSCs have shown promise in treating a variety of diseases clinically. However, most clinical trials/studies reported thus far are relatively smaller in scale. The collected evidence is insufficient to support the routine use of WJ-MSC therapy in the clinic. Thus, rigorous clinical trials are needed in the future to obtain more information on WJ-MSC therapy safety and efficacy.

Effect of cell density on formation of three-dimensional cartilaginous constructs using fibrin & human osteoarthritic chondrocytes.

Background & objectives: Seeding density is one of the major parameters affecting the quality of tissue-engineered cartilage. The objective of this study was to evaluate different seeding densities of osteoarthritis chondrocytes (OACs) to obtain the highest quality cartilage. Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development. Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II. Interpretation & conclusions: Our results confirmed that cell density was a significant factor affecting cell behaviour and aggregate production, and this was important for establishing good quality cartilage.

Development of an In Vitro Cardiac Ischemic Model Using Primary Human Cardiomyocytes.

Developing experimental models to study ischemic heart disease is necessary for understanding of biological mechanisms to improve the therapeutic approaches for restoring cardiomyocytes function following injury. The aim of this study was to develop an in vitro hypoxic/re-oxygenation model of ischemia using primary human cardiomyocytes (HCM) and define subsequent cytotoxic effects. HCM were cultured in serum and glucose free medium in hypoxic condition with 1% O2 ranging from 30 min to 12 h. The optimal hypoxic exposure time was determined using Hypoxia Inducible Factor 1α (HIF-1α) as the hypoxic marker. Subsequently, the cells were moved to normoxic condition for 3, 6 and 9 h to replicate the re-oxygenation phase. Optimal period of hypoxic/re-oxygenation was determined based on 50% mitochondrial injury via 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay and cytotoxicity via lactate dehydrogenase (LDH) assay. It was found that the number of cells expressing HIF-1α increased with hypoxic time and 3 h was sufficient to stimulate the expression of this marker in all the cells. Upon re-oxygenation, mitochondrial activity reduced significantly whereas the cytotoxicity increased significantly with time. Six hours of re-oxygenation was optimal to induce reversible cell injury. The injury became irreversible after 9 h as indicated by > 60% LDH leakage compared to the control group cultured in normal condition. Under optimized hypoxic reoxygenation experimental conditions, mesenchymal stem cells formed nanotube with ischemic HCM and facilitated transfer of mitochondria suggesting the feasibility of using this as a model system to study molecular mechanisms of myocardial injury and rescue.

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.

Cardiomyogenic differentiation of human sternal bone marrow mesenchymal stem cells using a combination of basic fibroblast growth factor and hydrocortisone.

The alarming rate of increase in myocardial infarction and marginal success in efforts to regenerate the damaged myocardium through conventional treatments creates an exceptional avenue for cell-based therapy. Adult bone marrow mesenchymal stem cells (MSCs) can be differentiated into cardiomyocytes, by treatment with 5-azacytidine, thus, have been anticipated as a therapeutic tool for myocardial infarction treatment. In this study, we investigated the ability of basic fibroblastic growth factor (bFGF) and hydrocortisone as a combined treatment to stimulate the differentiation of MSCs into cardiomyocytes. MSCs were isolated from sternal marrow of patients undergoing heart surgery (CABG). The isolated cells were initially monitored for the growth pattern, followed by characterization using ISCT recommendations. Cells were then differentiated using a combination of bFGF and hydrocortisone and evaluated for the expression of characteristic cardiac markers such as CTnI, CTnC, and Cnx43 at protein level using immunocytochemistry and flow cytometry, and CTnC and CTnT at mRNA level. The expression levels and pattern of the cardiac markers upon analysis with ICC and qRT-PCR were similar to that of 5-azacytidine induced cells and cultured primary human cardiomyocytes. However, flow cytometric evaluation revealed that induction with bFGF and hydrocortisone drives MSC differentiation to cardiomyocytes with a marginally higher efficiency. These results indicate that combination treatment of bFGF and hydrocortisone can be used as an alternative induction method for cardiomyogenic differentiation of MSCs for future clinical 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.

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.

Cytotoxic evaluation of biomechanically improved crosslinked ovine collagen on human dermal fibroblasts.

Earlier studies in our laboratory demonstrated that collagen extracted from ovine tendon is biocompatible towards human dermal fibroblast. To be able to use this collagen as a scaffold in skin tissue engineering, a mechanically stronger scaffold is required that can withstand manipulation before transplantation. This study was conducted to improve the mechanical strength of this collagen sponge using chemical crosslinkers, and evaluate their effect on physical, chemical and biocompatible properties. Collagen sponge was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA). Tensile test, FTIR study and mercury porosimetry were used to evaluate mechanical properties, chemical property and porosity, respectively. MTT assay was performed to evaluate the cytotoxic effect of crosslinked collagen sponge on human dermal fibroblasts. The FTIR study confirmed the successful crosslinking of collagen sponge. Crosslinking with EDC and GA significantly increased the mechanical strength of collagen sponge, with GA being more superior. Crosslinking of collagen sponge significantly reduced the porosity and the effect was predominant in GA-crosslinked collagen sponge. The GA-crosslinked collagen showed significantly lower, 60% cell viability towards human dermal fibroblasts compared to that of EDC-crosslinked collagen, 80% and non-crosslinked collagen, 100%. Although the mechanical strength was better when using GA but the more toxic effect on dermal fibroblast makes EDC a more suitable crosslinker for future skin tissue engineering.

