Reporter Chondrocyte Optimization of Bioinks for Zonal 3D Bioprinting of Cartilage

Osteoarthritis (OA) is the most common form of arthritis, often thought of as a disease of the elderly, but post traumatic OA predominantly impacts younger individuals. Articular cartilage is the tissue that coats the end of your bones in synovial joints. Since cartilage has limited healing capacity, defects, or injuries to it progressively erodes down to the subchondral bone. Unfortunately, current treatment options all have limitations, particularly for younger patients. Cartilage has a specific zonal architecture that is distinguished by the different cell morphologies and arrangements, biochemical composition, and mechanical properties. 3D bioprinting is a tissue engineering technique that involves the simultaneous extrusion of biomaterials and cells to fabricate constructs. The layer-by-layer nature of 3D bioprinting and the frequent use of hydrogels as biomaterials make it a promising technique to engineer zonal articular cartilage. The goal of this dissertation was to develop and use novel human reporter chondrocytes to determine optimal combinations of biomaterials to 3D bioprint both the middle-deep and surface zones of articular cartilage. Human articular chondrocytes were transduced with either a type II collagen promoter- or lubricin promoter-driven Gaussia luciferase. Upon promoter stimulation, luciferase is secreted by the cells enabling a high-throughput, temporal, assessment of either type II collagen or lubricin. The human chondrocyte reporter system was combined with a Design of Experiment approach which streamlined the process of biomaterial optimization. To 3D bioprint the deep zone, an optimal combination of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) was determined based on type II collagen promoter-driven luminescence, chondrocyte mobility and biomaterial stiffness. While an optimal combination of GelMA and oxidized methacrylated alginate (OMA) was determined for the surface zone based on lubricin promoter-driven luminescence, lubricin secretion, and construct shape fidelity. Together these results highlight the effectiveness of human reporter chondrocyte optimization for 3D bioprinting zonal cartilage

Changes In Adipose Tissue Cellular Composition During Obesity And Aging As A Cause Of Metabolic Dysregulation

Adipose tissue represents complex endocrine organ containing several different cellular populations including adipocytes, pre-adipocytes, mesenchymal stem cells, macrophages and lymphocytes. It is well establishing that these populations are not static but alter during obesity and aging. Changes in cellular populations alter inflammatory status and other common metabolic complications arise, therefore adipose tissue cellular composition helps dictate its endocrine and regulatory function. During excessive weight gain in obese individuals and as we age there is shift towards increase populations of inflammatory macrophages with a decrease of regulatory T cell. This altered cellular composition promote chronic low grade inflammation negatively affecting mesenchymal stem cell progenitor self-renewal, which result in deterioration of adipogenesis and increased cellular stress in adipocytes. All these changes promote metabolic disorders including age- or obese-related insulin resistance leading to type 2 diabetes

Suitability of Gelatin Methacrylate and Hydroxyapatite Hydrogels for 3D-Bioprinted Bone Tissue

Background: Complex bone defects are challenging to treat. Autografting is the gold standard for regenerating bone defects; however, its limitations include donor-site morbidity and increased surgical complexity. Advancements in 3D bioprinting (3DBP) offer a promising alternative for viable bone grafts. In this experiment, gels composed of varying levels of gelatin methacrylate (GelMA) and hydroxyapatite (HA) and gelatin concentrations are explored. The objective was to increase the hydroxyapatite content and find the upper limit before the printability was compromised and determine its effect on the mechanical properties and cell viability. Methods: Design of Experiments (DoE) was used to design 13 hydrogel bioinks of various GelMA/HA concentrations. These bioinks were assessed in terms of their pipettability and equilibrium modulus. An optimal bioink was designed using the DoE data to produce the greatest stiffness while still being pipettable. Three bioinks, one with the DoE-designed maximal stiffness, one with the experimentally defined maximal stiffness, and a literature-based control, were then printed using a 3D bioprinter and assessed for print fidelity. The resulting hydrogels were combined with human bone-marrow-derived mesenchymal stromal cells (hMSCs) and evaluated for cell viability. Results: The DoE ANOVA analysis indicated that the augmented three-level factorial design model used was a good fit (p p > 0.05). There was, however, a significantly lower cell viability in the gel composed of 12.3% GelMA, 15.7% HA, and 2% gelatin compared to the other gels with a lower HA concentration (p < 0.05), showing that a higher HA content or print pressure may be cytotoxic within hydrogels. Conclusions: Extrusion-based 3DBP offers significant advantages for bone–tissue implants due to its high customizability. This study demonstrates that it is possible to create printable bone-like grafts from GelMA and HA with an increased HA content, favorable mechanical properties (145 kPa), and a greater than 80% cell viability

MicroRNA regulation in colorectal cancer tissue and serum.

Colorectal cancer is recognized as the fourth leading cause of cancer-related deaths worldwide. Thus, there is ongoing search for potential new biomarkers allowing quicker and less invasive detection of the disease and prediction of the treatment outcome. Therefore, the aim of our study was to identify colorectal cancer specific miRNAs expressed in cancerous and healthy tissue from the same patient and to further correlate the presence of the same miRNAs in the circulation as potential biomarkers for diagnosis. In the current study we detected a set of 40 miRNAs differentially regulated in tumor tissue when comparing with healthy tissue. Additionally, we found 8 miRNAs differentially regulated in serum of colorectal cancer patients. Interestingly, there was no overlap in miRNAs regulated in tissue and serum, suggesting that serum regulated miRNAs may be not actively secreted from colorectal tumor cells. However, four of differentially expressed miRNAs, including miR-21, miR-17, miR-20a and miR-32 represent the miRNAs characteristic for different tumor types, including breast, colon, lung, pancreas, prostate and stomach cancer. This finding suggests important groups of miRNAs which can be further validated as markers for diagnosis of tumor tissue and regulation of carcinogenesis