Abstract
One way to improve orthopaedic materials is to understand the exact architectural parameters that influence bone cell behaviour. In this study substrates with highly controlled surface features were created using photo-lithographic processes. These surfaces were contrasted for their ability to influence osteoblast activity and inter-cellular communication.
An etched silicon wafer was created by photolithography and used to hot-emboss grooved substrates (10-30micrometers wide/ 5-16micrometers deep) in poly-carbonate (PC). Smaller features were created on polydimethylsiloxane (PDMS) by casting over a photo-resist patterned silicon wafer. Rat osteoblasts were routinely cultured on flat or micro-fabricated substrates or in media supplemented with osteogenic stimuli for 35 days. Alkaline phosphatase activity was colourimetri-cally localised, and mineralised matrix visualised with Von Kossa staining. Connexin-43 was immunolocalised with a CY-2 conjugated antibody. Intracellular communication was studied using a dye (Lucifer yellow) transfer technique and fluorescence microscopy.
Osteoblasts were aligned on the grooved surface. In 10micrometers grooves, cells were in single rows while at 30micrometers the rows were two/three cells wide. Culture of osteoblasts on these surfaces under osteogenic conditions demonstrated alkaline phosphatase activity comparable to flat surfaces but after 28-35 days there was little evidence of bone-like nodules on the grooved substrates. We hypothesized that on grooved substrates cell:cell communication is compromised thus gap-junctions were studied. Image analysis showed that there was lower connexin-43 expression in cells on grooved substrates and fewer discrete gap junction complexes compared to flat surfaces (p< 0.05 ANOVA.). There were also differences between the grooves with con-nexin-43 most abundant on the widest (30micrometers) and deepest grooves (16micrometers). Analysis of dye transfer demonstrated that whilst cell:cell coupling was maintained within grooves it was reduced at the boundaries of the groove. A surface of asymmetric arrays of micro-columns (diameter 5micrometers) was fabricated to retain lateral interactions between osteoblasts whilst still aligning cells. Osteoblast differentiation now resulted in the formation of numerous bone-like nodules and matrix was aligned in the direction of the shortest column distances.
Maintaining appropriate cell:cell communication structures is pivotal in the process of osteoblast differentiation and the design of novel biomaterial surfaces should ensure that cells can maintain these critical interactions.
Correspondence should be addressed to Mr Carlos Wigderowitz, Senior Lecturer, University Department of Orthopaedic and Trauma Surgery, Ninewells Hospital and Medical School, Dundee DD1 9SY.
