Development of a self-assembled meniscal replacement
Huey, Daniel Joseph
Athanasiou, Kyriacos A.
Doctor of Philosophy
Injuries to the inner-portion of the meniscus, common with today's active lifestyles, have little ability for intrinsic repair due to the lack of vascularity. Current treatments only alleviate the symptoms of meniscal damage and do nothing to prevent the eventual osteoarthritic changes to the articular surfaces of the knee joint. To prevent these changes by restoring the structure and functionality of the meniscus, the generation of biochemically and biomechanically robust tissue engineered constructs for tissue replacement is desirable. This thesis investigated methods to engineer and enhance a self-assembled meniscal replacement using both a leporine and bovine cell source. First, the leporine cell source was considered as it represents the potential for future small animal, allogenic, in vivo studies. The use of a chondrogenically-tuned expansion procedure, involving a chemically defined medium and high density monolayer culture, was employed to expand leporine articular chondrocytes (ACs). Not only did this protocol outperform traditional expansion in terms of promotion of a cartilaginous phenotype, but constructs formed with expanded ACs had higher GAG/WW and collagen 2/collagen 1 than constructs formed with primary ACs. To further enhance cartilaginous quality and potential clinical translatability, the effects of passage number, cryopreservation, and redifferentiation culture prior to self-assembly were studied for both leporine ACs and meniscus cells (MCs). This study found that by increasing the passage number to obtain more cells from the same amount of starting material, the biochemical and biomechanical properties of constructs were not detrimentally affected. Cryopreservation and aggregate pre-culture redifferentiation were found to enhance biomechanical properties of AC and MC self-assembled constructs. The remaining tissue engineering studies in this thesis employed immature bovine ACs and MCs because these cells have been successfully applied in the self-assembly process to create constructs of complex shapes. In addition, a study was performed to assess the immunogenicity of xenogenic, bovine and allogenic, leporine ACs and MCs when co-cultured with leporine peripheral blood mononuclear cells (PBMCs). The mixed lymphocyte reaction assay showed that an immune response was not elicited by either bovine or leporine cells. This result suggests that the use of bovine cells for leporine meniscal replacement may be a feasible option. Studies assessing chemical and mechanical stimulation of anatomically-shaped meniscus constructs formed from bovine ACs and MCs followed. First, effects of temporally coordinated chemical stimuli, chondroitinase ABC (C-ABC) and transforming growth factor p1 (TGF-beta1), were studied on anatomically-shaped meniscal constructs. A stimulation regimen, consisting of TGF-beta1 applied continuously and C-ABC applied after 1 wk of culture, was found to synergistically enhance the radial tensile modulus and compressive relaxation modulus; in addition, this regimen additively increased the compressive instantaneous modulus and collagen/WW. Next, the effects of combining the previously determined chemical stimulation regimen with physiologic mechanical stimulation were studied. The shape of the construct and compression stimulator allowed for application of simultaneous compression and tension stimulation which mimicked the types of forces experienced by native menisci. This study found that the application of mechanical stimulation from days 10-15 resulted in significant enhancement of all measured biochemical and biomechanical properties. Further, combined chemical and mechanical stimulation resulted in additive increases to collagen/WW and all biomechanical properties. Finally, the effects of self-assembly well topography and compliance were studied. This study indicated that a smooth topography and higher compliance resulted in constructs possessing higher GAG/WW, collagen/WW, and tensile modulus. In conclusion, this thesis identified (1) expansion, cryopreservation, and pre-self-assembly redifferentiation as factors able to enhance the cartilage-forming capability of leporine ACs and MCs, (2) determined that the use of bovine ACs and MCs in leporine meniscal engineering could be feasible due to lack of immunogenicity, and (3) discovered chemical and mechanical stimulation treatments that were able to enhance the functional properties of bovine AC and MC meniscus constructs to values in the range of native tissue. In the future, the translation of these techniques to clinical usage could reduce the risk of osteoarthritis following meniscus injuries by providing functional replacement tissue.