Insulin, Ascorbate, and Glucose Have a Much Greater Influence Than Transferrin and Selenous Acid on the In Vitro Growth of Engineered Cartilage in Chondrogenic Media

The primary goal of this study was to characterize the response of chondrocyte-seeded agarose constructs to varying concentrations of several key nutrients in a chondrogenic medium, within the overall context of optimizing the key nutrients and the placement of nutrient channels for successful growth of cartilage tissue constructs large enough to be clinically relevant in the treatment of osteoarthritis (OA). To this end, chondrocyte-agarose constructs (phi4x2.34 mm, 30x106 cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0x to 30x), selenous acid (0x to 10x), ascorbate (0x to 30x), and glucose (0x to 3x). The quality of resulting engineered tissue constructs was evaluated by their compressive modulus (E-Y), tensile modulus (E+Y), hydraulic permeability (k), and content of sulfated glycosaminoglycans (sGAG) and collagen (COL); DNA content was also quantified. Three control groups from two separate castings of constructs (1x concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E-Y=518 plus or minus 78, 401 plus or minus 113, 236 plus or minus 67 kPa; E+Y=1420 plus or minus 430, 1140 plus or minus 490, 1240 plus or minus 280 kPa; k=2.3 plus or minus 0.8x10-3, 5.4 plus or minus 7.0x10-3, 3.3 plus or minus 1.3x10-3 mm4/N times s; sGAG=7.8 plus or minus 0.3, 6.3 plus or minus 0.4, 4.1 plus or minus 0.5%/ww; COL=1.3 plus or minus 0.2, 1.1 plus or minus 0.3, 1.4 plus or minus 0.4%/ww; and DNA=11.5 plus or minus 2.2, 12.1 plus or minus 0.6, 5.2 plus or minus 2.8 μg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3x without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30x) was detrimental to E-Y, and 30x insulin was detrimental to both E+Y and E-Y. The presence of glucose was similarly essential, and matrix elaboration was significantly dependent on its concentration (p less than 10-6), with loss of functional properties, composition, and cellularity observed at less than or equal to 0.3x; excessive glucose supplementation (up to 3x) showed no detrimental effects. In contrast, transferrin and selenous acid had no influence on matrix elaboration. These findings suggest that adequate distributions of insulin, ascorbate, and glucose, but not necessarily of transferrin and selenous acid, must be ensured within large engineered cartilage constructs to produce a viable substitute for joint tissue lost due to OA

Does Uptake of Pharmaceuticals Vary Across Earthworm Species?

This study compared the uptake and depuration of four commonly used pharmaceuticals (carbamazepine, diclofenac, fluoxetine and orlistat) in two earthworm species (Lumbricus terrestris and Eisenia fetida). L. terrestris are a larger species and often found in deep burrows whereas E. fetida prefer to reside near the soil surface. Species burrowing habits and sizes may alter uptake by earthworms. All four pharmaceuticals were taken up into both L. terrestris and E. fetida tissue after 21 days exposure to spiked soil. Bioconcentration factors (BCFs) ranged between 1.72 and 29.83 for L. terrestris and 1.14 and 63.03 for E. fetida. For carbamazepine and diclofenac, BCFs were similar whereas for fluoxetine and orlistat, BCFs in E. fetida were more than double those seen in L. terrestris. Results indicate that uptake into earthworms cannot be generalised between species and that the influence of species traits can vary depending on the nature of the study chemical

