Development of three-dimensional tissue engineered bone-oral mucosal composite models

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications

Effects of inorganic and organic nitrogen and phosphorus on the growth and toxicity of two Alexandrium species from Hong Kong

The effects of three nitrogen (N) and two phosphorus (P) inorganic and organic forms on the growth, toxin content and composition, toxin production, and chemical composition of Alexandrium catenella and Alexandrium tamarense isolated from coastal waters of Hong Kong were determined. The toxin production rate and cellular toxin content for A. catenella were at least 10-fold higher than A. tamarense. The highest net production rate (R-tox) of the two Alexandrium species was generally achieved in the exponential phase. However, the highest cellular toxin content occurred in the stationary phase in all cultures, partly due to the enhancement of cell volume caused by P limitation, except for urea grown cultures where cellular toxin content remained low throughout the growth stage. For A. catenella, NH4 induced the highest growth rate (0.59 d(-1)), toxin production rate (mu(tox), 1.0 mu mol L-1 d(-1); NH4 2.5 pmol cell(-1) d(-1)) and cellular toxin content (2.8 pmol cell(-1)) among the three nitrogen sources regardless of inorganic and organic P. The form of phosphorus had limited effect on A. catenella. In contrast, the response of A. tamarense to different forms of nitrogen and phosphorus was more complex. NH4 induced the highest cellular toxin content (445 fmol cell-1), while NO3 yielded the highest toxin production rate (mu(tox), 0.71 nmol L-1 d(-1); R-tox, 140 fmol cell(-1) d(-1)) and urea produced the highest growth rate (0.57 d(-1)). For A. tamarense, the highest toxin production rate occurred under organic phosphorus. The relationship between toxin accumulation and the form of nitrogen varied with the phosphorus source. A. catenella cultures grown on NO3 and NH4 have about 80-90% C1/2 toxins and 5-15% GTX 1/4 toxins compared to 65-75% C1/2 toxins and 25-35% GTX 1/4 toxins in cultures grown on urea. Our results suggest that during summer when Alexandrium uses NH4 from local sewage effluent as its preferred nitrogen source, it might become more toxic in combination with episodically occurring P limitation in Hong Kong waters. (C) 2012 Elsevier B.V. All rights reserved