Grace D. O’Connell

Theoretical and Uniaxial Experimental Evaluation of Human Annulus Fibrosus Degeneration

The highly organized structure and composition of the annulus fibrosus provides the tissue with mechanical behaviors that include anisotropy and nonlinearity. Mathematical models are necessary to interpret and elucidate the meaning of directly measured mechanical properties and to understand the structure-function relationships of the tissue components, namely, the fibers and extrafibrillar matrix. This study models the annulus fibrosus as a combination of strain energy functions describing the fibers, matrix, and their interactions. The objective was to quantify the behavior of both nondegenerate and degenerate annulus fibrosus tissue using uniaxial tensile experimental data. Mechanical testing was performed with samples oriented along the circumferential, axial, and radial directions. For samples oriented along the radial direction, the toe-region modulus was 2x stiffer with degeneration. However, no other differences in measured mechanical properties were observed with degeneration. The constitutive model fit well to samples oriented along the radial and circumferential directions (R(2)> or =0.97). The fibers supported the highest proportion of stress for circumferential loading at 60%. There was a 70% decrease in the matrix contribution to stress from the toe-region to the linear-region of both the nondegenerate and degenerate tissue. The shear fiber-matrix interaction (FMI) contribution increased by 80% with degeneration in the linear-region. Samples oriented along the radial and axial direction behaved similarly under uniaxial tension (modulus=0.32 MPa versus 0.37 MPa), suggesting that uniaxial testing in the axial direction is not appropriate for quantifying the mechanics of a fiber reinforcement in the annulus. In conclusion, the structurally motivated nonlinear anisotropic hyperelastic constitutive model helps to further understand the effect of microstructural changes with degeneration, suggesting that remodeling in the subcomponents (i.e., the collagen fiber, matrix and FMI) may minimize the overall effects on mechanical function of the bulk material with degeneration.

MULTI-LAMELLAR AND MULTI-AXIAL MATURATION OF CELL-SEEDED FIBER- REINFORCED TISSUE ENGINEERED CONSTRUCTS

The architecture of load-bearing fibrous tissues is optimized to enable a specific set of mechanical functions. This organization arises from a complex process of cell patterning, matrix deposition, and functional maturation [1]. In their mature state, these tissues span multiple length scales, encompassing nanoscale interactions of cells with extracellular matrix to the centimeter length scales of the anatomic tissue volume and shape. Two structures that typify dense fibrous tissues are the meniscus of the knee and the annulus fibrosus (AF) of the intervertebral disc (IVD). The mechanical function of the wedge-shaped knee meniscus is based on its stiff prevailing circumferential collagen architecture that resists tensile deformation [2,3]. Adding to its complexity, radial tie fibers and sheets are interwoven amongst these fibers, increasing stiffness in the transverse direction and binding the tissue together [4]. In the annulus fibrosus, multiple anisotropic lamellae are stacked in concentric rings with their prevailing fiber directions alternating above and below the horizontal axis in adjacent layers [5]. The high circumferential tensile properties of this laminate structure allow it to resist bulging of the nucleus pulposus with compressive loading of the spine. Given their structural properties, unique form, and demanding mechanical environments, the knee meniscus and the AF region of the IVD represent two of the most challenging tissues to consider for functional tissue engineering.Copyright

Characterization of Depth-Dependent Mechanical Properties in Bio-Titanium Hybrid Osteochondral Tissue Engineered Constructs

With cartilage autografts and allografts in short supply, tissue engineered osteochondral (OC) grafts offer an alternative [1]. These constructs are comprised of a chondrocyte-seeded hydrogel region and a porous, bone-like base. Our laboratory has shown growth of more robust osteochondral constructs on clinically-relevant metal substrates (eg. tantalum) as opposed to devitalized bone, and these constructs have been evaluated in vivo [1,2]. Due to the presence of the base, it is expected that transport of nutrients and chemical factors in OC constructs will differ from transport in chondral-only constructs (Fig. 1, bottom-left). Depth-dependent mechanical properties of chondral-only constructs have been measured, yielding a “U-shaped” strain profile, in which the construct is stiffest on the edges and softest in the center. However, depth-dependent properties have not been measured in tissue engineered OC grafts [3].Copyright

