In vivo study to assess fat embolism resulting from the Reamer-Irrigator-Aspirator 2 system compared to a novel aspirator-based concept for intramedullary bone graft harvesting.

INTRODUCTION: Fat embolism (FE) following intramedullary (IM) reaming can cause severe pulmonary complications and sudden death. Recently, a new harvesting concept was introduced in which a novel aspirator is used first for bone marrow (BM) aspiration and then for subsequent aspiration of morselized endosteal bone during sequential reaming (A + R + A). In contrast to the established Reamer-Irrigator-Aspirator (RIA) 2 system, the new A + R + A concept allows for the evacuation of fatty BM prior to reaming. In this study, we hypothesized that the risk of FE, associated coagulopathic reactions and pulmonary FE would be comparable between the RIA 2 system and the A + R + A concept. MATERIALS AND METHODS: Intramedullary bone graft was harvested from intact femora of 16 Merino sheep (age: 1-2 years) with either the RIA 2 system (n = 8) or the A + R + A concept (n = 8). Fat intravasation was monitored with the Gurd test, coagulopathic response with D-dimer blood level concentration and pulmonary FE with histological evaluation of the lungs. RESULTS: The total number and average size of intravasated fat particles was similar between groups (p = 0.13 and p = 0.98, respectively). D-dimer concentration did not significantly increase within 4 h after completion of surgery (RIA 2: p = 0.82; A + R + A: p = 0.23), with an interaction effect similar between groups (p = 0.65). The average lung area covered with fat globules was similar between groups (p = 0.17). CONCLUSIONS: The use of the RIA 2 system and the novel A + R + A harvesting concept which consists of BM evacuation followed by sequential IM reaming and aspiration of endosteal bone, resulted in only minor fat intravasation, coagulopathic reactions and pulmonary FE, with no significant differences between the groups. Our results, therefore, suggest that both the RIA 2 system and the new A + R + A concept are comparable technologies in terms of FE-related complications.

3D-Printed Medical-Grade Polycaprolactone (mPCL) Scaffold for the Surgical Treatment of Vaginal Prolapse and Abdominal Hernias.

The expected outcome after a scaffold augmented hernia repair is the regeneration of a tissue composition strong enough to sustain biomechanical function over long periods. It is hypothesised that melt electrowriting (MEW) medical-grade polycaprolactone (mPCL) scaffolds loaded with platelet-rich plasma (PRP) will enhance soft tissue regeneration in fascial defects in abdominal and vaginal sheep models. A pre-clinical evaluation of vaginal and abdominal hernia reconstruction using mPCL mesh scaffolds and polypropylene (PP) meshes was undertaken using an ovine model. Each sheep was implanted with both a PP mesh (control group), and a mPCL mesh loaded with PRP (experimental group) in both abdominal and vaginal sites. Mechanical properties of the tissue-mesh complexes were assessed with plunger tests. Tissue responses to the implanted meshes were evaluated via histology, immunohistochemistry and histomorphometry. At 6 months post-surgery, the mPCL mesh was less stiff than the PP mesh, but stiffer than the native tissue, while showing equitable collagen and vascular ingrowth when compared to PP mesh. The results of this pilot study were supportive of mPCL as a safe and effective biodegradable scaffold for hernia and vaginal prolapse repair, hence a full-scale long-term study (over 24-36 months) with an adequate sample size is recommended.

In vivo characterization of 3D-printed polycaprolactone-hydroxyapatite scaffolds with Voronoi design to advance the concept of scaffold-guided bone regeneration.

