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The effects of titanium-coated micromachined grooved substrata on osteogenesis in vitro Khakbaznejad, Alireza

Abstract

Although previous studies have indicated that titanium-coated micromachined grooved substrata increase osteogenesis both in vitro and in vivo, the underlying cell behaviours have not been given much attention. One suggestion is that osteogenesis may be induced by appropriate cell orientation. It has also been suggested that the increased production of bonelike tissue on micromachined grooves results from the formation of a favourable microenvironment between the walls of the grooves. A third possibility is based on the hypothesis that extracellular matrix, mainly collagen, orientation plays an important role in increasing bone-like tissue formation. It was hypothesized that grooved surfaces, through orienting osteoblast-like cells, promoted osteogenesis in vitro. This study investigated cell and collagen orientation and the formation of bone-like nodules in response to surface topographies. Osteogenic cells (OGC) from newborn rat calvariae were plated (2 x 10⁵ cells/cm²) on smooth surface controls and test substrata in which smooth surfaces (gaps) were flanked by groups of parallel grooves of 47μm pitch and 3μm, 10μm, or 30μm depth. The medium used was a-MEM with 15% fetal calf serum (v/v). The medium was supplemented with β-glycerophosphate (10mM) and ascorbic acid (50μg/ml) after the eighth day and changed three times weekly thereafter. Von Kossa staining technique, propidium iodide staining of nuclei, and picro-sirius staining of collagen fibers were used to study bone-like tissue formation, cell orientation, and collagen orientation respectively. Histological sectioning and scanning electron microscopy (SEM) of the cultures were carried out to further examine cell orientation on the micromachined grooved surfaces. The results of the study indicated that both micromachined grooves and the smooth gaps within them produced more bone-like nodules than the smooth surface controls. It was found that the cells within the grooves generally aligned themselves with the direction of the grooves whereas the cells above the ridge of the grooves formed cell layers of parallel orientation at an angle to the grooves. The orientation angle (OA) of the cell layers above the ridge level increased with distance from the grooves. In the smooth gaps, the cell layer closest to the titanium surface had the best alignment with the direction of the flanking grooves, but the OA of the cell layers above it increased with distance from the titanium surface. The cells on the smooth surface controls showed no preferred orientation. Collagen fibers in the grooves were generally oriented with the grooves whereas collagen fibers in the smooth gaps had several distinguishable orientations including parallel to the grooves, perpendicular to the grooves, and diagonal to the grooves. Collagen fibers on the smooth surfaces were in arrays of parallel fibers in a criss-cross pattern. The current study provides further evidence that micromachined grooved substrata promote bone-like tissue formation of osteogenic cells in culture. Moreover, bone-like nodule formation is associated with an interplay of several factors including cell orientation, number of cell layers, and collagen fiber organization.

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