Nicht behandelte Knorpelschäden führen langfristig zum Verschleiß des Gelenks – der Arthrose. Eine vielversprechende Möglichkeit zur Behandlung von Knorpelschäden ist die Matrix-assoziierte Chondrozytenimplantation. In der vorliegenden Dissertationsarbeit sollte die neuartige Matrix NOVOCART® Inject der Firma TETEC® auf ihre Eignung für die Therapie von Knorpeldefekten untersucht und ihre potentiellen Effekte im Vergleich zu dem bereits in der klinischen Anwendung befindlichen Biomaterial Fibrin charakterisiert werden. Für diese Studie wurden bei insgesamt 12 Göttinger Minipigs jeweils zwei vollschichtige Defekte (Ø 6mm) auf den medialen Femurkondylen beider Knie gesetzt und mit humanen Chondrozyten, die entweder in NOVOCART® Inject oder Fibringel eingebettet waren, gefüllt. Zellfreie Träger dienten jeweils als Kontrolle. Nach zwei und vier Wochen wurde die Defektfüllung, Biokompatibilität, Wirtszellanlockung und der Verbleib der implantierten Zellen sowie die Regeneratqualität histologisch und auf Genexpressionsebene evaluiert. Es konnte nachgewiesen werden, dass NOVOCART® Inject auch ohne Membranabdeckung eine gute Defektfüllung erzielt. Es ist biokompatibel und weist keine signifikant erhöhten Entzündungszeichen gegenüber Fibringel auf. Des Weiteren konnte eine vergleichbare Regeneratqualität im Vergleich zum Fibringel im porzinen Tiermodell erreicht werden. Unabhängig vom verwendeten Trägermaterial überdauerten die xenogen transplantierten Zellen im vorliegenden Defektmodell nur kurzfristig. Wir fanden Hinweise auf eine Immunreaktion unter Beteiligung von Makrophagen, die ursächlich für das Verschwinden der implantierten Zellen sein könnten. Die Ergebnisse unserer Untersuchungen stellen folglich die häufig diskutierte Theorie des immunpriviligierten Status der Chondrozyten in Frage.

Tissue engineering is an emerging field aimed to combine biological, engineering and material methods to create a biomimetic three dimensional (3D) environment to control cells proliferation and functional tissue formation. In such an artificial structural environment, a scaffold, made from biomaterial(s), plays an essential role by providing a mechanical support and biological guidance platform. Hence, fabrication of tissue scaffolds is of a fundamental importance, yet a challenging task, in tissue engineering. This task becomes more challenging if living cells need to be encapsulated in the scaffolds so as to fabricate scaffolds with structures to mimic the native ones, mainly due to the issue of process-induced cell damage. This research aims to develop novel methods to model the process of fabricating cell-encapsulated scaffolds and process-induced cell damage. Particularly, this research focuses on the scaffold fabrication process based on the dispensing-based rapid prototyping technique - one of the most promising scaffold fabrication methods nowadays, by which a 3D scaffold is fabricated by laying down multiple, precisely formed layers in succession. In the dispensing-based scaffold fabrication process, the flow behavior of biomaterials solution can significantly affect the flow rate of material dispensed, thus the structure of scaffold fabricated. In this research, characterization of flow behavior of materials was studied; and models to represent the flow behaviour and its influence on the scaffold structure were developed. The resultant models were shown able to greatly improve the scaffold fabrication in terms of process parameter determination. If cells are encapsulated in hydrogel for scaffold fabrication, cell density can affect the mechanical properties of hydrogel scaffolds formed. In this research, the influence of cell density on mechanical properties of hydrogel scaffolds was investigated. Furthermore, finite element analysis (FEA) of mechanical properties of scaffolds with varying cell densities was performed.The results show that the local stress and strain energy on cells varies at different cell densities. The method developed may greatly facilitate hydrogel scaffolds design to minimize cell damage in scaffold and promote tissue regeneration. . In the cell-encapsulated scaffold fabrication process, cells inevitably suffer from mechanical forces and other process-induced hazards. In such a harsh environment, cells deform and may be injured, even damaged due to mechanical breakage of cell membrane. In this research, three primary physical variables: shear stress, exposure time, and temperature were examined and investigated with regard to their effects on cell damage. Cell damage laws through the development phenomenal models and computational fluidic dynamic (CFD) models were established; and their applications to the cell-encapsulated scaffold fabrication process were pursued. The results obtained show these models and modeling methods not only allow one to optimize process parameters to preserve cell viability but also provide a novel strategy to probe cell damage mechanism in microscopic view.

