Due to mechanical loading, cartilage experiences distortional change, volumetric change, and fluid flow. Research has shown cells to be responsive to unconfined compression, a load that produces all three conditions. To isolate the factor(s) responsible for chondrogenesis, the first goal of this research was to design and implement a device for the application of shear deformation to cells. Secondly, using this device, Stage 23/24 chick limb bud cells were suspended in 2% alginate and subjected to 20% shear deformation at 1 Hz. for two hours daily for three days. Gene expression, DNA content, sGAG content, and cartilage nodule formation were determined after eight days in culture and compared to results obtained for non-loaded cells. Results indicated that shear deformation at the applied level did not have a significant effect on chondrogenesis in Stage 23/24 chick limb bud cells, suggesting that this cell type is not extremely sensitive to distortional change.

The global objective of this research was to identify the proteins secreted by stem cells in response to mechanical stress. Since it has been shown in previous studies that conditioned medium from compressed chick limb bud cells cultured in alginate can initiate chondrogenesis in non-compressed cells, it was hypothesized that the conditioned medium contains valuable growth/differentiation factors. Due to cartilage?s limited capacity for repair, factors that stimulate stem-cell mediated regeneration are highly sought. To discern these proteins, conditioned medium was collected from cyclically compressed stage 23/24 chick limb buds suspended in alginate. The proteins were extracted, separated by 2-D gel electrophoresis, and evaluated by mass spectroscopy. While a few regulators of chondrogenesis were observed, such as FGF receptor, actin, and IP3 receptor, many potential peptides were not found in the database. However, this study showed that ascertaining proteins produced by chondrocytes in response to mechanical stimulation should be pursued.

<p>  </p> <p>Congenital diseases of the kidney are the leading cause of chronic kidney disease in pediatric patients. Tissue engineering models used to investigate these diseases are limited by an immature phenotype. Models cultured in an extracellular matrix (ECM), a network of proteins and glycosaminoglycans surrounding cells and providing structural support that mimic the matrix found in development will be likely more mature. However, developing kidney ECM composition and structural dynamics are unknown. To address this gap, we studied ECM composition using mass spectrometry and organization by visualizing the ECM in 3D.</p> <p>In this work, we used mass spectrometry to resolve ECM basement membrane and interstitial matrix dynamics between embryonic, perinatal, and adult kidneys. Surprisingly, we observed a transient upregulation of interstitial matrix structures that corresponded to dynamic 3D structures in the cortex (vertical fibers) and at the corticomedullary junction (medullary ray sheath fibers). Notably, in a model of abnormal <em>Foxd1</em>+ stromal cells, the vertical fibers were disorganized, and medullary ray sheath fibers were no longer associated with blood vessels, suggesting the dynamic 3D structures depended on stromal cell modulation.</p> <p>One of the effects of abnormal kidney development is decreased amniotic fluid, which limits embryonic movement and subsequent limb development. In additional studies, we looked at the implications of the lost motility in the muscular dysgenesis (<em>mdg</em>) mouse on the development of the myotendinous junction (MTJ). The MTJ links contractile muscle with tendon. We found the ECM protein COL22A1 was specific to the developing MTJ as early as embryonic day (E)13.5. The development of the MTJ from a linear structure to a cap-like structure with invaginations in adolescent mice depended on muscle contraction. Furthermore, we used a model to decouple the muscle-tendon-bone complex at an ectopic lateral triceps insertion (<em>Prrx1Cretg/+; Tbx3fl/fl</em>). We observed disorganized tendon and MTJ markers at the termination of the ectopic lateral triceps muscle but negligible cartilage markers. Together, this indicated MTJ maturation depended on motility but not on the enthesis.</p> <p>The information gleaned from our studies on how stromal cells affect dynamic 3D interstitial ECM structures and composition change during kidney development can be used as a template for 3D kidney culture systems. Combined with forelimb MTJ development, our results indicate the importance of the interstitial matrix in tissue morphogenesis.</p>