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

A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues

Mechanobiologic signals regulate cellular responses under physiologic and pathologic conditions. Using synthetic biology and tissue engineering, we developed a mechanically responsive bioartificial tissue that responds to mechanical loading to produce a preprogrammed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. We engineered these cells into living tissues that respond to mechanical loading by producing the anti-inflammatory biologic drug interleukin-1 receptor antagonist. Chondrocyte TRPV4 is activated by osmotic loading and not by direct cellular deformation, suggesting that tissue loading is transduced into an osmotic signal that activates TRPV4. Either osmotic or mechanical loading of tissues transduced with TRPV4-responsive circuits protected constructs from inflammatory degradation by interleukin-1α. This synthetic mechanobiology approach was used to develop a mechanogenetic system to enable long-term, autonomously regulated drug delivery driven by physiologically relevant loading

Damage mechanics in tissue engineered cartilage: biochemical data and computational models

This file contains biochemical data (collagen damage fraction, GAG and collagen (%ww, and %D0ww), cell content (millions of cells/construct), J (swelling ratio) and solid volume fraction over the 105 day culture for the control and chondroitinase-treated (control, CABC, respectively). FEBio optimized model for the growth of tissue engineered cartilage

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

Final Evaluation Report Computer Associates International, Inc. CA-ACF2 with MVS/ESA NATIONAL COMPUTER SECURITY CENTER

ions : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 7 2.4 Trusted Computing Base : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 10 2.4.1 Trusted Computing Base Structure : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 10 2.4.2 Trusted Computing Base Interface : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 12 2.4.3 Security Policy : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 13 3 Hardware Architecture 15 3.1 Architecture Overview : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 15 3.1.1 Processors : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 15 3.2 ESA/370 Architecture : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 16 3.2.1 Hardware Privilege Levels : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 16 3.2.2 Registers : : : : : : : : : : : : :..