* Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability.

When cultured with sufficient nutrient supply, engineered cartilage synthesizes proteoglycans rapidly, producing an osmotic swelling pressure that destabilizes immature collagen and prevents the development of a robust collagen framework, a hallmark of native cartilage. We hypothesized that mechanically constraining the proteoglycan-induced tissue swelling would enhance construct functional properties through the development of a more stable collagen framework. To test this hypothesis, we developed a novel "cage" growth system to mechanically prevent tissue constructs from swelling while ensuring adequate nutrient supply to the growing construct. The effectiveness of constrained culture was examined by testing constructs embedded within two different scaffolds: agarose and cartilage-derived matrix hydrogel (CDMH). Constructs were seeded with immature bovine chondrocytes and cultured under free swelling (FS) conditions for 14 days with transforming growth factor-ß before being placed into a constraining cage for the remainder of culture. Controls were cultured under FS conditions throughout. Agarose constructs cultured in cages did not expand after the day 14 caging while FS constructs expanded to 8 × their day 0 weight after 112 days of culture. In addition to the physical differences in growth, by day 56, caged constructs had higher equilibrium (agarose: 639 ± 179 kPa and CDMH: 608 ± 257 kPa) and dynamic compressive moduli (agarose: 3.4 ± 1.0 MPa and CDMH 2.8 ± 1.0 MPa) than FS constructs (agarose: 193 ± 74 kPa and 1.1 ± 0.5 MPa and CDMH: 317 ± 93 kPa and 1.8 ± 1.0 MPa for equilibrium and dynamic properties, respectively). Interestingly, when normalized to final day wet weight, cage and FS constructs did not exhibit differences in proteoglycan or collagen content. However, caged culture enhanced collagen maturation through the increased formation of pyridinoline crosslinks and improved collagen matrix stability as measured by α-chymotrypsin solubility. These findings demonstrate that physically constrained culture of engineered cartilage constructs improves functional properties through improved collagen network maturity and stability. We anticipate that constrained culture may benefit other reported engineered cartilage systems that exhibit a mismatch in proteoglycan and collagen synthesis.

Nutrient Channels Aid the Growth of Articular Surface-Sized Engineered Cartilage Constructs.

Symptomatic osteoarthritic lesions span large regions of joint surfaces and the ability to engineer cartilage constructs at clinically relevant sizes would be highly desirable. We previously demonstrated that nutrient transport limitations can be mitigated by the introduction of channels in 10 mm diameter cartilage constructs. In this study, we scaled up our previous system to cast and cultivate 40 mm diameter constructs (2.3 mm overall thickness); 4 mm diameter and channeled 10 mm diameter constructs were studied for comparison. Furthermore, to assess whether prior results using primary bovine cells are applicable for passaged cells-a more clinically realistic scenario-we cast constructs of each size with primary or twice-passaged cells. Constructs were assessed mechanically for equilibrium compressive Young's modulus (EY), dynamic modulus at 0.01 Hz (G*), and friction coefficient (µ); they were also assessed biochemically, histologically, and immunohistochemically for glycosaminoglycan (GAG) and collagen contents. By maintaining open channels, we successfully cultured robust constructs the size of entire human articular cartilage layers (growing to ∼52 mm in diameter, 4 mm thick, mass of 8 g by day 56), representing a 100-fold increase in scale over our 4 mm diameter constructs, without compromising their functional properties. Large constructs reached EY of up to 623 kPa and GAG contents up to 8.9%/ww (% of wet weight), both within native cartilage ranges, had G* >2 MPa, and up to 3.5%/ww collagen. Constructs also exhibited some of the lowest µ reported for engineered cartilage (0.06-0.11). Passaged cells produced tissue of lower quality, but still exhibited native EY and GAG content, similar to their smaller controls. The constructs produced in this study are, to our knowledge, the largest engineered cartilage constructs to date which possess native EY and GAG, and are a testament to the effectiveness of nutrient channels in overcoming transport limitations in cartilage tissue engineering.

Optimizing nutrient channel spacing and revisiting TGF-beta in large engineered cartilage constructs.

