Characterization of mechanical and biochemical properties of developing embryonic tendon

Abstract

Tendons have uniquely high tensile strength, critical to their function to transfer force from muscle to bone. When injured, their innate healing response results in aberrant matrix organization and functional properties. Efforts to regenerate tendon are challenged by limited understanding of its normal development. Consequently, there are few known markers to assess tendon formation and parameters to design tissue engineering scaffolds. We profiled mechanical and biological properties of embryonic tendon and demonstrated functional properties of developing tendon are not wholly reflected by protein expression and tissue morphology. Using force volume-atomic force microscopy, we found that nano-and microscale tendon elastic moduli increase nonlinearly and become increasingly spatially heterogeneous during embryonic development. When we analyzed potential biochemical contributors to modulus, we found statistically significant but weak correlation between elastic modulus and collagen content, and no correlation with DNA or glycosaminoglycan content, indicating there are additional contributors to mechanical properties. To investigate collagen cross-linking as a potential contributor, we inhibited lysyl oxidasemediated collagen cross-linking, which significantly reduced tendon elastic modulus without affecting collagen morphology or DNA, glycosaminoglycan, and collagen content. This suggests that lysyl oxidase-mediated cross-linking plays a significant role in the development of embryonic tendon functional properties and demonstrates that changes in cross-links alter mechanical properties without affecting matrix content and organization. Taken together, these data demonstrate the importance of functional markers to assess tendon development and provide a profile of tenogenic mechanical properties that may be implemented in tissue engineering scaffold design to mechanoregulate new tendon regeneration.musculoskeletal | second harmonic generation T endon is a principal tissue involved in movement, functioning primarily to transfer loads from muscle to bone. Acute and chronic tendon injuries are significant clinical problems due to poor innate healing ability and drawbacks associated with surgical repair (1, 2). In 2006, musculoskeletal symptoms were the second most frequent reason for physician visits in the United States, resulting in over 130 million visits at a cost of nearly $850 billion (3). Almost half of these visits involved tendons and ligaments, with incidence expected to rise with an aging population. Thus, efforts have focused on engineering new tissues for replacement, although this has been challenged by a paucity of markers with which to assess functional tendon development and few known cues to direct differentiation and new tissue formation.Characterization of tendon formation in embryonic or engineered tissue has typically relied on molecular markers, as well as matrix composition and organization (4-9). Although useful for assessing cell differentiation and ECM deposition during tissue formation, t...

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

“…The current study could not show a toxic or inhibiting effect on cells, which is in line with other studies (19,56), but it cannot be excluded that BAPN might directly interfere with collagen fibrillogenesis or other regulating proteins. However, it has been shown in vitro that BAPN does not bind directly to collagen fibrils (57,58) and does not interfere with procollagen processing (59), which argues against a direct effect of BAPN on fibrillogenesis.…”

“…Yet it cannot entirely be excluded that BAPN interferes with one of these molecules or other asyet-unknown regulators. Nevertheless, the well described LOX-inhibition mechanism (16,17), the increased collagen solubility, and reduced mechanical strength of tendon constructs (19,29) in combination with unaltered gene expression and protein content of established key regulators coincide with the so-far-established function of BAPN.…”

Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

“…tendons as measured in studies of chick tenogenesis (ranging from *0.1 to 2.0 MPa during mid-to late-stage development). 59,60 While moduli of tendons rapidly increase for approximately three orders of magnitude during postnatal maturation, [61][62][63] we and others have shown that implanting TECs in vivo increases their linear modulus 2-3 orders of magnitude, 23,24,64 suggesting that these TECs may be suitable for injury repair.…”

Vitamin C–enriched gelatin supplementation before intermittent activity augments collagen synthesis

“…Like developing tendons and ligaments, these tissues have more cells and less matrix (22), synthesize collagen at a faster rate (23), have more developmental isoforms of collagen (24), and are much weaker than adult ligaments (21). Despite these differences, engineered ligaments respond to physiologic stimuli in a manner that is quite similar to the native tissue (25)(26)(27)(28).…”