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.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

Biomechanical and histological changes secondary to aging in the human rotator cuff: A preliminary analysis.

The high failure rate of rotator cuff repair surgeries is positively correlated with age, yet the biomechanical changes to the tendons of the rotator cuff with age have not been described. As such, we sought to benchmark and characterize the biomechanical and histopathological properties with the accompanying gene expression of human rotator cuff tendons as a function of age and histopathological degeneration. All four rotator cuff tendons from fresh human cadaver shoulders underwent biomechanical, histopathological, and gene expression analyses. Following cadaver availability, samples were grouped into Younger (i.e., less than 36 years of age, n = 2 donors) and Aged (i.e., greater than 55 years of age, n = 3 donors) as a means of characterizing and quantifying the age-related changes exhibited by the tendons. Biomechanical testing and subsequent computational modeling techniques revealed both differences in properties between tendons and greater Young's moduli in the Younger tendons (supraspinatus 3.06x, infraspinatus 1.76x, subscapularis 1.25x, and teres minor 1.32x). Histopathological scoring using the semi-quantitative Bonar scoring scheme revealed a positive correlation with age across all tendons (r = 0.508, p < 0.001). These data contextualize the biomechanical and histopathological changes to tendons that occurs naturally with aging, highlighting the innate differences in biomechanical properties of all four rotator cuff tendons, as well as the difference in their degenerative trajectories. Additionally, the histopathological scoring revealed moderate signs of degeneration within the Younger supraspinatus tendons, suggesting tissue quality may decrease in this specific tendon in patients less than 40 years old, before clinical symptoms or tears.

Exosome Cell Origin Affects In Vitro Markers of Tendon Repair in Ovine Macrophages and Tenocytes.

Tendon injuries and disease are resistant to surgical repair; thus, adjunct therapies are widely investigated, especially mesenchymal stromal cells (MSCs) and, more recently, their extracellular vesicles (MSCdEVs), for example, exosomes. Thought to act on resident and infiltrating immune cells, the role of MSCdEVs in paracrine signaling is of great interest. This study investigated how MSCdEVs differ from analogs derived from resident (tenocyte) populations (TdEV). As macrophages play a significant role in tendon maintenance and repair, macrophage signaling was compared by cytokine quantification using a multiplexed immunoassay and tenocyte migration by in vitro scratch-wound analysis. TdEV-treated macrophages decreased IL-1 and increased MIP-1 and CXCL8 expression. In addition, macrophage signaling favored collagen synthesis and tenocyte bioactivity, while reducing proangiogenic signaling when TdEVs were used in place of MSCdEVs. These in vitro data demonstrate a differential influence of exosomes on macrophage signaling, according to cell source, supporting that local cell-derived exosomes may preferentially drive healing by different means with possible different outcomes compared to MSCdEVs. Impact Statement Adipose-derived mesenchymal stromal cell (AdMSC) exosomes (EVs) can improve tendon mechanical resilience, tissue organization, and M2 macrophage phenotype predominance in response to tendon injury. This active area of investigation drives great interest in the function of these exosomes as adjunct therapies for tendon disease, particularly rotator cuff tendinopathy. However, little is known about the effects of EVs as a function of cell source, nor regarding their efficacy in preclinical translational ovine models. Herein we demonstrate a differential effect of exosomes as a function of cell source, tenocyte compared to AdMSCs, on macrophage signaling and tenocyte migration of ovine cells.

Enhanced bone formation in locally-optimised, low-stiffness additive manufactured titanium implants: An in silico and in vivo tibial advancement study.