Cartilage tissue engineering is a promising approach to treat osteoarthritis. However, current techniques produce tissues too small for clinical relevance. Increasingly close-packed channels have helped overcome nutrient transport limitations in centimeter-sized chondrocyte-agarose constructs, yet optimal channel spacings to recapitulate native cartilage compositional and mechanical properties in constructs this large have not been identified. Transient active TGF-ß treatment consistently reproduces native compressive Young׳s modulus (EY) and glycosaminoglycan (GAG) content in constructs, but standard dosages of 10ng/mL exacerbate matrix heterogeneity. To ultimately produce articular layer-sized constructs, we must first optimize channel spacing and investigate the role of TGF-ß in the utility of channels. We cultured ∅10mm constructs with 0, 12, 19, or 27 nutrient channels (∅1mm) for 6-8 weeks with 0, 1, or 10ng/mL TGF-ß; subsequently we analyzed them mechanically, biochemically, and histologically. Constructs with 12 or 19 channels grew the most favorably, reaching EY=344±113kPa and GAG and collagen contents of 10.8±1.2% and 2.2±0.2% of construct wet weight, respectively. Constructs with 27 channels had significantly less deposited GAG than other groups. Channeled constructs given 1 or 10ng/mL TGF-ß developed similar properties. Without TGF-ß, constructs with 0 or 12 channels exhibited properties that were indistinguishable, and lower than TGF-ß-supplemented constructs. Taken together, these results emphasize that nutrient channels are effective only in the presence of TGF-ß, and indicate that spacings equivalent to 12 channels in ∅10mm constructs can be employed in articular-layer-sized constructs with reduced dosages of TGF-ß.

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties.

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3-4mm×2.3mm, with cell seeding densities ranging from 15 to 90×10(6)cells/mL. Subsets of constructs were subjected to transient or sustained TGF-ß treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young's moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-ß treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

Continuum theory of fibrous tissue damage mechanics using bond kinetics: application to cartilage tissue engineering.

This study presents a damage mechanics framework that employs observable state variables to describe damage in isotropic or anisotropic fibrous tissues. In this mixture theory framework, damage is tracked by the mass fraction of bonds that have broken. Anisotropic damage is subsumed in the assumption that multiple bond species may coexist in a material, each having its own damage behaviour. This approach recovers the classical damage mechanics formulation for isotropic materials, but does not appeal to a tensorial damage measure for anisotropic materials. In contrast with the classical approach, the use of observable state variables for damage allows direct comparison of model predictions to experimental damage measures, such as biochemical assays or Raman spectroscopy. Investigations of damage in discrete fibre distributions demonstrate that the resilience to damage increases with the number of fibre bundles; idealizing fibrous tissues using continuous fibre distribution models precludes the modelling of damage. This damage framework was used to test and validate the hypothesis that growth of cartilage constructs can lead to damage of the synthesized collagen matrix due to excessive swelling caused by synthesized glycosaminoglycans. Therefore, alternative strategies must be implemented in tissue engineering studies to prevent collagen damage during the growth process.

Heterogeneous engineered cartilage growth results from gradients of media-supplemented active TGF-ß and is ameliorated by the alternative supplementation of latent TGF-ß.

Transforming growth factor beta (TGF-ß) has become one of the most widely utilized mediators of engineered cartilage growth. It is typically exogenously supplemented in the culture medium in its active form, with the expectation that it will readily transport into tissue constructs through passive diffusion and influence cellular biosynthesis uniformly. The results of this investigation advance three novel concepts regarding the role of TGF-ß in cartilage tissue engineering that have important implications for tissue development. First, through the experimental and computational analysis of TGF-ß concentration distributions, we demonstrate that, contrary to conventional expectations, media-supplemented exogenous active TGF-ß exhibits a pronounced concentration gradient in tissue constructs, resulting from a combination of high-affinity binding interactions and a high cellular internalization rate. These gradients are sustained throughout the entire culture duration, leading to highly heterogeneous tissue growth; biochemical and histological measurements support that while biochemical content is enhanced up to 4-fold at the construct periphery, enhancements are entirely absent beyond 1 mm from the construct surface. Second, construct-encapsulated chondrocytes continuously secrete large amounts of endogenous TGF-ß in its latent form, a portion of which undergoes cell-mediated activation and enhances biosynthesis uniformly throughout the tissue. Finally, motivated by these prior insights, we demonstrate that the alternative supplementation of additional exogenous latent TGF-ß enhances biosynthesis uniformly throughout tissue constructs, leading to enhanced but homogeneous tissue growth. This novel demonstration suggests that latent TGF-ß supplementation may be utilized as an important tool for the translational engineering of large cartilage constructs that will be required to repair the large osteoarthritic defects observed clinically.

