MicroRNA Nano-Shuttles: Engineering Extracellular Vesicles as a Cutting-Edge Biotechnology Platform for Clinical Use in Therapeutics.

Extracellular vesicles (EVs) are nano-sized, membranous transporters of various active biomolecules with inflicting phenotypic capabilities, that are naturally secreted by almost all cells with a promising vantage point as a potential leading drug delivery platform. The intrinsic characteristics of their low toxicity, superior structural stability, and cargo loading capacity continue to fuel a multitude of research avenues dedicated to loading EVs with therapeutic and diagnostic cargos (pharmaceutical compounds, nucleic acids, proteins, and nanomaterials) in attempts to generate superior natural nanoscale delivery systems for clinical application in therapeutics. In addition to their well-known role in intercellular communication, EVs harbor microRNAs (miRNAs), which can alter the translational potential of receiving cells and thus act as important mediators in numerous biological and pathological processes. To leverage this potential, EVs can be structurally engineered to shuttle therapeutic miRNAs to diseased recipient cells as a potential targeted 'treatment' or 'therapy'. Herein, this review focuses on the therapeutic potential of EV-coupled miRNAs; summarizing the biogenesis, contents, and function of EVs, as well as providing both a comprehensive discussion of current EV loading techniques and an update on miRNA-engineered EVs as a next-generation platform piloting benchtop studies to propel potential clinical translation on the forefront of nanomedicine.

Treatment with PB125® Increases Femoral Long Bone Strength in 15-Month-Old Female Hartley Guinea Pigs.

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a transcription factor that serves as a master regulator of anti-inflammatory agents, phase I xenobiotic, and phase II antioxidant enzymes, all of which provide a cytoprotective role during disease progression. We hypothesized that oral administration of a purported phytochemical Nrf2-activator, PB125®, would increase long bone strength in aging Hartley guinea pigs, a model prone to musculoskeletal decline. Male (N = 56) and female (N = 56) guinea pigs were randomly assigned to receive daily oral treatment with either PB125® or vehicle control. Animals were treated for a consecutive 3-months (starting at 2-months of age) or 10-months (starting at 5-months of age) and sacrificed at 5-months or 15-months of age, respectively. Outcome measures included: (1) ANY-maze™ enclosure monitoring, (2) quantitative microcomputed tomography, and (3) biomechanical testing. Treatment with PB125® for 10 months resulted in increased long bone strength as determined by ultimate bending stress in female Hartley guinea pigs. In control groups, increasing age resulted in significant effects on geometric and structural properties of long bones, as well as a trending increase in ultimate bending stress. Furthermore, both age and sex had a significant effect on the geometric properties of both cortical and trabecular bone. Collectively, this work suggests that this nutraceutical may serve as a promising target and preventive measure in managing the decline in bone mass and quality documented in aging patients. Auxiliary to this main goal, this work also capitalized upon 5 and 15-month-old male and female animals in the control group to characterize age- and sex-specific differences on long bone geometric, structural, and material properties in this animal model.

Employing direct electromagnetic coupling to assess acute fracture healing: An ovine model assessment.

OBJECTIVES: This study explored the efficacy of collecting temporal fracture site compliance data via an advanced direct electromagnetic coupling (DEC) system equipped with a Vivaldi-type antenna, novel calibration technique, and multi-antenna setup (termed maDEC) as an approach to monitor acute fracture healing progress in a translational large animal model. The overarching goal of this approach was to provide insights into the acute healing dynamics, offering a promising avenue for optimizing fracture management strategies. METHODS: A sample of twelve sheep, subjected to ostectomies and intramedullary nail fixations, was divided into two groups, simulating normal and impaired healing scenarios. Sequential maDEC compliance or stiffness measurements and radiographs were taken from the surgery until euthanasia at four or eight weeks and were subsequently compared with post-sacrifice biomechanical, micro-CT, and histological findings. RESULTS: The results showed that the maDEC system offered straightforward quantification of fracture site compliance via a multiantenna array. Notably, the rate of change in the maDEC-measured bending stiffness significantly varied between normal and impaired healing groups during both the 4-week (p = 0.04) and 8-week (p = 0.02) periods. In contrast, radiographically derived mRUST healing measurements displayed no significant differences between the groups (p = 0.46). Moreover, the cumulative normalized stiffness maDEC data significantly correlated with post-sacrifice mechanical strength (r2 = 0.80, p < 0.001), micro-CT measurements of bone volume fraction (r2 = 0.60, p = 0.003), and density (r2 = 0.60, p = 0.003), and histomorphometric measurements of new bone area fraction (r2 = 0.61, p = 0.003) and new bone area (r2 = 0.60, p < 0.001). CONCLUSIONS: These data indicate that the enhanced maDEC system provides a non-invasive, accurate method to monitor fracture healing during the acute healing phase, showing distinct stiffness profiles between normal and impaired healing groups and offering critical insights into the healing process's progress and efficiency.

