Thesis (doctoral)--Universität Leipzig, 1908. / Lebenslauf. Includes bibliographical references.

No description available.

Continuous monitoring during tissue culture is important for the success of engineered tissue development. It is also challenging due to lack of suitable established monitoring techniques. In this study, microdialysis, a sampling technique for measuring the unbound solute concentrations in the tissues and organs of the living body, was adopted to monitor functional tissue growth in a bioreactor with explanted bovine caudal intervertebral discs (IVD) as the test tissue. Apart from cell metabolic activities, cell and tissue biological functions were investigated for the development of microdialysis for monitoring purposes. Methodologies of microdialysis with large pore size membrane probes for sampling macromolecular bio-functional markers were established. The effects of pumping methods, including 'push', 'pull' or 'push-and-pull', and the effect of the resulting transmembrane pressure on the fluid balance, and the relative recovery of small molecules and of macromolecules (proteins) were experimentally studied. The validity of the internal reference in-situ calibration was examined in detail. It was concluded that a push-and-pull system was the only effective method to eliminate fluid loss or gain. The relative recovery of small solutes was hardly affected by the applied pumping methods; however the relative recovery of macromolecules was significantly influenced by them. The in situ calibration technique using Phenol Red can provide reliable results for small molecules including glucose and lactic acid. Using lOkDa and 70kDa fluorescent dextrans as the internal standard for in situ calibration of large molecules of similar size, it was found that the pull pump system did not work well but that the push-and-pull pumping method did work well. A novel bioreactor system for in vitro IVD culture with static load and microdialysis monitoring was developed. Explanted IVDs were cultured under three different loads for up to 7 days. A single microdialysis probe with 3000 kDa membrane was inserted into each of the IVDs at a defined location. The in situ calibration technique was proved valid in the experiments and membrane fouling was not significant. The tissue metabolism and extracellular matrix turnover during 7 day culture were continuously monitored to investigate the effect of different loads. Microdialysis proved to be a feasible and efficient method for multi-parameter monitoring of tissue culture. Substantial effort was directed towards the identification of functional macromolecular markers in conjunction with microdialysis sampling. Amongst several proteins sampled, chitinase-3-like protein 1 (CHI3L1), a major soluble protein secreted by cultured IVD cells in alginate beads and by cultured IVD explants was identified following its successful isolation. Then it was established as a suitable functional marker. The effect of physico-chemical and mechanical stimuli (e.g. osmolarity, pH, oxygen tension and mechanical load) on secretion of CHI3L1 by cultured IVD cells and chondrocytes in alginate beads and by cultured IVD explant were investigated. CHI3L1 release was sensitive to physico-chemical stimulation. The production of CHI3L1 was directly correlated with the cell metabolism and this could be readily monitored with microdialysis.

No description available.

