The goal of this project is to investigate the effect of unloading on the tendon-to-bone attachment at a number of hierarchical scales. At the nanometer scale, I proposed to examine changes in the organization of the bone mineral with unloading via Transmission electron microscopy-electron energy loss spectroscopy (TEM-EELS). At a scale on the order of micrometers, I was interested in employing Raman and high-energy nano-x-ray diffraction (XRD) to identify modifications made to the mineral content, composition, structure, and organization in the interfacial tissue. Micro-mechanical testing would be employed to examine changes in the mechanics at the micron scale. At the millimeter scale, micro-computed tomography (µCT) and tensile testing were employed to determine the effects of unloading on bone quantity and quality and tissue mechanics.

In the past three years, significant discoveries have been made explaining how the structure and mechanics of the tendon-to-bone attachment vary with unloading. Botulinum Toxin A (Btx) injections into the supraspinatus muscle were employed as an Earthbound model of disuse and unloading. This past year, I found that although there was no significant difference in the elastic properties of the attachment size with unloading, there were significant differences in the failure properties, both the strength and toughness. This modification in the failure properties appears to be two pronged: (1) a compromised complaint zone and (2) loss of enthesial bone. Micro-mechanical testing of the attachment site indicated that it contains a region of high deformation which may serve to dissipate stresses and minimize failure. However, the experimental assays performed here have shown that there are significant modifications to the compliant zone including a decrease in crystal size and carbonate content as well as an increase in mineral misalignment. Taken together, these effects will lead to stiffening of the energy absorbing compliance zone and an increase in fracture risk. In addition, unloaded samples have exhibited an increase in the bone loss nearing 40%. This loss of trabecular structure, which acts as a support for the attachment site, will increase bending at the attachment site and increase the tissue deformation and extent of failure. These results help to explain the measured increase in tissue removal during attachment failure of unloaded samples compared to loaded samples. In comparison, the elastic properties of the tendon-to-bone attachment, including the stiffness and resilience, remains unchanged indicating that the load bearing structures remain intact. However, the cross-sectional area of the tendon decreases with unloading while the attachment footprint increases. We propose that the change in cross-sectional area is due to changes in the non-load bearing tendon sheath. Combined with the decrease in crystal size and mineral carbonate content, these results indicate that unloading may induce a local acidic environment via glycolysis that dissolves tendon and bone structures. However, these have no effect on the tissue elastic properties.

Finally, this study has provided some unique information about the organization of mineral across the attachment. At the micron scale, the mineral exhibits a gradient in size and orientation that is correlated to its location with respect to the collagen matrix. In both loaded and unloaded samples, the mineral crystals go from being uniquely extrafibrillar and free to grow in a disordered fashion in the regions of low mineralization to being intrafibrillar, constrained, and aligned in the regions of high mineralization. This shift in mineral organization directly affects percolation thresholds resulting in changes in the residual and root mean square strain as well as the local mechanical properties. These results indicate that then tendon-to-bone attachment uses a variety of structural techniques to finely control the local mechanical properties.

Micrometer scale analysis of the attachment area showed that both loaded and unloaded samples exhibit gradients in mineral spanning approximately 10 µm. Along this gradient, the residual strain and root mean square strain become more compressive with increasing mineralization. This increase in compressive stress may indicate that crystals are becoming more confined and reaching a percolation threshold. The level of this threshold would be controlled by the structural differences in crystal shape, orientation, and location and precisely control the local mechanics. This indicates that the attachment uses a complex number of variables to control local mechanics. Overall, all of the proposed tasks have been achieved over the past 3 years. We now have a much deeper understanding of how the tendon-to-bone attachment reacts to unloading from both a structural and mechanical perspective.

The goal of this project is to investigate the effect of unloading on the tendon-to-bone attachment at a number of hierarchical scales. At the nanometer scale, I proposed to examine changes in the organization of the bone mineral relative to the collagen fibrils with unloading via Transmission electron microscopy-electron energy loss spectroscopy (TEM-EELS). At a scale on the order of micrometers, I was interested in employing Raman and high-energy nano-x-ray diffraction (XRD) to identify modifications made to the mineral content, composition, structure and organization in the interfacial tissue. Micro-mechanical testing would be employed to examine changes in the mechanics at the micron scale. At the millimeter scale. Micro-computed tomography (µCT) and tensile testing were employed to determine the effects of unloading on bone quantity and quality and tissue mechanics.