Tissue loss and end-stage organ failure caused by disease or injury are two of the most costly problems encountered in modern medicine. To combat these problems, a relatively new field, called tissue engineering, has emerged. This field combines the medical and engineering fields in hopes of establishing an effective method to restore, maintain, or improve damaged tissue. In order to best replace the diseased tissue, many approaches to fabricating new tissue have focused on trying to replicate native tissue. The overall hypothesis of this dissertation is that a direct-write, BioAssembly Tool (BAT) can be utilized to fabricate viable constructs of cells and matrix that have a specified spatial organization and are truly three-dimensional (3D). The results of the studies within this dissertation demonstrate that the BAT can generate viable, spatially organized constructs comprised of cells and matrix by carefully controlling the environmental parameters of the system. A joint hypothesis associated with this dissertation is that 3D microscopy and image processing techniques can be combined to generate accurate representative stacks of images of the tissue within 3D, tissue engineered constructs. The results of the studies examining this hypothesis demonstrate that by taking into account the attenuation with depth in the imaged construct as well as by looking at the intensity and gradient of each voxel, accurate and reproducible thresholding can be achieved. Furthermore, this tool can be utilized to aid in the characterization of 3D tissue engineered constructs. Based on these studies, 3D microscopy and image processing shows promise in accurately representing the cellular volume within a tissue. More importantly, 3D, direct-write technology, specifically the BioAssembly Tool, could be used in the fabrication of viable, spatially organized constructs that can then be implanted into a patient to provide healthy tissue in the place of diseased or damaged tissue.

Polymer scaffold use has become commonplace in tissue engineering strategies. Scaffolds provide sturdy interfaces that securely anchor tissue engineered constructs to their designated locations. Researchers have used scaffolds to provide support to developing tissues as well as a growth template to aid the development of the desired phenotypic structure. In addition to using scaffolds for their mechanical support, scaffolds can be used as a diagnostic tool by attaching sensors. Strain gauge sensors have been attached to scaffolds to monitor compression and elongation. These polybutylterphalate (PBT) scaffolds were used in a cartilage tissue-engineering project for femoral cartilage repair. The aim of this project was to measure native cartilage pressure in normal canine stifle joints using strain gauge scaffolds. By using pressure sensitive films to confirm joint surface pressures determined with strain gauge measurements, "sensate" scaffolds were created to be able to provide in vivo joint loading measurements. An understanding of the in vivo pressures in the menisco-femoral joint space will facilitate the development of tissue engineered cartilage by determining chondrocyte mechanical triggers as well as helping define reasonable expectations for engineered articular cartilage tissue that is required for successful cartilage repair.

Osteoarthritis is the end result of damage to articular cartilage, which lacks the ability to self-repair. Tissue engineering of cartilage is a promising field of study that aims to promote healing of cartilage in vivo by manipulation of the chondrocytes that maintain the tissue, or through in vitro production of new cartilage for implantation into cartilage defects. Tissue-engineered cartilage constructs require mechanical stimulation to produce matrix components in quantities and proportions similar to native cartilage tissue, and adenosine triphosphate (ATP) is thought to be an autocrine/paracrine biochemical mediator of these mechanical forces on the cell, after its release from chondrocytes under mechanical stress. This study determined culture conditions for chondrocytes in 3D agarose scaffolds from mature donors undergoing total joint arthroplasty for the treatment of osteoarthritis, then supplemented these cells in vitro with exogenous ATP in concentrations varying from 50 nM to 1 mM in the presence of the radioisotopes [35S] and [3H]-proline, with radioisotope incorporation acting as markers of proteoglycan and collagen synthesis respectively. The basal concentrations of ATP in the chondrocyte cultures as well as the ATP half-life in the cultures were determined by lucifer/luciferase assay and luminometry. The P2Y receptor expression on the populations of chondrocytes from 8 donors was determined by flow cytometry, with largely varied individual expression and heterogeneity of P2Y1 and P2Y2 receptors. Exogenous ATP was found to increase synthesis of matrix components by 200% of the control cultures at doses of 100 nM to 1 µM. Patients with worse arthritis patterns, who were on chronic narcotic medications and who smoked were more likely to have a negative response to the exogenous ATP supplementation. The basal concentration of ATP in the cultures was less than 1 nM, and the ATP half-life varied from 1-2 hours, depending on the expression of P2Y1 receptors expressed by the donor’s chondrocyte population (R2 = 0.99). Supplementation of exogenous ATP to tissue-engineered cartilage in vitro appears to be a promising technique for improving the matrix synthesis of these constructs. / Thesis (Master, Mechanical and Materials Engineering) -- Queen's University, 2011-01-28 10:49:47.118

