Biology subjects

Kean, T. J.

Publications and source records attributed to Kean, T. J..

5 recordsLinked to original sources

bioRxiv2023

Disease Modifying Osteoarthritis Drug Discovery Using A Temporal Phenotypic Reporter In Primary Human Chondrocytes

Osteoarthritis is a significant and growing problem with no disease modifying drugs in the clinic. Current screening platforms typically use 2D culture, immortalized or non-human cells in a hyperoxic environment. To challenge this paradigm and identify new drugs, we engineered primary human chondrocytes with a secreted luciferase reporter under the control of the articular cartilage marker, type II collagen. We then successfully screened a natural product library using a high throughput model with COL2A1-Gaussia luciferase primary human chondrocyte reporter cells in 3D aggregates under physioxia. We identified several candidate compounds that increased type II collagen over controls, with aromoline being the best candidate. Aromoline is a bisbenzylisoquinoline alkaloid that has been studied for its anti-proliferative, anti-inflammatory, and anti-microbial properties, and we are the first to explore its effects on chondrocytes and chondrogenesis. In silico analysis of predicted targets narrowed by RNA-Seq data on expression provided an unexpected initial candidate target protein: the dopamine receptor D4 (DRD4). The researchers confirmed upregulation in the expression of DRD4 and type II collagen after treatment with aromoline. This novel approach combining in silico and in vitro methods provides a platform for drug discovery in a challenging and under-researched area. In conclusion, a novel drug (aromoline) and target receptor (dopamine receptor D4) were identified as stimulating type II collagen, with the goal to treat or prevent osteoarthritis.

pharmacology and toxicology↗

bioRxiv2022

Micronutrient Optimization Using Design of Experiments Approach in Tissue Engineered Articular Cartilage for Production of Type II Collagen

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen expression. Five conditions predicted to produce the greatest Luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen expression. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Youngs tensile modulus compared to engineered cartilage in basal media alone. Collagen cross-linking analysis confirmed formation of type II-type : II collagen and type II-type : IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

cell biology↗

bioRxiv2022

Optimizing bioink composition for human chondrocyte expression of lubricin

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, 14% GelMA/2% OMA, 14% GelMA and 16% GelMA were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion.

bioengineering↗

bioRxiv2020

Physioxia stimulates extra-cellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage

Cartilage tissue has been recalcitrant to tissue engineering approaches. In this study, human chondrocytes were formed into self-assembled cartilage sheets, cultured in physiologic (5%) and atmospheric (20%) oxygen conditions and underwent biochemical, histological and biomechanical analysis at one- and two-months. The results indicated that sheets formed at physiological oxygen tension were thicker, contained greater amounts of glycosaminoglycans (GAGs) and type II collagen, and had greater compressive and tensile properties than those cultured in atmospheric oxygen. In all cases, cartilage sheets stained throughout for extracellular matrix components. Type II-IX-XI collagen heteropolymer formed in the neo-cartilage and fibrils were stabilized by trivalent pyridinoline cross-links. Collagen cross-links were not significantly affected by oxygen tension but increased with time in culture. Physiological oxygen tension and longer culture periods both served to increase extracellular matrix components. The foremost correlation was found between compressive stiffness and the GAG to collagen ratio. SummaryTissue-engineered cartilage formed from human articular chondrocytes produces thicker, stiffer, more extracellular-matrix rich cartilage tissue when grown under physiological (5%) vs. atmospheric oxygen (20%) tension.

cell biology↗

bioRxiv2019

High-throughput, temporal and dose dependent, effect of vitamins and minerals on chondrogenesis

Tissue engineered hyaline cartilage is plagued by poor mechanical properties largely due to inadequate type II collagen expression. Of note, commonly used defined chondrogenic media lack 14 vitamins and minerals, some of which are implicated in chondrogenesis. Type II collagen promoter-driven Gaussia luciferase was transfected into ATDC5 cells to create a chondrogenic cell with a secreted-reporter. The reporter cells were used in an aggregate-based chondrogenic culture model to develop a high-throughput analytic platform. This high-throughput platform was used to assess the effect of vitamins and minerals, alone and in combination with TGF{beta}1, on type II collagen expression. Significant combinatorial effects between vitamins, minerals and TGF{beta}1 in terms of type II collagen expression and metabolism were discovered. An optimal continual supplement of copper and vitamin K in the presence of TGF{beta}1 gave a 2.5-fold increase in collagen expression over TGF{beta}1 supplemented media alone.\n\nSummaryCurrent defined chondrogenic culture media lack several vitamins and minerals. Type II collagen is the quintessential marker of articular hyaline cartilage, and is commonly deficient in engineered tissue. A type II collagen promoter driven secreted luciferase construct has been transduced into ATDC5 cells and used to assess vitamin and mineral effects on chondrogenesis in a high-throughput format.

cell biology↗