Matrix Production in Large Engineered Cartilage Constructs Is Enhanced by Nutrient Channels and Excess Media Supply.

Cartilage tissue engineering is a promising approach to resurfacing osteoarthritic joints. Existing techniques successfully engineer small-sized constructs with native levels of extracellular matrix (glycosaminoglycans [GAG] or collagen). However, a remaining challenge is the growth of large-sized constructs with properties similar to those of small constructs, due to consumption and transport limitations resulting in inadequate nutrient availability within the interior of large constructs. This study employed system-specific computational models for estimating glucose requirements of large constructs, with or without channels, to enhance nutrient availability. Based on glucose requirements for matrix synthesis in cartilage constructs, computational simulations were performed to identify the media volume (MV) and the number of nutrient channels (CH) needed to maintain adequate glucose levels within tissue constructs over the 3-day period between media replenishments. In Study 1, the influence of MV (5, 10, 15 mL/construct) and number of nutrient channels (CH: 0, 3, 7, 12 per construct) on glucose availability was investigated computationally for ∅10 × 2.34 mm cylindrical constructs. Results showed that the conventionally used MV 5 led to deleterious glucose depletion after only 40 h of culture, and that MV 15 was required to maintain sufficient glucose levels for all channel configurations. Study 2 examined experimentally the validity of these predictions, for tissue constructs cultured for 56 days. Matrix elaboration was highest in MV 15/CH 12 constructs (21.6% ± 2.4%/ww GAG, 5.5% ± 0.7%/ww collagen, normalized to wet weight (ww) on day 0), leading to the greatest amount of swelling (3.0 ± 0.3 times day-0 volume), in contrast to the significantly lower matrix elaboration of conventional culture, MV 5/CH 0 (11.8% ± 1.6%/ww GAG and 2.5% ± 0.6%/ww collagen, 1.6 ± 0.1 times day-0 volume). The computational analyses correctly predicted the need to increase the conventional media levels threefold to support matrix synthesis in large channeled engineered constructs. Results also suggested that more elaborate computational models are needed for accurate predictive tissue engineering simulations, which account for a broader set of nutrients, cell proliferation, matrix synthesis, and swelling of the constructs.

Nutrient channels and stirring enhanced the composition and stiffness of large cartilage constructs.

A significant challenge in cartilage tissue engineering is to successfully culture functional tissues that are sufficiently large to treat osteoarthritic joints. Transport limitations due to nutrient consumption by peripheral cells produce heterogeneous constructs with matrix-deficient centers. Incorporation of nutrient channels into large constructs is a promising technique for alleviating transport limitations, in conjunction with simple yet effective methods for enhancing media flow through channels. Cultivation of cylindrical channeled constructs flat in culture dishes, with or without orbital shaking, produced asymmetric constructs with poor tissue properties. We therefore explored a method for exposing the entire construct surface to the culture media, while promoting flow through the channels. To this end, chondrocyte-seeded agarose constructs (∅10mm, 2.34mm thick), with zero or three nutrient channels (∅1mm), were suspended on their sides in custom culture racks and subjected to three media stirring modes for 56 days: uniaxial rocking, orbital shaking, or static control. Orbital shaking led to the highest construct EY, sulfated glycosaminoglycan (sGAG), and collagen contents, whereas rocking had detrimental effects on sGAG and collagen versus static control. Nutrient channels increased EY as well as sGAG homogeneity, and the beneficial effects of channels were most marked in orbitally shaken samples. Under these conditions, the constructs developed symmetrically and reached or exceeded native levels of EY (~400kPa) and sGAG (~9%/ww). These results suggest that the cultivation of channeled constructs in culture racks with orbital shaking is a promising method for engineering mechanically competent large cartilage constructs.

