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.

Effects of applied cadmium on its accumulation, dry matter production and net photosynthesis in okra and amelioration of cadmium toxicity through lime application

Aim: Vegetables grown in cadmium contaminated soils accumulate cadmium in their tissues and are risky for consumption. The aim of the study was to get an insight into the effect of different levels of cadmium in soil, on accumulation in different plant parts of okra and its effect on overall growth, biomass production and photosynthesis rate so that suitable management option is explored to produce safe vegetable in cadmium contaminated soils. Methodology: The study was conducted in replicated pots with three soil pH (5.46, 6.54 and 7.45) attained through addition of CaCO3 as main treatment and four Cd levels viz., 0, 3, 6 and 9 mg kg-1 of soil as sub-treatment. Okra (Abelmoschus esculentus) was taken as the test crop. The experiment was conducted in a net house. Results: The Cd concentrations were minimum in fruits (0.54, 0.31 and 0.14 mg kg-1) and higher in leaves at acidic pH (5.5), while in soil limed to slightly acidic (6.5) and alkaline pH (7.5) roots retained maximum Cd among plant parts. Net photosynthesis and biomass production decreased significantly with higher Cd doses at acidic pH (5.5). The rate of decline in net photosynthesis was lesser at higher soil pH. The transfer factors decreased with increase in soil pH. The DTPA extractable soil Cd decreased from 8.5 to 2% when soil pH increased rendering the Cd less available for plant uptake. Interpretation: Liming can be an effective ameliorative measure to mitigate Cd toxicity in acidic soils and can ensure safe consumption. Lowest accumulation of cadmium in fruit part suggests okra to be a potential vegetable crop for Cd polluted soils.

Comparative Study on Cartilage Tissue Collected From Less- and Severely-Affected Region of Osteoarthritic Knee

Culture expanded chondrocytes isolated from non-load bearing region of osteoarthritic (OA) joint has been used to construct tissue engineered cartilage for treatment purposes. The aim of the study was to compare the histological properties of the cartilage tissue and morphological properties of the chondrocytes isolated from less and severely affected OA knee. Human articular cartilage was obtained as redundant tissue from consented patients with late-stage OA undergoing total knee replacement surgery at Universiti Kebangsaan Malaysia Medical Centre (UKMMC). Articular cartilage was graded according to Dougados and Osteoarthritis Research Society International (OARSI) classification. Articular cartilage was classified into less affected (LA; Grade 0-1) and severely affected (SA; Grade 2-3). Cartilage tissue from less and severely affected region was stained with Safranin O staining. Isolated chondrocytes from each group were cultured until passage 4 (P4). Their growth patterns, cell areas, and circularity were compared. LA-cartilage tissue shows uniform spread of safranin O staining indicating intact extracellular matrix (ECM) component. However, SA-cartilage shows significant reduction and unstable staining due to its degraded ECM. LA-chondrocytes showed an aggregated growth compared to SA-chondrocyte that remains monolayer. Moreover, LA-chondrocytes have significantly higher cell area with wider spreading at passage 0 and 4 compared to SA-chondrocytes. It was also found that chondrocyte circularity increased with passage, and circularity of LA-chondrocytes was significantly higher than that of the SA-chondrocytes at passage 3. This study demonstrated the considerable difference in the cellular properties for less and severely affected chondrocytes and implication of these differences in cell-based therapy needed to be explored.