In the past two years, significant discoveries have been made explaining how the structure and mechanics of the tendon-to-bone attachment vary with unloading. Botulinum Toxin A (Btx) injections into the supraspinatus muscle were employed as an Earthbound model of disuse and unloading. At the millimeter scale, I found that 3 weeks of unloading resulted in a ~30% loss in bone volume as measured by µCT. Surprisingly, this loss was accompanied by a significant increases in tendon elastic modulus. This change in modulus is likely due to a changes in the soft tissue composition during unloading or structural changes at lower hierarchical scales. The tendon-to-bone attachment exhibits a gradient in mineral content at the micrometer scale. The width of this mineral gradient remains unaffected by unloading. However, nano-XRD studies indicate that the gradient region exhibits a variety of changes in mineral organization and structure. Traversing from the unmineralized tendon to the mineralized bone, the mineral crystals exhibit increased size, alignment, and compressive strain in both control and unloaded samples. However, unloading results in greater misalignment of the mineral crystals and smaller crystal sizes. Inputting these changes in crystal size and alignment into a basic rotational model it was determined that unloading leads to a decrease in work of rotation with unloading, leading to a decrease in tissue toughness. Raman measurements of the mineralized tissues showed that the mineral composition is modified due to unloading resulting in a decrease in carbonate substitution in the mineral structure. Together these results show that the effects of unloading are not limited to a single hierarchical scale but affect the interfacial tissue at a number of length scales. By understanding what structural and mechanical features are modified by unloading we can better focus our efforts when developing strategies for injury prevention and repair.

For the next year, I will continue to examine the structure and mechanics across the length scales. I have established a collaboration with Prof. Anthony Lau at the College of New Jersey examining the structural efficiency of the bone architecture. Comparing Btx treated samples to space flight samples will serve to validate Btx treatment as a microgravity analogue.

To better understand the increased modulus of the tendon-to-bone attachment I am also undertaking a study examining changes to the failure mode and failure area with unloading. Finally, at the nano-meter scale, we have preliminary evidence that mineral crystals are located both within and on the surface of collagen fibrils in regions of high mineralization but become uniquely extra-fibrillar with decreased mineralization. Samples have been embedded and sectioned to perform a full study examining nano-scale mineral distribution via TEM-EELS. I believe that we will see a decrease in extra-fibrillar mineral with unloading which would help explain the decrease in crystal size seen by XRD.

At the micrometer scale, I was interested in examining the gradient region of the tendon to bone attachment where the composition transitions from fully mineralized to unmineralized. X-ray fluorescence and Raman spectroscopy results both indicate that there is no change in the width of this graded region with unloading. However, the composition, structure, and organization of the mineral crystals in the graded region is modified. Nano-X-ray diffraction measurements performed across the graded region show that trends in the mineral size, orientation and stiffness are the same for loaded and unloaded samples. Moving from the unmineralized to mineralized regions of the attachment the crystal size, compressive residual strain, and crystal alignment increase. However, the unloaded samples exhibit greater misalignment and decreased crystal size. Rotational models showed that these changes induced by unloading resulted in a decreased work of rotation upon loading suggesting a decrease in toughness.

At the nanometer scale, Transmission Electron Microscopy–Electron Energy Loss Spectroscopy (TEM-EELS) has been used to examine the location of mineral crystals relative to the collagen fibrils in healthy tendon-to-bone attachments. I intend to pursue this preliminary study to understand how unloading modifies the nano-structure of the tendon-to-bone attachment. Together, the results gathered in the past year provide important information in understanding what features of the tendon-to-bone attachment are affected by unloading. This understanding will allow us to better focus our efforts when developing strategies for reducing rotator cuff tears and other musculoskeletal injuries.

Rotator cuff injuries often occur at the site of the tendon-to-bone attachment. The sensitivity of the musculoskeletal system to its loading environment may augment the risk of injury at insertion sites due to extended periods of microgravity or unloading. Long-term changes in mechanical loading on joints, such as may be experienced during extended space travel, will lead to modifications in the tissues' structural and therefore mechanical properties. The goal of this study is to investigate the effect of unloading on the microstructure and mechanics of the tendon-to-bone attachment at the nanometer and micrometer scale.

At the nanometer scale, I proposed to determine the location of mineral relative to the collagen fibril using Scanning Transmission Electron Microscopy (STEM) and identify load-transfer behavior between the two phases via synchrotron x-ray diffraction (XRD). At the micrometer scale, I proposed to study the mineral organization across the attachment site using synchrotron XRD and measure the micromechanics using a novel scanning electron microscopy-atomic force microscopy (SEM/AFM) system. Unloading of the tissue was done by injection of Botulinum Toxin (BtxA) which causes local paralysis.

In the past year, I have focused on developing the techniques and acquiring data related to the micrometer scale structure and mechanics. Synchrotron XRD was used to examine mineral organization in supraspinatus-humerus tendon-to-bone attachments. Mineral orientation, size, strain, and strain distribution were calculated from the XRD patterns taken across the attachment. Moving from the unmineralized to the mineralized tissue the mineral orientation became more aligned. The crystal size increased with mineralization while the strain and strain distribution decreased. These changes in crystal organization suggest that as mineralization increases the crystals become more organized within the collagen matrix and form a continuous mineral matrix. With this baseline established, preliminary data examining only the mineralized region of unloaded shoulders were obtained. They indicated that BtxA treatment has significant effects on crystal size, strain, and orientation. To test the micromechanics of the tendon-to-bone attachment, I used a novel technique which combines SEM for imaging and AFM for mechanical testing. This technique has been previously used on mineralized tissues and polymer fibers, but never for unmineralized or graded tissues. A fair amount of time has been dedicated to identifying the issues and challenges associated with testing attachment samples and developing a new testing protocol. The new completed protocol uses Laser capture microscopy and cryo-focused ion beam to prepare small beams of the tendon-to-bone insertion. These small beams are then tested mechanically until failure using an AFM system mounted within an SEM. The SEM allow for the visualization of the sample deformation. Data from the SEM and AFM can then be analyzed to obtain stress-strain and local strain information. All of the current findings support the original research plan; as a result the original specific aims will continue to be pursued. Both techniques for the micrometer scale testing have now been validated.