Synthesis rates and binding kinetics of matrix products in engineered cartilage constructs using chondrocyte-seeded agarose gels.

Large-sized cartilage constructs suffer from inhomogeneous extracellular matrix deposition due to insufficient nutrient availability. Computational models of nutrient consumption and tissue growth can be utilized as an efficient alternative to experimental trials to optimize the culture of large constructs; models require system-specific growth and consumption parameters. To inform models of the [bovine chondrocyte]-[agarose gel] system, total synthesis rate (matrix accumulation rate+matrix release rate) and matrix retention fractions of glycosaminoglycans (GAG), collagen, and cartilage oligomeric matrix protein (COMP) were measured either in the presence (continuous or transient) or absence of TGF-ß3 supplementation. TGF-ß3's influences on pyridinoline content and mechanical properties were also measured. Reversible binding kinetic parameters were characterized using computational models. Based on our recent nutrient supplementation work, we measured glucose consumption and critical glucose concentration for tissue growth to computationally simulate the culture of a human patella-sized tissue construct, reproducing the experiment of Hung et al. (2003). Transient TGF-ß3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-ß3 supplementation groups over control, with the highest binding affinity observed in the transient supplementation group; both COMP synthesis and retention were lower than those for GAG and collagen. These results informed the modeling of GAG deposition within a large patella construct; this computational example was similar to the previous experimental results without further adjustments to modeling parameters. These results suggest that these nutrient consumption and matrix synthesis models are an attractive alternative for optimizing the culture of large-sized constructs.

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 (ø4×2.34 mm, 30×10(6) cells/mL) were subjected to varying supplementation levels of insulin (0× to 30× relative to standard supplementation), transferrin (0× to 30×), selenous acid (0× to 10×), ascorbate (0× to 30×), and glucose (0× to 3×). 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 (1× concentrations of all medium constituents) were used. After 42 days of culture, values in each of these controls were, respectively, E(-Y)=518±78, 401±113, 236±67 kPa; E(+Y)=1420±430, 1140±490, 1240±280 kPa; k=2.3±0.8×10(-3), 5.4±7.0×10(-3), 3.3±1.3×10(-3) mm(4)/N·s; sGAG=7.8±0.3, 6.3±0.4, 4.1±0.5%/ww; COL=1.3±0.2, 1.1±0.3, 1.4±0.4%/ww; and DNA=11.5±2.2, 12.1±0.6, 5.2±2.8 µg/disk. The presence of insulin and ascorbate was essential, but their concentrations may drop as low as 0.3× without detrimental effects on any of the measured properties; excessive supplementation of ascorbate (up to 30×) was detrimental to E(-Y), and 30× 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<10(-6)), with loss of functional properties, composition, and cellularity observed at ≤0.3×; excessive glucose supplementation (up to 3×) 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.

Dynamic mechanical compression of devitalized articular cartilage does not activate latent TGF-ß.

A growing body of research has highlighted the role that mechanical forces play in the activation of latent TGF-ß in biological tissues. In synovial joints, it has recently been demonstrated that the mechanical shearing of synovial fluid, induced during joint motion, rapidly activates a large fraction of its soluble latent TGF-ß content. Based on this observation, the primary hypothesis of the current study is that the mechanical deformation of articular cartilage, induced by dynamic joint motion, can similarly activate the large stores of latent TGF-ß bound to the tissue extracellular matrix (ECM). Here, devitalized deep zone articular cartilage cylindrical explants (n=84) were subjected to continuous dynamic mechanical loading (low strain: ±2% or high strain: ±7.5% at 0.5Hz) for up to 15h or maintained unloaded. TGF-ß activation was measured in these samples over time while accounting for the active TGF-ß that remains bound to the cartilage ECM. Results indicate that TGF-ß1 is present in cartilage at high levels (68.5±20.6ng/mL) and resides predominantly in the latent form (>98% of total). Under dynamic loading, active TGF-ß1 levels did not statistically increase from the initial value nor the corresponding unloaded control values for any test, indicating that physiologic dynamic compression of cartilage is unable to directly activate ECM-bound latent TGF-ß via purely mechanical pathways and leading us to reject the hypothesis of this study. These results suggest that deep zone articular chondrocytes must alternatively obtain access to active TGF-ß through chemical-mediated activation and further suggest that mechanical deformation is unlikely to directly activate the ECM-bound latent TGF-ß of various other tissues, such as muscle, ligament, and tendon.

