Biology subjects

June, R. K.

Publications and source records attributed to June, R. K..

25 records · Page 2Linked to original sources

bioRxiv2021

A comparison of shear- and compression-induced mechanotransduction in SW1353 chondrocytes

Mechanotransduction is a biological phenomenon where mechanical stimuli are converted to biochemical responses. A model system for studying mechanotransduction are the chondrocytes of articular cartilage. Breakdown of this tissue results in decreased mobility, increased pain, and reduced quality of life. Either disuse or overloading can disrupt cartilage homeostasis, but physiological cyclical loading promotes cartilage homeostasis. To model this, we exposed SW1353 cells to cyclical mechanical stimuli, shear and compression, for different durations of time (15 and 30 min). By utilizing liquid chromatography-mass spectroscopy (LC-MS), metabolomic profiles were generated detailing metabolite features and biological pathways that are altered in response to mechanical stimulation. In total, 1,457 metabolite features were detected. Statistical analyses identified several pathways of interest. Taken together, differences between experimental groups were associated with inflammatory pathways, lipid metabolism, beta-oxidation, central energy metabolism, and amino acid production. These findings expand our understanding of chondrocyte mechanotransduction under varying loading conditions and time periods.

bioengineering↗

bioRxiv2020

Development and analytical validation of a finite element model of fluid transport through osteochondral tissue

Fluid transport between cartilage and bone is critical to joint health. The objective of this study was to develop and analytically validate a finite element model of osteochondral tissue capable of modeling cartilage-bone fluid transport. A biphasic viscoelastic model using an ellipsoidal fiber distribution was created with three distinct layers of cartilage (superficial zone, middle zone, and deep zone) along with a layer of subchondral bone. For stress-relaxation in unconfined compression, our results for compressive stress, radial stress, effective fluid pressure, and elastic recoil were compared with established biphasic analytical solutions. Our model also shows the development of fluid pressure gradients at the cartilage-bone interface during loading. Fluid pressure gradients developed at the cartilage-bone interface with consistently higher pressures in cartilage following initial loading to 10% strain, followed by convergence towards equal pressures in cartilage and bone during the 400s relaxation period. These results provide additional evidence that fluid is transported between cartilage and bone during loading and improves upon estimates of the magnitude of this effect through incorporating a realistic distribution and estimate of the collagen ultrastructure. Understanding fluid transport between cartilage and bone may be key to new insights about the mechanical and biological environment of both tissues in health and disease.

bioengineering↗

bioRxiv2020

Metabolomic Profiling and Mechanotransduction of Single Chondrocytes Encapsulated in Alginate Microgels

Human articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened resulting in increased chondrocyte stress. As chondrocytes are responsible for cartilage synthesis and maintenance, it is important to understand how mechanical loads affect cellular responses of chondrocytes. Many studies have examined the chondrocyte response to in vitro mechanical loading by embedding in stiff agarose. However, these experiments are mostly performed in the absence of PCM which may obscure important responses to mechanotransduction. Here, we demonstrate that drop-based microfluidics allows culture of single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes form PCM over 10 days in these single cell microenvironments. Single cell microgels and monolayer controls were encapsulated in high stiffness agarose to mimic the cartilage PCM. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.

bioengineering↗

bioRxiv2020

A 3-D Constitutive Model for Finite Element Analyses of Agarose with a Range of Gel Concentrations

