Ihh Signaling and Muscle Forces are Required for Enthesis Development

<p>Tendon-to-bone repair is clinically challenging and plagued by high failure rates. The attachment of relatively stiff bone (~20GPa) to more compliant tendon (~200MPa) represents a fundamental engineering challenge. In the native tendon-bone attachment, termed the "enthesis," transitional tissue contains gradients of structure and composition that effectively reduce stress concentrations at the boundary between hard and soft tissue. This transitional tissue is replaced by scar after injury and repair, resulting in a mechanically inferior attachment. The goal of this thesis is to study biological and mechanical cues that are critical to the development of the structure and function of the native tendon, which could inspire novel repair strategies to improve tendon-to-bone healing.</p><p>To accomplish this, we characterized mineralization patterns in the murine supraspinatus enthesis throughout postnatal development on the micro-scale using Raman spectroscopy and at the nano-scale using transmission electron microscopy - electron energy loss spectroscopy. Mineralization of this tissue occured postnatally via endochondral ossification. We observed a constant and approximately linear increase in the mineral-to-collagen ratio at the mineralizing front within the enthesis at all developmental stages. Using a multi-scale linear elastic model of the tendon enthesis, we demonstrated that the mineral gradient amplifies stresses near mineralizing cells early in development while reducing stress concentrations at the mature tendon-bone interface.</p><p>Next, we investigated tendon enthesis development in the absence of muscle forces. This localized paralysis model resulted in joint level deformities and mineralization defects. We observed a dramatic decrease in the enthesis biomechanical properties accompanied by structural and compositional changes. Collagen fiber alignment was reduced and mineralization defects were observed using Raman spectroscopy and X-ray diffraction.</p><p>In order to probe the biological mechanisms that might influence development of this tissue, we hypothesized that factors critical to endochondral bone formation will also influence enthesis mineralization. Using a murine reporter of active Indian hedgehog (Ihh) signaling, we identified a population of cells present early in development that populate the mature enthesis. Lineage tracing analyses indicated that this cell population remained at the mature enthesis while down-regulating Ihh signaling in mineralized regions. In the case of reduced muscle forces, Ihh signaling was slightly elevated in this model compared to controls. Eliminating Ihh signaling throughout development using a conditional Smoothened (Smo) knockout mouse model specific to tendon lineage cells resulted in dramatic mineralization defects in the enthesis and reduced biomechanical behavior of the attachment.</p><p>Taken together, this thesis demonstrates that Ihh signaling and muscle loading are necessary for mineralization and maturation of a mechanically robust tendon-to-bone attachment.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Computational and Molecular Biophysics

Year dc:date.available

2014

Author dc:creator

• Schwartz, Andrea Gitomer

Contributors dc:contributor

• Stavros Thomopoulos

• indian hedgehog
• mineral
• tendon

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7N58JCF

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-2261