When damaged through either trauma or disease, articular cartilage exhibits little to no capacity for self-repair, thus prompting surgical intervention to alleviate joint pain. Damaged articular cartilage can degenerate to an arthritic state, leaving the subject with a painful and debilitating condition. Few clinical interventions have proven effective in restoring joint function over the long-term, prompting the exploration of new strategies including tissue engineering. Cells are combined with growth factors in an environment suitable for matrix production and assembly into in vitro derived articular cartilage tissue. Tissue engineering offers the promise of providing an “off-the-shelf” tissue with the matrix composition and mechanical properties similar to that of native tissue which can be implanted into a defect site with the hopes of restoring function. The cells and bioactive factors within the tissue maintain normal tissue homeostasis and grow with the subject, ideally offering a permanent, life-time solution and improved quality of life.
A tissue-engineered articular cartilage construct with the matrix composition and mechanical properties near that of native tissue has yet to be produced. While many strategies to achieve this goal exist, a dominant one is the use of bioreactor systems. Besides being amenable to large-scale bio-processing, bioreactors can provide a mechanical stimulus and/or improve mass transport in order to stimulate tissue development. Mechanical stimuli applied in vitro, such as compression, tension and shear, are inspired by the in vivo setting, as articular cartilage resides in a complex and demanding mechanical environment. Reproducing this environment is hypothesized to condition neo-tissue to become more like native tissue while developing in vitro.
The overall objective of this thesis is to employ a novel flow bioreactor to improve matrix composition and mechanical properties of cartilage constructs for loadbearing applications. Improving the matrix components and mechanical stability of the tissue to be more similar to that of native tissue may improve the ability of the tissue to function in vivo. Our central hypothesis is that exposure of three-dimensional (3D) scaffold-free, tissue-engineered cartilage constructs to physiologically-inspired forces in a novel bioreactor system will enhance cartilage matrix synthesis, leading to more robust mechanical properties.
The novel, dual-chambered, parallel-plate bioreactor system was developed to provide a quantifiable, steady shear stress stimulus to a slab of scaffold-free engineered cartilage. While fluid-induced shear stress has been used in other bioreactor systems, this is the first study involving a parallel-plate bioreactor with a block of neo-tissue. The parallel-plate nature of this system is significant because when properly designed, a predetermined level of stimulus is applied to >95% of the tissue’s length. This is in stark contrast to spinner flask-like or perfusion systems which apply an unknown or widely heterogeneous shear to the tissue [1, 2]. Perfusion systems are particularly difficult to analyze as fluid flows through a complex maze of pores in scaffold-based tissue. To facilitate analysis of shear stress on the engineered tissue, a scaffold-free tissue was used in our system. Tissues of this type have the added clinical benefit of possessing matrix components derived solely from the cells. In addition, this bioreactor possess two chambers that are separated by the tissue, allowing for multiple stimuli (biochemical or mechanical) to be applied in a differential fashion for potential induction of heterogeneity, similar to the native cartilage-to-bone transition.
In Chapter 3, the bioreactor design is presented along with a feasibility study. Shear stress was applied to the developing scaffold-free tissue in order to improve the matrix composition and tensile mechanical properties. After only three days of continuous shear stress stimulus, a significant increase was measured in type II collagen and total collagen content as well as in the tensile mechanical properties in shear-exposed tissues (0.1-Pa) when compared to static controls. Tissues were stained throughout for type II collagen and sulfated glycosaminoglycans (sGAG), without evidence of type I collagen deposition. Type I collagen should be specifically avoided in engineered articular cartilage constructs, as this protein is well known to not be present in normal tissue and is relegated to fibrocartilaginous or degenerative tissues . Only the tensile mechanical properties were tested in this system, as compressive analysis was not reliable given the thin nature (~300 µm) of the tissues. While this study showed feasibility of the bioreactor to support and stimulate engineered cartilage, the matrix composition and mechanical properties still fell short of that seen in native tissue.