Three-dimensional (3D)-printed medical-grade polycaprolactone (mPCL) composite scaffolds have been the first to enable the concept of scaffold-guided bone regeneration (SGBR) from bench to bedside. However, advances in 3D printing technologies now promise next-generation scaffolds such as those with Voronoi tessellation. We hypothesized that the combination of a Voronoi design, applied for the first time to 3D-printed mPCL and ceramic fillers (here hydroxyapatite, HA), would allow slow degradation and high osteogenicity needed to regenerate bone tissue and enhance regenerative properties when mixed with xenograft material. We tested this hypothesis in vitro and in vivo using 3D-printed composite mPCL-HA scaffolds (wt 96%:4%) with the Voronoi design using an ISO 13485 certified additive manufacturing platform. The resulting scaffold porosity was 73% and minimal in vitro degradation (mass loss <1%) was observed over the period of 6 months. After loading the scaffolds with different types of fresh sheep xenograft and ectopic implantation in rats for 8 weeks, highly vascularized tissue without extensive fibrous encapsulation was found in all mPCL-HA Voronoi scaffolds and endochondral bone formation was observed, with no adverse host-tissue reactions. This study supports the use of mPCL-HA Voronoi scaffolds for further testing in future large preclinical animal studies prior to clinical trials to ultimately successfully advance the SGBR concept.

An in vivo study to investigate an original intramedullary bone graft harvesting technology.

BACKGROUND: Harvesting bone graft (BG) from the intramedullary canal to treat bone defects is largely conducted using the Reamer-Irrigator-Aspirator (RIA) system. The RIA system uses irrigation fluid during harvesting, which may result in washout of osteoinductive factors. Here, we propose a new harvesting technology dedicated to improving BG collection without the potential washout effect of osteoinductive factors associated with irrigation fluid. This novel technology involves the conceptual approach of first aspirating the bone marrow (BM) with a novel aspirator prototype, followed by reaming with standard reamers and collecting the bone chips with the aspirator (reaming-aspiration method, R-A method). The aim of this study was to assess the harvesting efficacy and osteoinductive profile of the BG harvested with RIA 2 system (RIA 2 group) compared to the novel harvesting concept (aspirator + R-A method, ARA group). METHODS: Pre-planning computed tomography (CT) imaging was conducted on 16 sheep to determine the femoral isthmus canal diameter. In this non-recovery study, sheep were divided into two groups: RIA 2 group (n = 8) and ARA group (n = 8). We measured BG weight collected from left femur and determined femoral cortical bone volume reduction in postoperative CT imaging. Growth factor and inflammatory cytokine amounts of the BGs were quantified using enzyme-linked immunosorbent assay (ELISA) methods. RESULTS: The use of the stand-alone novel aspirator in BM collection, and in harvesting BG when the aspirator is used in conjunction with sequential reaming (R-A method) was proven feasible. ELISA results showed that the collected BG contained relevant amounts of growth factors and inflammatory cytokines in both the RIA 2 and the ARA group. CONCLUSIONS: Here, we present the first results of an innovative concept for harvesting intramedullary BG. It is a prototype of a novel aspirator technology that enables the stepwise harvesting of first BM and subsequent bone chips from the intramedullary canal of long bones. Both the BG collected with the RIA 2 system and the aspirator prototype had the capacity to preserve the BG's osteoinductive microenvironment. Future in vivo studies are required to confirm the bone regenerative capacity of BG harvested with the innovative harvesting technology.

Convergence of scaffold-guided bone regeneration principles and microvascular tissue transfer surgery.

A preclinical evaluation using a regenerative medicine methodology comprising an additively manufactured medical-grade ε-polycaprolactone ß-tricalcium phosphate (mPCL-TCP) scaffold with a corticoperiosteal flap was undertaken in eight sheep with a tibial critical-size segmental bone defect (9.5 cm3, M size) using the regenerative matching axial vascularization (RMAV) approach. Biomechanical, radiological, histological, and immunohistochemical analysis confirmed functional bone regeneration comparable to a clinical gold standard control (autologous bone graft) and was superior to a scaffold control group (mPCL-TCP only). Affirmative bone regeneration results from a pilot study using an XL size defect volume (19 cm3) subsequently supported clinical translation. A 27-year-old adult male underwent reconstruction of a 36-cm near-total intercalary tibial defect secondary to osteomyelitis using the RMAV approach. Robust bone regeneration led to complete independent weight bearing within 24 months. This article demonstrates the widely advocated and seldomly accomplished concept of "bench-to-bedside" research and has weighty implications for reconstructive surgery and regenerative medicine more generally.

In vitro and in vivo investigation of a zonal microstructured scaffold for osteochondral defect repair.

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [ϵ-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen™, and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

Bone Regeneration Exploiting Corticoperiosteal Tissue Transfer for Scaffold-Guided Bone Regeneration.

