Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: Influence of crosslinking with diphenylphosphorylazide on matrix reorganization, growth, and biosynthetic and proteolytic activities

Abstract

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials frequently are used as cell transplant devices. In this study we determined the behavior of mouse fibroblasts cultured for up to 6 weeks in control sponges treated by severe dehydration and used commercially as hemostatic agents and in two sponges (DPPA 2 and 3) crosslinked by diphenylphosphorylazide, a method developed in our laboratory. Growth capacity, biosynthetic and proteolytic activities, and matrix reorganization were followed over time in cultures and compared with similar data for fibroblasts in monolayer culture on plastic and in floating or attached collagen gels. Control sponges with and without seeded mouse fibroblasts showed rapid partial denaturation or contraction, weight loss, and severe calcification (13-18% Ca) after 6 weeks. In contrast, the crosslinked sponges showed only slightly decreased size and weight, and the calcification was inhibited (0.2% Ca) in the presence of cells. Mouse fibroblasts seeded on the crosslinked sponge surface at 50,000-200,000 cells/cm(2) progressively penetrated the matrix and proliferated to give the same constant cell density after 3 weeks (around 600,000 cells/sponge). A specific, two- to threefold decrease in collagen synthesis was observed between 1 and 3 or 6 weeks, due mainly to a decrease in the fraction secreted into the medium (25-30% instead of 45-50%). No collagenase 3 activity was detected in the culture medium under any condition or time whereas 25% gelatinase A was found by gelatin zymography to be in an active form in cultures within sponges as compared with less than 10% in monolayers and more than 50% in floating collagen gel. A small amount of gelatinase B was observed after 1 week in sponge cultures and was completely absent thereafter. These results show that the biosynthetic and proteolytic behavior of mouse fibroblasts cultured in crosslinked collagen scaffolds is different from that in monolayers or in floating collagen gels and more similar to that previously described in attached collagen gels.

Macrophage-mediated degradation of crosslinked collagen scaffolds

“…Crosslinking collagen increases its resistance to degradation, and this has been shown using a variety of methods of crosslinking: UV radiation, diphenylphosphorylazide, hydrazine and glutaraldehyde [24,40,41]. The differences presented here, showing that EDC crosslinked samples are more resistant to degradation than DHT crosslinked samples, corroborates previously published data [42].…”

Post-self-assembly experimentation on extruded collagen fibres for tissue engineering applications

“…The fibres produced herein exhibited denaturation temperatures ranging from 45 to 50°C, which is higher than any other non-cross-linked collagen preparation and even higher than the body temperature, making such matrices suitable for tissue engineering applications. It has been suggested that when collagen molecules aggregate to form fibres, the shrinkage temperature is high due to the increased energy of crystallization derived from the interaction between the close packed molecules in the fibre form [9,55,[73][74][75][76][77][78][79]. In the same manner, differences in thermal stability between the alcohol-treated fibres and the rest treatments can be attributed to the different packing of the fibrils within the fibre.…”

Local and regional mechanical characterisation of a collagen-glycosaminoglycan scaffold using high-resolution finite element analysis

“…However, on a scaffoldlevel, previous investigations have demonstrated that scaffold volume fraction can influence cell infiltration: the larger the pore size (lower volume fraction) the more the cells disperse throughout the scaffold due the increased fluid movement [Byrne et al, 2008;Tierney et al, 2008]; though interestingly without fluid influences, and when contact guidance is measured (cell motility along scaffold struts), cells migrate less in larger pores (151µm to 96µm) . Similarly, biosynthesis and degradation rates have also been shown to vary according to scaffold properties, such as cross-link densities and stiffness, which in part is related to relative density [Chevallay et al, 2000;Lee et al, 2001;Petite et al, 1994;Powell et al, 2006;Vickers et al, 2006;Weadcock et al, 1996]. Thus, it is likely the regional variances do impact on cell activity and tissue growth.…”