Porous scaffolds made from a biodegradable copolymer of trimethylene carbonate and glycolide were evaluated for tissue-engineered medical products. We examined the scaffold coated with cell adhesion protein and fibronectin and cultured under a dynamic mixing condition to enhance the growth of chondrocytes. Our hypothesis was that the combination of coating and dynamic mixing would be beneficial to the viability of the chondrocytic cells. Fibronectin was selected as the model protein because of its availability and routine assaying methods. Sterile samples of scaffolds of about 1 mm in thickness were coated with fibronectin at 37 degrees C for 1.5 h. Four groups of scaffolds were used: uncoated static or dynamic, and coated static or dynamic. Scaffold samples were placed in either a Petri dish or a spinner flask (static vs. dynamic groups) after inoculation with rat chondrocytes of an initial cell density of 1.29 x 10(5) cell/mL. After 7, 14, 21, and 28 days, each sample was fixed, embedded, and sectioned at 5 micro thickness. The sections were double-label immunostained using antibodies against cellular fibronectin synthesized by adherent cells as a measure of cell viability. A Hoechst 33258 nuclear stain was used to measure the number of cells attached to the scaffold at each time interval. The slides were examined using a fluorescence microscope to determine the cell ingrowth. At least 25 fields/treatment group (except the 7 day group) were measured. The data showed that cell in-growths into the porous scaffolds were higher at all time periods for the coated dynamic group than those for the other three groups.

The objective of this study was to develop a synthetic bone graft in a paste form. Reported here are the results of the evaluation of a paste of chitosan glutamate (Protosan) and hydroxyapatite (referred to as a paste) used in a critical size defect model in rats. Eight-millimeter--diameter cranial defects were made in rat calvaria following a protocol approved by the animal review committee. Five groups were studied: (1) empty control, (2) defect filled with paste only, (3) defect filled with the paste containing bone-marrow aspirate, (4) defect filled with paste containing BMP-2, and (5) defect filled with paste containing osteoblasts cultured from bone-marrow aspirate. The sacrifice intervals were 9 and 18 weeks. Calvaria containing the defect were harvested, and the bone mineral density (BMD) was determined by dual energy X-ray absorptiometry. Push-out strength measurements were also performed. The BMD values of empty control were significantly lower than those of other groups at both 9 and 18 weeks. The mechanical properties, that is, push-out strengths and area under the push-out load and displacement were not significantly different between the samples. Histological examination of Goldner-trichromestained undecalcified sections showed the presence of mineralized bone spicules in the defect areas that were more prominent in those filled with paste and osteoblasts cultured from bone-marrow aspirate. Hence, this study demonstrated that the paste of chitosan glutamate and hydroxyapatite-containing osteoblasts cultured from bone-marrow aspirate would be an effective material to repair bone defects.

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