A mechanotransduction study of chondrogenesis using a novel tubular scaffold.

摘要

The lack of vascularization and poor regenerative capabilities of articular cartilage provides impetus for developing methodologies for replicating cartilage in vitro. Normal loading of the tissue, through normal activity of the joint, creates mechanical, electrical, and physiochemical signals that help to direct the activity of chondrocytes. Therefore, in order to create a realistic cartilage replacement, biomechanical and biochemical properties of cartilage must be characterized and recreated in an in vitro environment. Micromass culture models constitute a majority of current in vitro cartilage studies but lack the biochemical and biomechanical stimuli that a cartilage scaffold model can provide. However, tubular scaffolds made from biomaterals allow for chondrocytes to have constant access to nutrients in media and provide stimulus for growth. Chicken limb buds mesenchyme (LB MSCs) are extracted from Hamburger & Hamilton stage 22 and stage 25 embryos. We have found that both stages of chick LB MSC thrive in a collagen type I tubular scaffold when supplied with media for up to 6 weeks and express proper cartilage markers. The extra-cellular matrix created, however, was lacking organizational structure. To further stimulate the growing LB MSC and the attempt to alter the protein structure, we applied mechanical stimulation in the form of tensile strain. During compression of cartilage, the non-conformal surfaces of the joints create tensile strain fields of up to 5%, which have been documented in the literature. Cells from each stage were cultured statically for 3-5 days to allow integration into the scaffold. Subsequently the experimental scaffold and integrated cells were exposed to a 5% circumferential tensile strain, by inserting a balloon catheter into the lumen and inflating the tube. From confocal images of the cells and matrix, the stage 22 LB MSC expressed collagen type II that appeared more organized, more fibril like, and evenly dispersed throughout the matrix when compared with the control; however, the stage 25 LB MSCs and matrix were different in that the collagen type II was not excreted outside of the cells to make fibrils. The drastic difference in response prompted a DNA microarray study between the two stages to compare the gene expression. Hallmark cartilage markers were examined using RT-PCR to show relative expression. Results suggested that stage 22 LB MSC were following known expression patterns for cartilage development and were not undergoing maturation; in contrast, stage 25 LB MSC were creating a more fibrous tissue and possibly undergoing differentiation.