A model of the initial complement mediated inflammatory response to tissue engineering (TE) scaffolds is developed in this study. TE has been given increasing attention in cases of large defects caused by severe trauma or disease based on the concept that the scaffold degrades and is replaced by tissue regeneration. Numerous attempts have been made to modify biomaterials chemical compositions, mechanical properties, and biomolecular incorporation to improve biocompatibility. The surface properties have been shown to affect host inflammatory responses and the ultimate fibrous capsule formation through foreign body reaction around implanted biomaterials. Although extensive research has been conducted regarding immune responses, such as macrophage activity, upon biomaterial particles and to a lesser extent on biomaterials surface, the inflammatory cascade and its effect on differentiation of preosteoblasts is not completely understood. Biomaterials are known to adsorb serum proteins and activate the complement system followed by subsequent inflammatory cascade. Complement activation through either direct deposition or interaction with adsorbed protein layer on biomaterial surface is believed to be the initiation process of inflammatory responses to implanted biomaterials. By setting the goal of constructing a tissue engineering model, the present study utilizes complement, macrophages, and preosteoblast-like cells to investigate the initial step of the inflammatory response and their effect on osteogenesis on biomaterial surface, poly(methylmethacrylate).
PMMA polymer with different tacticities effects on MC3T3-E1 cell proliferation and differentiation were evaluated with cell proliferation and viability assay (MTS assay). In addition, their effect on MC3T3-E1 cell differentiation was assessed with MTS assay and osteogenic activity markers quantifications. No significant differences between different tacticity PMMA and tissue culture polystyrene were found.
The effect of complement on macrophage, RAW264.7, activation on biomaterial surface was investigated in present study. Three cytokines, TNF-α, IL-6, and IL-1β, expression were quantified with ELISA to evaluate macrophage activation on PMMA surfaces and particles. Cytokine expressions on PMMA surface were not significantly different from control group. At 6 hour, 1-10 μm PMMA beads induced significantly increased TNF-α expression. Mouse complement pretreatment resulted in increased expression of IL-6 and decreased expression of TNF-α while IL-1β was barely detectable in present experimental conditions.
Co-culture system is constructed by means of transferring RAW264.7 cell culture media to preosteoblast culture with corresponding material surfaces or particulates. The mRNA expression of Runx2, ON, and Collagen Type I are quantified with real time PCR to evaluate osteogenic activity of MC3T3-E1 cells under osteogenic condition. MC3T3-E1 cells cultured with osteogenic media supplemented with RAW264.7 cell culture media with LPS showed elongated morphology under light microscope. Real time PCR study for mRNA expression shows significantly lower Runx2 and SPARC expression in LPS stimulated co-culture system. Complement involvement in co-culture system generally decreased all three mRNA expressions.
Conclusion: within the limitations of this study, complement treatment decreases TNF-α and increases IL-6 expression of RAW cells interacting with PMMA surfaces and particles. Culture media from LPS activated macrophage induced MC3T3-E1 cells differentiating toward fibroblasts even with osteogenic media. Complement-macrophage system inhibits osteogenic activity of MC3T3-E1 cells on PMMA surfaces and particles.