Hydrogels have seen widespread application across biomedical sciences and there is considerable interest in using hydrogels, including agarose, for creating in vitro three-dimensional environments to grow cells and study mechanobiology and mechanotransduction. Recent advances in the preparation of agarose gels enable successful encapsulation of viable cells at gel concentrations as high as 5%. Agarose with a range of gel concentrations can thus serve as an experimental model mimicking changes in the 3-D microenvironment of cells during disease progression and can facilitate experiments aimed at probing the corresponding mechanobiology, e.g. the evolving mechanobiology of chondrocytes during the progression of osteoarthritis. Importantly, whether stresses (forces) or strains (displacement) drive mechanobiology and mechanotransduction is currently unknown. We can use experiments to quantify mechanical properties of hydrogels, and imaging to estimate microstructure and even strains; however, only computational models can estimate intra-gel stresses in cell-seeded agarose constructs because the required in vitro experiments are currently impossible. Finite element modeling is well-established for (computational) mechanical analyses, but accurate constitutive models for modeling the 3-D mechanical environments of cells within high-stiffness agarose are currently unavailable. In this study we aimed to establish a 3-D constitutive model of high-stiffness agarose with a range of gel concentrations. We applied a multi-step, physics-based optimization approach to separately fit subsets of model parameters and help achieve robust convergence. Our constitutive model, fitted to experimental data on progressive stress-relaxations, was able to predict reaction forces determined from independent experiments on cyclical loading. Our model has broad applications in finite element modeling aimed at interpreting mechanical experiments on agarose specimens seeded with cells, particularly in predicting distributions of intra-gel stresses. Our model and fitted parameters enable more accurate finite element simulations of high-stiffness agarose constructs, and thus better understanding of experiments aimed at mechanobiology, mechanotransduction, or other applications in tissue engineering.

bioengineering↗

bioRxiv2020

The microbiome mediates subchondral bone loss and metabolomic changes after acute joint trauma

ObjectiveTo compare the early responses to joint injury in conventional and germ-free mice. DesignPost traumatic osteoarthritis PTOA was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n=8 GF and n=10 conventional mice. To examine the effects of injury, n=5 GF and n=9 conventional control mice were used. Mice were euthanized seven days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (CT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using CT assessed early OA progression in GF and conventional mice. ResultsCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system. ConclusionsThese results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

immunology↗

bioRxiv2020

The TAT Protein Transduction Domain as an Intra-articular Drug-Delivery Technology

ObjectiveIntra-articular drug delivery holds great promise for the treatment of joint diseases such as osteoarthritis. The objective of this study was to evaluate the TAT peptide transduction domain (TAT-PTD) as a potential intra-articular drug delivery technology for synovial joints. DesignExperiments examined the ability of TAT conjugates to associate with primary chondrocytes and alter cellular function both in vitro and in vivo. Further experiments examined the ability of the TAT-PTD to bind to human osteoarthritic cartilage. ResultsThe results show that the TAT-PTD associates with chondrocytes, is capable of delivering siRNA for chondrocyte gene knockdown, and that the recombinant enzyme TAT-Cre is capable of inducing in vivo genetic recombination within the knee joint in a reporter mouse model. Lastly, binding studies show that osteoarthritic cartilage preferentially uptakes the TAT-PTD from solution. ConclusionsThe results suggest that the TAT-PTD is a promising delivery strategy for intra-articular therapeutics.

bioengineering↗

bioRxiv2018

Physiological Dynamic Compression Regulates Central Energy Metabolism in Primary Human Chondrocytes

Chondrocytes use the pathways of central metabolism to synthesize molecular building blocks and energy for cartilage homeostasis. An interesting feature of the in vivo chondrocyte environment is the cyclical loading generated in various activities (e.g. walking). However, it is unknown if central metabolism is altered by mechanical loading. We hypothesized that physiological dynamic compression alters central metabolism in chondrocytes to promote production of amino acid precursors for matrix synthesis. We measured the expression of central metabolites (e.g. glucose, its derivatives, and relevant co-factors) for primary human osteoarthritic chondrocytes in response to 0-30 minutes of compression. To analyze the data, we used principal components analysis and ANOVA simultaneous components analysis, as well as metabolic [fl]ux analysis. Compression induced metabolic responses consistent with our hypothesis. Additionally, these data show that chondrocyte samples from different patient donors exhibit different sensitivity to compression. Most important, we [fi]nd that grade IV osteoarthritic chondrocytes are capable of synthesizing non-essential amino acids and precursors in response to mechanical loading. These results suggest that further advances in metabolic engineering of chondrocyte mechanotransduction may yield novel translational strategies for cartilage repair.

systems biology↗