Limb bud mesenchyme cultured under tensile strain remodel collagen type I tubes to produce fibrillar collagen type II

Abstract

In this work, we studied the effects of tensile strain on limb bud mesenchymal cells (MSC) cultured on a collagen type I tubular scaffold. A novel bioreactor was designed to culture the cells while subjecting the tubular scaffold to tensile stress and strain. Control samples included unseeded and MSC-seeded tubes cultured for 2 weeks under unloaded, no-strain conditions, and unseeded tubes subjected to prolonged tensile stress and strain. Mechanical properties of tube specimens were measured under oscillatory compressive stress. Following mechanical testing, scaffolds were fixed for immunohistochemistry or frozen for mRNA extraction. The storage modulii of both seeded/unstrained and seeded/strained tubes were significantly less than that of unseeded tubes, suggesting that MSC disrupted the structure and elasticity of the tubes' collagen type I. At a frequency of 1.0 Hz, the loss tangent of seeded/strained tubes was more than 2.5 times greater than that of seeded/unstrained tubes, and almost 6 times greater than that of unseeded tubes. Confocal microscopy and qRT-PCR results demonstrated that collagen type II and aggrecan expression was upregulated in the seeded/strained tubes. The images also show, for the first time, that culture under tensile strain induces MSC to remodel the collagen type I tube with collagen type II and aggrecan expression into fibrils dispersed throughout the matrix. The seeded/unstrained tubes manifested less collagen type II with a more random expression pattern. Compared to seeded/unstrained tubes, qRT-PCR for collagen type II in the seeded/strained tubes showed a 4-fold increase in the message for collagen type II and a 13-fold increase in the message for aggrecan. These results demonstrate that MSC cultured for at least some period under tensile strain are able to remodel collagen type I scaffolds to produce a more viscous construct having many of the mechanical and biological features of engineered cartilage.