Browsing by Author "Athanasiou, Kyriacos A."
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Item Biomechanics of the single chondrocyte and its developing extracellular matrix(2010) Ofek, Gidon; Athanasiou, Kyriacos A.Degradation of articular cartilage results in poor joint movement and afflicts millions of patients each year. Since this tissue is incapable of self-repair, developing new approaches to treat injured cartilage would be a tremendous boon to patient quality of life, as well as have important economic ramifications. To address this debilitating condition, this thesis investigated the biomechanical nature of single chondrocytes and studied their emergent biophysical environment. Detailed insight into the role of intracellular structures on chondrocyte mechanical characteristics is a vital first step in understanding the etiology of cartilage degradation and identifying potential treatments. This thesis demonstrated that actin, intermediate filaments, and microtubules each play a unique function in cellular compressive stiffness, Poisson's ratio and its strain dependence, as well as recovery behavior in response to a range of applied strains. The in situ stiffness of the nucleus was found to be minimally greater than that of the cytoplasm, countering current theories in chondrocyte biomechanics and identifying a potential new avenue for mechanotransduction. A videocapture method was also developed to examine the response of single chondrocytes to direct shear, whose results were further correlated with alterations in actin and focal adhesions. This thesis then examined the effect of two key components of cartilage regenerative processes, phenotypic modulation and growth factors, on cellular mechanics. A 'mechanical range' was observed for single cells along a chondrogenic lineage and a subpopulation of differentiated stem cells was identified with similar characteristics as chondrocytes. Moreover, growth factors were found to induce changes in chondrocyte stiffness and volumetric properties. The second major component of this thesis examined the developing biophysical milieu of chondrocytes. Through a novel 'self-assembly' tissue engineering approach, the evolving matrix composition and mechanical properties of cartilage neotissue were examined. Moreover, several notable similarities were identified between tissue maturation in self-assembled cartilage and known developmental processes for native tissue. This thesis sheds light on how chondrocytes respond to physicochemical stimuli, the role of biophysical factors in the maintenance of the cellular phenotype, and the composition of the emergent chondrocyte environment. This work can greatly aid researchers toward developing effective treatments for deteriorated cartilage.Item Characterization and tissue engineering of the temporomandibular joint disc(2005) Detamore, Michael Scott; Athanasiou, Kyriacos A.The temporomandibular joint (TMJ), commonly known as the jaw joint, can cause a great deal of suffering for those afflicted with TMJ disorders. Everyday activities like chewing, yawning, and sometimes even talking and laughing, can be agonizing, and personal life and work life often suffer. Approximately 3--4% of the population seek treatment for TMJ disorders, and almost 70% of these patients suffer from displacement of the TMJ disc. The TMJ disc is a poorly understood and scarcely studied structure in comparison to other musculoskeletal tissues. Prior to this thesis, a gap existed between the tissue engineering community and the TMJ characterization community. The objective of this work was therefore to perform the characterization studies vital to tissue engineering efforts, and to provide the initial steps toward an engineered TMJ disc construct. An argument was formed that provides a rationale for tissue engineering the TMJ disc, citing deficiencies in the current treatments for advanced stages of internal derangement. The TMJ disc has been shown to be mechanically non-homogeneous and highly anisotropic, which has been attributed to non-homogeneous extracellular matrix and cell sub-population distribution and anisotropic collagen orientation. The greatest variation by region in the TMJ disc is between the intermediate zone and the bands (anterior and posterior). The intermediate zone contains a higher proportion of fibrochondrocytes and higher quantities of type II collagen, chondroitin sulfate, keratan sulfate and dermatan sulfate proteoglycan compared to the anterior and posterior bands. Moreover, the intermediate zone is over an order of magnitude softer and weaker under mediolateral tension compared to these bands. Pioneering efforts in TMJ disc tissue engineering have been made, exploring growth factor effects and exploiting bioreactor technology. Insulin-like growth factor-I was selected from a group of four growth factors as the most promising for TMJ disc tissue engineering, most notably for its benefits associated with collagen synthesis. Moreover, a rotating bioreactor was shown to influence morphology and structure of engineered constructs, accelerating scaffold contraction and producing a much more heterogeneous matrix distribution compared to static culture.Item Characterization of chondrocytic differentiation and optimization of a self-assembling process for tissue engineering of articular cartilage(2008) Revell, Christopher Morton; Athanasiou, Kyriacos A.Articular cartilage degeneration, which occurs due to trauma or disease, results in the formation of tissue with inferior structural and mechanical properties. Tissue engineering has been proposed as a method to aid in the repair of damaged tissue. This thesis describes advancements in our laboratory's articular cartilage tissue engineering approach performed in three specific aims. One major limitation in cartilage tissue engineering is