Contemporary reconstructive approaches for critical size bone defects carry significant disadvantages. As a result, clinically driven research has focused on the development and translation of alternative therapeutic concepts. Scaffold-guided tissue regeneration (SGTR) is an emerging technique to heal critical size bone defects. However, issues synchronizing scaffold vascularization with bone-specific regenerative processes currently limit bone regeneration for extra large (XL, 19 cm3) critical bone defects. To address this issue, we developed a large animal model that incorporates a corticoperiosteal flap (CPF) for sustained scaffold neovascularization and bone regeneration. In 10 sheep, we demonstrated the efficacy of this approach for healing medium (M, 9 cm3) size critical bone defects as demonstrated on plain radiography, microcomputed tomography, and histology. Furthermore, in two sheep, we demonstrate how this approach can be safely extended to heal XL critical size defects. This article presents an original CPF technique in a well-described preclinical model, which can be used in conjunction with the SGTR concept, to address challenging critical size bone defects in vivo. Impact statement This article describes a novel scaffold-guided tissue engineering approach utilizing a corticoperiosteal flap for bone healing in critical size long bone defects. This approach will be of use for tissue engineers and surgeons exploring vascularized tissue transfer as an option to regenerate large volumes of bone for extensive critical size bone defects both in vivo and in the clinical arena.

Poly-ε-Caprolactone/Fibrin-Alginate Scaffold: A New Pro-Angiogenic Composite Biomaterial for the Treatment of Bone Defects.

We hypothesized that a composite of 3D porous melt-electrowritten poly-ɛ-caprolactone (PCL) coated throughout with a porous and slowly biodegradable fibrin/alginate (FA) matrix would accelerate bone repair due to its angiogenic potential. Scanning electron microscopy showed that the open pore structure of the FA matrix was maintained in the PCL/FA composites. Fourier transform infrared spectroscopy and differential scanning calorimetry showed complete coverage of the PCL fibres by FA, and the PCL/FA crystallinity was decreased compared with PCL. In vitro cell work with osteoprogenitor cells showed that they preferentially bound to the FA component and proliferated on all scaffolds over 28 days. A chorioallantoic membrane assay showed more blood vessel infiltration into FA and PCL/FA compared with PCL, and a significantly higher number of bifurcation points for PCL/FA compared with both FA and PCL. Implantation into a rat cranial defect model followed by microcomputed tomography, histology, and immunohistochemistry after 4- and 12-weeks post operation showed fast early bone formation at week 4, with significantly higher bone formation for FA and PCL/FA compared with PCL. However, this phenomenon was not extrapolated to week 12. Therefore, for long-term bone regeneration, tuning of FA degradation to ensure syncing with new bone formation is likely necessary.

Scaffold-guided bone regeneration in large volume tibial segmental defects.

Large volume losses in weight bearing long bones are a major challenge in clinical practice. Despite multiple innovations over the last decades, significant limitations subsist in current clinical treatment options which is driving a strong clinical demand for clinically translatable treatment alternatives, including bone tissue engineering applications. Despite these shortcomings, preclinical large animal models of large volume segmental bone defects to investigate the regenerative capacity of bone tissue engineering strategies under clinically relevant conditions are rarely described in literature. We herein present a newly established preclinical ovine animal model for the treatment of XL volume (19 cm3) segmental tibial defects. In eight aged male Merino sheep (age > 6 years) a mid-diaphyseal tibial segmental defect was created and stabilized with a 5.6 mm Dynamic Compression Plate (DCP). We present short-term (3 months) and long-term (12-15 months) results of a pilot study using medical grade Polycaprolactone-Tricalciumphosphate (mPCL-TCP) scaffolds combined with a dose of 2 mg rhBMP-7 delivered in Platelet-Rich- Plasma (PRP). Furthermore, detailed analyses of the mechanical properties of the scaffolds as well as interfragmentary movement (IFM) and DCP-surface strain in vitro and a comprehensive description of the surgical and post-surgery protocol and post-mortem analysis is given.

Novel resin tissue array system reduces sample preparation time, labour and reagent costs in bone tissue histology.