In order to build upon these results, the duration of flow was extended out to seven days, aiming to further improving the matrix composition and mechanical properties. As detailed in Chapter 4, while the mechanical properties, type II and total collagen content were again increased in shear-exposed tissues compared to controls, the beneficial effects came at the expense of several detrimental effects. A distinct band of type I collagen was noted on the shear-exposed surface of the tissue as well as a depletion of sGAG from the matrix. An increasing release of sGAG to the media was also evident over time and varied with increasing shear stress. Type I collagen was noted in the static controls as well, but significantly increased with the application of shear stress. Co-localized with this band of type I collagen were flattened, fibroblast-like cells reminiscent of a type I collagen-producing phenotype as opposed to the rounded, type II producing cells throughout the rest of the matrix. In this study, an additional flow group was added at a two-decade level (0.001-Pa) below that of the previously used flow group (0.1-Pa). This group was added to not only account for differences in media volume between static and flow-exposed groups, but also to begin to examine any dose-dependent effects of shear stress on tissue development. Also in this study, quantitative PCR was instituted to analyze the gene expression profile of the tissues over time. Generally, type I and type II collagen were transiently up-regulated as early as 24 h in the highest shear group compared to the others. This gene expression profile was corroborated by the quantitative protein and qualitative histology data. Taken together, these studies showed that shear stress magnitude and duration modulates matrix composition and tensile mechanical properties.
With a solid understanding of how fluid flow and shear stress modulates the matrix composition and mechanical properties of engineered cartilage, the lack of thickness associated with the tissues was addressed next. Two benefits of thicker constructs (1-2 mm) are that they (1) are more clinically relevant and thus add impact to the study, and (2) are amenable to compressive testing. Therefore, Chapter 5 describes the effects of seeding density and time in culture on matrix composition and compressive mechanical properties of engineered cartilage of clinically relevant thickness. In this study, tissues were created in small “cell-culture inserts” which are essentially a static, 1/10th scale version of the bioreactor environment. While increasing the seeding density did increase the thickness of the tissues, it was not in a linear dose-dependent manner, indicating a loss of efficiency with increasing seeding density. In fact, the lowest seeding density performed as well as any other group or better in terms of sGAG and collagen production on a per-cell basis. Compressive mechanical properties were improved over time in nearly all seeding densities, with the greatest dynamic modulus appearing at the three week time point of approximately 325 kPa at seeding densities of 4X and 6X. One of the most interesting results of the study came about when the increased seeding densities were tried in the bioreactor: at higher seeding densities (6X and 8X greater than normal), tissues formed blebs and wrinkles and easily delaminated off the membrane. Hypothesizing that the pores of the membrane were saturated with matrix, membranes with larger pore sizes were used and found to improve attachment and gross appearance of the tissues. Thus, this change (i.e., increasing the pore size of the membranes used in the bioreactor) was instituted immediately for future bioreactor flow studies.
In the final series of experiments as described in Chapter 6, thicker tissues (~1 mm) were developed in the bioreactor and subjected to multiple levels of shear stress using steady and intermittent waveforms. Tissues were analyzed for matrix composition as well as tensile and compressive mechanical properties after three and seven days of flow, to parallel that which was examined in earlier, thin-tissue studies. Generally, the effects of shear stress and time in culture were similar for many metrics of matrix composition and mechanical properties, but to a lesser extent. This could perhaps be attributed to the time required for the thicker tissues to develop and mature. In an effort to exceed the beneficial results obtained in thin tissues while avoiding the detrimental effects, intermittent flows were examined. Tissues were subjected to flows alternating between different levels of shear stress for multiple duty cycles. Unfortunately, no synergistic effects were seen with the intermittent flow, with most intermittent conditions scoring in between the two different shear levels. On a more positive note, these results underscore the fidelity with which one can use shear stress to affect the development of engineered cartilage.
