Establishing in vivo and ex vivo chick embryo models to investigate fetal tendon healing.

Injured adult tendons heal fibrotically and possess high re-injury rates, whereas fetal tendons appear to heal scarlessly. However, knowledge of fetal tendon wound healing is limited due in part to the need for an accessible animal model. Here, we developed and characterized an in vivo and ex vivo chick embryo tendon model to study fetal tendon healing. In both models, injury sites filled rapidly with cells and extracellular matrix during healing, with wound closure occurring faster in vivo. Tendons injured at an earlier embryonic stage improved mechanical properties to levels similar to non-injured controls, whereas tendons injured at a later embryonic stage did not. Expression levels of tendon phenotype markers, collagens, collagen crosslinking regulators, matrix metalloproteinases, and pro-inflammatory mediators exhibited embryonic stage-dependent trends during healing. Apoptosis occurred during healing, but ex vivo tendons exhibited higher levels of apoptosis than tendons in vivo. Future studies will use these in vivo and ex vivo chick embryo tendon injury models to elucidate mechanisms of stage-specific fetal tendon healing to inform the development of therapeutic approaches to regeneratively heal adult tendons.

Recombinant lysyl oxidase effects on embryonic tendon cell phenotype and behavior.

Lysyl oxidase (LOX) plays an important role in the elaboration of tendon mechanical properties during embryonic development by mediating enzymatic collagen crosslinking. We previously showed recombinant LOX (rLOX) treatment of developing tendon significantly increased LOX-mediated collagen crosslink density to enhance tendon mechanical properties at different stages of tissue formation. Working toward the future development of rLOX-based therapeutic strategies to enhance mechanical properties of tendons that are compromised, such as after injury or due to abnormal development, this study characterized the direct effects of rLOX treatment on embryonic tendon cells from different stages of tissue formation. Tendon cell morphology, proliferation rate, proliferative capacity, and metabolic activity were not affected by rLOX treatment. Tenogenic phenotype was stable with rLOX treatment, reflected by no change in cell morphology or tendon marker messenger RNA (mRNA) levels assessed by reverse-transcription polymerase chain reaction. Collagen mRNA levels also remained constant. Matrix metalloproteinase-9 expression levels were downregulated in later stage tendon cells, but not in earlier stage cells, whereas enzyme activity levels were undetected. Bone morphogenetic protein-1 (BMP-1) expression was upregulated in earlier stage tendon cells, but not in later stage cells. Furthermore, BMP-1 activity was unchanged when intracellular LOX enzyme activity levels were upregulated in both stage cells, suggesting exogenous rLOX may have entered the cells. Based on our data, rLOX treatment had minimal effects on tendon cell phenotype and behaviors. These findings will inform future development of LOX-focused treatments to enhance tendon mechanical properties without adverse effects on tendon cell phenotype and behaviors.

Tendon mechanical properties are enhanced via recombinant lysyl oxidase treatment.

Tendon mechanical properties are significantly compromised in adult tendon injuries, tendon-related birth defects, and connective tissue disorders. Unfortunately, there currently is no effective treatment to restore native tendon mechanical properties after postnatal tendon injury or abnormal fetal development. Approaches to promote crosslinking of extracellular matrix components in tendon have been proposed to enhance insufficient mechanical properties of fibrotic tendon after healing. However, these crosslinking agents, which are not naturally present in the body, are associated with toxicity and significant reductions in metabolic activity at concentrations that enhance tendon mechanical properties. In contrast, we propose that an effective method to restore tendon mechanical properties would be to promote lysyl oxidase (LOX)-mediated collagen crosslinking in tendon during adult tissue healing or fetal tissue development. LOX is naturally occurring in the body, and we previously demonstrated LOX-mediated collagen crosslinking to be a critical regulator of tendon mechanical properties during new tissue formation. In this study, we examined the effects of recombinant LOX treatment on tendon at different stages of development. We found that recombinant LOX treatment significantly enhanced tensile and nanoscale tendon mechanical properties without affecting cell viability or collagen content, density, and maturity. Interestingly, both tendon elastic modulus and LOX-mediated collagen crosslink density plateaued at higher recombinant LOX concentrations, which may have been due to limited availability of adjacent lysine residues that are near enough to be crosslinked together. The plateau in crosslink density at higher concentrations of recombinant LOX treatments may have implications for preventing over-stiffening of tendon, though this requires further investigation. These findings demonstrate the exciting potential for a LOX-based therapeutic to enhance tendon mechanical properties via a naturally occurring crosslinking mechanism, which could have tremendous implications for an estimated 32 million acute and chronic tendon and ligament injuries each year in the U.S.