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 30 wt.%) 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 Sircol™ 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. Sircol™ 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.