Alterations of biaxial viscoelastic properties of the right ventricle in pulmonary hypertension development in rest and acute stress conditions.

Introduction: The right ventricle (RV) mechanical property is an important determinant of its function. However, compared to its elasticity, RV viscoelasticity is much less studied, and it remains unclear how pulmonary hypertension (PH) alters RV viscoelasticity. Our goal was to characterize the changes in RV free wall (RVFW) anisotropic viscoelastic properties with PH development and at varied heart rates. Methods: PH was induced in rats by monocrotaline treatment, and the RV function was quantified by echocardiography. After euthanasia, equibiaxial stress relaxation tests were performed on RVFWs from healthy and PH rats at various strain-rates and strain levels, which recapitulate physiological deformations at varied heart rates (at rest and under acute stress) and diastole phases (at early and late filling), respectively. Results and Discussion: We observed that PH increased RVFW viscoelasticity in both longitudinal (outflow tract) and circumferential directions. The tissue anisotropy was pronounced for the diseased RVs, not healthy RVs. We also examined the relative change of viscosity to elasticity by the damping capacity (ratio of dissipated energy to total energy), and we found that PH decreased RVFW damping capacity in both directions. The RV viscoelasticity was also differently altered from resting to acute stress conditions between the groups-the damping capacity was decreased only in the circumferential direction for healthy RVs, but it was reduced in both directions for diseased RVs. Lastly, we found some correlations between the damping capacity and RV function indices and there was no correlation between elasticity or viscosity and RV function. Thus, the RV damping capacity may be a better indicator of RV function than elasticity or viscosity alone. These novel findings on RV dynamic mechanical properties offer deeper insights into the role of RV biomechanics in the adaptation of RV to chronic pressure overload and acute stress.

Experimental models of bone marrow lesions in ovine femoral condyles.

OBJECTIVE: To develop an in vivo experimental model for bone marrow lesions (BMLs) in ovine femorotibial joints. STUDY DESIGN: Randomized, prospective experimental study. ANIMALS: Eighteen healthy, skeletally-mature Dorper cross ewes. METHODS: One medial femoral condyle was penetrated with a 1.1 mm pin, and the contralateral medial femoral condyle was treated with transcutaneous extracorporeal shockwave (ESW) at 0.39 ± 0.04 mJ/mm2 . Clinical examination, magnetic resonance imaging (MRI), computed tomography (CT), and histopathological analyses were used to detect and characterize the development and progression of BMLs in the medial femoral condyle at 4, 8, and 12 weeks post-surgery. RESULTS: Pin penetration induced a BML detected on MRI within 2 weeks and lasted at least 12 weeks. BMLs were not observed in ESW-treated condyles. Histologically, BMLs were characterized by hemorrhage and inflammatory cellular infiltrate, and progressed to more dense fibrous tissue over time. Pathological changes were not observed in the articular cartilage overlying the region of BMLs. CONCLUSIONS: Direct, focal trauma to all layers of the osteochondral unit was sufficient to create an experimentally-induced BML which persisted for at least 90 days. The protocol used for ESW in this study did not induce BMLs. CLINICAL SIGNIFICANCE: Experimental induction of BMLs is possible and mimicked naturally occurring disease states. Volumetric imaging is a sensitive method for characterization of the dynamic nature of these lesions.

Biomechanical evaluation of a novel repair strategy for intervertebral disc herniation in an ovine lumbar spine model.

Following herniation of the intervertebral disc, there is a need for advanced surgical strategies to protect the diseased tissue from further herniation and to minimize further degeneration. Accordingly, a novel tissue engineered implant for annulus fibrosus (AF) repair was fabricated via three-dimensional fiber deposition and evaluated in a large animal model. Specifically, lumbar spine kinetics were assessed for eight (n = 8) cadaveric ovine lumbar spines in three pure moment loading settings (flexion-extension, lateral bending, and axial rotation) and three clinical conditions (intact, with a defect in the AF, and with the defect treated using the AF repair implant). In ex vivo testing, seven of the fifteen evaluated biomechanical measures were significantly altered by the defect. In each of these cases, the treated spine more closely approximated the intact biomechanics and four of these cases were also significantly different to the defect. The same spinal kinetics were also assessed in a preliminary in vivo study of three (n = 3) ovine lumbar spines 12 weeks post-implantation. Similar to the ex vivo results, functional efficacy of the treatment was demonstrated as compared to the defect model at 12 weeks post-implantation. These promising results motivate a future large animal study cohort which will establish statistical power of these results further elucidate the observed outcomes, and provide a platform for clinical translation of this novel AF repair patch strategy. Ultimately, the developed approach to AF repair holds the potential to maintain the long-term biomechanical function of the spine and prevent symptomatic re-herniation.

