Three-dimensional compound tissue engineered scaffold fabricated by cell assembly system

Aim: To overcome the shortcomings of fabricating cell and material composites in tissue engineering, this study is designed to form three-dimension (3D) chondrocytes and materials compound scaffolds with predesigned parameters by using cell controlled assembling system, and evaluate the morphology and proliferation of chondrocytes; in the formed cell/gel scaffolds cultured in vitro. Methods: The experiments were completed in the State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University and Center of Organism Manufacturing, Department of Mechanical Engineering, Tsinghua University from June 2006 to March 2007. 1 Alginate and gelatin were selected to form mixed hydrogel, which was used as biomaterial of scaffold. The total concentration of hydrogel was 7% with the alginate/ gelatin ratio of 3:4. After crosslinked with CaCl₂ solution and cryodesiccation, the inner structure of mixed materials was observed by scanning electron microscope (SEM). 2 Articular cartilage was harvested from young (3-5 days old) New Zealand white rabbits, and then was digested by collagen type II to release chondrocytes. Then chondrocytes were cultured in vitro to expand for use. 3 The chondrocyte suspension was added into the mixed hydrogel. Then this mixture of hydrogel and cell was used to form 3D composite scaffold by cell assembling system with rapid prototyping, and the composite scaffold was cultured in vitro. 4 After cell culturing for 1, 7, 14 and 21 days, MTT assay was used to analyse proliferation of chondrocytes in gel. At 7 days of culture, the morphology of cells was observed by histological analysis, in which the cell/gel scaffold was stained with hematoxylin-eosin (HE). At 14 days of culture, immunohistochemical analysis was used to detect the expression of type II collagen for chondrocytes in gel. Results: 1 The result of SEM observation revealed that, the inner structure of mixed hydrogel was reseau with interstice. 2 The porous grid-like cell/gel scaffold was formed by cell assembling system with the size of 10 mm×10 mm× 6 mm. Cell viability after this procedure was assessed by the trypan blue exclusion assay and was confirmed to be over 90%. 3 MTT assay indicated that, the number of chondrocytes in the cell/gel scaffold increased with time. The absorbance at 21 days was about 3.5 times the value for that at 1 day. 4 HE staining result at the culture of 7 days revealed that, the chondrocytes in gel had spherical shape, which was the normal morphology of functional chondrocytes. Furthermore, the cell which was still in division could be observed. 5 The result of immunohistochemical analysis indicated that, all the chondrocytes cultured in gel were active and maintained the abilities to secret collagen II. Conclusion: The 3D compound tissue engineered scaffolds are formed by cell assembly system with the predesigned parameters. Chondrocytes have little damage through the process of assembling. The embedded chondrocytes can normally grow, proliferate, maintain th chondrocytic phenotype and cartilage activity, which suggest that this might be a feasible approach for cartilage repair.