In conclusion, this work produced a novel bioreactor system capable of modulating the matrix composition and mechanical properties of tissue-engineered cartilage. Results also support the notion that bioreactor-mediated mechanical conditioning can be beneficial to the acceleration of tissue development in an in vitro setting. Undoubtedly, the full potential of this bioreactor system has yet to be fulfilled. For example, compressive or hydrostatic pressurization can be used in lieu of or in conjunction with shear stress to condition the tissues. The use of growth factors and / or scaffolds to produce a cartilage or osteochondral construct have yet to be explored in this system, but the versatility inherent in the design warrants further exploration. Additional future directions are discussed in Chapter 7, with the ultimate goal being the development of a clinically relevant tissue-engineered articular cartilage construct for the restoration of function.
|1982||Armstrong CG, Mow VC. Variations in the intrinsic mechanical properties of human articular cartilage with age, degeneration, and water content. J Bone Joint Surg. 1982;64A(1):88-94.|
|1998||Soltz MA, Ateshian GA. Experimental verification and theoretical prediction of cartilage interstitial fluid pressurization at an impermeable contact interface in confined compression. J Biomech. October 1998;31(10):927-934.|
|1995||Hung T Clark, Pollack R Solomon, Reilly TM, Brighton T Carl. Real-time calcium response of cultured bone cells to fluid flow. Clin Orthop Relat Res. 1995;313:256-269.|
|1999||McAllister TN, Frangos JA. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways. J Bone Miner Res. June 1999;14(6):930-936.|
|1995||Buschmann MD, Gluzband YA, Grodzinsky AJ, Hunziker EB. Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture. J Cell Sci. April 1995;108(4):1497-1508.|
|1980||Mow VC, Kuei SC, Lai WM, Armstrong CG. Biphasic creep and stress relaxation of articular cartilage in compression: theory and experiments. J Biomech Eng. February 1980;102(1):73-84.|
|1998||Jacobs CR, Yellowley CE, Davis BR, Zhou Z, Cimbala JM, Donahue HJ. Differential effect of steady versus oscillating flow on bone cells. J Biomech. November 1998;31(11):969-976.|
|1989||Sah RL-Y, Kim Y-J, Doong J-YH, Grodzinsky AJ, Plass AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res. 1989;7(5):619-636.|
|1991||Lai WM, Hou JS, Mow VC. A triphasic theory for the swelling and deformation behaviors of articular cartilage. J Biomech Eng. August 1991;113(3):245-258.|
|1985||Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science. March 22, 1985;227(4693):1477-1479.|
|1987||Akizuki S, Mow VC, Muller F, Pita JC, Howell DS. Tensile properties of human knee joint cartilage, II: correlations between weight bearing and tissue pathology and the kinetics of swelling. J Orthop Res. 1987;5(2):173-186.|
|1986||Akizuki S, Mow VC, Müller F, Pita JC, Howell DS, Manicourt DH. Tensile properties of human knee joint cartilage, I: influence of ionic conditions, weight bearing, and fibrillation on the tensile modulus. J Orthop Res. 1986;4(4):379-392.|
|1988||Eyre DR, Dickson IR, Van Ness K. Collagen cross-linking in human bone and articular cartilage: age-related changes in the content of mature hydroxypyridinium residues. Biochem J. June 1988;252(2):495-500.|
|1961||Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch Biochem Biophys. May 1961;93(2):440-447.|
|1999||Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. April 2, 1999;284(5411):143-147.|
|2000||Robling AG, Burr DB, Turner CH. Partitioning a daily mechanical stimulus into discrete loading bouts improves the osteogenic response to loading. J Bone Miner Res. August 2000;15(8):1596-1602.|
|1986||Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. September 4, 1986;883(2):173-177.|
|2000||Mauck RL, Soltz MA, Wang CCB, Wong DD, Chao P-HG, Valhmu WB, Hung CT, Ateshian GA. Functional tissue engineering of articular cartilage through dynamic loading of chondrocyte-seeded agarose gels. J Biomech Eng. June 2000;122(3):252-260.|