Resin histology plays an essential role in the analysis of hard tissues, such as bone and teeth, as well as in the context of metallic implant analysis. However, the techniques of resin embedding, followed by ground sectioning, are very costly due to significantly increased reagent cost and labour time when compared to the conventional paraffin histology approach. In the present study, a novel resin array system was developed to increase the affordability of a project analysing rat femur tissues containing metallic or polymeric implants. The resin array system enabled the simultaneous embedding of the femur samples in groups of eight samples compared to the conventional resin method where samples are processed individually. The ground sections produced with the resin array system allowed uniform ROI selection, ground section thickness, staining consistency, and histological analysis with Goldner's trichrome stain, offering a substantial opportunity for reproducible immunohistochemistry which is unable to be achieved when processing samples embedded individually. The application of this novel resin array system significantly reduced resource usage when compared to doing the same analysis on individual samples. A reduction of approximately 40% was achieved for both total labour time and total reagent cost through the use of the array system compared with individual embedding. This novel resin array system has widespread applicability to many bone, hard tissue, and metallic implant studies, offering substantial conservation of research funds and increased accessibility to advanced techniques for commercial partners due to more cost-effective sample preparation and more accurate, reproducible data.

A Preclinical Animal Model for the Study of Scaffold-Guided Breast Tissue Engineering.

Scaffold-guided breast tissue engineering (SGBTE) has the potential to transform reconstructive breast surgery. Currently, there is a deficiency in clinically relevant animal models suitable for studying novel breast tissue engineering concepts. To date, only a small number of large animal studies have been conducted and characterization of these large animal models is poorly described in the literature. Addressing this gap in the literature, this publication comprehensively describes our original porcine model based on the current published literature and the experience gained from previous animal studies conducted by our research group. In a long-term experiment using our model, we investigated our SGBTE approach by implanting 60 additively manufactured bioresorbable scaffolds under the panniculus carnosus muscle along the flanks of 12 pigs over 12 months. Our model has the flexibility to compare multiple treatment modalities where we successfully investigated scaffolds filled with various treatments of immediate and delayed fat graft and augmentation with platelet rich plasma. No wound complications were observed using our animal model. We were able to grow clinically relevant volumes of soft tissue, which validates our model. Our preclinical large animal model is ideally suited to assess different scaffold or hydrogel-driven soft tissue regeneration strategies. Impact statement The ability to regenerate soft tissue through scaffold-guided tissue engineering concepts can transform breast reconstructive surgery. We describe an original preclinical large animal model to study controlled and reproducible scaffold-guided breast tissue engineering (SGBTE) concepts. This model features the flexibility to investigate multiple treatment conditions per animal, making it an efficient model. We have validated our model with a long-term experiment over 12 months, which exceeds other shorter published studies. Our SGBTE concept provides a more clinically relevant approach in terms of breast reconstruction. Future studies using this model will support the translation of SGBTE into clinical practice.

The Current Versatility of Polyurethane Three-Dimensional Printing for Biomedical Applications.

Reconstructive surgery aims to restore tissue defects by replacing them with similar autologous tissue to achieve good clinical outcomes. However, often the defect is too large or the tissue available is limited, requiring synthetic materials to restore the anatomical shape and partial function. The utilization of three-dimensional (3D) printing allows for the manufacture of implants with complex geometries and internal architecture that more closely matches the required clinical needs. Synthetic polymers offer certain advantages over natural polymers as biomedical materials due to their ability to more closely mimic the mechanical and chemical properties of the native tissue. Synthetic polymer materials such as poly(lactic acid) and acrylonitrile butadiene styrene are easily 3D printed to generate 3D objects due to their flexibility in their chemical and mechanical properties and physical form. Polyurethanes (PUs) are widely used as short- and long-term, implantable medical devices due to their good mechanical properties, biocompatibility, and hemocompatibility. This article provides an overview on the advancement of 3D printable PU-based materials for biomedical applications. A summary of the chemical structure and synthesis of PUs is provided to explain how PUs may be processed into medical devices using additive manufacturing techniques. Currently, PUs are being explored by several 3D printing approaches, including fused filament fabrication, bioplotting, and stereolithography, to fabricate complex implants with precise patterns and shapes with fine resolution. PU scaffolds using 3D printing have shown good cell viability and tissue integration in vivo. The important limitations of PU printing are identified to stimulate future research. PUs offer a biocompatible, synthetic polymeric material that can be 3D printed to manufacture implants that are tailored to meet specific anatomical, mechanical, and biological requirements for biomedical applications.