Direct electromagnetic coupling to determine diagnostic bone fracture stiffness.

Background: Rapid prediction of adverse bone fracture healing outcome (e.g., nonunion and/or delayed union) is essential to advise adjunct therapies to reduce patient suffering and improving healing outcome. Radiographic diagnostic methods remain ineffective during early healing, resulting in average nonunion diagnosis times surpassing six months. To address this clinical deficit, we developed a novel diagnostic device to predict fracture healing outcome by noninvasive telemetric measurements of fracture bending stiffness. This study evaluated the hypothesis that our diagnostic antenna system is capable of accurately measuring temporal fracture healing stiffness, and advises the utility of this data for expedited prediction of healing outcomes during early (≤3 weeks) fracture recovery. Methods: Fracture repair was simulated, in reverse chronology, by progressively destabilizing cadaveric ovine metatarsals (n=8) stabilized via locking plate fixation. Bending stiffness of each fracture state were predicted using a novel direct electromagnetic coupling diagnostic system, and results were compared to values from material testing (MT) methods. While direct calculation of fracture stiffness in a simplistic cadaver model is possible, comparable analysis of the innumerable permutations of fracture and treatment type is not feasible. Thus, clinical feasibility of direct electromagnetic coupling was explored by parametric finite element (FE) analyses (n=1,632 simulations). Implant mechanics were simulated throughout the course of healing for cases with variations to fracture size, implant type, implant structure, and implant material. Results: For all fracture states, stiffness values predicted by the direct electromagnetic coupling system were not significantly different than those quantified by in vitro MT methods [P=0.587, P=0.985, P=0.975; for comparing intact, destabilized, and fully fractured (FF) states; respectively]. In comparable models, the total implant deflection reduction (from FF to intact states) was less than 10% different between direct electromagnetic coupling measurements (82.2 µm) and FE predictions (74.7 µm). For all treatment parameters, FE analyses predicted nonlinear reduction in bending induced implant midspan deflections for increasing callus stiffness. Conclusions: This technology demonstrates potential as a noninvasive clinical tool to accurately quantify healing fracture stiffness to augment and expedite healing outcome predictions made using radiographic imaging.

Strain-dependent stress relaxation behavior of healthy right ventricular free wall.

The increasing evidence of stress-strain hysteresis in large animal or human myocardium calls for extensive characterizations of the passive viscoelastic behavior of the myocardium. Several recent studies have investigated and modeled the viscoelasticity of the left ventricle while the right ventricle (RV) viscoelasticity remains poorly understood. Our goal was to characterize the biaxial viscoelastic behavior of RV free wall (RVFW) using two modeling approaches. We applied both quasi-linear viscoelastic (QLV) and nonlinear viscoelastic (NLV) theories to experimental stress relaxation data from healthy adult ovine. A three-term Prony series relaxation function combined with an Ogden strain energy density function was used in the QLV modeling, while a power-law formulation was adopted in the NLV approach. The ovine RVFW exhibited an anisotropic and strain-dependent viscoelastic behavior relative to anatomical coordinates, and the NLV model showed a higher capacity in predicting strain-dependent stress relaxation than the QLV model. From the QLV fitting, the relaxation term associated with the largest time constant played the dominant role in the overall relaxation behavior at most strains from early to late diastole, whereas the term associated with the smallest time constant was pronounced only at low strains at early diastole. From the NLV fitting, the parameters showed a nonlinear dependence on the strain. Overall, our study characterized the anisotropic, nonlinear viscoelasticity to capture the elastic and viscous resistances of the RVFW during diastole. These findings deepen our understanding of RV myocardium dynamic mechanical properties. STATEMENT OF SIGNIFICANCE: Although significant progress has been made to understand the passive elastic behavior of the right ventricle free wall (RVFW), its viscoelastic behavior remains poorly understood. In this study, we originally applied both quasi-linear viscoelastic (QLV) and nonlinear viscoelastic (NLV) models to published experimental data from healthy ovine RVFW. Our results revealed an anisotropic and strain-dependent viscoelastic behavior of the RVFW. The parameters from the NLV fitting showed nonlinear relationships with the strain, and the NLV model showed a higher capacity in predicting strain-dependent stress relaxation than the QLV model. These findings characterize the anisotropic, nonlinear viscoelasticity of RVFW to fully capture the total (elastic and viscous) resistance that is critical to diastolic function.

