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

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

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

Establishment and characterisation of an open mini- thoracotomy surgical approach to an ovine thoracic spine fusion model

Background A large animal model is required for assessment of minimally invasive, tissue engineering based approaches to thoracic spine fusion, with relevance to deformity correction surgery for human adolescent idiopathic scoliosis. Here we develop a novel open mini–thoracotomy approach in an ovine model of thoracic interbody fusion which allows assessment of various fusion constructs, with a focus on novel, tissue engineering based interventions. Methods The open mini-thoracotomy surgical approach was developed through a series of mock surgeries, and then applied in a live sheep study. Customized scaffolds were manufactured to conform with intervertebral disc space clearances required of the study. Twelve male Merino sheep aged 4 to 6 years and weighing 35 – 45 kg underwent the abovementioned procedure and were divided into two groups of six sheep at survival timelines of 6 and 12 months. Each sheep underwent a 3-level discectomy (T6/7, T8/9 and T10/11) with randomly allocated implantation of a different graft substitute at each of the three levels; (i) polycaprolactone (PCL) based scaffold plus 0.54μg rhBMP-2, (ii) PCL-based scaffold alone or (iii) autograft. The sheep were closely monitored post- operatively for signs of pain (i.e. gait abnormalities/ teeth gnawing/ social isolation). Fusion assessments were conducted post-sacrifice using Computed Tomography and hard-tissue histology. All scientific work was undertaken in accordance with the study protocol has been approved by the Institute's committee on animal research. Results. All twelve sheep were successfully operated on and reached the allotted survival timelines, thereby demonstrating the feasibility of the surgical procedure and post-operative care. There were no significant complications and during the post-operative period the animals did not exhibit marked signs of distress according to the described assessment criteria. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL-based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluation of the respective groups. Conclusion. This novel open mini-thoracotomy surgical approach to the ovine thoracic spine represents a safe surgical method which can reproducibly form the platform for research into various spine tissue engineered constructs (TEC) and their fusion promoting properties

Evaluation of the effect of tamoxifen citrate on model of osteoporosis in dog: biomechanical and histopathological studies

Summary The effect of tamoxifen citrate on bone mass in immobilization osteoporosis was studied in 10 dogs. Osteoporosis was induced by fiberglass cast immobilization of the right hind-limb for 28 days, while the left hind-limb served as a non-immobilized control. Five dogs received tamoxifen citrate (1.5 mg/kg per os) once daily for 28 days; five dogs received no treatment. All dogs were euthanized on day 28 and tibiae were harvested. Bone biomechanical properties and microscopic structures of tibiae from casted and uncasted limbs were studied. Significant differences in the percent of decreased values of examined mechanical properties were found between untreated and tamoxifen-treated dogs. No remarkable histopathological changes indicative of osteoporosis were detected in the tibiae of casted limb of tamoxifen-treated dogs. These findings indicated that short term tamoxifen therapy may have promising effects on prevention of osteoporosis in dog