Osteoarthritis is a debilitating and irreversible disease afflicting the synovial joints. It is characterized by pain and hindered mobility. Given that osteoarthritis has no cure, current treatments focus on pain management. Ultimately, however, a patient's pain and immobility necessitates joint replacement surgery. An attractive alternative to this treatment paradigm, tissue engineering is a promising strategy for resurfacing the osteoarthritis-afflicted cartilage surface with a biochemically and biomechanically similar tissue to the healthy native cartilage tissue. The most successful cartilage tissue engineered systems to date can repeatably grow constructs ~4 mm in diameter with native proteoglycan and compressive mechanical properties. Unfortunately, as symptomatic cartilage typically presents only once lesions span large regions of the joint (~25 mm in diameter), these small construct are of limited use in clinical practice. Numerous attempts to simply grow a construct large enough to span the size of an osteoarthritic lesion have shown that the growth of large engineered tissues develop heterogeneous properties, emphasizing the need for culture protocols to enhance tissue homogeneity and robustness. In particular, as nutrient limitations drive heterogeneous growth in engineered cartilage, developing strategies to improve nutrition throughout the construct are critical for clinical translation of the technology. To this end, our lab has successfully supplemented nutrient channels within large engineered cartilage constructs to improve the functional properties of developing tissue. However, it is unknown what the optimal nutrient channel spacing is for growing large cartilage constructs of anatomical scale. Additionally, the fundamental factors and mechanisms which drive tissue heterogeneity have not been implicated, making the results of channel-spacing optimizations difficult to translate across different systems. Computational models of growth, faithful to the physics and biology of engineered tissue growth, may serve as an insightful and efficient tool for optimally designing culture protocols and construct geometries to ensure homogeneous matrix deposition. Such computational tools, however, are not presently available, owing to the unresolved mechanical and biological growth phenomena within developing engineered cartilage. This dissertation seeks to develop and implement computational models for predicting the biochemical and biomechanical growth of engineered tissues and apply these models to optimizing tissue culture strategies. These models are developed in two stages: 1) based on our recent characterization of the nutrient demands of engineered cartilage, models are developed to simulate the spatial biochemical deposition of matrix within tissue constructs and, subsequently, 2) based on models of biochemical matrix deposition we develop models for simulating the mechanical growth of tissue constructs. To accomplish these tasks, we first develop models simulating glucose availability within large tissue constructs using system-specific modeling based on our recent characterization of the glucose demands of engineered cartilage. These models led to early insight that we had to enhance the supply of glucose within large tissue constructs to ensure maximal matrix synthesis throughout culture. Experimental validations confirmed that increasing glucose supply enhanced matrix deposition and growth in large tissue constructs. However, even despite the increased glucose supply, increasing the size of constructs demonstrated that severe matrix heterogeneities were still present. Subsequent nutrient characterization led to the finding that TGF-ß transport was significantly hindered within large tissue constructs. Incorporating the influence of glucose and TGF-ß into the computational model growth kinetics. Using both nutrients, models recreated the heterogeneous matrix deposition evident in our earlier work and could account for the role of cell seeding density and construct geometry on tissue growth. The insights gathered from this modeling analysis led to important changes in our culture protocols: we could reduce the dose of TGF-ß from 10 ng/mL to 1 ng/mL for constructs cultured with channels, saving considerable expense while still maintaining a high level of matrix synthesis throughout the construct. In the presence of sufficient nutrition, we witnessed an unprecedented level of matrix deposition and physical growth of the constructs. In fact, by using developmentally physiologic cell seeding densities (120 million cells/mL) and providing adequate nutrition, constructs physically grew to 9-times their originally cast size. Despite such encouraging growth, tissue function properties plateaued at sub-physiologic levels. For insight into the connection between matrix deposition and tissue mechanics, we extended the computational growth models to consider the mechanisms underlying physical growth. Interestingly, we found that a large matrix synthesis mismatch between proteoglycans and collagen gave rise to the excessive tissue swelling. Computational models of this matrix synthesis mismatch predicted the high tissue swelling displayed experimentally only when a damage-able collagen fiber material was implemented. Together, the experimental and modeling evidence suggested a new mechanism of cartilage growth: the high proteoglycan deposition creates a swelling pressure within the nascent tissue which outcompetes the restraining force of newly deposited collagens; this rapid tissue swelling disrupts a functional collagen network from forming. Subsequent analysis suggested that the disruption of the collagen network prevented the formation of collagen crosslinks, stymieing the development of native functional properties. Based on this insight into the mechanisms of cartilage growth, we developed a culture systems to improve tissue functional properties. Modeling analysis indicated two novel routes for improving tissue mechanics: either through 1) reducing the swelling response (synthesis and deposition) of proteoglycans or 2) enhancing and reinforcing the newly synthesized collagen to prevent disruptions brought on by tissue swelling. We developed a cage culture system for resisting the swelling pressure of deposited proteoglycans and reenforcing the deposition of new collagens. Using this cage system, we grew tissue constructs with enhanced functional properties using two separate scaffold systems – agarose and a cartilage-derived matrix hydrogel – suggesting this mechanism of growth is fundamental to engineered cartilage development. This work has generated a novel paradigm towards engineering cartilage constructs using biomimetic strategies. Performing simulations with the validated, computational growth models allowed anatomically-sized cartilage constructs to grow into the largest, homogeneous cartilage constructs grown to date. Models presented a new level of insight into the nutrient demands of developing tissues, allowing for the first time the successful development of large tissue constructs grown with developmentally physiologic cell seeding densities. In this way, tissue constructs growth followed a biomimetic approach, based on the high cell densities and cartilage canals and vasculature present during fetal cartilage development. Adequate nutrition led to high levels of tissue growth not previously experienced in vitro, a result of adequately nourishing primary chondrocytes, a cell type which preferentially deposits proteoglycans over collagen. We therefore developed a cage-based growth system to resist the proteoglycan-induced tissue swelling in a manner similar to the fetal development of cartilage where the resident cells synthesize more collagens than proteoglycans. Together, the use of nutrient growth models, high cell seeding densities, and culture systems to strengthen the collagen-framework of de novo cartilage proved beneficial for engineering anatomically-sized cartilage constructs. The fundamental mechanisms identified here are likely to be universal across a number of engineered cartilage systems and will be adapted to more clinically-relevant cell sources in future our work.

Current cartilage repair methods are ineffective in restoring the mechanical and biological functions of native hyaline cartilage. Therefore, using the paracrine actions of stem cell therapies to stimulate endogenous cartilage regeneration has gained momentum. Adipose stem cells (ASCs) are an attractive option for this endeavor because of their accessibility, chondrogenic potential, and secretion of factors that promote connective tissue repair. In order to use the factors secreted by ASCs to stimulate cartilage regeneration, the signaling pathways that affect postnatal cartilage development and morphology need to be understood. Next, approaches need to be developed to tailor the secretory profile of ASCs to promote cartilage regeneration. Finally, delivery methods that localize ASCs within a defect site while facilitating paracrine factor secretion need to be optimized. The overall objective of this thesis was to develop an ASC therapy that could be effectively delivered in cartilage defects and stimulate regeneration via its paracrine actions. The general hypothesis was that the secretory profile of ASCs can be tailored to enhance cartilage regeneration and be effectively delivered to regenerate cartilage in vivo. The overall approach used the growth plate as an initial model to study changes in postnatal cartilage morphology and the molecular mechanisms that regulate it, different media treatments and microencapsulation to tailor growth factor production, and alginate microbeads to deliver ASCs in vivo to repair cartilage focal defects.