Vivaldi Antennas for Contactless Sensing of Implant Deflections and Stiffness for Orthopaedic Applications.

The implementation of novel coaxial dipole antennas has been shown to be a satisfactory diagnostic platform for the prediction of orthopaedic bone fracture healing outcomes. These techniques require mechanical deflection of implanted metallic hardware (i.e., rods and plates), which, when loaded, produce measurable changes in the resonant frequency of the adjacent antenna. Despite promising initial results, the coiled coaxial antenna design is limited by large antenna sizes and nonlinearity in the resonant frequency data. The purpose of this study was to develop two Vivaldi antennas (a.k.a., "standard" and "miniaturized") to address these challenges. Antenna behaviors were first computationally modeled prior to prototype fabrication. In subsequent benchtop tests, metallic plate segments were displaced from the prototype antennas via precision linear actuator while measuring resultant change in resonant frequency. Close agreement was observed between computational and benchtop results, where antennas were highly sensitive to small displacements of the metallic hardware, with sensitivity decreasing nonlinearly with increasing distance. Greater sensitivity was observed for the miniaturized design for both stainless steel and titanium implants. Additionally, these data demonstrated that by taking resonant frequency data during implant displacement and then again during antenna displacement from the same sample, via linear actuators, that "antenna calibration procedures" could be used to enable a clinically relevant quantification of fracture stiffness from the raw resonant frequency data. These improvements mitigate diagnostic challenges associated with nonlinear resonant frequency response seen in previous antenna designs.

Intubation biomechanics: Computational modeling to identify methods to minimize cervical spine motion and spinal cord strain during laryngoscopy and tracheal intubation in an intact cervical spine.

STUDY OBJECTIVE: To minimize the risk of cervical spinal cord injury in patients who have cervical spine pathology, minimizing cervical spine motion during laryngoscopy and tracheal intubation is commonly recommended. However, clinicians may better aim to reduce cervical spinal cord strain during airway management of their patients. The aim of this study was to predict laryngoscope force characteristics (location, magnitude, and direction) that would minimize cervical spine motions and cord strains. DESIGN: We utilized a computational model of the adult human cervical spine and spinal cord to predict intervertebral motions (rotation [flexion/extension] and translation [subluxation]) and cord strains (stretch and compression) during laryngoscopy. INTERVENTIONS: Routine direct (Macintosh) laryngoscopy conditions were defined by a specific force application location (mid-C3 vertebral body), magnitude (48.8 N), and direction (70 degrees). Sixty laryngoscope force conditions were simulated using 4 force locations (cephalad and caudad of routine), 5 magnitudes (25-200% of routine), and 3 directions (50, 70, 90 degrees). MAIN RESULTS: Under all conditions, extension at Oc-C1 and C1-C2 were greater than in all other cervical segments. Decreasing force magnitude to values reported for indirect laryngoscopes (8-17 N) decreased cervical extension to ~50% of routine values. The cervical cord was most likely to experience potentially injurious compressive strain at C3, but force magnitudes ≤50% of routine (≤24.4 N) decreased strain in C3 and all other cord regions to non-injurious values. Changing laryngoscope force locations and directions had minor effects on motion and strain. CONCLUSIONS: The model predicts clinicians can most effectively minimize cervical spine motion and cord strain during laryngoscopy by decreasing laryngoscope force magnitude. Very low force magnitudes (<5 N, ~10% of routine) are necessary to decrease overall cervical extension to <50% of routine values. Force magnitudes ≤24.4 N (≤50% of routine) are predicted to help prevent potentially injurious compressive cord strain.

Multiscale Contrasts Between the Right and Left Ventricle Biomechanics in Healthy Adult Sheep and Translational Implications.

