The aetiopathogenesis of medial coronoid disease (MCD) remains obscure, despite its high prevalence. The role of changes to subchondral bone or articular cartilage is much debated. Although there is evidence of micro-damage to subchondral bone, it is not known whether this is a cause or a consequence of MCD, nor is it known whether articular cartilage is modified in the early stages of the disease. The aim of the present study was to use equilibrium partitioning of an ionic contrast agent with micro-computed tomography (microCT) to investigate changes to both the articular cartilage and the subchondral bone of the medial coronoid processes (MCP) of growing Labrador retrievers at an early stage of the disease and at different bodyweights. Of 14 purpose-bred Labrador retrievers (15-27 weeks), six were diagnosed with bilateral MCD and one was diagnosed with unilateral MCD on the basis of microCT studies. The mean X-ray attenuation of articular cartilage was significantly higher in dogs with MCD than in dogs without MCD (P<0.01). In all dogs, the mean X-ray attenuation of articular cartilage was significantly higher at the lateral (P<0.001) than at the proximal aspect of the MCP, indicating decreased glycosaminoglycan content. Changes in parameters of subchondral bone micro-architecture, namely the ratio of bone volume to tissue volume (BV/TV), bone surface density (BS/TV), bone surface to volume ratio (BS/BV), trabecular thickness (Tb.Th; mm), size of marrow cavities described by trabecular spacing (Tb.Sp; mm), and structural model index (SMI), differed significantly by litter (P<0.05) due to the difference in age and weight, but not by the presence/absence of MCD (P>0.05), indicating that subchondral bone density is not affected in early MCD. This study demonstrated that cartilage matrix and not subchondral bone density is affected in the early stages of MCD.
The use of gelatin microspheres (GMs) as a cell carrier has been extensively researched. One of its limitations is that it dissolves rapidly in aqueous settings, precluding its use for long-term cell propagation. This circumstance necessitates the use of crosslinking agents to circumvent the constraint. Thus, this study examines two different methods of crosslinking and their effect on the microsphere's physicochemical and cartilage tissue regeneration capacity. Crosslinking was accomplished by physical (dehydrothermal [DHT]) and natural (genipin) crosslinking of the three-dimensional (3D) GM. We begin by comparing the microstructures of the scaffolds and their long-term resistance to degradation under physiological conditions (in an isotonic solution, at 37°C, pH = 7.4). Infrared spectroscopy indicated that the gelatin structure was preserved after the crosslinking treatments. The crosslinked GM demonstrated good cell adhesion, viability, proliferation, and widespread 3D scaffold colonization when seeded with human bone marrow mesenchymal stem cells. In addition, the crosslinked microspheres enhanced chondrogenesis, as demonstrated by the data. It was discovered that crosslinked GM increased the expression of cartilage-related genes and the biosynthesis of a glycosaminoglycan-positive matrix as compared with non-crosslinked GM. In comparison, DHT-crosslinked results were significantly enhanced. To summarize, DHT treatment was found to be a superior approach for crosslinking the GM to promote better cartilage tissue regeneration.
Stem cell differentiation is guided by contact with the physical microenvironment, influence by both topography and mechanical properties of the matrix. In this study, the combined effect of substratum nano-topography and mechanical stiffness in directing mesenchymal stem cell (MSC) chondrogenesis was investigated. Three polyesters of varying stiffness were thermally imprinted to create nano-grating or pillar patterns of the same dimension. The surface of the nano-patterned substrate was coated with chondroitin sulfate (CS) to provide an even surface chemistry, with cell-adhesive and chondro-inductive properties, across all polymeric substrates. The surface characteristic, mechanical modulus, and degradation of the CS-coated patterned polymeric substrates were analyzed. The cell morphology adopted on the nano-topographic surfaces were accounted by F-actin distribution, and correlated to the cell proliferation and chondrogenic differentiation outcomes. Results show that substratum stiffness and topographical cues affected MSC morphology and aggregation, and influenced the phenotypic development at the earlier stage of chondrogenic differentiation. Hyaline-like cartilage with middle/deep zone cartilage characteristics was generated on softer pillar surface, while on stiffer nano-pillar material MSCs showed potential to generate constituents of hyaline/fibro/hypertrophic cartilage. Fibro/superficial zone-like cartilage could be derived from nano-grating of softer stiffness, while stiffer nano-grating resulted in insignificant chondrogenesis. This study demonstrates the possibility of refining the phenotype of cartilage generated from MSCs by manipulating surface topography and material stiffness.