Chondroitinase-ABC Digestion and Dynamic Loading Increase Tension-Compression Nonlinearity in Tissue-Engineered Cartilage

Native articular cartilage exhibits tension-compression nonlinearity (TCN), where the compressive modulus is lower than its relatively high tensile modulus [1–2]. TCN produces in restricted lateral expansion of the tissue upon axial compression. We previously demnostrated that osmotic swelling can be used to measure the TCN of engineered cartilage by placing the tissue in an initial state of tensile strain. Incremental application of compression can be used to study the tissue’s mechanical properties as it transitions from tension to compression [3]. Although engineered cartilage is able to achieve the Young’s modulus (EY) and glycosaminoglycan (GAG) content of native tissue, the collagen content and dynamic modulus (G*) consistently underperform the native tissue. Removing GAG with chondroitinase ABC (cABC) has been shown to significantly decrease the tissue properties immediately after digestion but the properties rebound, with improved collagen content and G* compared to undigested controls [4]. Furthermore, we have previously shown that cABC digestion significantly increases TCN in engineered cartilage [3]. Dynamic loading (DL) has been shown to significantly increase the mechanical properties without significantly altering biochemical composition of engineered cartilage, however the mechanism through which DL modulates the mechanical strength of engineered cartilage may be due in part to improved extracellular matrix (ECM) organization [5]. We therefore hypothesize that cABC digestion and DL will improve the tensile properties of engineered cartilage.Copyright

Prolonged Treatment of Ultra-Low Dose Chondroitinase ABC Improves Matrix Production in Engineered Cartilage

Articular cartilage is the load bearing soft tissue of diarthrodial joints, and mechanical loading maintains the integrity of the tissue. The predominant extracellular matrix constituents, proteoglycans and collagen, allow cartilage to support the high compressive and tensile loads experienced in diurnal loading. Our laboratory has been successful in cultivating engineered cartilage constructs with a compressive equilibrium modulus and glycosaminglycan (GAG) content near native values [1, 2]. Many approaches to cultivating engineered cartilage have been limited by low collagen production in vitro, an impediment for attaining native functional load-bearing properties [3].© 2013 ASME

Differences in Engineered Cartilage From Human Chondrocytes and Mesenchymal Stem Cells in Pellet and Construct Culture

Articular cartilage serves as the load-bearing material of joints. One approach to functional tissue engineering is to recapitulate the biochemical and mechanical function of healthy native cartilage in vitro, prior to implantation. We have been successful in cultivating engineered cartilage with compressive mechanical properties and glycosaminoglycan (GAG) content near native values by encapsulating chondrocytes or stem cells in a clinically relevant hydrogel [1, 2]. Clinical application of functional engineered cartilage will likely use of chondrocytes (AC) from osteoarthritic tissue or mesenchymal stem cells (MSCs), which have been shown to have chondrogenic potential. That is, it is may be more feasible to differentiate healthy MSCs towards a chondrogenic lineage than to ‘reprogram’ ACs acquired from an osteoarthritic joint.Copyright

Lipid Mictrotubes as a Nutrient Reservoir or Enzyme Delivery Vehicle in Engineered Cartilage