Cardiac biomechanics play a significant role in the progression of structural heart diseases (SHDs). SHDs alter baseline myocardial biomechanics leading to single or bi-ventricular dysfunction. But therapies for left ventricle (LV) failure patients do not always work well for right ventricle (RV) failure patients. This is partly because the basic knowledge of baseline contrasts between the RV and LV biomechanics remains elusive with limited discrepant findings. The aim of the study was to investigate the multiscale contrasts between LV and RV biomechanics in large animal species. We hypothesize that the adult healthy LV and RV have distinct passive anisotropic biomechanical properties. Ex vivo biaxial tests were performed in fresh sheep hearts. Histology and immunohistochemistry were performed to measure tissue collagen. The experimental data were then fitted to a Fung type model and a structurally informed model, separately. We found that the LV was stiffer in the longitudinal (outflow tract) than circumferential direction, whereas the RV showed the opposite anisotropic behavior. The anisotropic parameter K from the Fung type model accurately captured contrasting anisotropic behaviors in the LV and RV. When comparing the elasticity in the same direction, the LV was stiffer than the RV longitudinally and the RV was stiffer than the LV circumferentially, suggesting different filling patterns of these ventricles during diastole. Results from the structurally informed model suggest potentially stiffer collagen fibers in the LV than RV, demanding further investigation. Finally, type III collagen content was correlated with the low-strain elastic moduli in both ventricles. In summary, our findings provide fundamental biomechanical differences between the chambers. These results provide valuable insights for guiding cardiac tissue engineering and regenerative studies to implement chamber-specific matrix mechanics, which is particularly critical for identifying biomechanical mechanisms of diseases or mechanical regulation of therapeutic responses. In addition, our results serve as a benchmark for image-based inverse modeling technologies to non-invasively estimate myocardial properties in the RV and LV.

Relationship Between Glottic View and Intubation Force During Macintosh and Airtraq Laryngoscopy and Intubation.

BACKGROUND: Because intubation-mediated cervical spine and spinal cord injury are likely determined by intubation force magnitude, understanding the determinants of intubation force magnitude is clinically relevant. With direct (Macintosh) laryngoscopy, when glottic view is less favorable, anesthesiologists apply greater force. We hypothesized that, when compared with direct (Macintosh) laryngoscopy, intubation force with an optical indirect laryngoscope (Airtraq) would be less dependent on glottic visualization. METHODS: Using data obtained in a prior clinical study, we tested whether the slope of the intubation force versus glottic view relationship differed between intubations performed in 14 patients who were intubated twice, once with a Macintosh and once with an Airtraq videolaryngoscope. Slopes were compared using least-squares linear regression and robust regression. RESULTS: The slope of the intubation force (N) versus glottic view (%) relationship with the Macintosh (-0.679 [standard error {SE}, 0.147]) was significantly more negative than that of the Airtraq (-0.076 [SE, 0.246]). The least-squares regression difference in slopes was -0.603 (SE, 0.287); P = .046. The robust regression difference in slopes was -0.747 (SE, 0.187); P = .0005. Thus, when compared with the Macintosh, intubation force magnitude with Airtraq laryngoscopy was less dependent on glottic visualization. CONCLUSIONS: Previously, we reported that intubation force with the Airtraq was less in magnitude compared with the Macintosh. Our current study adds that intubation force also is less dependent on glottic view with Airtraq compared with the Macintosh.

A Large Animal Model for Orthopedic Foot and Ankle Research.

Trauma to the soft tissues of the ankle joint distal syndesmosis often leads to syndesmotic instability, resulting in undesired movement of the talus, abnormal pressure distributions, and ultimately arthritis if deterioration progresses without treatment. Historically, syndesmotic injuries have been repaired by placing a screw across the distal syndesmosis to provide rigid fixation to facilitate ligament repair. While rigid syndesmotic screw fixation immobilizes the ligamentous injury between the tibia and fibula to promote healing, the same screws inhibit normal physiologic movement and dorsiflexion. It has been shown that intact screw removal can be beneficial for long-term patient success; however, the exact timing remains an unanswered question that necessitates further investigation, perhaps using animal models. Because of the sparsity of relevant preclinical models, the purpose of this study was to develop a new, more translatable, large animal model that can be used for the investigation of clinical foot and ankle implants. Eight (8) skeletally mature sheep underwent stabilization of the left and right distal carpal bones following transection of the dorsal and interosseous ligaments while the remaining two animals served as un-instrumented controls. Four of the surgically stabilized animals were sacrificed 6 weeks after surgery while the remaining four animals were sacrificed 10 weeks after surgery. Ligamentous healing was evaluated using radiography, histology, histomorphometry, and histopathology. Overall, animals demonstrated a high tolerance to the surgical procedure with minimal complications. Animals sacrificed at 10 weeks post-surgery had a slight trend toward mildly decreased inflammation, decreased necrotic debris, and a slight increase in the healing of the transected ligaments. The overall degree of soft tissue fibrosis/fibrous expansion, including along the dorsal periosteal surfaces/joint capsule of the carpal bones was very similar between both timepoints and often exhibited signs of healing. The findings of this study indicate that the carpometacarpal joint may serve as a viable location for the investigation of human foot and ankle orthopedic devices. Future work may include the investigation of orthopedic foot and ankle medical devices, biologic treatments, and repair techniques in a large animal model capable of providing translational results for human treatment.