A preclinical large-animal model for the assessment of critical-size load-bearing bone defect reconstruction.

Critical-size bone defects, which require large-volume tissue reconstruction, remain a clinical challenge. Bone engineering has the potential to provide new treatment concepts, yet clinical translation requires anatomically and physiologically relevant preclinical models. The ovine critical-size long-bone defect model has been validated in numerous studies as a preclinical tool for evaluating both conventional and novel bone-engineering concepts. With sufficient training and experience in large-animal studies, it is a technically feasible procedure with a high level of reproducibility when appropriate preoperative and postoperative management protocols are followed. The model can be established by following a procedure that includes the following stages: (i) preoperative planning and preparation, (ii) the surgical approach, (iii) postoperative management, and (iv) postmortem analysis. Using this model, full results for peer-reviewed publication can be attained within 2 years. In this protocol, we comprehensively describe how to establish proficiency using the preclinical model for the evaluation of a range of bone defect reconstruction options.

Investigation of Sustained BMP Delivery in the Prevention of Medication-Related Osteonecrosis of the Jaw (MRONJ) in a Rat Model.

Medication-related osteonecrosis of the jaw (MRONJ) poses an ongoing challenge for clinicians and researchers. Currently, there is a lack of preventative measures available for at-risk patients undergoing tooth extractions, especially those with prior bisphosphonate treatment due to osteoporosis or bone metastasis diagnoses. Here, these issues are addressed using a preventative tissue engineering strategy against MRONJ development. This study evaluates the efficacy of a poly(ethylene glycol)-heparin hydrogel as a tool for the delivery of arginylglycylaspartic acid (RGD) and recombinant human bone morphogenic protein-2 (rhBMP-2). Three groups of skeletally mature rats each receive two doses of intravenous zoledronic acid prior to surgery and undergo extraction of the right first mandibular molar with gingival closure. Experimental groups either have the sockets left empty, filled with hydrogel minus rhBMP-2, or filled with hydrogel plus rhBMP-2. Eight weeks postoperatively specimens are analyzed using radiological, histological, and scanning electron microscopy (SEM) techniques. µCT analysis shows increased bone formation with hydrogel/rhBMP-2 delivery compared to the empty socket. Hydrogel-treated groups display increased presence of osteocytes and increased osteoclastic action compared to the empty sockets. These results represent the first step toward improved delivery of rhBMP-2 and a potential MRONJ preventative for patients undergoing bisphosphonate treatment.

A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation.

Convergence of Scaffold-Guided Bone Reconstruction and Surgical Vascularization Strategies-A Quest for Regenerative Matching Axial Vascularization.

The prevalent challenge facing tissue engineering today is the lack of adequate vascularization to support the growth, function, and viability of tissue engineered constructs (TECs) that require blood vessel supply. The research and clinical community rely on the increasing knowledge of angiogenic and vasculogenic processes to stimulate a clinically-relevant vascular network formation within TECs. The regenerative matching axial vascularization approach presented in this manuscript incorporates the advantages of flap-based techniques for neo-vascularization yet also harnesses the in vivo bioreactor principle in a more directed "like for like" approach to further assist regeneration of the specific tissue type that is lost, such as a corticoperiosteal flap in critical sized bone defect reconstruction.

Additively manufactured biphasic construct loaded with BMP-2 for vertical bone regeneration: A pilot study in rabbit.