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.
Articular cartilage is well known for its simple uniqueness of avascular and aneural structure that has limited capacity to heal itself when injured. The use of three dimensional construct in tissue engineering holds great potential in regenerating cartilage defects. This study evaluated the in vitro cartilaginous tissue formation using rabbit's bone marrow mesenchymal stem cells (BMSCs)-seeded onto poly(lactic-co-glycolic acid) PLGA/fibrin and PLGA scaffolds. The in vitro cartilaginous engineered constructs were evaluated by gross inspection, histology, cell proliferation, gene expression and sulphated glycosaminoglycan (sGAG) production at week 1, 2 and 3. After 3 weeks of culture, the PLGA/fibrin construct demonstrated gross features similar to the native tissue with smooth, firm and glistening appearance, superior histoarchitectural and better cartilaginous extracellular matrix compound in concert with the positive glycosaminoglycan accumulation on Alcian blue. Significantly higher cell proliferation in PLGA/fibrin construct was noted at day-7, day-14 and day-21 (p<0.05 respectively). Both constructs expressed the accumulation of collagen type II, collagen type IX, aggrecan and sox9, showed down-regulation of collagen type I as well as produced relative sGAG content with PLGA/fibrin construct exhibited better gene expression in all profiles and showed significantly higher relative sGAG content at each time point (p<0.05). This study suggested that with optimum in vitro manipulation, PLGA/fibrin when seeded with pluripotent non-committed BMSCs has the capability to differentiate into chondrogenic lineage and may serve as a prospective construct to be developed as functional tissue engineered cartilage.
This is a retrospective study examining the outcome of paediatric patients with subglottic stenosis who underwent partial cricotracheal resection (PCTR) as a primary open procedure from 2004 to 2012. There were 5 patients identified aged from 3 to 18 years old. All the subglottic stenosis were acquired type. All of them were secondary to prolonged intubation. Three patients were classified as Myer-Cotton grade III and the other two were Myer-Cotton grade IV. Two of the patients had concomitant bilateral vocal cord immobility. All patients underwent two staged PCTR . All patients underwent two staged PCTR, and one patient underwent posterior cordectomy apart from partial CTR at different setting. All patients were successfully decannulated at various durations postoperatively. Although this is an early experience in our institution, PCTR has shown to be effective and safe procedure in patients with subglottic stenosis especially those with Myer-Cotton grade III and IV.
A case of a 21 year old gentleman is described, with no history of preceding trauma, presenting with intermittent dysphagia to solids and fluids for 4 years. Neck examination at rest was normal. However on deep inspiration, the right thyroid lamina protrudes or becomes more prominent. The patient is able to return the larynx to its normal position with manual manipulation. Laryngeal examination with fibreoptic scope during rest and deep breath shows gross rotation of the laryngeal structures for more than 60 degrees on deep breath, with the vocal cords axis rotated to the left side. Management was conservative.
A motorcyclist was involved in a motor vehicle accident and presented with respiratory distress and neck swelling with surgical emphysema. He sustained gross tracheal injury, severe pneumothoraces and lung contusions. As intubation was successful, the tracheal injury was not addressed immediately in view of the other severe respiratory problems. Evidence of aspiration lead to further investigations which confirmed the diagnosis 22 days post trauma. Thyrotracheal anastomosis was carried out without stenting. A complete cricotracheal separation is a rare event and can be easily overlooked in the emergency department.
