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

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.

Three-dimensional culture of annulus fibrosus cells within PDLLA/Bioglass composite foam scaffolds: assessment of cell attachment, proliferation and extracellular matrix production.

The objective of the present study was to assess cell attachment, proliferation and extracellular matrix (ECM) production by bovine annulus fibrosus (BAF) cells cultured in vitro in PDLLA/Bioglass composite foams. PDLLA foams incorporated with different percentages (0, 5 and 30wt%) of Bioglass particles were prepared by thermally induced phase separation (TIPS) process and characterized by scanning electron microscopy (SEM). BAF cell morphology and attachment within the PDLLA/Bioglass foams were analysed using SEM. An assessment of cell proliferation was conducted using the WST-1 assay. The amount of sulphated glycosaminoglycans (sGAG) were quantified using the 1,9-dimethylmethylene blue (DMMB) assay after 4 weeks in culture. Furthermore, the amount of collagen synthesis was determined using a hydroxyproline assay, and the presence of collagen types I and II was investigated using Western blotting. Our results reveal that PDLLA/Bioglass foam scaffolds can provide an appropriate microenvironment for BAF cell culture which enhances cell proliferation and promotes the production of sGAG, collagen type I and collagen type II. These findings provide preliminary evidence for the use of PDLLA/Bioglass composite scaffolds as cell-carrier materials for future treatments of intervertebral discs with damaged AF regions.

Control of stem cell fate and function by engineering physical microenvironments.

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell­cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niche environments and regulate stem cell fate and function.