The fibrocartilaginous menisci dwell between the articular surfaces of the knee and play a crucial role in healthy joint loading, functioning to transmit forces, absorb shock, and enhance the stability of the joint. Traumatic injury and/or degenerative changes disrupt the mechanical function of these tissues and lead to the early onset and accelerated development of osteoarthritis. The current standard of treatment is meniscectomy, or resection of the damaged portion of the meniscus, a procedure that fails to re-establish normal knee mechanics or prevent the initiation of osteoarthritic cascades. Given the high prevalence of meniscal injury, to date, a repair strategy that restores meniscus mechanical function remains a preeminent need in orthopaedic medicine.
Thus, the overarching goal of this thesis is to develop strategies and technologies for replacing damaged or diseased meniscus with tissue engineered, mechanically-competent fibrocartilage. Once implanted, this biologic tissue would be maintained by the body, avoiding the wear issues and limited lifespan of artificial implants and the numerous drawbacks associated with allografts. The functional properties of the meniscus stem from its highly ordered extracellular matrix, primarily composed of co-aligned collagen fibers which enable the tissue to bear high tensile loads. Towards recapitulating the structural features of the meniscus, this work focuses on scaffolds composed of aligned arrays of polymeric nanofibers fabricated with the electrospinning process. These biocompatible and biodegradable nanofibers can be formulated to mimic the length scale and organization of collagenous tissues, and as such, serve as a suitable foundation for
investigates aspects of scaffold design, cell source selection, and modulation of the mechanical environment with the aim of engineering fibrocartilage that approximates the organization, composition, and mechanical function of the native meniscus.
To establish the functional metrics and target characteristics of tissue engineered fibrocartilage, Chapter 2 describes the structure, composition, and physiologic function of the native meniscus. Providing the motivation for this work, the failure modes of the meniscus are described, as well as the historical clinical approaches to repairing this tissue. The current state of meniscus tissue engineering is covered in detail, reviewing the different scaffolding materials that have been explored, potential cell sources suitable for reconstituting this tissue, and investigations into optimizing tissue formation with the use of bioreactors. Finally, an overview of the challenges associated with implementing engineered meniscus tissue is presented.
Demonstrating the utility of nanofibrous scaffolds for engineering anisotropic fibrous tissues such as fibrocartilage, Chapter 3 investigates the effect of nanofiber alignment on the organization of cells and cell-deposited collagen. Cells were seeded onto two distinct scaffold architectures formed from the same material: scaffolds where the nanofibers were randomly organized, or ones where the fibers were uniformly aligned in the same direction. With culture, collagen deposition parallel to the aligned nanofibers was observed, while only disorganized collagen was identified in nonaligned scaffolds. This chapter demonstrates that nanofiber alignment dictates the organization of collagen with
profound consequences on the load-bearing properties of the resultant tissue, and provides the foundation for the remainder of the work.
A significant limitation observed in these studies was the slow rates at which cells colonize these three-dimensional matrices. This problem arises from the dense packing of fibers during the electrospinning process which leads to small pore sizes. To increase pore sizes and hasten cell ingress, Chapters 4 and 5 develop composite scaffolds that contain both slow-eroding structural fibers, and removable elements that serve to hold space during the formation of the scaffold. Chapter 4 details the design and mechanics of these composite scaffolds, and shows that the use of sacrificial fiber elements leads to improvements in cell infiltration over short-term culture. In Chapter 5, the longer-term ramifications of sacrificial fiber inclusion on construct maturation were explored. With high sacrificial fiber content, increases in collagen distribution and content led to larger increases in mechanical properties. To underscore the widespread applicability of these composites, scaffold colonization by host cells was investigated in a rat subcutaneous model.
Findings from Chapters 4 and 5 demonstrate the utility of combining multiple types of polymer fibers into a composite, and motivate the addition of a fiber population that erodes concomitant with tissue formation. Thus, Chapter 6 focuses on engineering a tri- polymer composite with temporally dynamic mechanical properties. While the experimental aspects of this chapter focused on integrating a choice polymer with specific degradation rates and mechanical properties, the approach of combining multiple
fibers each with unique characteristics into a single composite could have bearing on numerous applications and tissues. To generalize this strategy, a theoretical model that describes the temporal mechanical behavior of composites was developed and validated with experimental data. This model was used to simulate the time-dependent stress-strain response of scaffolds of hypothetical formulations, and introduces a novel approach to the intelligent design of nanofibrous scaffolds with dynamic mechanical properties.
