DISEÑO DE INGENIERÍA

The musculoskeletal system is the fundamental scaffold that provides support for body infrastructure and allows for physical movement. As such, orthopaedic injuries significantly impact both quality of life and life expectancy. While some tissues, such as bone, can remodel to pre-injury equivalence after damage, dense connective tissues, including the knee meniscus, annulus fibrosus of the intervertebral disc, tendon, and ligament, exhibit poor healing capacity in adults. The microstructure of these tissues (at the cell level) is dominated by aligned collagen bundles, which provide mechanical anisotropy and greater tensile strength in the loading direction, allowing function in mechanically demanding environments. For example, the meniscus promotes stifle joint congruency and stability by distributing load over a large surface area. It is mainly comprised of circumferentially arranged collagen bundles that serve to resist tensile hoop stresses in the knee. Tears that disrupt this collagen architecture predispose the joint to altered biomechanics and eventually osteoarthritis. Despite the common occurrence of such injuries, there are limited restorative strategies available (e.g. allograft replacement), and in many cases damage culminates in a total joint replacement. An ideal alternative would be to reestablish native tissue function by promoting cell growth, organized extracellular matrix (ECM) production, and, most importantly, integration of the newly formed tissue via the creation of an instructive regenerative microenvironment.

The overall goal of this thesis is to expedite dense connective tissue repair by identifying and manipulating the cellular and ECM impediments to wound healing. This body of work focuses on the knee meniscus, but the principles outlined within are applicable to other fiber-reinforced soft tissues. Chapter 2 will introduce meniscus structure and function, describe the biological limitations to intrinsic repair, and review strategies to augment the

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healing response. Interestingly, the meniscus exhibits a robust repair capacity early in development, but this potential declines with aging. To overcome the restricted blood supply and sparse cell population of the mature meniscus, most experimental therapies attempt to directly boost the vascularity and cellularity at the defect. Others strategies seek to increase the anabolic activity of resident cells via the provision of biochemical and mechanical cues.

While several of these studies appear promising, few address the biophysical barrier of the ECM itself, which may affect the interstitial migration of endogenous cells to the injury site and subsequent repair. The cellular and extracellular determinants of 3D migration, a complex set of interdependent factors absent from 2D migration, will be detailed in Chapter 3. This chapter begins by discussing the basic mechanisms of cell locomotion in confined 3D environments and constraints to mobility posed by the cell and its environment. The remaining sections explore the implication of 3D migration in fibrous tissue repair and tissue engineering, processes that rely on a sufficient population of cells to produce, remodel, and maintain the ECM. While tissues and scaffolds are optimized for mechanical function, their dense and stiff microenvironments may hinder cell mobility and infiltration. Recent advances in biomaterial design that mitigate these issues are also highlighted.

Building on these concepts, we hypothesized that the density and stiffness of the adult meniscal ECM prevent cell migration to the wound margin, and that removing these inhibitions will facilitate cell migration and tissue repair. To achieve this, we developed a novel electrospun polymer nanofiber system from which bioactive factors are released to initiate and enhance repair. The challenges of this project are two-fold: first, to create a local wound environment favorable to cell migration by delivering a matrix-degrading

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enzyme, and second, to enforce and instruct matrix deposition in the regenerate tissue that bridges the wound interface with a structural scaffold. Chapter 4 investigates the effect of partial digestion of the wound interface with the enzyme collagenase on meniscal repair using a tissue explant model. Additionally, a technique to fabricate bioactive, enzyme- delivering nanofibers that can modulate tissue properties is presented. Chapter 5, which continues this work in vivo, assesses the use of composite collagenase-releasing scaffolds to enhance meniscal repair in a subcutaneous implantation model (rat) and an orthotopic meniscal defect model (sheep). These studies demonstrate that partial degradation of the wound margin leads to local micromechanical and microstructural changes in the ECM, which becomes more compliant and porous. A targeted burst release of collagenase from nanofibrous scaffolds localizes this effect to the tissue margin. Importantly, enzyme treatment results in higher cell density and physical integration of the meniscal wound interface, suggesting that endogenous cells are capable of repair when the steric impediments of the ECM are reduced.

To better comprehend this mechanism on the microscale, a method to visualize and quantify interstitial cell migration through native tissue is developed in Chapter 6. Here, the influence of the tissue microenvironment on migratory efficacy is investigated using fetal, adult, and adult meniscal tissue substrates with and without pre-digestion with collagenase. Results suggest that age-related biophysical changes to the microenvironment affect cell migration, which may partly explain the differential repair capacity of fetal and adult tissues. This work confirms that cell mobility is extremely limited in the adult meniscus, but can be enhanced by modulating tissue stiffness and density via enzymatic digestion.

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In addition to enabling cell mobility, scaffolds may be further functionalized to deliver biochemical signals that guide native cells to the injury site. Chapter 7 explores the application of a soluble chemoattractant to trigger directed cell migration to the wound site after matrix degradation. A novel tissue-based migration chamber to study interstitial chemotaxis is designed for this purpose. To determine whether aging also changes a cell’s intrinsic mobility, the ability of fetal and adult meniscal cells to translocate through increasingly small pores is evaluated, along with other biophysical features of the cell. Platelet-derived growth factor-AB (PDGF-AB) is identified as a chemoattractant for cells of both ages, as well as its dose-dependent and pore-size dependent impact on cell migration. Further, fetal cells appear more mobile than adult cells, potentially due to their smaller and more deformable nuclei. Finally, this chapter details the development of a tri- component nanofibrous scaffold that delivers collagenase, PDGF-AB, or both biofactors in a temporally controlled manner. Cell infiltration into these nanofibrous composites is assessed in vitro prior to their evaluation in a subcutaneous implantation model. As expected, cell migration into scaffolds is limited by nanofiber density and inter-fiber pore size. In vivo results indicate that local PDGF-AB delivery alone can enhance cellularity at the wound interface in the short-term, but that collagenase-mediated reprogramming of the ECM may be essential for cell invasion of the scaffold in the long-term. Taken together, these findings illustrate the importance of biophysical barriers to interstitial migration, as well as the feasibility of delivering multiple agents in a staged fashion to prime the wound microenvironment for repair.

Finally, Chapter 8 provides a summary of the findings herein and describes their impact on dense connective tissue repair and the current understanding of cell behavior in physiologic 3D environments. The limitations and future directions of this research are also discussed, a testament to the exploratory nature of this work.

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CHAPTER 2: BASIC SCIENCE OF MENISCUS REPAIR: LIMITATIONS AND