Neural network auto-segmentation of serial-block-face scanning electron microscopy images exhibit collagen fibril structural differences with tendon type and health.

A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

Overload in a Rat In Vivo Model of Synergist Ablation Induces Tendon Multiscale Structural and Functional Degeneration.

Tendon degeneration is typically described as an overuse injury with little distinction made between magnitude of load (overload) and number of cycles (overuse). Further, in vivo, animal models of tendon degeneration are mostly overuse models, where tendon damage is caused by a high number of load cycles. As a result, there is a lack of knowledge of how isolated overload leads to degeneration in tendons. A surgical model of synergist ablation (SynAb) overloads the target tendon, plantaris, by ablating its synergist tendon, Achilles. The objective of this study was to evaluate the structural and functional changes that occur following overload of plantaris tendon in a rat SynAb model. Tendon cross-sectional area (CSA) and shape changes were evaluated by longitudinal MR imaging up to 8 weeks postsurgery. Tissue-scale structural changes were evaluated by semiquantified histology and second harmonic generation microscopy. Fibril level changes were evaluated with serial block face scanning electron microscopy (SBF-SEM). Functional changes were evaluated using tension tests at the tissue and microscale using a custom testing system allowing both video and microscopy imaging. At 8 weeks, overloaded plantaris tendons exhibited degenerative changes including increases in CSA, cell density, collagen damage area fraction (DAF), and fibril diameter, and decreases in collagen alignment, modulus, and yield stress. To interpret the differences between overload and overuse in tendon, we introduce a new framework for tendon remodeling and degeneration that differentiates between the inputs of overload and overuse. In summary, isolated overload induces multiscale degenerative structural and functional changes in plantaris tendon.

Stress deprivation of tendon explants or Tpm3.1 inhibition in tendon cells reduces F-actin to promote a tendinosis-like phenotype.

Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Tendon Multiscale Structure, Mechanics, and Damage Are Affected by Osmolarity of Bath Solution.

One of the most common bath solutions used in musculoskeletal mechanical testing is phosphate buffered saline (PBS). In tendon, swelling induced by physiological PBS results in decreased tendon modulus and induces microstructural changes. It is critical to evaluate the multiscale mechanical behavior of tendon under swelling to interpret prior work and provide information to design future studies. We compared the effects of physiological PBS and 8% polyethylene glycol and saline bathing solutions on tendon multiscale tendon mechanics and damage as well as microstructure with TEM in order to understand the effect of swelling on tendon. At the tissue level, tendons in PBS had a lower modulus than SPEG samples. PBS samples also showed an increased amount of non-recoverable sliding, which is an analog for microscale damage. SPEG had a higher microscale to tissue-scale strain ratio, showing the fibrils experienced less strain attenuation. From the TEM data, we showed the fibril spacing of SPEG samples was more similar to fresh control than PBS. We concluded that swelling alters multiscale mechanics and damage in addition to tendon microstructure. Future mechanical testing should consider using SPEG as a bath solution with an osmotic pressure which preserves fresh tissue water content.

Evaluation of transverse poroelastic mechanics of tendon using osmotic loading and biphasic mixture finite element modeling.

Tendon's viscoelastic behaviors are important to the tissue mechanical function and cellular mechanobiology. When loaded in longitudinal tension, tendons often have a large Poisson's ratio (ν>2) that exceeds the limit of incompressibility for isotropic material (ν=0.5), indicating that tendon experiences volume loss, inducing poroelastic fluid exudation in the transverse direction. Therefore, transverse poroelasticity is an important contributor to tendon material behavior. Tendon hydraulic permeability which is required to evaluate the fluid flow contribution to viscoelasticity, is mostly unavailable, and where available, varies by several orders of magnitude. In this manuscript, we quantified the transverse poroelastic material parameters of rat tail tendon fascicles by conducting transverse osmotic loading experiments, in both tension and compression. We used a multi-start optimization method to evaluate the parameters using biphasic finite element modeling. Our tendon samples had a transverse hydraulic permeability of 10-4 to 10-5 mm4. (Ns)-1 and showed a significant tension-compression nonlinearity in the transverse direction. Further, using these results, we predict hydraulic permeability during longitudinal (fiber-aligned) tensile loading, and the spatial distribution of fluid flow during osmotic loading. These results reveal novel aspects of tendon mechanics and can be used to study the physiomechanical response of tendon in response to mechanical loading.

An inter-species computational analysis of vibrotactile sensitivity in Pacinian and Herbst corpuscles.

Vibration sensing is ubiquitous among vertebrates, with the sensory end organ generally being a multilayered ellipsoidal structure. There is, however, a wide range of sizes and structural arrangements across species. In this work, we applied our earlier computational model of the Pacinian corpuscle to predict the sensory response of different species to various stimulus frequencies, and based on the results, we identified the optimal frequency for vibration sensing and the bandwidth over which frequencies should be most detectable. We found that although the size and layering of the corpuscles were very different, almost all of the 19 species studied showed very similar sensitivity ranges. The human and goose were the notable exceptions, with their corpuscle tuned to higher frequencies (130-170 versus 40-50 Hz). We observed no correlation between animal size and any measure of corpuscle geometry in our model. Based on the results generated by our computational model, we hypothesize that lamellar corpuscles across different species may use different sizes and structures to achieve similar frequency detection bands.

Micropipette aspiration of the Pacinian corpuscle.

The Pacinian corpuscle (PC) is a cutaneous mechanoreceptor sensitive to high-frequency vibrations (20-1000Hz). The PC is of importance due to its integral role in somatosensation and the critical need to understand PC function for haptic feedback system development. Previous theoretical and computational studies have modeled the physiological response of the PC to sustained or vibrating mechanical stimuli, but they have used estimates of the receptor's mechanical properties, which remain largely unmeasured. In this study, we used micropipette aspiration (MPA) to determine an apparent Young's modulus for PCs isolated from a cadaveric human hand. MPA was applied in increments of 5mm H2O (49Pa), and the change in protrusion length of the PC into the pipette was recorded. The protrusion length vs. suction pressure data were used to calculate the apparent Young's modulus. Using 10 PCs with long-axis lengths of 2.99±0.41mm and short-axis lengths of 1.45±0.22mm, we calculated a Young's modulus of 1.40±0.86kPa. Our measurement is on the same order of magnitude as those approximated in previous models, which estimated the PC to be on the same order of magnitude as skin or isolated cells, so we recommend that a modulus in the kPa range be used in future studies.