Long-term applicability of current surgical interventions for the repair of articular cartilage is jeopardized by the formation of mechanically inferior repair tissue. Cartilage tissue engineering offers the possibility of developing functional repair tissue, similar to that of native cartilage, enabling long-lasting repair of cartilage defects. Current techniques, however, rely on the need for a large number of cells, requiring substantial harvesting of donor tissue or a separate cell expansion phase. As routine cell expansion methods tend to elicit negative effects on cell function, the following study describes an approach to generate large-sized engineered cartilage constructs (≥ 3 cm2) directly from a small number of immature rabbit chondrocytes (approximately 20,000), without the use of a scaffold. After characterizing the hyaline-like engineered constructs, the in vivo repair capacity was assessed in a chondral defect model in the patellar groove of rabbits. In vitro remodeling of the constructs developed in the bioreactor occurred as early as 3 weeks, with the histological staining exhibiting zonal differences throughout the depth of the tissue. With culturing parameters optimized (3 weeks growth under 15 mM NaHCO3), constructs were grown and implanted into critical-sized 4 mm chondral defects. Assessed after 1, 3 and 6 months (n=6), implants were scored macroscopically to evaluate integration and survival of the implants. Out of 18 rabbits, 16 received normal or nearly normal over-all repair assessment. Histological and immunohistochemical evaluation showed good integration with surrounding cartilage and underlying subchondral bone. Architectural remodeling of the constructs was present at each time point, with the presence of flattened chondrocytes at the implant surface and columnar arrangement of chondrocytes in deeper zones. The observation of in vivo remodeling was also supported by the changes in biochemical composition of the constructs. At each time point, constructs had a collagen to proteoglycan ratio similar to that of native cartilage (3:1 collagen to proteoglycan). In contrast, the repair tissue for each control group was inferior to that produced with treated defects. These initial results hold promise for the generation of engineered articular cartilage for the clinical repair of cartilage defects without the limitations of current surgical repair strategies. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-01-31 01:03:15.276

Tissue engineering is a promising approach for repairing focal defects in articular cartilage. However, using current technologies tissue engineered cartilage displays insufficient biochemical, mechanical and structural properties which compromise the efficacy of implantable material. Researchers have utilized mechanical stimulation as a means to enhance these shortcomings, but few studies have applied mechanical stimulation in a complex manner similar to forces experienced by the tissue in vitro. It is hypothesized that application of simulated joint loading (SJL), a small moving contact area over the surface of tissue engineered constructs, will affect the expression and accumulation of superficial zone specific constituents, leading to improvements in construct functionality. Optimal factors of SJL (i.e. compressive load, frequency and duration) were determined via reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). The optimal combination of factors, chosen via peak levels of superficial zone genes, was discovered to be 9.81 mN, 1 Hz and 15 minutes. A study to determine spatial expression of select superficial zone genes stimulated at optimal factors was investigated via ISH, and contrasted with a finite element model (FEM) of constructs stimulated at optimal parameters displayed a correlation between gene expression and surface and sub-surface stress and strain. SJL at optimal factors (9.81 mN, 1 Hz, 15 minutes) was applied to chondrocyte-agarose hydrogel cultures in long-term studies over a period of four weeks and protein accumulation, expression and mechanical properties was investigated. Intermittent long-term application of SJL enhanced the expression and accumulation of structural proteins and enhanced the compressive and shear properties of tissue engineered constructs over a period of three weeks. Combined, the results illustrate the effectiveness of SJL as a method to affect the short-term expression, long-term accumulation and mechanical properties of superficial zone constituents. Both short and long term experiments illustrated a strain dependent behavior. The mechanism behind these results is unclear, but transduction of strain events by mechanoreceptive elements of integrins, ion channels and the pericellular matrix are potential mediators. This study demonstrated the effectiveness of SJL as a stimulation method, and demonstrated the potential to affect regional expression through alterations of SJL contact mechanics. / Thesis (Ph.D, Mechanical and Materials Engineering) -- Queen's University, 2012-02-23 18:45:12.42

Tissue engineering is a promising approach for repairing focal defects in articular cartilage. However, using current technologies tissue engineered cartilage displays insufficient biochemical, mechanical and structural properties which compromise the efficacy of implantable material. Researchers have utilized mechanical stimulation as a means to enhance these shortcomings, but few studies have applied mechanical stimulation in a complex manner similar to forces experienced by the tissue in vitro. It is hypothesized that application of simulated joint loading (SJL), a small moving contact area over the surface of tissue engineered constructs, will affect the expression and accumulation of superficial zone specific constituents, leading to improvements in construct functionality. Optimal factors of SJL (i.e. compressive load, frequency and duration) were determined via reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). The optimal combination of factors, chosen via peak levels of superficial zone genes, was discovered to be 9.81 mN, 1 Hz and 15 minutes. A study to determine spatial expression of select superficial zone genes stimulated at optimal factors was investigated via ISH, and contrasted with a finite element model (FEM) of constructs stimulated at optimal parameters displayed a correlation between gene expression and surface and sub-surface stress and strain. SJL at optimal factors (9.81 mN, 1 Hz, 15 minutes) was applied to chondrocyte-agarose hydrogel cultures in long-term studies over a period of four weeks and protein accumulation, expression and mechanical properties was investigated. Intermittent long-term application of SJL enhanced the expression and accumulation of structural proteins and enhanced the compressive and shear properties of tissue engineered constructs over a period of three weeks. Combined, the results illustrate the effectiveness of SJL as a method to affect the short-term expression, long-term accumulation and mechanical properties of superficial zone constituents. Both short and long term experiments illustrated a strain dependent behavior. The mechanism behind these results is unclear, but transduction of strain events by mechanoreceptive elements of integrins, ion channels and the pericellular matrix are potential mediators. This study demonstrated the effectiveness of SJL as a stimulation method, and demonstrated the potential to affect regional expression through alterations of SJL contact mechanics. / Thesis (Ph.D, Mechanical and Materials Engineering) -- Queen's University, 2012-02-23 18:45:12.42

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.