The Interventricular Septum Is Biomechanically Distinct from the Ventricular Free Walls.

The interventricular septum contributes to the pumping function of both ventricles. However, unlike the ventricular wall, its mechanical behavior remains largely unknown. To fill the knowledge gap, this study aims to characterize the biaxial and transmural variation of the mechanical properties of the septum and compare it to the free walls of the left and right ventricles (LV/RV). Fresh hearts were obtained from healthy, adult sheep. The septal wall was sliced along the mid-line into two septal sides and compared to the epicardial layers of the LV- and RV-free walls. Biaxial tensile mechanical tests and constitutive modeling were performed to obtain the passive mechanical properties of the LV- and RV-side of the septum and ventricular walls. We found that both sides of the septum were significantly softer than the respective ventricular walls, and that the septum presented significantly less collagen than the ventricular walls. At low strains, we observed the symmetric distribution of the fiber orientations and a similar anisotropic behavior between the LV-side and RV-side of the septum, with a stiffer material property in the longitudinal direction, rather than the circumferential direction. At high strains, both sides showed isotropic behavior. Both septal sides had similar intrinsic elasticity, as evidenced by experimental data and constitutive modeling. These new findings offer important knowledge of the biomechanics of the septum wall, which may deepen the understanding of heart physiology.

Intubation Biomechanics: Clinical Implications of Computational Modeling of Intervertebral Motion and Spinal Cord Strain during Tracheal Intubation in an Intact Cervical Spine.

BACKGROUND: In a closed claims study, most patients experiencing cervical spinal cord injury had stable cervical spines. This raises two questions. First, in the presence of an intact (stable) cervical spine, are there tracheal intubation conditions in which cervical intervertebral motions exceed physiologically normal maximum values? Second, with an intact spine, are there tracheal intubation conditions in which potentially injurious cervical cord strains can occur? METHODS: This study utilized a computational model of the cervical spine and cord to predict intervertebral motions (rotation, translation) and cord strains (stretch, compression). Routine (Macintosh) intubation force conditions were defined by a specific application location (mid-C3 vertebral body), magnitude (48.8 N), and direction (70 degrees). A total of 48 intubation conditions were modeled: all combinations of 4 force locations (cephalad and caudad of routine), 4 magnitudes (50 to 200% of routine), and 3 directions (50, 70, and 90 degrees). Modeled maximum intervertebral motions were compared to motions reported in previous clinical studies of the range of voluntary cervical motion. Modeled peak cord strains were compared to potential strain injury thresholds. RESULTS: Modeled maximum intervertebral motions occurred with maximum force magnitude (97.6 N) and did not differ from physiologically normal maximum motion values. Peak tensile cord strains (stretch) did not exceed the potential injury threshold (0.14) in any of the 48 force conditions. Peak compressive strains exceeded the potential injury threshold (-0.20) in 3 of 48 conditions, all with maximum force magnitude applied in a nonroutine location. CONCLUSIONS: With an intact cervical spine, even with application of twice the routine value of force magnitude, intervertebral motions during intubation did not exceed physiologically normal maximum values. However, under nonroutine high-force conditions, compressive strains exceeded potentially injurious values. In patients whose cords have less than normal tolerance to acute strain, compressive strains occurring with routine intubation forces may reach potentially injurious values.

High throughput computational evaluation of how scaffold architecture, material selection, and loading modality influence the cellular micromechanical environment in tissue engineering strategies.

BACKGROUND: In tissue engineering (TE) strategies, cell processes are regulated by mechanical stimuli. Although TE scaffolds have been developed to replicate tissue-level mechanical properties, it is intractable to experimentally measure and prescribe the cellular micromechanical environment (CME) generated within these constructs. Accordingly, this study aimed to fill this lack of understanding by modeling the CME in TE scaffolds using the finite element method. METHODS: A repeating unit of composite fiber scaffold for annulus fibrosus (AF) repair with a fibrin hydrogel matrix was prescribed a series of loading, material, and architectural parameters. The distribution of CME in the scaffold was predicted and compared to proposed target mechanics based on anabolic responses of AF cells. RESULTS: The multi-axial loading modality predicted the greatest percentage of cell volumes falling within the CME target envelope (%PTE) in the study (65 %PTE for 5.0% equibiaxial tensile strain with 50 kPa radial-direction compression; 7.6 %PTE without radial pressure). Additionally, the architectural scale had a moderate influence on the CME (maximum of 17 %PTE), with minimal change in the tissue-level properties of the scaffold. Scaffold materials and architectures had secondary influences on the predicted regeneration by modifying the tissue-level scaffold mechanics. CONCLUSIONS: Scaffold loading modality was identified as the critical factor for TE the AF. Scaffold materials and architecture were also predicted to modulate the scaffold loading and, therefore, control the CME indirectly. This study facilitated an improved understanding of the relationship between tissue-level and cell-level mechanics to drive anabolic cell responses for tissue regeneration.