A tibial tuberosity advancement (TTA), used to treat lameness in the canine stifle, provides a framework to investigate implant performance within an uneven loading environment due to the dominating patellar tendon. The purpose of this study was to reassess how we design orthopaedic implants in a load-bearing model to investigate potential for improved osseointegration capacity of fully-scaffolded mechanically-matched additive manufactured (AM) implants. While the mechanobiological nature of bone is well known, we have identified a lower limit in the literature where investigation into exceedingly soft scaffolds relative to trabecular bone ceases due to the trade-off in mechanical strength. We developed a finite element model of the sheep stifle to assess the stresses and strains of homogeneous and locally-optimised TTA implant designs. Using additive manufacturing, we printed three different low-stiffness Ti-6Al-4 V TTA implants: 0.8 GPa (Ti1), 0.6 GPa (Ti2) and an optimised design with a 0.3 GPa cortex and 0.1 GPa centre (Ti3), for implantation in a 12-week in vivo ovine pilot study. Static histomorphometry demonstrated uniform bone ingrowth in optimised low-modulus Ti3 samples compared to homogeneous designs (Ti1 and Ti2), and greater bone-implant contact. Mineralising surfaces were apparent in all implants, though mineral apposition rate was only consistent throughout Ti3. The greatest bone formation scores were seen in Ti3, followed by Ti2 and Ti1. Results from our study suggest lower stiffnesses and higher strain ranges improve early bone formation, and that by accounting for loading environments through rational design, implants can be optimised to improve uniform osseointegration. STATEMENT OF SIGNIFICANCE: The effect of different strain ranges on bone healing has been traditionally investigated and characterised through computational models, with much of the literature suggesting higher strain ranges being favourable. However, little has been done to incorporate strain-optimisation into porous orthopaedic implants due to the trade-off in mechanical strength required to induce these microenvironments. In this study, we used finite element analysis to optimise the design of additive manufactured (AM) titanium orthopaedic implants for different strain ranges, using a clinically-relevant surgical model. Our research suggests that there is potential for locally-optimised AM scaffolds in the use of orthopaedic devices to induce higher strains, which in turn encourages de novo bone formation and uniform osseointegration.

Modified Alendronate Mitigates Mechanical Degradation of the Rotator Cuff in an Osteoporotic Ovine Model.

BACKGROUND: Osteoporosis is an independent risk factor for failure after arthroscopic rotator cuff repair. Since rerupture rates after rotator cuff repair are associated with decreased bone mineral density and bone microarchitecture, adaptations of biomechanical properties of the rotator cuff enthesis in patients with osteoporosis remain unclear. Additionally, the effects of osteogenic therapy carrier drugs used for the treatment of osteoporosis on rotator cuff structure and properties have not been previously documented. PURPOSE: To investigate the changes to soft tissue biomechanics and insertional structure secondary to osteoporosis with and without an osteogenic therapy carrier (ie, modified alendronate). STUDY DESIGN: Controlled laboratory study. METHODS: Biomechanical, histopathological, and microcomputed tomography analyses were performed on 20 shoulders obtained from 10 osteoporotic sheep randomly allocated to modified bisphosphonate (ie, alendronate) or control (ie, osteoporotic without treatment) groups; 6 shoulders from healthy sheep were utilized for comparison purposes. RESULTS: Tendons from the control group exhibited a 57% decrease in undeformed Young modulus as compared with the healthy group (P = .010). Tendons from the modified bisphosphonate treatment group exhibited a 229% increase in initial Young modulus as compared with the control group (P = .010). Marked changes within the tendon insertional organization were noted in both the control and the modified bisphosphonate treatment group samples as evidenced by increased interdigitation of the bone-mineralized fibrocartilaginous junction. The control samples exhibited a markedly paucicellular insertion, whereas the modified bisphosphonate treated tendons exhibited a hypercellular insertional region as compared with the healthy group. Both groups exhibited significantly (P < .01) decreased bone quality underlying the infraspinatus insertion, as evidenced by all microcomputed tomography outcome parameters. CONCLUSION: This work illuminates changes to rotator cuff tendon secondary to osteoporosis. Specifically, it revealed decreased tendon modulus and altered insertional structure in the osteoporotic samples. Secondarily, these data revealed increases in tendon modulus accompanied by increased cellularity within the tendon insertion region after systemic modified bisphosphonate injections. CLINICAL RELEVANCE: Bisphosphonate treatment may have a positive effect on the healing of the enthesis after rotator cuff repair.

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.

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.

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.

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.

Biomechanical, Morphological, and Biochemical Characteristics of Articular Cartilage of the Ovine Humeral Head.

