Abstract
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.
Highlights
A systematic analysis of the Global Burden of Diseases Study 2013 indicated the top 10 leading causes of YLDs included low back pain (LBP) [1]
Poly(lacticcoglycolic acid), atelocollagen and fibrin hybrid scaffold seeded with annulus fibrosus cells enhances the formation of cartilaginous tissue engineered construct in vitro
The tissue engineered constructs (TECs) were evaluated by comparing the groups in terms of the cellular morphology and attachment using scanning electron microscopy (SEM) (Jeol, Japan), cells viability using a modified MTT cells proliferation assay on day 4, 7, 14 and 21, cartilaginous sulphated Glycosaminoglycan matrix production using KAssay® sGAG assay kit and DNA content using PicoGreen® DNA quantification assay at each time point of week 1, 2 and 3
Summary
A systematic analysis of the Global Burden of Diseases Study 2013 indicated the top 10 leading causes of YLDs included low back pain (LBP) [1]. Poly(lacticcoglycolic acid), atelocollagen and fibrin hybrid scaffold seeded with annulus fibrosus cells enhances the formation of cartilaginous tissue engineered construct in vitro The incorporation of atelocollagen and fibrin has been reported to facilitate and enhance cell proliferation, ECM production and cartilaginous tissue formation in vitro [2].
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