the paucity of donor tissue. To address this concern, dermal fibroblasts were investigated for their potential use in cartilage tissue engineering. Specifically, cartilage-specific proteins were examined for their ability to modulate the morphological characteristics of dermal fibroblasts toward the characteristically spherical morphology of articular chondrocytes. Optimal coating conditions were identified for stimulating fibroblasts into a more chondrocytic morphology with significantly increased cell height and lower surface area-volume ratios. Another limitation of cartilage tissue engineering is the production of tissue with sufficient biochemical and biomechanical properties. To this end, a self-assembling process was used to engineer neotissue with articular chondrocytes. Optimization of various parameters of this process was performed to increase this method's functionality toward clinical applicability and translatability. Three studies were performed: (1) Removal of serum was achieved, an essential requirement toward translatability, with a concomitant increase in functionality as evidenced by a 5-fold increase in compressive stiffness over serum-containing controls. (2) Temporal assessment identified 4 wks as an optimal time for both in vitro culture and the application of external stimuli to assist in enhancing functional properties. (3) Optimized parameters were then combined to examine the minimum quantity of cells required for the production of self-assembled constructs. It was found that the number of cells could be reduced by 32% while maintaining construct salient properties. To increase the functionality of self-assembled constructs, exogenous stimulation was investigated in this aim. Treatment of developing constructs with Chondroitinase ABC resulted in a more functional biochemical network which led to a 50% increase in tensile stiffness. Finally, a direct compression bioreactor was used to examine the effects of mechanical stimulation on self-assembled constructs. Properties of self-assembled constructs indicated that certain compression regimens imparted an immediate increase in GAG which contributed to increased compressive properties. Ultimately, direct compression with 17% strain applied at 0.1 Hz allowed constructs to reach 12% GAG/ww and an aggregate modulus of 290 kPa. The results of this thesis in toto advance the field of articular cartilage tissue engineering through optimizing conditions toward the use of an alternative cell source and its significant contributions to the understanding and progression of the self-assembling process. Moreover, successes of this thesis have led to the production of neocartilage constructs with ECM and compressive stiffness values elevated above native immature bovine cartilage.Item Characterization of the meniscus for future tissue engineering efforts(2005) Sweigart, Mark Andrew; Athanasiou, Kyriacos A.The meniscus, a fibrocartilagenous tissue found between the femur and tibia, is responsible for shock absorption, load transmission, and stability within the knee joint. Damage to this tissue can lead to osteoarthritic changes, suggesting that the meniscus protects the knee joint from degenerative joint disease. Historically, repair techniques consisted of excision or suturing of the damaged tissue. Unfortunately, neither of these techniques successfully repairs the damaged tissue; tissue engineering is one possible solution. When attempting to tissue engineer a tissue, it is ideal to start in a small animal model, such as a rabbit, before attempting the repair process in a larger animal model. The objective of this work was to characterize the medial rabbit meniscus ultrastructurally, biomechanically, biochemically, and cellularly and to perform biomechanical characterization on larger animal models for future scale up efforts. The medial rabbit meniscus was found to have a higher hydration level, greater amount of sulfated glycosaminoglycans, and lower level of hydroxyproline at the inner 1/3 of the tissue, which confirmed the more chondrocytic nature of this region. It was also found that the anterior portion of the tissue, particularly in the inner 1/3, had a higher hydration level, sulfated glycosaminoglycan level, aggregate modulus, permeability, shear modulus, and a lower hydroxyproline level than the central and posterior locations. It is believed that this topographical variation is due to the bent-knee resting stance of the rabbit and its propensity to jump. It was also determined that significant variations exist in the compressive creep properties, both intraspecies and interspecies, in a variety of animal models, indicating caution when comparing animal models and determining which animal model to use in future tissue engineering efforts. The characterization in this study can serve as a "gold standard" reference for future meniscal tissue engineering efforts and be used as a baseline for future large animal tissue engineering efforts.Item Characterization of the Temporomandibular Joint Disc and Fibrocartilage Engineering using Human Embryonic Stem Cells(2012) Willard, Vincent P.; Athanasiou, Kyriacos A.Fibrocartilages in the body, including the temporomandibular joint (TMJ) disc and knee meniscus, lack intrinsic healing capacity following trauma or disease. Current treatments only address the symptoms of fibrocartilage damage and do nothing to prevent further degradation of the joint. A tissue engineered replacement, with biochemical and biomechanical properties approaching those of native tissue, could provide a solution. This thesis investigates two components critical to the generation of a tissue engineered TMJ disc: 1) characterization of the native disc to identify a suitable animal model and create design parameters, and 2) development of approaches to use human embryonic stem cells (hESCs) in