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

Injuries to articular cartilage are debilitating and typically require surgical intervention due to the tissue's inability to self-repair. Current surgical options for focal cartilage defects are successful only in treating the associated symptoms, but produce inconsistent clinical outcomes in the long term, and are implicated in degenerative joint changes years later. As an alternative, 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, which might influence the integration of the implanted tissue with the surrounding cartilage, 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. Using in situ hybridization and itnmunohistochemistry, tissue engineered cartilage (TEC) hydrogels that were cultured in a novel bioreactor that simulates rotational Couette flow demonstrated an increase in aggrecan and type II collagen mRNA expression and protein synthesis near the surface of the hydrogel exposed to the flow, compared to deeper regions within the hydrogel and control hydrogels not stimulated by Couette flow. Furthermore, the alignment of the collagen fibers in the superficial layer of the bioreactor-cultured TEC hydrogels was significantly enhanced compared to controls. When quantified using Fast Fourier Transform (FFT) algorithms, the collagen alignment in the surface region of the bioreactor-cultured TEC hydrogels was remarkably similar to the alignment pattern of collagen in the superficial zone of native articular cartilage.;To formally test the hypothesis that the aforementioned effects of Couette flow are related to interstitial flow fields, the interstitial flow velocity profiles within TEC hydrogels were experimentally measured as a function of flow rate through a parallel plate (Poiseuille flow) bioreactor using a flow visualization technique based on Fluorescence Recovery After Photobleaching (FRAP). 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. More importantly, the experimental measurements of the interstitial fluid velocity fit theoretical predictions described in the context of the Biphasic theory of cartilage mechanics, and exhibited depth-gradients in the interstitial flow velocity and the associated shear stress profiles. These findings suggest that the depth-dependent interstitial flow fields (velocity and shear stress) could enhance convective transport and mechanically stimulate the cells to induce the observed compositional and structural anisotropy in the TEC hydrogels, although the relative contribution of enhanced transport versus mechanical stimulation remain to be elucidated. Computational fluid dynamics (CFD) 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. Since the permeability is inversely related to the frictional drag induced by relative fluid flow past the porous solid matrix, 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 mechanostimutation regimens for cartilage tissue engineering.;The last 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-α 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-α on Agc and Col2al 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-κB nuclear translocation. While the exact mechanism leading to this inhibition and the link between the mechanosignaling cascade and the inflammatory pathway have not been evaluated in this work, the experimental platform and associated protocols described herein provide opportunities for future work towards this goal.