We studied two similar cases of severe subglottic stenosis secondary to trauma with different treatment modalities. One had laryngotracheal reconstruction with cartilage augmentation and the other had laryngotracheal resection anastomosis. We wish to compare the two popular choices of surgery for laryngotracheal stenosis from two cases with similar nature and severity thus highlighting the complexity of managing severe subglottic stenosis in adult.
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.
Tissue engineering applies the principle of engineering and life sciences towards the development of biological substitute that restore, maintain or improve tissue or organ function. Scientists grow tissues or organs in vitro and implant them when the body is unable to prompt into healing itself. This presentation aims to highlight the potential clinical application of engineered tissues being researched on at the Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre.
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.
We reviewed the recurrence rate and possible factors influencing recurrence of preauricular sinus after excision. Seventy-one patients with 73 preauricular sinuses seen at our centre from year 2000 to 2005 were reviewed in this study. The overall recurrence rate was 14.1%. Twelve sinuses needed to be drained for an abscess prior to a definitive surgery. Different modalities used in demonstrating the sinus tract between methylene blue alone and probing together with methylene blue, showed different outcomes, which were statistically significant with a p value of < 0.05(chi-square test). A preauricular sinus with a previous history of infection or actively infected during the definitive surgery may have a higher tendency of recurrence. Meanwhile demonstrating the sinus tract by probing with lacrimal probe/sinus probe followed by injection of methylene blue reduces the recurrence rate (p < 0.05 with chi-square test).
Acquired subglottic stenosis is a compication or neonatal endotracheal intubation. Although it is rare, it contributes significantly to the morbidity and physical well being of post extubated neonates. A 20-day old neonate, ventilated for meconium aspiration syndrome and persistent fetal circulation, presented with marked stridor and respiratory embarrassment. A stenosed subglottic area was found on rigid bronchoscopy. Anterior cricoid split was performed to relieve the obstruction. He is asymptomatic post operatively.
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.
We have previously formulated an optimized human chondrocytes growth medium based on 2% fetal bovine serum supplementation. For clinical usage, the animal serum must be replaced by patient own serum. We investigated the effects of human serum concentration for human nasal septum chondrocytes monolayer culture and cartilage reconstruction. Human serum demonstrated a dose dependent manner in promoting chondrocytes growth and cartilage engineering.
To date there is no optimal approach to reconstruct an external ear. However, advances in tissue engineering technologies have indicated that in vitro autologous elastic cartilage might be of great importance in the future treatment of these patients. The aim of this study was to observe monolayer expansion of auricular cartilage and to evaluate engineered cartilage using standard histochemical study.
Patient own fibrin may act as the safest, cheapest and immediate available biodegradable scaffold material in clinical 1 tissue engineering. This study investigated the feasibility of using patient own fibrin isolated from whole blood to construct a new human cartilage, skin and bone. Constructed in vitro tissues were implanted on the dorsal part of the nude mice for in vivo maturation. After 8 weeks of implantation, the engineered tissues were removed for histological analysis. Our results demonstrated autologous fibrin has great potential as clinical scaffold material to construct various human tissues.
Treatment of articular cartilage lesions remains a clinical challenge. The uses of prosthetic joint replace allograft and/or autograft transplant carry a risk of complications due to infection, loosening of its component, immunological rejection and morbidity at the donor site. There has been an increasing interest in the management of cartilage damages, owing to the introduction of new therapeutic options. Tissue engineering as a method for tissue restoration begins to provide a potential alternative therapy for autologous grafts transplantations. We aimed to evaluate how well a tissue engineered neocartilage implant, consist of human articular chondrocytes cultured with the presence of autologous serum and mixed in a fresh fibrin derived from patient, would perform in subcutaneous implantation in athymic mice.