fibrocartilage tissue engineering. The first step to achieving this goal was to identify an animal model for the human TMJ disc based on quantitative biochemical and biomechanical properties. To this end, rabbit, goat, pig, cow, and human discs were analyzed, and the pig disc was shown to possess properties most similar to the human. The next step was to further characterize the pig TMJ, as many aspects of the joint were still poorly understood. Though the TMJ disc is anchored to the surrounding bony tissue on all sides by discal attachments, little was known about their properties. Biochemical and histological analysis was performed on these attachments and indicated that they are similar to the disc but possess distinct regional matrix content related to joint biomechanics. Finally, though the contribution of collagen to the mechanical properties of the TMJ disc was well characterized, the contribution of the glycosaminoglycans (GAGs) was unknown. By removing sulfated GAGs with chondroitinase ABC, it was found that these molecules contribute to the viscoelastic compressive properties of the disc, but only in regions with the highest native GAG content. The second aspect of this thesis involved producing fibrocartilage tissue from hESCs. The pluripotency and unlimited self-renewal of these cells makes them ideally suited for producing fibrocartilages that contain a spectrum of matrix components. This work began by investigating what factors are necessary for fibrochondrogenic differentiation of hESCs in embryoid bodies (EBs). Growth factors and co-cultures with primary fibrochondrocytes were both shown to be potent modulators of fibrochondrogenesis, although differentiation of hESCs consistently produced a heterogeneous cell population. To purify populations of fibrochondrocytes differentiated form hESCs, two inexpensive and novel techniques were investigated. First, density gradient separation was the first technique attempted. This technique was able to isolate distinct subpopulations of cells, some of which were mechanically similar to native chondrocytes. Second, a chondrogenic tuning technique was applied to differentiated hESCs. Following fibrochondrogenesis in EBs, cells were expanded in monolayer in chondrocyte specific media before being used for tissue engineering. Chondrogenic tuning produced several distinct cell populations during expansion, and, as a result, a spectrum of different cartilaginous tissues was achieved for tissue engineering. Three of the cell populations produced tissues similar to the native TMJ disc, outer meniscus, and inner meniscus. Overall, this thesis identified an animal model for TMJ characterization and in vivo studies, furthered understanding of structure-function relationships of the TMJ disc and its attachments, and developed a technique for producing a spectrum of engineered fibrocartilages from hESCs.Item Chondrocyte biomechanics and chondrogenesis(2007) Koay, Eugene J.; Athanasiou, Kyriacos A.This thesis contributes to two aspects of cartilage research: understanding the biomechanical characteristics of single chondrocytes, and tissue engineering musculoskeletal cartilages with human embryonic stem cells (hESCs). Both of these areas of investigation are motivated by the significant economic and social burdens of cartilage afflictions. Mechanical forces directly influence the biological processes of native chondrocytes; understanding and controlling this phenomenon begins at the single cell level. Thus, the first aspect of this thesis analyzes the biomechanical nature of the single chondrocyte by developing (1) a creep cytoindentation apparatus (CCA) to obtain the viscoelastic properties of single chondrocytes and (2) a new method to investigate the response of single chondrocytes to direct compression. The CCA was built, validated, and used to show that chondrocytes are three orders of magnitude less stiff than reported values for their surrounding extracellular matrix, suggesting that the cellular mechanical milieu is markedly different from the rest of the tissue. Chondrocytes were also found to alter their biomechanical properties in response to physicochemical stimuli while exhibiting a mechanical yield behavior. These results counter current theories of cartilage that assume fixed cellular properties and illuminate a possible threshold between physiological and detrimental mechanical stimuli. The second aspect of this thesis involves the design and application of a strategy to engineer musculoskeletal cartilages with hESCs. These cells, which have been scarcely studied to date, possess unique properties, such as pluripotency and self-renewal, that make them advantageous for tissue engineering. A novel modular experimental approach was established, involving hESC chondrogenic differentiation followed by a scaffold-less engineering strategy called self-assembly. Results showed that the properties of hESC-derived cartilage can be modulated through biochemical growth factors, differentiation timelines, and hypoxia, raising the possibility of engineering tissue with hESCs for various cartilage applications, including fibrocartilages and hyaline articular cartilage. Additionally, serum-free, chemically defined conditions were developed for the entire modular approach, aiding future translation of this research to a cartilage therapy. This thesis generates new directions for understanding and utilizing mechanical forces to stimulate chondrocytes and opens avenues to further investigate a powerful cell source for tissue engineering in hESCs.Item Chondrocyte self-assembly and culture in bioreactors(2005) Hu, Jerry (Chi-Yuan); Athanasiou, Kyriacos A.Articular cartilage is an avascular tissue that does not respond adequately to injuries, leaving permanent chondral defects or fibrocartilage filled osteochondral defects that cannot