Tissue-engineered cartilage using a hydrogel scaffold is capable of achieving native compressive properties and glycosaminglycan (GAG) content [1]. However, these tissues are limited in their collagen production and closer inspection of the localized mechanical properties demonstrates that mature constructs consist of a stiffer periphery region surrounding a softer core [1, 2]. Nutrient diffusion becomes increasingly more challenging as the cells in the construct periphery deposit extracellular matrix. Altering the scaffold porosity by adding microscopic porogens can improve the nutrient diffusion into the center of the construct [3]. Furthermore, chondroitinase ABC (chABC) has been shown to improve collagen production of mature engineered cartilage (i.e. tissue cultured for 2–4 weeks before chABC digestion). Lipid microtubes, designed to slowly release chABC for spinal chord injury repair can be incorporated into our agarose hydrogel scaffold in a chABC-loaded or unloaded form. The objective of this study was to explore the use of lipid microtubes in our scaffold as a tubular porogen and as a vehicle to deliver chABC throughout the scaffold to improve nutrient diffusion and collagen production into our engineered constructs.© 2012 ASME

Beneficial Effects of Chondroitinase ABC Release From Lipid Microtubes Encapsulated in Chondrocyte-Seeded Hydrogel Constructs

Tissue-engineered cartilage using a hydrogel scaffold is capable of achieving native compressive properties and glycosaminglycan (GAG) content [1]; however, promoting collagen growth towards native values has been challenging. As the cells in the cartilage constructs deposit matrix over time in culture, transport of nutrients to the construct center becomes increasingly hindered [2]. Digestion of mature tissue engineered constructs with chondroitinase (chABC) temporarily suppresses the GAG content, allowing an increase in the collagen content and eventually improving the mechanical properties after GAG content recovers [1]. However, adding chABC into the feeding media limits its effectiveness to the construct’s periphery, reflecting enzyme diffusion gradients. Additionally, long-term use of chABC, without re-application, is limited since its enzymatic activity degrades within 5 days at 37°C [3]. Lee and co-workers have developed a method for delivering thermostabilized chABC using sugar trehalose and hydrogel-microtubes for applications desiring extended enzyme release [4]. Lipid microtubes loaded with thermostabilized chABC may be incorporated into an agarose hydrogel scaffold to provide long-term release of the enzyme uniformly throughout the construct [3]. The objective of this study was to test the hypothesis that chABC-filled microtubes will enhance in vitro development of engineered cartilage.© 2011 ASME

Experimental and Theoretical Evaluation of Failure Properties for Immature Tissue Engineered Cartilage

Articular cartilage functions as a low friction load bearing soft tissue. The contact area and load distribution is highly location dependent in the knee joint [1]. Regeneration and repair strategies for osteoarthritis include tissue-engineered cartilage, which will need to bear high mechanical stresses and strains. There has been variable success in developing tissue-engineered cartilage with compressive mechanical properties comparable to native tissue (modulus = 40kPa – 1000kPa) [2–4]. There has also been some debate on the costs and benefits of implanting immature constructs and allowing them to elaborate their properties in situ, or culturing them in vitro and implanting them only after they have elaborated sufficiently functional properties. The former strategy may benefit from using the body as a bioreactor and might promote better construct integration, though constructs may bee to frail to sustain the physiological loading environment. The failure properties of engineered cartilage have not been widely evaluated, and may greatly affect the successful implantation of engineered cartilage as a repair strategy for the knee joint. The objective of this study was twofold: 1) to evaluate the failure properties of agarose hydrogels used as scaffolds in our tissue engineering studies, and 2) to evaluate whether joint congruence might sufficiently shield immature constructs to prevent their early failure. The long-term hypothesis of this study is that engineering analyses, based on an informed failure criterion for tissue constructs, might allow proper pre-assessment of failure risk for a given set of construct properties, dimensions, and joint congruence.Copyright

Priming of Synovium-Derived Mesenchymal Stem Cells for Cartilage Tissue Engineering

The clinical potential of stem cells has driven forward efforts toward their optimization for tissue engineering applications. The intimal layer of the synovium is composed of two cell types, macrophages and fibroblast-like cells. The fibroblast-like cells, often referred to as synovial-derived mesenchymal stem cells (sMSCs), have the capability to differentiate down a chondrogenic lineage1. In addition, in vivo tests have shown that synovial cells may be recruited from the synovial membrane to aid in the repair of articular cartilage defects2.Copyright