BME PhD Defense: Tony Chen

The Role of Interstitial Fluid Flow as a Mediator of Matrix Anisotropy and as a Protective Mechanism Against Inflammation in Cartilage Tissue Engineering

Supervised by Professor Hani Awad

Abstract

Functional tissue engineering may soon offer translational approaches for cartilage repair by producing implantable artificial tissues that recapitulate the composition, structure, and function of the native tissue. While current tissue engineering bioreactors that introduce various physiologically inspired mechanical stimuli are able to reproduce the mechanical properties of native cartilage, recreating the tissue's structural and compositional anisotropy has to date remained elusive. Furthermore, the fate of the engineered construct during the inflammatory phase that ensues upon implantation in the joint has been less studied, as most of the literature focuses on developing engineered cartilage in idealized culture conditions in vitro, and for the most part neglects the interplay between inflammation and repair mechanobiology.

This dissertation sets to recreate aspects of the characteristic zonal anisotropy of articular cartilage using custom-designed bioreactors based on Couette and Poiseuille flow with the hypothesis that interstitial fluid flow can influence matrix synthesis and the alignment of collagen fibers. TEC hydrogels that were cultured in a novel bioreactor simulating rotational Couette flow demonstrated aggrecan and type II collagen anisotropy compared to statically cultured hydrogels. Furthermore, the alignment of the collagen fibers in the superficial layer of the bioreactor-cultured TEC hydrogels was significantly enhanced compared to controls. Experimental measurements of the fluid velocity profile over a range of flow rates demonstrated that Poiseuille flow induced measurable interstitial flow fields near the flow-exposed surface of the hydrogel. These experimental measurements and computational fluid dynamics modeling revealed the novel observation that the increase in interfacial shear stress with increased flow rate exponentially decreased the apparent permeability of the TEC hydrogel. The resulting interstitial flow fields and associated shear stresses and convective transport must therefore depend on this non-linear permeability reduction, which should be taken into consideration when designing flow bioreactors and mechanostimulation regimens for cartilage tissue engineering.

The final part of the dissertation addresses the hypothesis that fluid flow induces protective effects in chondrocytes against inflammatory cytokines. First we demonstrated that the inflammatory cytokine TNF- significantly suppressed the aggrecan (Agc) and type II collagen (Col2a1) gene expression in high-density chondrocyte monolayer cultures, while Poiseuille flow in a custom parallel plate flow chamber had no effects on Agc and Col2a1 gene expression levels. Interestingly, when pre-stimulated with fluid flow, the chondrocytes were desensitized to the suppressive effects of subsequent treatment with TNF- on Agc and Col2a1 gene expression. Western blot and immunohistochemistry analyses of the potential signaling pathways involved suggested that this protective effect of fluid flow is mediated through the inhibition of NF-kB nuclear translocation.