Cell viability, proliferation and extracellular matrix production of human annulus fibrosus cells cultured within PDLLA/Bioglass composite foam scaffolds in vitro.

Abstract

The objective of this study was to assess cell viability, attachment, morphology, proliferation, and collagen and sulphated glycosaminoglycan (s-GAG) production by human annulus fibrosus (HAF) cells cultured in vitro in poly(d,l-lactide) (PDLLA)/Bioglass composite foams. PDLLA foams with different percentages (0, 5 and 30wt.%) of Bioglass particles were prepared by thermally induced phase separation (TIPS) and characterized by scanning electron microscopy (SEM). HAF cell viability in the PDLLA/Bioglass foam was analysed using Live/Dead staining. HAF cell attachment was observed using SEM. An assessment of cell proliferation was conducted using the WST-1 assay. The level of s-GAG and collagen produced by HAF cells was quantified using the 1,9-dimethylmethylene blue (DMMB) assay and Sircoltrade mark assay after 4 weeks of culture. The presence of collagen types I and II within the PDLLA/Bioglass composite foams was analysed using immunohistochemistry. Live/dead staining showed that many viable HAF cells were present on the top surface of the foams as well as penetrating into the internal pore structure, suggesting that the PDLLA/Bioglass composite materials are non-toxic and that the presence of Bioglass particles within PDLLA scaffolds does not inhibit HAF cell growth. The SEM observations revealed that more clusters of HAF cells were attached to the pore walls of both the PDLLA/5BG foam and the PDLLA/30BG foam when compared with the PDLLA/0BG foam. WST-1 assay performed over a period of 4 weeks showed an increased tendency of HAF cells to proliferate within both the PDLLA/5BG foam and the PDLLA/30BG foam when compared with both the tissue culture plastic control and the PDLLA/0BG foam, indicating the presence of Bioglass in the foam has a positive effect on HAF cell proliferation. Sircoltrade mark and DMMB assays showed that HAF cells cultured within the PDLLA/30BG foam had a greater ability to deposit collagen and proteoglycan when compared with the control and the PDLLA/0BG foam after 4 weeks in culture, suggesting that the increase of Bioglass content may induce microenvironmental changes which promote the production of extracellular matrix containing abundant collagen and s-GAG. The immunohistochemical analysis of collagen production demonstrated that collagen produced in all cultures was predominantly of type I. These findings provide preliminary evidence for the use of PDLLA/Bioglass composite as cell-carrier materials for future treatments of the intervertebral disc with damaged AF region.