bear physiological stress, eventually failing. Current articular cartilage tissue engineering methods employ homogeneously cell-seeded scaffolds that do not recreate the zonal structure or the biomechanical function of the native tissue. Thus, the goal of this study was to take the first steps in re-creating the zonal structure, and thus function, of articular cartilage by examining the effects of zone, passage, diffusion, seeding density, and mechanical stimuli on chondrocytes. A technique for isolating zonal chondrocytes was developed and verified. Chondrocytes from the superficial and growth zones were found to be phenotypically different, though this difference diminished rapidly in passage. A critical seeding density was found for the culture of chondrocyte constructs in bioreactors. From this finding, a novel self-assembling process was developed. The self-assembling process was shown to form articular cartilage constructs more than 1 mm thick with 1/3 the stiffness of native tissue after 12 weeks. Zonal chondrocytes cultured using the self-assembling process retained phenotypic differences. Fifth passage, dedifferentiated chondrocytes were shown to cease collagen type I expression when self-assembled. Lastly, the self-assembling process was shown to benefit from intermittent hydrostatic pressure stimulation. This study can serve as a launching point for a series of projects whose governing hypothesis is that articular cartilage can be regenerated by following a cell-based in vitro tissue engineering approach.Item Dermis-derived cells for tissue engineering applications(2014-01-28) Athanasiou, Kyriacos A.; Deng, Ying; Hu, Jerry; Rice University; United States Patent and Trademark OfficeMethods for inducing differentiation of dermis-derived cells to serve as a source of chondrocytes and associated methods of use in forming tissue engineered constructs. One example of a method is a method for inducing differentiation of cells into chondrocytes comprising providing aggrecan sensitive isolated dermis cells and seeding the cells onto an aggrecan coated surface.Item Development of a self-assembled meniscal replacement(2010) Huey, Daniel Joseph; Athanasiou, Kyriacos A.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.Item Exogenous stimulation of meniscus cells for the purpose of tissue engineering the knee meniscus(2009) Gunja, Najmuddin Juzer; Athanasiou, Kyriacos A.Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate the symptoms, but lead to premature osteoarthritis due to reduced stability and changes in knee biomechanics. An alternative treatment to overcome these problems involves functional tissue engineering. This thesis examined several exogenous factors to enhance the capability of meniscus cells (MCs) to synthesize relevant ECM markers and improve the functionality of constructs in vitro. First, the effect of passage on the phenotype of MCs in monolayer was investigated, and rapid changes were observed in collagen I, collagen II, and COMP expression. Collagen I and aggrecan protein coatings assisted in reversing expression levels of certain ECM markers; however, collagen II expression could not be reversed. Next, 3D tissue engineering studies were conducted using a cell-scaffold approach with MCs seeded on PLLA meshes. Anabolic stimuli that aided in meniscus regeneration included (1) hypoxia and bFGF, which resulted in synergistic increases in the total glycosaminoglycan content and compressive properties of constructs; (2) 10 MPa static hydrostatic pressure (HP), which resulted in increases in collagen content and the relaxation modulus of constructs; and (3) 10 MPa static HP and TGF-beta1, which resulted in additive increases in collagen content, and synergistic increases in the compressive moduli of constructs. Finally, a self-assembly, scaffoldless approach was employed for meniscus regeneration using co-cultures of MCs and articular chondrocytes (ACs). A high density of cells were seeded on non-adherent agarose molds and allowed to coalesce into a construct without a scaffold. Different co-culture ratios of MCs and ACs resulted in a spectrum of fibrocartilages that recapitulated some biochemical and biomechanical properties of the rabbit meniscus. Cell culturing conditions were optimized with the identification of a smooth 1% agarose mold that resulted in geometrically-mimetic meniscus constructs. In conclusion, this thesis quantified phenotypic changes in MCs over passage, and used scaffold-based and scaffoldless approaches to regenerate constructs with biochemical and biomechanical properties in the range of native tissue values. Successful replacement of a damaged meniscus will improve the quality of patient life and reduce the risk of osteoarthritis.Item Extracellular matrix characterization and tissue engineering of the temporomandibular joint disc(2005) Almarza, Alejandro Jose; Athanasiou, Kyriacos A.The temporomandibular joint (TMJ) disc is a specialized fibrocartilaginous tissue located between the mandibular condyle and the glenoid fossa-articular eminence of the temporal bone. It has been observed that up to 70% of patients with temporomandibular joint disorders (TMDs) suffer from displacement of the disc. When the displaced disc becomes an obstacle to movement and degeneration is severe, surgeons have no choice but to replace the disc with various allopastic or biological materials. Tissue engineering may provide a better alternative for discectomy patients. Toward this end, the work described in this thesis provides a systematic approach for tissue engineering the TMJ disc. Initial studies first characterized the biochemical composition of the porcine TMJ disc. It was determined that the majority of the porcine TMJ disc matrix is collagen, while glycosaminoglycans are present in small quantities. Once this biochemical standard was