Evaluation of lumbar spinal fusion utilizing recombinant human platelet derived growth factor-B chain homodimer (rhPDGF-BB) combined with a bovine collagen/ß-tricalcium phosphate (ß-TCP) matrix in an ovine model.

BACKGROUND CONTEXT: While the clinical effectiveness of recombinant human Platelet Derived Growth Factor-B chain homodimer combined with collagen and ß-tricalcium phosphate (rhPDGF-BB + collagen/ß-TCP) treatment for indications involving hindfoot and ankle is well-established, it is not approved for use in spinal interbody fusion, and the use of autograft remains the gold standard. PURPOSE: The purpose of this study was to compare the effects of rhPDGF-BB + collagen/ß-TCP treatment on lumbar spine interbody fusion in an ovine model to those of autograft bone and collagen/ß-TCP treatments using biomechanical, radiographic, and histological assessment techniques. STUDY DESIGN: Thirty-two skeletally mature Columbian Rambouillet sheep were used to evaluate the safety and effectiveness of rhPDGF-BB + collagen/ß-TCP matrix in a lumbar spinal fusion model. Interbody polyetheretherketone (PEEK) cages contained either autograft, rhPDGF-BB + collagen/ß-TCP, collagen/ß-TCP matrix, or left empty. METHODS: Animals were sacrificed 8- or 16-weeks post-surgery. Spinal fusion was evaluated via post-sacrifice biomechanical, micro-computed tomography (µCT), and histological analysis. Outcomes were statistically compared using a two-way analysis of variance (ANOVA) with an alpha value of 0.05 and a Tukey post-hoc test. RESULTS: There were no statistically significant differences between groups within treatment timepoints for flexion-extension, lateral bending, or axial rotation range of motion, neutral zone, neutral zone stiffness, or elastic zone stiffness. µCT bone volume fraction was significantly greater between treatment groups independent of timepoint where Autograft and rhPDGF-BB + collagen/ß-TCP treatments demonstrated significantly greater bone volume fraction as compared to collagen/ß-TCP (P = .026 and P = .038, respectively) and Empty cage treatments (P = .002 and P = .003, respectively). µCT mean bone density fraction was most improved in rhPDGF-BB + collagen/ß-TCP specimens at the 8 week and 16-week timepoints as compared to all other treatment groups. There were no statistically significant differences in histomorphometric measurements of bone, soft tissue, or empty space between rhPDGF-BB + collagen/ß-TCP and autograft treatments. CONCLUSIONS: The results of this study indicate that the use of rhPDGF-BB combined with collagen/ß-TCP promotes spinal fusion comparable to that of autograft bone. CLINICAL SIGNIFICANCE: The data indicate that rhPDGF-BB combined with collagen/ß-TCP promotes spinal fusion comparably to autograft bone treatment and may offer a viable alternative in large animal spinal fusion. Future prospective clinical studies are necessary to fully understand the role of rhPDGF-BB combined with collagen/ß-TCP in human spinal fusion healing.

Diagnostic prediction of ovine fracture healing outcomes via a novel multi-location direct electromagnetic coupling antenna.

BACKGROUND: Expedient prediction of adverse bone fracture healing (delayed- or non-union) is necessary to advise secondary treatments for improving healing outcome to minimize patient suffering. Radiographic imaging, the current standard diagnostic, remains largely ineffective at predicting nonunions during the early stages of fracture healing resulting in mean nonunion diagnosis times exceeding six months. Thus, there remains a clinical deficit necessitating improved diagnostic techniques. It was hypothesized that adverse fracture healing expresses impaired biological progression at the fracture site, thus resulting in reduced temporal progression of fracture site stiffness which may be quantified prior to the appearance of radiographic indicators of fracture healing (i.e., calcified tissue). METHODS: A novel multi-location direct electromagnetic coupling antenna was developed to diagnose relative changes in the stiffness of fractures treated by metallic orthopaedic hardware. The efficacy of this diagnostic was evaluated during fracture healing simulated by progressive destabilization of cadaveric ovine metatarsals treated by locking plate fixation (n=8). An ovine in vivo comparative fracture study (n=8) was then utilized to better characterize the performance of the developed diagnostic in a clinically translatable setting. In vivo measurements using the developed diagnostic were compared to weekly radiographic images and postmortem biomechanical, histological, and micro computed tomography analyses. RESULTS: For all cadaveric samples, the novel direct electromagnetic coupling antenna displayed significant differences at the fracture site (P<0.05) when measuring a fully fractured sample versus partially intact and fully intact fracture states. In subsequent in vivo fracture models, this technology detected significant differences (P<0.001) in fractures trending towards delayed healing during the first 30 days post-fracture. CONCLUSIONS: This technology, relative to traditional X-ray imaging, exhibits potential to greatly expedite clinical diagnosis of fracture nonunion, thus warranting additional technological development.