The use of autologous fibrin as a scaffold for cultivating autologous conjunctiva in the treatment of conjunctival defect.

The purpose of this study was to compare the use of autologous fibrin to human amniotic membrane (HAM) as a scaffold in cultivating autologous conjunctiva for transplantation in treatment of conjunctival defect. An experimental study was performed using 18 adult New Zealand white strain rabbits which were divided into 3 groups. Each group consists of 6 rabbits. The conjunctiva on the temporal site was excised to create a conjunctival epithelial defect. The excised area in the Group 1 was transplanted with autologous conjunctiva cultivated on autologous fibrin; Group 2 was transplanted with autologous conjunctiva cultivated on HAM and Group 3 was left bare. The rabbits were followed up at regular intervals until 6 weeks. The mean period of complete conjunctival epithelization was 11.50 ± 8.22 days for the autologous fibrin group, 15.33 ± 11.80 days for the HAM group and 25.33 ± 5.32 days in the bare sclera group. The epithelization rate for the autologous fibrin group was faster compared to the other two groups. However all the results were not statistically significant (p value >0.05). There were no postoperative complications noted during the follow up. Autologous fibrin is comparable to HAM as a scaffold for cultivation of conjunctiva in the treatment of conjunctival defect.

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.

The association between GJB2 mutation and GJB6 gene in non syndromic hearing loss school children.

Recently, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non syndromic hearing impairment of unknown cause. The aims of this study are to determine the association between GJB2 mutation and GJB6 and to report the variation of mutations in deaf students who have heterozygous GJB2. This retrospective study was conducted at Universiti Kebangsaan Malaysia Medical Center (UKMMC). Data was collected from previous files and records from Tissue Engineering and Human Genetic Research Group Laboratory. Approval from Ethical Committee was obtained prior to the study. A total of 138 students have been screened in previous studies in UKMMC for the presence of GJB2 mutations as a cause for hearing loss. Thirty four of the 138 subjects have GJB2 mutations; 2 showed homozygous mutations whereas another 32 were heterozygous for GJB2 gene mutation. Only 31 DNA samples of students presented with sensorineural hearing loss with heterozygous mutation in GJB2 gene were included in this study. The sequencing results obtained were analyzed. The degree of hearing loss of those students with association between GJB2 mutation and GJB6 mutation will be discussed. Five out of 31 subjects (16.2%) have mutations in their GJB6 gene, suggesting a digenic inheritance of GJB2/GJB6 mutation. In total, four novel mutations were identified; E137D (n=1), R32Q (n=1), E101K (n=1) and Y156H (n=1) and one mutation deletion; 366delT (n=1). All students with association GJB2 mutation and GJB6 showed severe to profound hearing loss in both ears. Interestingly this study not detected the large deletion of 342 kb in GJB6 gene suggesting that the mutation is very rare in this region compared to certain parts of the world.

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.

Sequential induction of marrow stromal cells by FGF2 and BMP2 improves their growth and differentiation potential in vivo.

BACKGROUND: repairing bone loss by autologous grafting requires that a patient's marrow stromal cells (MSCs) be collected and cultured until the number of cells is adequate for implantation. Currently used techniques allow a slow proliferation rate and produce a culture that contains only small amounts of pluripotent stem cells that will become osteoblasts in culture. OBJECTIVE: to develop culture conditions that permit a rapid increase in the number of MSCs while retaining or improving their potential for complete differentiation in vivo. RESULTS: sequential applications of low doses of basic fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) improved the growth and differentiation potential of MSCs. FGF2 also elevated sensitivity of the cells to BMP2. BMP2 increased the syntheses of alkaline phosphatase (ALP), collagen type I and bone sialoprotein, while FGF2 increased the expression of osteocalcin (OC). Full induction as determined by the formation of mineralised nodules in vitro was observed within 7 days. Seeding the induced cells onto scaffolds and then implanting them into nude mice resulted in newly formed bone 4 weeks later. The results of real-time polymerase chain reaction (PCR) and Western blotting suggested that FGF2 increased the pool of committed osteoblasts by up-regulating the Cbfa1/Runx2 gene. The later stages of bone formation seemed to be induced by Cbfa1/Runx2-downstream factors such as BMP2, ALP, collagen type I, bone sialoprotein and OC. CONCLUSION: the culture system that was developed increased both the proliferation of MSC and the proportion that developed into pre-osteoblasts.

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.