established, an appropriate scaffold composed of poly(glycolic) acid (PGA) non-woven meshes for TMJ disc tissue engineering was identified. Dynamic spinner flask seeding was determined to be the most effective method for seeding TMJ disc cells on PGA, based on an increased production of collagen. The addition of two growth factors, whether insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), or transforming growth factor-beta1 (TGF-beta1), at high concentrations of 100 ng/ml for IGF-I and bFGF and 30 ng/ml for TGF-beta1, improved the cellularity of constructs after six weeks in culture, but did not improve matrix production. Ascorbic acid concentration was found to be an important factor affecting attachment of passaged TMJ disc cells onto PGA. A concentration of 25 mug/ml of medium was observed to be more beneficial than no ascorbic acid and 50 mug/ml of medium. A high cell seeding density of 75 million cells per ml of construct produced twice more collagen than previous attempted seeding densities. In the last portion of this work, the effects of hydrostatic pressure were examined as part of an in vitro tissue engineering approach. A constant hydrostatic pressure regimen of 10 MPa for 4 hrs was shown to increase collagen expression in 2D and 3D, and increase collagen production. Surprisingly and perhaps counter-intuitively, an intermittent exposure of the hydrostatic pressure regimen at 1 Hz was observed to be detrimental to TMJ disc cells. The results of this work established general criteria, both in terms of biochemical and biomechanical factors, toward addressing the complex problem of regeneration of the TMJ disc.Item Functional tissue engineering of the temporomandibular joint disc(2008) Johns, Deirdre Ellen; Athanasiou, Kyriacos A.Temporomandibular joint (TMJ) disorders arise from disease or trauma and may result in degeneration of the soft tissues. Tissue engineering may provide a solution to disorders of the TMJ without the side effects seen with artificial materials, such as improper incorporation with the surrounding tissues or immunological rejection of the artificial replacement. Several experiments were completed toward the goal of creating a functional TMJ disc replacement using a cell-based approach; the cell types that were primarily examined in this work were TMJ disc cells and costal chondrocytes. Attempts were made to improve the properties of scaffolds seeded with TMJ disc cells, and while proliferation was increased for the monolayer expansion phase of the approach, improvements were not seen in the properties of the three-dimensional constructs by adding L-proline to the culture. Due to the limited success of the TMJ disc cell constructs and the donor scarcity of this cell type, alternative cell sources were investigated in a scaffoldless tissue engineering method to improve the functionality and translatability of the engineered constructs. Chief among the cell types investigated, costal chondrocytes (CCs) consistently produced constructs with considerable amounts of extracellular matrix that were relevant to regenerating TMJ disc fibrocartilage. From this initial success, other aspects in using CCs for TMJ disc tissue engineering were investigated, specifically, passaging the CCs and adding exogenous stimuli. Examining passaged costal and articular chondrocytes showed that while the process of passaging and expanding chondrocytes caused an increase in collagen type I over type II, constructs made from passaged chondrocytes had higher collagen content and tensile properties than primary chondrocyte constructs. The observation that passaged cells were just as, if not more, capable of producing functional constructs also enhanced the translatability of this method by addressing the issue of donor tissue scarcity. Therefore, CCs at a variety of passages were examined in construct culture. Passaged CC constructs consistently produced more glycosaminoglycans per wet weight than primary cell constructs. Passaged CC constructs were then examined in the presence of exogenous stimuli to further improve their properties. At the regimens examined, hydrostatic pressure did not affect the constructs. In contrast, insulin-like growth factor-I improved construct properties over the no growth factor control. Overall, this thesis presents considerable support for the use of passaged costal chondrocytes for the purposes of improving functionality and clinical translatability of constructs for TMJ disc tissue engineering.Item Growth factor effects on single chondrocyte biomechanics and gene expression(2006) Leipzig, Nic Davis; Athanasiou, Kyriacos A.Studying chondrocyte responses to mechanical forces and how growth factors modify these responses allows for exploration of the underlying principles of cartilage physiology and disease. The work described in this thesis aims to understand single chondrocyte response to mechanical stimulation and how soluble growth factors can modulate this response. This knowledge is valuable for the formulation of successful cartilage tissue repair and replacement strategies, as well as etiopathogenesis of and treatments for the disease osteoarthritis. The first portion of this thesis established unconfined compression as a method for creep testing single adherent chondrocytes. Fitting of three continuum biomechanics models showed that a continuum viscolelastic model best described the creep behavior of single chondrocytes. Unconfined creep compression was next used to test single superficial and middle/deep zone chondrocytes. The effects of growth factor treatment (TGF-beta1, IGF-I, and TGF-beta1 + IGF-I) and seeding time (3 and 18 hr) were also tested. Creep testing demonstrated that all growth factor treatments stiffened the cytoskeleton of single cells, and staining showed increased F-actin due to growth factors. Techniques were