The application of prolonged mechanical loading to cartilage alters the osmolality of the extracellular environment, with osmotic challenge known to alter the gene expression and the metabolic activity of chondrocytes. However, the mechanisms by which osmolality controls chondrocyte activity remain unclear. Previous study on various cell types, including chondrocytes, showed that hyper-osmotic challenge induces the condensation of chromatin, with highly condensed chromatin often associated with gene poor regions of DNA and gene silencing. The present study investigated the effect of osmotic challenge on chromatin organisation, genome wide gene-expression and the cellular and nuclear deformability of chondrocytes. In order to observe a broad effect of osmotic challenge on the nuclei, the chondrocytes were subjected to a range of hypo- and hyper-osmotic challenge and imaged by confocal microscopy. Chromatin condensation was quantified by the Sobel edge algorithm in MATLAB. Hyper-osmotic challenge on chondrocytes induced an increase in chromatin condensation. Interestingly, the most marked condensation occurred within the osmolality range of articular cartilage in vivo. The effect of osmotic challenge varied between the monolayer cultured and agarose seeded chondrocytes, which may be due to the differences in cytoskeleton organisation between the two culture conditions. Additionally, chromatin condensation induced by hyper-osmotic challenge was shown to be reversible. Marked differences were observed in the deformability of the cell and nucleus in chondrocytes post osmotic challenge, compared to the 300 mOsm/kg conditions typically used for in vitro isolated chondrocyte studies. From the microarray study, the application of 500 mOsm/kg for both 1 and 5 hours altered the gene expression, including the expression of histone related genes, with a higher number of genes affected by the 5 hours hyper-osmotic challenge. The findings of this study suggest that osmotically-induced alterations in nuclei morphology and chromatin structure may provide a direct biophysical mechanism that controls chondrocytes activity.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2007. / Radhakrishna, Harish, Committee Member ; LaPlaca, Michelle, Committee Member ; Nerem, Robert, Committee Member ; Garcia, Andres, Committee Member ; Levenston, Marc, Committee Chair.

Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2008. / Guldberg, Robert, Committee Chair ; Boyan, Barbara, Committee Member ; O'Keefe, Regis, Committee Member ; Vito, Ray, Committee Member ; Bellankonda, Ravi, Committee Member.

Damaged cartilage can cause severe pain and restricted mobility, with few long term treatments available. The developing field of tissue engineering offers an alternative to the currently used full joint replacement. Restoring damaged cartilage through tissue engineering would enable an active lifestyle to be recovered and retained, without restrictions to joint mobility. This is increasingly important as the prevalence of osteoarthritis rises. Tissue engineering requires biomaterial scaffolds that mimic the function of the tissue while cells develop, and so the scaffold must provide the appropriate biological, chemical and mechanical stimuli. In this work, methods were developed to enable the design of scaffolds that mimic the microstructure and mechanical properties of articular cartilage. Electrospinning was investigated as a method to mimic the nanoscale collagen fibres within cartilage extracellular matrix. A parametric study was conducted to determine how changes to a gelatin solution affect the mechanical properties of the non-woven fibrous mesh. The solution properties had a clear impact on the morphology of the fibres, but the effect on the mesh mechanical properties was convoluted. The results demonstrated the need for greater understanding of the 3D morphology of electrospun meshes, to establish how these may be altered in order to design scaffolds with desirable mechanical properties. The fracture mechanics of soft materials are complex, and are generally overlooked when designing tissue engineering scaffolds. The complexities have led to a lack of standardised testing, making comparisons between studies impractical. In this work, fracture testing methods were compared, using a viscoelastic polymer to mimic some of the complexities of soft tissue mechanics. Mode III trouser tear tests and mode I pure shear tests were found to provide reliable measurements. Due to the ease of testing small samples, trouser tear testing was concluded to be the most advantageous for determining the fracture resistance of soft tissue engineering scaffolds. Finally, electrospun meshes were combined with hydrogels to create biomimetic scaffolds, which were characterised using tensile and trouser tear fracture tests. Fibre-reinforcement was shown to enhance the mechanical properties of a weak hydrogel, but diminished those of a strong, tough polyacrylamide (PAAm)-alginate hydrogel. The PAAm-alginate hydrogel exhibited mechanical properties close to those of natural articular cartilage, but without the microstructure that would enhance its suitability for use as a cartilage tissue engineering scaffold. An alternative method for reinforcing PAAm-alginate was proposed, which shows promise for producing a biocompatible scaffold that mimics both the mechanics and the microstructure of articular cartilage. Ultimately, this thesis aimed to improve the design of biomimetic scaffolds for cartilage tissue engineering, and advance mechanical characterisation techniques within this field.

Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.

Thesis (Ph.D)--Chemical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Boyan, Barbara; Committee Chair: Wick, Timothy; Committee Member: Brockbank, Kelvin; Committee Member: Nenes, Athanasios; Committee Member: Sambanis, Athanassios. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2006. / Harish Radhakrishna, Committee Member ; Christopher Jacobs, Committee Member ; Andres Garcia, Committee Member ; Marc E. Levenston, Committee Chair ; Barbara Boyan, Committee Member.

Articular cartilage has limited intrinsic healing capacity due to its dense and avascular structure. Clinical approaches have been developed to address the limitations associated with the poor ability of articular cartilage to regenerate. Current clinically approved techniques, however, can result in repair tissue that lacks appropriate hyaline cartilage biochemical and biomechanical properties, which lead to uncertain long-term clinical outcomes. Using tissue engineering strategies and a range of scaffolding materials, cell types, growth factors, culture conditions, and culture times, engineered tissues have been produced with compositional and biomechanical properties that approximate that of native tissue. In these studies, a considerable number of samples are typically sacrificed to evaluate compositional and mechanical properties, such as the amount of deposited collagen and sulfated glycosaminoglycan (sGAG) in the constructs. The number of sacrificed samples, as well as the amount of time and resources spent to evaluate the sacrificed samples using current gold standards, motivates an alternative method for evaluation of compositional properties. Vibrational spectroscopy, including infrared, has been considered as an alternative technique for assessment of tissues over the last 15-20 years. Infrared spectroscopy is based on absorbance of infrared light by tissue functional groups at specific vibrational frequencies, and thus, no external contrast is required. Vibrational spectroscopy is typically performed in two frequency regions, the mid infrared region (750-4000 cm-1), where penetration depth is limited to approximately 10 microns, and the near infrared (NIR) region (4000-12000 cm-1). In the NIR region, penetration of light is on the order of millimeters or centimeters, which makes it ideal for obtaining data through the full depth of engineered constructs. Here we employ NIR spectroscopy to nondestructively monitor the development of tissue-engineered constructs over culture period. / Bioengineering

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