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.

Development and Validation of the Patient/Caregiver Reported Hydroxyurea Evaluation of Adherence for Life (HEAL) Scale.

Introduction: Hydroxyurea reduces the incidence of vaso-occlusive episodes, stroke, and respiratory, cardiac, and renal damage in sickle cell disease by increasing fetal hemoglobin. However, because suboptimal adherence to hydroxyurea limits its effectiveness, understanding patient-specific barriers to hydroxyurea adherence could help improve adherence and health outcomes in patients with sickle cell disease. The aim of this single-site, prospective, IRB-approved study was to validate a 24-item patient- and caregiver-reported hydroxyurea treatment adherence questionnaire, the Hydroxyurea Evaluation of Adherence for Life (HEAL) scale. Methods: A sample of 24 adults with sickle cell disease and 16 caregivers of children with sickle cell disease completed the HEAL scale, and a subset of the original sample provided a second HEAL scale for test-retest reliability. HEAL scale results were validated against global adherence ratings from participants and health-care providers, records of access to pill bottles, and laboratory values for fetal hemoglobin and absolute neutrophil count. Results and Discussion: Results demonstrated excellent internal consistency for the HEAL Total score and eight (3-item) subscale scores (Dose, Remember, Plan, Cost, Understand, Effectiveness, Laboratory, and Pharmacy), as well as strong test-retest reliability for all HEAL scores except the Cost subscale. HEAL Total scores correlated significantly with validity measures, including global adherence ratings and lab values. The HEAL scale offers significant clinical potential for understanding adherence in individual sickle cell disease patients and significant research potential for characterizing adherence in persons with sickle cell disease who are treated with hydroxyurea.

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.

No child left behind: Building a comprehensive sickle cell disease care oasis in the Lake County, Indiana care desert.

BACKGROUND: Sickle cell disease (SCD) leads to end-organ damage and shortened life expectancy. The second highest incidence of SCD in Indiana is in Lake County, but until 2017, there was no SCD expert within 65 miles. The Indiana Hemophilia and Thrombosis Center (IHTC) developed the Sickle Care coordination OutReach and Education (SCORE) program in 2017 to bring high-quality, guideline-based care to children with SCD. PROCEDURE: The St. Vincent IRB deemed this retrospective analysis of SCORE clinic care from 2017 to 2020 exempt. Data on the number of transcranial Dopplers (TCD) performed, HU dosing escalation, and vaccine rates were collected along with the number of school and home visits completed. RESULTS: Fifty-three children, adolescents, and young adults completed 288 SCORE clinic visits during the study period; over 75% completed at least three clinic visits. Mean HU dose increased significantly with SCORE clinic care. TCD screening rates increased every year starting in 2018 through 2020 when we added local care coordination. One hundred seventy-three vaccines were administered in SCORE outreach clinics. The PPSV23 vaccines had a 100% acceptance rate, and seasonal influenza had a 75.8% acceptance rate. CONCLUSION: Access to care coordination services and local hematology specialty care alleviates barriers to care and enables comprehensive SCD care delivery to children in need. Prior to the establishment of the SCORE clinic, 75% of children in Lake County were not receiving recommended stroke screening. The SCORE clinic model demonstrates feasibility and impact when delivering on the promise of high-quality care for children with SCD.

Ring the Bell for Sickle Cell: Encouraging Advocacy in an Underserved Community.

Sickle cell disease (SCD) was once a disease of childhood because of a limited life expectancy. Due to medical advances, it is now common for people with SCD to live into adulthood. Funding and resources for adults with SCD, however, remain limited. Adult patients would benefit from increased access to medical care, mental health care services, and workforce development. The Indiana Sickle Cell Consortium, a group of medical providers and community-based organizations, worked closely with people living with SCD and their family members to create a campaign advocating for state funding for programs for adults with SCD. This campaign culminated with the passage of a bill that provides $250,000 in funding for program development for adults with SCD. The bill also directs the Indiana Department of Health to carry out a needs assessment for people with SCD in Indiana. However, continued efforts are needed to reduce health disparities for people with SCD. The Indiana Sickle Cell Consortium will continue advocacy efforts in future legislative cycles and bring attention to the health inequities that affect people with SCD.