developed to isolate specific adherent single chondrocytes and assay their gene expression with real-time RT-PCR. These techniques were used to detect significant differences in the gene expression of single zonal chondrocytes exposed to IGF-I. Finally, single cells were statically compressed with or without TGF-beta1 and IGF-I and their resulting gene expression was measured. Static compression elicited catabolic gene expression in control single cells. TGF-beta1 and IGF-I provided mechanoprotection and differentially prevented this catabolic response. Cytoimmunohistochemistry of single chondrocytes fixed in compression demonstrated that nearly all axial strain experienced by the cell is experienced by the nucleus. Also, cells compressed at higher levels of force had increasingly deformed nuclei with larger spaces in the chromatin. These results suggest that transcription is modified directly by nuclear strains through force-mediated changes of the chromatin. This work provides the first evidence of mechanical forces modifying gene expression and provides a starting point for future studies where precise thresholds of mechanical stimulation required to elicit desired metabolic changes in single cells will be determined.Item Growth factors in tissue engineering the knee meniscus(2004) Pangborn, Christine A.; Athanasiou, Kyriacos A.Tissue engineering is a promising solution to creating a replacement meniscus. The structure and composition of the meniscus lends the tissue its ability to withstand tension and compression. Enhancing extracellular matrix production in an engineered construct, may improve mechanical properties of the construct. The goal of this study was to determine the growth factors that would most increase the production of collagen and glycosaminoglycans (GAGs) produced by meniscal fibrochodrocytes. Cells were studied in monolayer and in three dimensional cultures. The growth factors and concentrations evaluated were: TGF-beta1 (1, 10, 100 ng/ml), IGF-I (5, 12.5, 50 ng/ml), PDGF-AB (10, 25, 100 ng/ml), and bFGF (10, 25, 100 ng/ml). TGF-beta1 (at 10 and 100 ng/ml) was the only growth factor that showed an increase in both collagen and GAG component uptake in both culture conditions as indicated by radiolabeling. TGF-beta1 showed the most increase in component uptake over the control and over the other growth factors and is recommended for use in tissue engineering the knee meniscus.Item Interferometry of chondrocytes and impact of articular cartilage(2006) Scott, Charles Corey; Athanasiou, Kyriacos A.Osteoarthritis and the subset of post-traumatic osteoarthritis both represent the end-stage of a degenerative process that can result from an initial tissue insult, particularly from a single mechanical impact. No current treatment has been shown to slow or stop its progression. Here, two approaches are taken to understand the physiology and pathology of articular cartilage. A cellular approach develops and uses a novel imaging technique for single cells and bioactive surfaces, while a tissue approach consists of understanding the acute and temporal effects of mechanical impact. Thus, the goals of this study are two-fold: (1) to develop vertical scanning interferometry (VSI) to obtain all salient features of chondrocytes and characterize bioactive surfaces, and (2) to develop methodologies for protecting diarthrodial joints from pathologic impact loading. VSI was validated and developed to obtain three-dimensional chondrocyte and fibroblast geometries, as well as to characterize protein-coated surfaces. VSI can now be applied to an array of studies involving single cell biomechanics, surface characterization, and cell adhesion and spreading. To examine pathology, an impact instrument was built and validated to apply repeatable impacts to articular cartilage. An explant model was characterized to understand the physiologic changes articular cartilage tissue experiences in culture over four weeks. Then, the acute and temporal effects of two levels of impact were characterized, consisting of a low level impact that did not show initial gross damage and a high level impact that caused immediate surface disruption. These studies illustrated that clinically undetectable impact injuries immediately show some subtle changes in extracellular matrix (ECM) glycosaminoglycan release and gene expression, but otherwise resemble the culture controls, while the high impact level caused gross damage. However, over a four week culture period, the subclinical impact proved to have started a degeneration cascade that significantly affected the biomechanical integrity, gene expression profile, biochemical makeup of the ECM, and chondrocyte viability. Therefore, impact injuries may account for a substantial proportion of the primary osteoarthritis cases. Further, if the start of the degeneration cascade of the low impact level can be stopped or reversed when only subtle changes are occurring, osteoarthritis prevention would be possible.Item Interspecies characterization and tissue engineering of the temporomandibular joint disc(2010) Kalpakei, Kerem Nathan; Athanasiou, Kyriacos A.Disorders of the temporomandibular joint (TMJ) are widespread, afflicting millions of people. The majority of these cases involve displacement or injury to the TMJ disc. Current treatments do not fully address severe cases of TMJ dysfunction; therefore, efforts to engineer functional tissues for repair or replacement are warranted. While previous studies have laid the groundwork for these efforts, significant challenges remain, including (1) identification of appropriate animal models, (2) development of methodologies for in vitro TMJ tissue engineering, and (3) refinement of tissue culture procedures for clinically relevant cells sources. This thesis contributes to overcoming these challenges by (1) exploring