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Purpose: To evaluate the in vitro formation of 3D tissue engineered constructs (TECs) using rabbits’ annulus fibrosus (AF) cells seeded on poly(lactic-co-glycolic acid) (PLGA) based scaffolds.  Methods: Porous disc-shaped PLGA was fabricated using solvent casting and salt leaching technique. It was crosslinked with atelocollagen to form “PA”scaffold group. Fibrin was added to PLGA and PLGA-atelocollagen composite to form “PF” and “PAF” scaffolds, respectively. The AF cells were seeded into the prefabricated scaffolds (1.0x105 cells per scaffold) to form the following TECs groups: AF+PLGA (AFP; control), AF+PLGA+atelocollagen (AFPA), AF+PLGA+fibrin (AFPF) and AF+PLGA+atelocollagen+fibrin (AFPAF). The resulting TECs were cultured for three-week and evaluated for cells viability using MTT assay, cellular morphology and attachment using SEM, cartilaginous matrix production using sGAG assay and DNA content using PicoGreen® assay.  Results: Significant number of viable cells was observed in the AFPAF group (987,985.7±286,858.9 cells)when compared to other TECs(AFP: 373,319.0±5,456.9; AFPA: 547,763.4±66,038.2; AFPF: 463,763.4±46,160.8 cells). Cellular morphology and attachment were comparable in all TECs. The AFPA has the highest sGAG accumulation (0.279±0.117mg/ml) but shows no statistical difference when compared to the other TECs (AFP: 0.083±0.038; AFPF: 0.237±0.131; AFPAF: 0.181±0.024 mg/ml).The AFPF has the highest DNA content (1,919.338±89.050 ng/ml) but shows no statistical difference when comparedto the other TECs (AFP: 485.659±27.468; AFPA: 845.987±82.134; AFPAF: 1,575.007±307.174 ng/ml). Hence, atelocollagen seemed to provide better environment for cellular attachment and proliferation. This unique collagenous material also promotes sGAG production and DNA content in TECs.  Conclusion: The incorporation of atelocollagen into PLGA scaffold enhances the formation of TECs in vitro.

frontiers in bioengineering and biotechnology

Poly(lactic-co-glycolic acid), atelocollagen and fibrin hybrid scaffold seeded with annulus fibrosus cells enhances the formation of cartilaginous tissue engineered construct in vitro