I have applied for access to the synchrotron beam line and hope to obtain data for the Btx treated samples in spring 2015. A new batch of samples was recently sent to London for micro-mechanical testing according to the new protocol. I hope to obtain a full set of mechanical data by December 2014. At the nanoscale I expect to begin the STEM experiments in October 2014 and complete them by January 2015. Preliminary tests for the synchrotron loading experiments will be performed at the Spring 2015 synchrotron access. Further access in the summer/fall 2015 will be requested to complete the experiments.

In the past year, I have focused on developing the techniques and acquiring data related to the micrometer scale structure and mechanics. Sections of healthy tendon-to-bone attachments were examined using nano-scale synchrotron XRD at Argonne National Laboratory. XRD patterns were obtained across the attachment from the unmineralized to the mineralized fibrocartilage. Mineral orientation, size, strain, and strain distribution were calculated from the XRD patterns. Moving from the unmineralized to the mineralized tissue the mineral orientation became more aligned. The crystal size increased with mineralization while the strain and strain distribution decreased. These changes in crystal organization suggest that as mineralization increases the crystals become more organized within the collagen matrix and form a continuous mineral matrix. Preliminary data examining only the mineralized region of unloaded shoulders indicate the BtxA treatment has significant effects on crystal size, strain, and orientation.

Access to the synchrotron has been requested to obtain additional data on the BtxA treated samples. To test the micromechanics of the tendon-to-bone attachment, I used a novel technique which combines SEM for imaging and AFM for mechanical testing. This technique has been previously used on mineralized tissues and polymer fibers, but never for unmineralized or graded tissues. A fair amount of time has been dedicated to identifying the issues and challenges associated with testing attachment samples and developing a new testing protocol. The new completed protocol uses Laser capture microscopy and cryo-focused ion beam to prepare small beams of the tendon-to-bone insertion. These small beams are then tested mechanically until failure using and AFM system mounted within an SEM. The SEM allow for the visualization of the sample deformation. Data from the SEM and AFM can then be analyzed to obtain stress-strain and local strain information. New beams of the attachment have recently been sent to London to be tested.

Rotator cuff injuries often occur at the site of tendon-to-bone attachments, also called the insertion site or enthesis. Long-term changes in mechanical loading on joints, such as may be experienced during extended space travel, may magnify the injury risk to the tendons. Even in the best conditions on Earth, these injuries do not heal well and will severely debilitate an injured astronaut. Unloading the musculoskeletal system leads to rapid bone resorption, loss of bone mass, and decreased mechanical properties.

However, much less is known about the results of extended weightlessness or unloading on the interfaces between hard and soft tissues. The tendon-to-bone attachment site achieves an effective connection between tendon and bone through a multi-scale structural organization. On the nanometer scale, mineralized collagen fibrils serve as templates for mineral deposition, which provides stiffness to the attachment. On the micrometer scale, these mineralized fibrils create gradients in both mineral content and collagen fibril orientation. These are responsible for dissipating stress concentrations, thus limiting the risk of failure. Changes to this structure caused by unloading can result in changes to the mechanics of the attachment. The overall objective of this project is to determine the effect of unloading on the structural, and in turn mechanical, properties of the tendon-to-bone attachment.

To achieve this goal, mouse rotator cuff attachments will be unloaded via botulinum toxin injections and examined using cutting edge techniques. At the nanoscale, we will determine the effects of unloading on the mineral organization in relation to the collagen fibril and in turn how it affects the mechanics. We expect that the unloading will cause a decrease in mineral at the attachment. This mineral is necessary to stiffen and toughen the attachment; therefore, unloading is expected to cause a decrease in these properties.

At the micrometer scale, previous work has shown that unloading causes a decrease in collagen fibril alignment. Since the collagen serves as the template for mineralization, we will examine how unloading affects the mineral organization and how that may affect the mechanics. We expect that increased collagen disorder will lead to increased mineral disorder. In turn, this increased disorder will decrease the load-bearing efficiency of the structure. As a result, the strength of the insertion will decrease with unloading.

With a better understanding of the effects of unloading on the tendon-to-bone insertion, we can start to develop preventative measures to maintain the health of the tendon-to-bone attachments in astronauts exposed to long-term microgravity.