Fibrocartilage Tissue Engineering: The Role of the Stress Environment on Cell Morphology and Matrix Expression

Abstract

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development. IntroductionM usculoskeletal injuries are a common cause of pain and disability, and result in significant healthcare costs.1 Many of these injuries require regeneration of fibrocartilage (tissue composed of fibrous and cartilaginous components) for effective healing.2-4 For example, meniscus healing is typically insufficient due to a lack of fibrocartilage regeneration.3 Similarly, tendon-to-bone healing and repair, as frequently required after rotator cuff injury, often fails due to a lack of fibrocartilage formation at the tendon-to-bone interface. 4 Little is known about natural fibrocartilage healing, and hence little can be done to improve it. We and others have hypothesized that rebuilding the fibrocartilaginous insertion site of the tendon or ligament into bone is critical for restoration of function and for prevention of re-injury. [4][5][6] Several studies provide evidence that the stress environment influences cell morphology and the fibrocartilage production. 7,8 Compressive loads in vivo have been shown to change tendon composition and structur...

Understanding Entheses: Bridging the Gap Between Clinical and Anthropological Perspectives

“…Tendons have very high tensile strength with a modulus of around 200 megapascals (Thomopoulos et al, 2011). However, their compressive strength is much lower as they lose shape and collapse.…”

“…This is attributable to immeasurable fibril beams having small second moments of area, or axial resistance to bending, whereby shear stiffness is low (Currey, 2002). In comparison, bone has considerably more shear stiffness with roughly an equal tensile strength of 20 gigapascals (Hems and Tillmann, 2000;Thomopoulos et al, 2011). However, tendon fibers have an elastic modulus ten times smaller than that of bone (Hems and Tillmann, 2000).…”

“…All of these entheses indirectly insert into bone through a structure consisting of four distinct zones that progressively shift between structural materials (Fig. 5), (Benjamin et al, , 2006Thomopoulos et al, 2006Thomopoulos et al, , 2011. Moving distally towards the insertion site, the first zone is a dense fibrous connective tissue containing Type I collagen and proteoglycans, forming the tendon proper.…”

“…It appears that a gradual shift from soft to hard tissue may enable efficient load transfer, and reduce stress concentrations (Benjamin et al, , 2006Thomopoulos et al, 2003Thomopoulos et al, , 2011; essentially balancing tensile force between two materials of widely varying elasticity (Hems and Tillmann, 2000). Doschak and Zernicke (2005) suggest these four regions correspond to the transitional zones of increased stiffness outlined on stress/strain curves for tendon.…”

“…However, there remains a need for additional well designed experiments assessing enthesial development and what, if any, contribution these markers have in occupation-based research. Despite these shortcomings, there is a wealth of research centered on bone's global and localized response to both mechanical and systemic influences, and a large body of research examining the functional roles of entheses and how they adapt to a changing mechanical environment (Frost, 2003;Thomopoulos et al, 2010;Thomopoulos et al, 2011). This article seeks to synthesize available clinical information within an anthropological context, providing a theoretical foundation from which future research can be grounded.…”