Modulating gradients in regulatory signals within mesenchymal stem cell seeded hydrogels: a novel strategy to engineer zonal articular cartilage.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Engineering organs and tissues with the spatial composition and organisation of their native equivalents remains a major challenge. One approach to engineer such spatial complexity is to recapitulate the gradients in regulatory signals that during development and maturation are believed to drive spatial changes in stem cell differentiation. Mesenchymal stem cell (MSC) differentiation is known to be influenced by both soluble factors and mechanical cues present in the local microenvironment. The objective of this study was to engineer a cartilaginous tissue with a native zonal composition by modulating both the oxygen tension and mechanical environment thorough the depth of MSC seeded hydrogels. To this end, constructs were radially confined to half their thickness and subjected to dynamic compression (DC). Confinement reduced oxygen levels in the bottom of the construct and with the application of DC, increased strains across the top of the construct. These spatial changes correlated with increased glycosaminoglycan accumulation in the bottom of constructs, increased collagen accumulation in the top of constructs, and a suppression of hypertrophy and calcification throughout the construct. Matrix accumulation increased for higher hydrogel cell seeding densities; with DC further enhancing both glycosaminoglycan accumulation and construct stiffness. The combination of spatial confinement and DC was also found to increase proteoglycan-4 (lubricin) deposition toward the top surface of these tissues. In conclusion, by modulating the environment through the depth of developing constructs, it is possible to suppress MSC endochondral progression and to engineer tissues with zonal gradients mimicking certain aspects of articular cartilage.Funding was provided by Science Foundation Ireland (President of Ireland Young Researcher Award: 08/Y15/B1336) and the European Research Council (StemRepair – Project number 258463)

Cytokine preconditioning of engineered cartilage provides protection against interleukin-1 insult

Research reported in this publication was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R01AR60361, R01AR061988, P41EB002520). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ART was supported by a National Science Foundation Graduate Fellowship

Accumulation of Exogenous Activated TGF-β in the Superficial Zone of Articular Cartilage

It was recently demonstrated that mechanical shearing of synovial fluid (SF), induced during joint motion, rapidly activates latent transforming growth factor β (TGF-β). This discovery raised the possibility of a physiological process consisting of latent TGF-β supply to SF, activation via shearing, and transport of TGF-β into the cartilage matrix. Therefore, the two primary objectives of this investigation were to characterize the secretion rate of latent TGF-β into SF, and the transport of active TGF-β across the articular surface and into the cartilage layer. Experiments on tissue explants demonstrate that high levels of latent TGF-β1 are secreted from both the synovium and all three articular cartilage zones (superficial, middle, and deep), suggesting that these tissues are capable of continuously replenishing latent TGF-β to SF. Furthermore, upon exposure of cartilage to active TGF-β1, the peptide accumulates in the superficial zone (SZ) due to the presence of an overwhelming concentration of nonspecific TGF-β binding sites in the extracellular matrix. Although this response leads to high levels of active TGF-β in the SZ, the active peptide is unable to penetrate deeper into the middle and deep zones of cartilage. These results provide strong evidence for a sequential physiologic mechanism through which SZ chondrocytes gain access to active TGF-β: the synovium and articular cartilage secrete latent TGF-β into the SF and, upon activation, TGF-β transports back into the cartilage layer, binding exclusively to the SZ

Accumulation of Exogenous Activated TGF-β in the Superficial Zone of Articular Cartilage

It was recently demonstrated that mechanical shearing of synovial fluid (SF), induced during joint motion, rapidly activates latent transforming growth factor β (TGF-β). This discovery raised the possibility of a physiological process consisting of latent TGF-β supply to SF, activation via shearing, and transport of TGF-β into the cartilage matrix. Therefore, the two primary objectives of this investigation were to characterize the secretion rate of latent TGF-β into SF, and the transport of active TGF-β across the articular surface and into the cartilage layer. Experiments on tissue explants demonstrate that high levels of latent TGF-β1 are secreted from both the synovium and all three articular cartilage zones (superficial, middle, and deep), suggesting that these tissues are capable of continuously replenishing latent TGF-β to SF. Furthermore, upon exposure of cartilage to active TGF-β1, the peptide accumulates in the superficial zone (SZ) due to the presence of an overwhelming concentration of nonspecific TGF-β binding sites in the extracellular matrix. Although this response leads to high levels of active TGF-β in the SZ, the active peptide is unable to penetrate deeper into the middle and deep zones of cartilage. These results provide strong evidence for a sequential physiologic mechanism through which SZ chondrocytes gain access to active TGF-β: the synovium and articular cartilage secrete latent TGF-β into the SF and, upon activation, TGF-β transports back into the cartilage layer, binding exclusively to the SZ