OBJECTIVE: Shoulder pain is commonly attributed to rotator cuff injury or osteoarthritis. Ovine translational models are used to investigate novel treatments aimed at remedying these conditions to prevent articular cartilage degeneration and subsequent joint degradation. However, topographical properties of articular cartilage in the ovine shoulder are undefined. This study investigates the biomechanical, morphological, and biochemical attributes of healthy ovine humeral head articular cartilage and characterizes topographical variations between surface locations. DESIGN: Ten humeral heads were collected from healthy skeletally mature sheep and each was segregated into 4 quadrants using 16 regions of interest (ROIs) across the articular surface. Articular cartilage of each ROI was analyzed for creep indentation, thickness, and sulfated glycosaminoglycan (sGAG) and collagen quantity. Comparisons of each variable were made between quadrants and between ROIs within each quadrant. RESULTS: Percent creep, thickness, and sGAG content, but not collagen content, were significantly different between humeral head quadrants. Subregion analysis of the ROIs within each surface quadrant revealed differences in all measured variables within at least one quadrant. Percent creep was correlated with sGAG (r = -0.32, P = 0.0001). Collagen content was correlated with percent creep (r = 0.32, P = 0.0009), sGAG (r = -0.19, P = 0.049), and thickness (r = -0.19, P = 0.04). CONCLUSIONS: Topographical variations exist in mechanical, morphologic, and biochemical properties across the articular surface of the ovine humeral head. Recognizing this variability in ovine humeral head cartilage will provide researchers and clinicians with accurate information that could impact study outcomes.

Rotator cuff repair using a bioresorbable nanofiber interposition scaffold: a biomechanical and histologic analysis in sheep.

BACKGROUND: The purpose of this study was to evaluate the mechanical, structural, and histologic quality of rotator cuff repairs augmented with an interposition electrospun nanofiber scaffold composed of polyglycolic acid (PGA) and poly-L-lactide-co-ε-caprolactone (PLCL) in an acute sheep model. METHODS: Forty acute infraspinatus tendon detachment and repair procedures were performed in a sheep infraspinatus model using a double-row transosseous-equivalent anchor technique either with an interposition nanofiber scaffold composed of polyglycolic acid-poly-L-lactide-co-ε-caprolactone or with no scaffold. Animals were euthanized at the 6-week (20 samples) and 12-week (20 samples) postoperative time points to assess the biomechanical and histologic properties of the repairs and to compare differences within each group. RESULTS: Within the scaffold-treated group, there was a significant increase in ultimate failure force (in newtons) from 6 to 12 weeks (P < .01), a significant increase in ultimate failure load from 6 to 12 weeks (P < .01), and a significant increase in ultimate failure stress (in megapascals) from 6 to 12 weeks (P < .01). At 6 weeks, the tendon-bone attachment was most consistent with an "indirect" type of insertion, whereas at 12 weeks, a visible difference in the progression and re-formation of the enthesis was observed. Compared with controls, animals in the scaffold-treated group displayed an insertion of the fibrous tendon with the humeral footprint that was beginning to be organized in a manner similar to the "native" direct/fibrocartilaginous insertion of the ovine infraspinatus tendon. In the majority of these animals treated with the scaffold, prominent perforating collagen fibers, similar to Sharpey fibers, were present and extending through a region of calcified fibrocartilage and attaching to the humeral footprint. No surgical complications occurred in any of the 40 sheep, including delayed wound healing or infection. CONCLUSIONS: In a sheep acute rotator cuff repair model, securing a nanofiber scaffold between the tendon and the bone using a double-row transosseous-equivalent anchor fixation technique resulted in greater failure strength. Additionally, at the enthesis, Sharpey fiber-like attachments (ie, collagen fibers extending from the tendon into the calcified fibrocartilage of the humerus) were observed, which were not seen in the control group.

Tendon midsubstance trauma as a means for the development of translatable chronic rotator cuff degeneration in an ovine model.