In addition to a well-designed biomaterial scaffold, an essential component to generating fibrocartilage in vitro is a cell type that can reconstitute the extracellular content of the meniscus. The two most accessible options are meniscal fibrochondrocytes (MFCs), the cells indigenous to the meniscus that assemble and maintain its extracellular matrix, and mesenchymal stem cells (MSCs), a multipotent cell type under widespread investigation for applications in musculoskeletal tissue engineering. The juvenile bovine MSCs and MFCs used in Chapter 3 (in order to compare healthy, donor-matched cell types) were found to synthesize a robust, fibrocartilaginous matrix on nanofibrous scaffolds. To move this technology towards clinical implementation, Chapter 7 examines the functional potential of human MFCs isolated from surgical waste tissue. These cells present a number of advantages: they possess the appropriate phenotype, would be autologous and so limit immune rejection, and can be obtained without an additional surgical site. Nanofibrous constructs were seeded with MFCs isolated from ten human donors and biochemical, mechanical, and histological features were assessed over long-term culture. While considerable donor-donor variability was noted, all ten cell lines synthesized load-
bearing fibrocartilaginous matrix, indicating MFCs isolated from surgical waste are a pertinent cell source for meniscus tissue engineering.
Chapter 8 assessed the ability of human marrow-derived MSCs to elaborate a mechanically functional, fibrocartilaginous matrix in a nanofibrous context. As MSCs can be readily harvested from bone marrow, their use for engineering replacement tissues would negate the need for multiple surgeries at the defect site. Based on results from Chapter 3 where juvenile bovine MSCs synthesized higher amounts of key fibrocartilaginous matrix components, we hypothesized that human MSCs would similarly outperform donor-matched MFCs. Instead, MSCs demonstrated limited proliferation and synthesized sparse extracellular matrix which led to negligible increases in construct mechanical properties as compared to donor-matched MFCs. Interestingly, there was no difference in matrix production of MSCs and MFCs when cultured in pellet form, highlighting the sensitivity of human MSCs to their three-dimensional microenvironment.
In the previous chapters, the highest tensile modulus achieved in an engineered construct after long-term free-swelling culture was approximately 30MPa, a value below adult meniscus by a factor of 2 or more. As these tissues require exposure to mechanical forces in vivo for proper formation and maintenance, it is perhaps not surprising that engineered tissue cultured in static, free-floating conditions failed to achieve native tissue properties. In an effort to hasten the in vitro maturation of these constructs and gain insights into how cells within a nanofibrous microenvironment respond to mechanical
stimulation, Chapters 9 and 10 investigate the primary loading modality of the meniscus: tensile deformation.
In Chapter 9, we begin at the cellular level by examining how tensile strains applied to the scaffold translate to cell and subcellular changes. Adult human MSCs and MFCs were sparsely seeded onto both aligned and nonaligned nanofibrous scaffolds which were deformed and held fixed at strains of up to 10% for analysis. Gross morphological changes in the cell as well as alterations in nuclear shape and organization were examined. With an applied deformation, the response of cells and their nuclei was found to be highly dependent upon the underlying scaffold architecture. Furthermore, by selectively removing cytoskeletal elements from these cells, the role of the actin, microtubule, and intermediate filament networks in mediating force transfer from the scaffold to the nucleus was interrogated.
Chapter 9 provides a basic understanding of how strains applied to aligned nanofibrous scaffolds affects adhering cells. We next scale up in complexity by examining cyclic tensile loading, which better approximates the dynamic mechanical environment of the meniscus. Chapter 10 focuses on the design and validation of a custom tensile bioreactor for applying cyclic loads to cell-seeded nanofibrous constructs during in vitro culture. Using this system, we asked whether daily administration of physiologic mechanical loading would positively impact the development of MSC-laden fibrocartilaginous constructs. Aligned nanofibrous were seeded with juvenile bovine MSCs and mechanically stimulated for four weeks. Dynamic loading led to increases in tensile
stiffness and total collagen content, and the expression of key matrix-associated genes were modulated with mechanical loading. These results have relevance to strategies for MSC-based tissue engineering, but also provide new insights on how stem cells can respond to external mechanical stimuli and modify their microenvironment.
Finally, Chapter 11 provides a summary of the major themes and findings stemming from this body of work, and proposes implications for engineering other anisotropic, load- bearing tissues. This is followed by an in-depth discussion of the key limitations to the described studies and drawbacks to the overall approach of engineering fibrocartilage with nanofibrous scaffolds. In addressing some of these limitations, future directions for this research are suggested, and some preliminary data is provided in support of these new avenues of investigation.