topographical and interspecies variation in functional properties of the TMJ disc, (2) developing an in vitro tissue engineering strategy capable of recapitulating native tissue characteristics, and (3) enhancing protocols for chondrogenesis of dermis-derived cells. The first aim of this thesis characterized the biomechanical and biochemical properties of human TMJ disc in relation to several animal models. Significant regional and interspecies variations were indentified, though certain characteristics were observed across all species. While the human disc displayed properties distinct from the other species, the pig was the most similar and was therefore identified as the most appropriate animal model. The second aim applied these findings as design criteria in the development of an in vitro tissue engineering strategy. Scaffoldless constructs derived from co-cultures of chondrocytes and fibrochondrocytes were enhanced through optimization of growth factor and serum supplementation, such that they recapitulated many characteristics of native TMJ cartilage. Finally, the third aim refined the differentiation process for chondroinduction of dermis-derived cells. Using an optimized, low-cost surface coating, chondrogenesis was significantly enhanced through incorporation of hypoxia during culture. These experiments address several aspects of fibrocartilage tissue engineering and represent a significant step towards in vivo application of these technologies.Item Investigation of surface mechanical environment as an optimization criterion for improved tissue engineering scaffolds(2008) Bucklen, Brandon; Liebschner, Michael A. K.; Athanasiou, Kyriacos A.; Grande-Allen, K. Jane; Gunaratne, Germane H.; Heinkenschloss, MatthiasTrabecular bone, the porous bone found predominately in the spine and ends of long bones, is a mechanically regulated tissue. The hierarchy of bone consists of several levels of structure such as raw collagen and calcium phosphate on the microscale to trabecular packets, which are constantly being remodeled by bone cells on the tissue level. The remodeling of bone is believed to be explained through the concept of functional adaptation-where bone is a maximum strength yet minimum weight material. In functional adaptation, phenomenological models are able to predict the density distributions and bone shapes that are witnessed in vivo to a certain degree. Functional adaptation assumes there is an equilibrium state in which no changes in bone mass or structure will occur at the bony surface. Topological and mass changes are incurred on a local level when equilibrium is not achieved. The combination of these local changes produces a self-organized structure -meaning that the global bone shape is explained by simple local rules. Unfortunately, neither tissue engineering nor medical device design has incorporated the knowledge base of functional adaptation of bone into their orthopedic designs. The objective of this dissertation work was to examine how the concept of functional adaptation could be applied to tissue engineering of bone in so much as it leads to the development of a computer-aided tissue engineering (CA TE) framework. The idea was to increase the specificity in which implant/scaffold architectural shape can be matched to tissue mechanical properties of the spine (or other locations), as well as matched to an individual patient who has experienced fracture. Because a variety of mechanical stimuli have been proposed in the functional adaptation literature, the first step of this work was to categorize the most probable variables that explain mechanical loading of trabecular bone in the spine. This was accomplished through reverse engineering cadaver specimens into J..t-finite element models. Two algorithms were developed for scaffold design, which makes use of the mechano-transductive principles specifically designed for the pre-determined mechanical variables. Finally, a framework for assembling scaffolds from local building blocks, which are derived from bone was proposed.Item Mechanical characterization, gene expression, and biosynthesis of the porcine TMJ disc for the purposes of tissue engineering(2006) Allen, Kyle D.; Athanasiou, Kyriacos A.The temporomandibular joint (TMJ) may be permanently damaged by disease or trauma; thus, TMJ cartilage is a prime target for tissue engineers. The damaged TMJ disc may become obstructive to normal jaw function, leading to severe tissue degradation and rendering the tissue useless in subsequent surgeries. Thus, post-operative TMJ patients may experience residual pain and dysfunction. Toward the tissue-engineered TMJ disc, this thesis describes contributions to our laboratory's tissue engineering approach. The TMJ disc's compressive properties are described topographically by a viscoelastic, incremental stress relaxation solution. The bands of the disc have large instantaneous moduli, approximately 3 times larger than central sections, while relaxation moduli and coefficients of viscosity revealed a lateral region with limited mechanical integrity. Second, the gene expression of TMJ disc cells changes as a function of passage, growth factor treatment, and exposure to specific proteins. The act of passaging cells rapidly changes TMJ disc gene expression. Aggrecan, collagen type I, and collagen type II gene expression decrease rapidly with passage; decorin and GAPDH expression increase. Recovery attempts via growth factors or protein coated surfaces were unsuccessful. The gene expression response to growth factor stimuli in either monolayer or pellet culture was not large enough to counter passage effects. In fact, pellet culture only further decreased aggrecan, biglycan, and collagen type I gene expression. With these data, tissue engineering studies were designed. Mesh-like scaffolds were examined, leading to