BACKGROUND: Chronic degeneration of rotator cuff tendons is a major contributing factor to the unacceptably high prevalence of rotator cuff repair surgery failures. The etiology of chronic rotator cuff degeneration is not well understood, and current therapies are not effective, necessitating preclinical research to fill this knowledge gap. Unfortunately, current large animal models rely on enthesis disruption as a means of model generation, which is not representative of human patients with chronic rotator cuff degeneration prior to full-thickness tears. Following, the goal of this study was to develop and characterize a translational large-animal model of chronic rotator cuff degeneration without enthesis release. METHODS: A midsubstance damage model [i.e., "combed fenestration" (CF)] in adult sheep was generated by creating 16 longitudinal cuts within the top third of the infraspinatus tendon thickness. Tendon integrity was characterized through exhaustive non-destructive biomechanical stress relaxation testing [peak stress, peak load, percent relaxation, and cross-sectional area (CSA)], followed by histopathological degeneration scoring and analysis (Bonar score), histomorphological analysis of collagen organization and fatty atrophy (percent adipose area), and gene expression analyses. RESULTS: The CF model tendons exhibited significantly decreased mechanical properties as evidenced by decreased peak stress (P<0.025) and increased percent relaxation (18-week vs. Control, P<0.035) at multiple strain magnitudes and across all timepoints. At all timepoints, the CF tendons exhibited pathological changes aligned with tendon degeneration, as evidenced by increased Bonar scoring (P<0.001) and decreased collagen organization (6-week vs. Control, P=0.013). Increases in intramuscular adipose content were also documented through histomorphology analysis (6- and 18-week vs. Control, P<0.077). Significant changes in gene expression were noted at all timepoints. CONCLUSIONS: These data reveal that this new ovine CF model of chronic rotator cuff degeneration results in tendons with decreased mechanical properties, degenerative pathology characteristics, and gene expression profiles that aligned with the degenerative changes that have been noted in humans with tendinopathy. For these reasons, we believe this novel large animal model of chronic rotator cuff degeneration is a translational platform in which to test devices, therapies, and/or technologies aimed at repairing damage to the shoulder.

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.

Biomechanical Comparison of Tibial Plateau Leveling Osteotomy Performed With a Novel Titanium Alloy Locking Plate Construct vs. an Established Stainless-Steel Locking Plate Construct.

A novel canine tibial plateau leveling osteotomy (TPLO) fixation device was recently developed with design features such as titanium alloy (TA) material, distal monocortical screw fixation, and a point contact undersurface specifically targeted to reduce surgical site infection rates by ensuring tissue perfusion under the plate. The strength of the novel TPLO construct was compared with that of a predicate stainless steel (SS) locking plate construct with bicortical screws in 16 paired cadaveric canine limbs. The mean loads to failure were 716.71 ± 109.50 N (range 455.69-839.69 N) and 629.50 ± 176.83 N (range 272.58-856.18 N) in the TA and SS groups, respectively. The average ratio of the loads to failure of the paired specimens was 1.18 (p = 0.031). No failure of the TA constructs involved the distal fixation with monocortical screws. Substantial mechanical equivalence of this novel TA monocortical/bicortical fixation construct to an established SS bicortical screw fixation construct is demonstrated. Clinical investigation of potential merits of this novel TA, monocortical/bicortical locking screw/plate system is now warranted.

Enthesis trauma as a means for the development of translatable chronic rotator cuff degeneration in an ovine model.

