Robert J. Nims

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-β3 supplementation. TGF-β3s 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-β3 produced the highest GAG synthesis rate, highest GAG retention ratio, and the highest binding affinity; collagen synthesis was elevated in TGF-β3 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.

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.

Computational modeling of chemical reactions and interstitial growth and remodeling involving charged solutes and solid-bound molecules

Mechanobiological processes are rooted in mechanics and chemistry, and such processes may be modeled in a framework that couples their governing equations starting from fundamental principles. In many biological applications, the reactants and products of chemical reactions may be electrically charged, and these charge effects may produce driving forces and constraints that significantly influence outcomes. In this study, a novel formulation and computational implementation are presented for modeling chemical reactions in biological tissues that involve charged solutes and solid-bound molecules within a deformable porous hydrated solid matrix, coupling mechanics with chemistry while accounting for electric charges. The deposition or removal of solid-bound molecules contributes to the growth and remodeling of the solid matrix; in particular, volumetric growth may be driven by Donnan osmotic swelling, resulting from charged molecular species fixed to the solid matrix. This formulation incorporates the state of strain as a state variable in the production rate of chemical reactions, explicitly tying chemistry with mechanics for the purpose of modeling mechanobiology. To achieve these objectives, this treatment identifies the specific theoretical and computational challenges faced in modeling complex systems of interacting neutral and charged constituents while accommodating any number of simultaneous reactions where reactants and products may be modeled explicitly or implicitly. Several finite element verification problems are shown to agree with closed-form analytical solutions. An illustrative tissue engineering analysis demonstrates tissue growth and swelling resulting from the deposition of chondroitin sulfate, a charged solid-bound molecular species. This implementation is released in the open-source program FEBio (www.febio.org). The availability of this framework may be particularly beneficial to optimizing tissue engineering culture systems by examining the influence of nutrient availability on the evolution of inhomogeneous tissue composition and mechanical properties, the evolution of construct dimensions with growth, the influence of solute and solid matrix electric charge on the transport of cytokines, the influence of binding kinetics on transport, the influence of loading on binding kinetics, and the differential growth response to dynamically loaded versus free-swelling culture conditions.

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.

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-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Youngs 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-β 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.

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

A growing body of research has highlighted the role that mechanical forces play in the activation of latent TGF-β 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-β 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-β 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-β activation was measured in these samples over time while accounting for the active TGF-β that remains bound to the cartilage ECM. Results indicate that TGF-β1 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-β1 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-β 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-β through chemical-mediated activation and further suggest that mechanical deformation is unlikely to directly activate the ECM-bound latent TGF-β of various other tissues, such as muscle, ligament, and tendon.

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 Youngs 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-β 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-β 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-β; 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-β developed similar properties. Without TGF-β, constructs with 0 or 12 channels exhibited properties that were indistinguishable, and lower than TGF-β-supplemented constructs. Taken together, these results emphasize that nutrient channels are effective only in the presence of TGF-β, and indicate that spacings equivalent to 12 channels in ∅10mm constructs can be employed in articular-layer-sized constructs with reduced dosages of TGF-β.

Effects of Media Stirring and Presence of Nutrient Channels on Functional Properties of Large Engineered Cartilage Constructs

Cartilage tissue engineering is a promising approach for the replacement of degraded joint cartilage in osteoarthritis (OA) patients. Current strategies employ smaller constructs (∼20 mm2), however OA generally does not become symptomatic until defects reach ≥ 5 cm2. Therefore, small constructs may not ultimately be clinically relevant for treatment of OA. Attempts to scale up construct size are met with challenges, as inhomogeneous properties develop as a result of poor nutrient availability at the construct center due to cellular consumption at the periphery [1]. Previously, the incorporation of ∅1 mm nutrient channels in large (∅10 mm) constructs was found to improve Young’s modulus (EY) and glycosaminoglycan (GAG) content and reproduce their native values [2]. Rotational mixing has been shown to improve properties of micro-channeled constructs [3]. As a major goal of our research is to optimize channel size and arrangement to improve the quality of large engineered cartilage constructs, it is essential to develop a simple but effective method for convecting media through channeled constructs. Therefore, this study seeks to compare the functional properties of large constructs that are subjected to different types of media stirring, by rocking or orbital motion, and to determine whether either of these conditions favors the quality of constructs with nutrient channels.Copyright