Different Passive Viscoelastic Properties Between the Left and Right Ventricles in Healthy Adult Ovine.

Ventricle dysfunction is the most common cause of heart failure, which leads to high mortality and morbidity. The mechanical behavior of the ventricle is critical to its physiological function. It is known that the ventricle is anisotropic and viscoelastic. However, the understanding of ventricular viscoelasticity is much less than that of its elasticity. Moreover, the left and right ventricles (LV&RV) are different in embryologic origin, anatomy, and function, but whether they distinguish in viscoelastic properties is unclear. We hypothesized that passive viscoelasticity is different between healthy LVs and RVs. Ex vivo cyclic biaxial tensile mechanical tests (1, 0.1, 0.01 Hz) and stress relaxation (strain of 3, 6, 9, 12, 15%) were performed for ventricles from healthy adult sheep. Outflow track direction was defined as the longitudinal direction. Hysteresis stress-strain loops and stress relaxation curves were obtained to quantify the viscoelastic properties. We found that the RV had more pronounced frequency-dependent viscoelastic changes than the LV. Under the physiological frequency (1 Hz), the LV was more anisotropic in the elasticity and stiffer than the RV in both directions, whereas the RV was more anisotropic in the viscosity and more viscous than the LV in the longitudinal direction. The LV was quasi-linear viscoelastic in the longitudinal but not circumferential direction, and the RV was nonlinear viscoelastic in both directions. This study is the first to investigate passive viscoelastic differences in healthy LVs and RVs, and the findings will deepen the understanding of biomechanical mechanisms of ventricular function.

Investigation of a Prevascularized Bone Graft for Large Defects in the Ovine Tibia.

In vivo bioreactors are a promising approach for engineering vascularized autologous bone grafts to repair large bone defects. In this pilot parametric study, we first developed a three-dimensional (3D) printed scaffold uniquely designed to accommodate inclusion of a vascular bundle and facilitate growth factor delivery for accelerated vascular invasion and ectopic bone formation. Second, we established a new sheep deep circumflex iliac artery (DCIA) model as an in vivo bioreactor for engineering a vascularized bone graft and evaluated the effect of implantation duration on ectopic bone formation. Third, after 8 weeks of implantation around the DCIA, we transplanted the prevascularized bone graft to a 5 cm segmental bone defect in the sheep tibia, using the custom 3D printed bone morphogenic protein 2 (BMP-2) loaded scaffold without prior in vivo bioreactor maturation as a control. Analysis by micro-computed tomography and histomorphometry found ectopic bone formation in BMP-2 loaded scaffolds implanted for 8 and 12 weeks in the iliac pouch, with greater bone formation occurring after 12 weeks. Grafts transplanted to the tibial defect supported bone growth, mainly on the periphery of the graft, but greater bone growth and less soft tissue invasion was observed in the avascular BMP-2 loaded scaffold implanted directly into the tibia without prior in vivo maturation. Histopathological evaluation noted considerably greater vascularity in the bone grafts that underwent in vivo maturation with an inserted vascular bundle compared with the avascular BMP-2 loaded graft. Our findings indicate that the use of an initial DCIA in vivo bioreactor maturation step is a promising approach to developing vascularized autologous bone grafts, although scaffolds with greater osteoinductivity should be further studied. Impact statement This translational pilot study aims at combining a tissue engineering scaffold strategy, in vivo prevascularization, and a modified transplantation technique to accelerate large segmental bone defect repair. First, we three-dimensional (3D) printed a 5 cm scaffold with a unique design to facilitate vascular bundle inclusion and osteoinductive growth factor delivery. Second, we established a new sheep deep circumflex iliac artery model as an in vivo bioreactor for prevascularizing the novel 3D printed osteoinductive scaffold. Subsequently, we transplanted the prevascularized bone graft to a clinically relevant 5 cm segmental bone defect in the sheep tibia for bone regeneration.