Tuning the Structure of Nylon 6,6 Electrospun Bundles to Mimic the Mechanical Performance of Tendon Fascicles.

Tendon and ligament injuries are triggered by mechanical loading, but the specific mechanisms are not yet clearly identified. It is well established however, that the inflection and transition points in tendon stress-strain curves represent thresholds that may signal the onset of irreversible fibrillar sliding. This phenomenon often results in a progressive macroscopic failure of these tissues. With the aim to simulate and replace tendons, electrospinning has been demonstrated to be a suitable technology to produce nanofibers similar to the collagen fibrils in a mat form. These nanofibrous mats can be easily assembled in higher hierarchical levels to reproduce the whole tissue structure. Despite the fact that several groups have developed electrospun tendon-inspired structures, an investigation of the inflection and transition point mechanics is missing. Comparing their behavior with that of the natural counterpart is important to adequately replicate their behavior at physiological strain levels. To fill this gap, in this work fascicle-inspired electrospun nylon 6,6 bundles were produced with different collector peripheral speeds (i.e., 19.7 m s-1; 13.7 m s-1; 7.9 m s-1), obtaining different patterns of nanofibers alignment. The scanning electron microcopy revealed a fibril-inspired structure of the nanofibers with an orientation at the higher speed similar to those in tendons and ligaments (T/L). A tensile mechanical characterization was carried out showing an elastic-brittle biomimetic behavior for the higher speed bundles with a progressively more ductile behavior at slower speeds. Moreover, for each sample category the transition and the inflection points were defined to study how these points can shift with the nanofiber arrangement and to compare their values with those of tendons. The results of this study will be of extreme interest for the material scientists working in the field, to model and improve the design of their electrospun structures and scaffolds and enable building a new generation of artificial tendons and ligaments.

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.

Adherence to Quality of Care Indicators and Location of Sickle Cell Care Within Indiana.

Newborn screening (NBS) follow-up programs for infants with sickle cell disease (SCD) are highly variable among states. Initiated in 2009, Sickle SAFE, the NBS follow-up program for infants with SCD in Indiana, follows infants through home visits and phone contact. The current study assessed the attainment rates for recently published quality indicators of pediatric SCD care for Sickle SAFE participants. Using retrospective data, we determined the proportion of children who received transcranial Doppler (TCD) screening, influenza, and pneumococcal vaccination and were prescribed hydroxyurea. We calculated the mean age at confirmatory testing, time to receipt of penicillin prophylaxis, and mean age when genetic counseling was offered. One hundred ninety-eight children born with SCD in Indiana between July 1, 2009 and June 30, 2017 were followed for at least 1 year. While 97.5% received at least one dose of conjugated pneumococcal vaccine, vaccination with the 23 valent pneumococcal vaccine varied by location (county) of care (Allen: 14.3%, Lake: 26.7%, St. Joseph: 40.0%, Marion: 73.3%). Overall TCD screening rate for eligible children was 53%; TCD screening rate varied widely by location of care (Lake: 25% vs. Marion: 63.8%). Similarly, hydroxyurea prescribing practices varied significantly by location of care (p < 0.001). Identified gaps in adherence to quality indicators in SCD care will serve as the basis for future quality improvement initiatives.

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.

A validated measure of adherence to antibiotic prophylaxis in children with sickle cell disease.

BACKGROUND: Antibiotic prophylaxis is a mainstay in sickle cell disease management. However, adherence is estimated at only 66%. This study aimed to develop and validate a Sickle Cell Antibiotic Adherence Level Evaluation (SCAALE) to promote systematic and detailed adherence evaluation. METHODS: A 28-item questionnaire was created, covering seven adherence areas. General Adherence Ratings from the parent and one health care provider and medication possession ratios were obtained as validation measures. RESULTS: Internal consistency was very good to excellent for the total SCAALE (α=0.89) and four of the seven subscales. Correlations between SCAALE scores and validation measures were strong for the total SCAALE and five of the seven subscales. CONCLUSION: The SCAALE provides a detailed, quantitative, multidimensional, and global measurement of adherence and can promote clinical care and research.