Vertical bone augmentation of the jaws is required when the height of bone is insufficient at the site of dental implant placement. In this proof of concept study, we investigated the potential of a biphasic polycaprolactone construct combined with a hyaluronic acid based hydrogel loaded with recombinant human bone morphogenetic growth factor-2 (BMP-2) for vertical bone regeneration. The biphasic scaffold consisted of an outer shell manufactured by fused deposition modelling, mimicking native cortical bone and providing mechanical and space maintenance properties essential for bone formation. Within this shell, a 90% porous melt electrospun microfibrous mesh mimicking the architecture of cancellous bone was incorporated in order to facilitate hydrogel loading and subsequent osteogenesis and angiogenesis. The in vitro performances of the biphasic construct demonstrated that BMP-2 was released in a sustained manner over several weeks and that cell viability was maintained in the hydrogel over 21 days. qRT-PCR demonstrated the upregulation of bone markers such as osteopontin, osteocalcin and collagen 1A1 at day 3 and 14 in the constructs loaded with BMP2. In vivo assessment of the biphasic scaffold was performed using a dose of 30 µg of BMP-2 in a rabbit calvarial vertical bone augmentation model. The histology and micro-CT analysis of the elevated space demonstrated that the hydrogel and the presence of BMP-2 enabled bone formation. However, this was limited to the immediate vicinity of the calvarial bone. The amount of newly formed bone was relatively small which was likely due to poor vascularisation of the extraskeletal space. The utilisation of this biomimetic biphasic construct with excellent space maintenance properties can be of interest in dentistry although the in vivo model requires refinement to demonstrated appropriate efficacy.

Establishing sheep as an experimental species to validate ultrasound-mediated blood-brain barrier opening for potential therapeutic interventions.

Rationale: Treating diseases of the brain such as Alzheimer's disease (AD) is challenging as the blood-brain barrier (BBB) effectively restricts access of a large number of potentially useful drugs. A potential solution to this problem is presented by therapeutic ultrasound, a novel treatment modality that can achieve transient BBB opening in species including rodents, facilitated by biologically inert microbubbles that are routinely used in a clinical setting for contrast enhancement. However, in translating rodent studies to the human brain, the presence of a thick cancellous skull that both absorbs and distorts ultrasound presents a challenge. A larger animal model that is more similar to humans is therefore required in order to establish a suitable protocol and to test devices. Here we investigated whether sheep provide such a model. Methods: In a stepwise manner, we used a total of 12 sheep to establish a sonication protocol using a spherically focused transducer. This was assisted by ex vivo simulations based on CT scans to establish suitable sonication parameters. BBB opening was assessed by Evans blue staining and a range of histological tests. Results: Here we demonstrate noninvasive microbubble-mediated BBB opening through the intact sheep skull. Our non-recovery protocol allowed for BBB opening at the base of the brain, and in areas relevant for AD, including the cortex and hippocampus. Linear time-shift invariant analysis and finite element analysis simulations were used to optimize the position of the transducer and to predict the acoustic pressure and location of the focus. Conclusion: Our study establishes sheep as a novel animal model for ultrasound-mediated BBB opening and highlights opportunities and challenges in using this model. Moreover, as sheep develop an AD-like pathology with aging, they represent a large animal model that could potentially complement the use of non-human primates.

Early mechanical stimulation only permits timely bone healing in sheep.

Bone fracture healing is sensitive to the fixation stability. However, it is unclear which phases of healing are mechano-sensitive and if mechanical stimulation is required throughout repair. In this study, a novel bone defect model, which isolates an experimental fracture from functional loading, was applied in sheep to investigate if stimulation limited to the early proliferative phase is sufficient for bone healing. An active fixator controlled motion in the fracture. Animals of the control group were unstimulated. In the physiological-like group, 1 mm axial compressive movements were applied between day 5 and 21, thereafter the movements were decreased in weekly increments and stopped after 6 weeks. In the early stimulatory group, the movements were stopped after 3 weeks. The experimental fractures were evaluated with mechanical and micro-computed tomography methods after 9 weeks healing. The callus strength of the stimulated fractures (physiological-like and early stimulatory) was greater than the unstimulated control group. The control group was characterized by minimal external callus formation and a lack of bone bridging at 9 weeks. In contrast, the stimulated groups exhibited advanced healing with solid bone formation across the defect. This was confirmed quantitatively by a lower bone volume in the control group compared to the stimulated groups.The novel experimental model permits the application of a well-defined load history to an experimental bone fracture. The poor healing observed in the control group is consistent with under-stimulation. This study has shown early mechanical stimulation only is sufficient for a timely healing outcome. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1790-1796, 2018.