BACKGROUND: Untreated rotator cuff tears lead to irreversible tendon degeneration, resulting in unacceptable repair prognosis. The inability of current animal models of degenerated rotator cuff tendons to more fully emulate the manifestation and degree of pathology seen in humans with a previously torn rotator cuff tendon (s) significantly impairs the development of novel therapeutics. Therefore, the objective of this study was to develop a large-animal translational model of enthesis damage to the rotator cuff tendons to mimic the chronic degenerative changes that occur in patients that demonstrate clinical manifestations of tendinopathy. METHODS: A partial enthesis tear model (i.e., sharp transection) in adult sheep was created by cutting the tendon fibers perpendicularly through the enthesis midpoint, while leaving the other portion of the tendon in-tact. To assess tendon integrity, non-destructive biomechanical tests were performed, followed by histopathological, histomorphological, and gene expression analysis. Samples of degenerated human rotator cuff tendons obtained from patients undergoing reverse total shoulder arthroplasty to use for comparative pathological analysis. RESULTS: In the sheep model, transected tendons at all timepoints had significantly decreased mechanical properties. Histopathologic evaluation and Bonar scoring revealed that the tendons in sheep underwent degenerative changes similar in magnitude and manifestation as the degenerated human tendon samples. Furthermore, similar levels of collagen disorganization were noted between the 6 and 12-week ovine samples and the degenerated human samples. CONCLUSIONS: These findings indicate that the new sheep model of rotator cuff injury reliably recapitulates the structural and cellular changes that occur clinically in humans with chronic rotator cuff tendon injuries and suggest that this new model is well suited to evaluation of new therapeutic interventions.

Longitudinal tendon healing assessed with multi-modality advanced imaging and tissue analysis.

BACKGROUND: The range of diagnostic modalities available to evaluate superficial digital flexor tendon (SDFT) injury includes magnetic resonance imaging (MRI), computed tomography (CT) and ultrasonography (US). Direct, comprehensive comparison of multi-modality imaging characteristics to end-point data has not previously been performed using a model of tendinopathy but is required to obtain a better understanding of each modality's diagnostic capabilities. OBJECTIVE: To compare CT, MRI and US evaluation to outcome measures for histologic, biochemical and biomechanical parameters using an equine surgical model of tendinopathy. STUDY DESIGN: Controlled experiment. METHODS: Lesions were surgically created in both forelimb SDFTs of eight horses and imaged using MRI, CT and US at seven time points over 12 months. Imaging characteristics were then correlated to end point histologic, biochemical and biomechanical data using lasso regression. Longitudinal lesion size was compared between imaging modalities. RESULTS: Lesion to tendon isoattenuation on CT evaluation correlated with the greatest levels of aggrecan deposition. A significant correlation between cellular density and percentage of tendon involvement on the T2-weighted sequence and signal intensity on the proton density fat saturated (PD FS) sequence was appreciated at the 12-month time point (P = .006, P = .02 respectively). There was no significant correlation between end-point data and US or contrast imaging characteristics. Cross sectional area lesion to tendon measurements were significantly largest on CT evaluation, followed by MRI and then US (P < .001). MAIN LIMITATIONS: Experimentally induced tendon injury with singular end-point data correlation. CONCLUSIONS: Lesion isoattenuation on CT evaluation suggested scar tissue deposition, while T2-weighted hyperintensity indicated hypercellular tendinopathy even in chronic stages of healing. Non contrast-enhanced MRI and CT evaluation correlated most closely to cellular characteristics of surgically damaged tendons assessed over a twelve month study period. Ultrasonographic evaluation underestimates true lesional size and should be interpreted with caution.

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.

Osteoinductive 3D printed scaffold healed 5 cm segmental bone defects in the ovine metatarsus.

Autologous bone grafts are considered the gold standard grafting material for the treatment of nonunion, but in very large bone defects, traditional autograft alone is insufficient to induce repair. Recombinant human bone morphogenetic protein 2 (rhBMP-2) can stimulate bone regeneration and enhance the healing efficacy of bone grafts. The delivery of rhBMP-2 may even enable engineered synthetic scaffolds to be used in place of autologous bone grafts for the treatment of critical size defects, eliminating risks associated with autologous tissue harvest. We here demonstrate that an osteoinductive scaffold, fabricated by combining a 3D printed rigid polymer/ceramic composite scaffold with an rhBMP-2-eluting collagen sponge can treat extremely large-scale segmental defects in a pilot feasibility study using a new sheep metatarsus fracture model stabilized with an intramedullary nail. Bone regeneration after 24 weeks was evaluated by micro-computed tomography, mechanical testing, and histological characterization. Load-bearing cortical bridging was achieved in all animals, with increased bone volume observed in sheep that received osteoinductive scaffolds compared to sheep that received an rhBMP-2-eluting collagen sponge alone.