the selection of PLLA. Scaffolds were then seeded with primary TMJ disc cells, exposed to a growth factor, and measured for mechanical and biochemical content at 6 weeks. TGF-β1 unmistakably demonstrated benefits toward TMJ disc tissue engineering. Constructs exposed to TGF-β1 had twice the matrix formation as TGF-β3 constructs and several fold more than IGF-I constructs and no treatment controls; furthermore, TGF-β1 constructs had improved tensile and compressive properties. The results of this work in toto establish key biomechanical validation criteria, cell culture and cell selection criteria, and a tissue engineering foundation from which constructs of significant volume, biochemical makeup, and mechanical properties may be derived. Moreover, this work addresses key components of the complex TMJ disc regeneration issue.Item Mechanical stimulation towards tissue engineering of the knee meniscus(2006) Aufderheide, Adam C.; Athanasiou, Kyriacos A.Tissue engineering has been proposed to alleviate injury to the knee meniscus, which leads to loss of function and damage to the surrounding articular cartilage. Relatively few studies have been performed to tissue engineer the meniscus; however, much guidance can be found by studying related tissues such as articular cartilage. One technique that has proved beneficial for producing tissue engineered articular cartilage constructs is mechanical stimulation. This thesis describes the investigation of scaffold choices and the development of culture techniques for producing meniscal constructs. In addition, a direct compression stimulator was developed, validated, and applied to the tissue engineered meniscal constructs. Poly-glycolic acid (PGA) and agarose were examined for use as scaffolds. It was found that agarose did not support fibrochondrocyte growth, and that while PGA supported cell production and proliferation, constructs were not mechanically robust after 7 wks in culture. A direct compression bioreactor was developed and validated using articular cartilage and meniscal explants. An attempt to produce better constructs than previously achieved in vitro in terms of mechanical properties, matrix production and organization, was successful using the self-assembly (SA) method. The SA method developed for the meniscus employs a ring-shaped agarose mold seeded with articular chondrocytes (AC) and meniscal fibrochondrocytes (MFC). It was found that a spectrum of biomechanical and biochemical properties could be achieved based on the seeding ratio of ACs:MFCs. It was determined that a 50:50 ratio of AC:MFC produced a construct that best replicated the cross-section of the native meniscus. A direct comparison of SA constructs to constructs formed with PGA was made via an investigation into the effect of dynamic direct compression stimulation. It was found that, after 4 wks in culture, PGA constructs lacked sufficient mechanical integrity to undergo loading. Wk 8 SA constructs were 3.4 times stronger and stiffer in the circumferential direction than in the radial direction. In addition, SA constructs had 3 times more GAG and 2 times more collagen than PGA constructs. The application of dynamic stimulation did not further increase mechanical properties or matrix production in SA constructs, but merits further study examining different loading regimens.Item Mechanobiology of single chondrocytes(2006) Shieh, Adrian C.; Athanasiou, Kyriacos A.The objective of this thesis was to expand the current understanding of how biomechanical factors mediate a variety of processes in articular cartilage, using an approach that focused on single cell biomechanics and mechanobiology. Single chondrocytes were mechanically tested, to derive salient biomechanical parameters that could aid in more accurate descriptions of the in vivo cellular mechanical environment. Building upon these results, single chondrocytes were then subjected to static and dynamic mechanical forces, and the resulting changes in the expression of key genes was measured using single cell real-time RT-PCR. These studies yielded several major findings relevant to chondrocytes and the nature of their responses to mechanical forces. Superficial zone chondrocytes were significantly stiffer than cells from the deeper layers of cartilage. This suggests that cells adapt to their mechanical environment by altering their properties, and that these zone-dependent differences could lead to varying cell responses to the same externally applied mechanical load. Chondrocytes were also found to have strain-dependent recovery properties. Specifically, the residual strain, volume fraction recovered, and recovery time after the cell was compressed were dependent on compressive strain. The most intriguing finding was that the dependence on compressive strain increased at approximately 25-30% strain, suggesting that this range of strain causes a fundamental change in cell biomechanical behavior. Furthermore, this strain range may represent an important threshold for discriminating whether a given mechanical stimulus has a beneficial or deleterious effect on chondrocytes. Finally, dynamic compression was shown to increase type II collagen and aggrecan gene expression compared to statically loaded single chondrocytes. This result was very exciting, as it demonstrated that studying the effects of mechanical forces on single cells was a viable approach. It was also shown that gene expression in single chondrocytes appears to be lognormally distributed. Thus, tests examining populations of cells may be biased by a small fraction of cells with very high levels of gene expression. These findings reinforce the notion that a single cell approach offers significant advantages over existing techniques, and may allow researchers to answer questions that were previously intractable with traditional methodologies.