Accumulation of exogenous activated TGF-ß 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 ß (TGF-ß). This discovery raised the possibility of a physiological process consisting of latent TGF-ß supply to SF, activation via shearing, and transport of TGF-ß into the cartilage matrix. Therefore, the two primary objectives of this investigation were to characterize the secretion rate of latent TGF-ß into SF, and the transport of active TGF-ß across the articular surface and into the cartilage layer. Experiments on tissue explants demonstrate that high levels of latent TGF-ß1 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-ß to SF. Furthermore, upon exposure of cartilage to active TGF-ß1, the peptide accumulates in the superficial zone (SZ) due to the presence of an overwhelming concentration of nonspecific TGF-ß binding sites in the extracellular matrix. Although this response leads to high levels of active TGF-ß 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-ß: the synovium and articular cartilage secrete latent TGF-ß into the SF and, upon activation, TGF-ß transports back into the cartilage layer, binding exclusively to the SZ.

Expression of doublecortin reveals articular chondrocyte lineage in mouse embryonic limbs.

The doublecortin (Dcx) gene encodes a microtubule-binding protein that was originally found in immature neurons. In this study, we used two mouse strains that express reporter genes (LacZ and enhanced green fluorescence protein, respectively) driven by the endogenous Dcx promoter. We found that Dcx was expressed in the mesenchymal cells in the mouse embryonic limb buds. A population of the mesenchymal cells continued Dcx expression after they differentiated into joint interzone cells and then articular chondrocytes. In contrast, the endochondral chondrocytes lost Dcx expression when the mesenchymal cells differentiated into endochondral chondrocytes. These data support a concept that the articular and endochondral chondrocytes originate from the same mesenchymal cells that express Dcx. In contrast to the notion that articular chondrocytes are derived from de-differentiated endochondral chondrocytes, our findings demonstrate that the lineages of articular and endochondral chondrocytes bifurcate at the stage of endochondral chondrogenesis.

Ex vivo deformations of the urinary bladder wall during whole bladder filling: contributions of extracellular matrix and smooth muscle.

As the complete understanding of urinary bladder function requires knowledge of organ level deformations, we conducted ex vivo studies of surface strains of whole bladders during controlled filling. The surface strains derived from displacements of surface markers applied to the posterior surface of excised rat bladders were tracked under slow filling with pressure and volume simultaneously recorded in the passive and completely inactivated states (i.e. with and without smooth muscle tone, respectively). Bladders evaluated in the passive state exhibited spontaneous contractions and larger average peak pressures (16.7 mm Hg compared to 6.4 mm Hg in the inactive state). Overall, the bladders exhibited anisotropic deformations and were stiffer in the circumferential direction, with average peak stretch values of approximately 2.3 and approximately 1.9 in the longitudinal and circumferential directions, respectively, for both states. Although bladders in the passive state were stiffer, they had similar average peak areal stretches of 4.3 in both states. However, differences early in the filling process as a result of a loss in smooth muscle tone in the inactive state resulted in longitudinal lengthening of 36%. Idealizing the bladder as a prolate spheroid, we estimated the wall stress-strain relation during filling and demonstrated that the intact bladder exhibited the classic stress-stretch relation, with a significantly protracted low stress region and peak stresses of 36 and 51 kPa in the longitudinal and circumferential directions, respectively. The present study fills a major gap in the urinary bladder biomechanics literature, wherein knowledge of the pressure-volume-wall stress-wall strain relation was explored for the first time in a functioning organ ex vivo.