Bone tissue engineering on patterned collagen films: an in vitro study

Abstract

This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane. Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27 microm, groove depth: 12 microm, ridge width: 2 microm) and macropatterned (groove width: 250 microm, groove depth: 250 microm, ridge width: 100 microm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation. Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used. This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.