In Vivo Efficacy of an Adhesive Bioresorbable Patch to Treat Aortic Dissections.

Endovascular repair of aortic dissection still presents significant limitations. Preserving the mechanical and biological properties set by the aortic microstructure is critical to the success of implantable grafts. In this paper, we present the performance of an adhesive bioresorbable patch designed to cover the entry tear of aortic dissections. We demonstrate the power of using a biomimetic scaffold in a vascular environment.

A humanised rat model of osteosarcoma reveals ultrastructural differences between bone and mineralised tumour tissue.

Current xenograft animal models fail to accurately replicate the complexity of human bone disease. To gain translatable and clinically valuable data from animal models, new in vivo models need to be developed that mimic pivotal aspects of human bone physiology as well as its diseased state. Above all, an advanced bone disease model should promote the development of new treatment strategies and facilitate the conduction of common clinical interventional procedures. Here we describe the development and characterisation of an orthotopic humanised tissue-engineered osteosarcoma (OS) model in a recently genetically engineered x-linked severe combined immunodeficient (X-SCID) rat. For the first time in a genetically modified rat, our results show the successful implementation of an orthotopic humanised tissue-engineered bone niche supporting the growth of a human OS cell line including its metastatic spread to the lung. Moreover, we studied the inter- and intraspecies differences in ultrastructural composition of bone and calcified tissue produced by the tumour, pointing to the crucial role of humanised animal models.

Breast cancer-secreted factors perturb murine bone growth in regions prone to metastasis.

Breast cancer frequently metastasizes to bone, causing osteolytic lesions. However, how factors secreted by primary tumors affect the bone microenvironment before the osteolytic phase of metastatic tumor growth remains unclear. Understanding these changes is critical as they may regulate metastatic dissemination and progression. To mimic premetastatic bone adaptation, immunocompromised mice were injected with MDA-MB-231-conditioned medium [tumor-conditioned media (TCM)]. Subsequently, the bones of these mice were subjected to multiscale, correlative analysis including RNA sequencing, histology, micro-computed tomography, x-ray scattering analysis, and Raman imaging. In contrast to overt metastasis causing osteolysis, TCM treatment induced new bone formation that was characterized by increased mineral apposition rate relative to control bones, altered bone quality with less matrix and more carbonate substitution, and the deposition of disoriented mineral near the growth plate. Our study suggests that breast cancer-secreted factors may promote perturbed bone growth before metastasis, which could affect initial seeding of tumor cells.

An Early Myeloma Bone Disease Model in Skeletally Mature Mice as a Platform for Biomaterial Characterization of the Extracellular Matrix.

Multiple myeloma (MM) bone disease is characterized by osteolytic bone tissue destruction resulting in bone pain, fractures, vertebral collapse, and spinal cord compression in patients. Upon initial diagnosis of MM, almost 80% of patients suffer from bone disease. Earlier diagnosis and intervention in MM bone disease would potentially improve treatment outcome and patient survival. New preclinical models are needed for developing novel diagnostic markers of bone structural changes as early as possible in the disease course. Here, we report a proof-of-concept, syngeneic, intrafemoral MOPC315.BM MM murine model in skeletally mature BALB/c mice for detection and characterization of very early changes in the extracellular matrix (ECM) of MM-injected animals. Bioluminescence imaging (BLI) in vivo confirmed myeloma engraftment in 100% of the animals with high osteoclast activity within 21 days after tumor cell inoculation. Early signs of aggressive bone turnover were observed on the outer bone surfaces by high-resolution microcomputed tomography (microCT). Synchrotron phase contrast-enhanced microcomputer tomography (PCE-CT) revealed very local microarchitecture differences highlighting numerous active sites of erosion and new bone at the micrometer scale. Correlative backscattered electron imaging (BSE) and confocal laser scanning microscopy allowed direct comparison of mineralized and nonmineralized matrix changes in the cortical bone. The osteocyte lacunar-canalicular network (OLCN) architecture was disorganized, and irregular-shaped osteocyte lacunae were observed in MM-injected bones after 21 days. Our model provides a potential platform to further evaluate pathological MM bone lesion development at the micro- and ultrastructural levels. These promising results make it possible to combine material science and pharmacological investigations that may improve early detection and treatment of MM bone disease.

Remodelling of human bone on the chorioallantoic membrane of the chicken egg: De novo bone formation and resorption.

Traditionally used as an angiogenic assay, the chorioallantoic membrane (CAM) assay of the chick embryo offers significant potential as an in vivo model for xenograft organ culture. Viable human bone can be cultivated on the CAM and increases in bone volume are evident; however, it remains unclear by what mechanism this change occurs and whether this reflects the physiological process of bone remodelling. In this study we tested the hypothesis that CAM-induced bone remodelling is a consequence of host and graft mediated processes. Bone cylinders harvested from femoral heads post surgery were placed on the CAM of green fluorescent protein (GFP)-chick embryos for 9 days, followed by micro computed tomography (µCT) and histological analysis. Three-dimensional registration of consecutive µCT-scans showed newly mineralised tissue in CAM-implanted bone cylinders, as well as new osteoid deposition histologically. Immunohistochemistry demonstrated the presence of bone resorption and formation markers (Cathepsin K, SOX9 and RUNX2) co-localising with GFP staining, expressed by avian cells only. To investigate the role of the human cells in the process of bone formation, decellularised bone cylinders were implanted on the CAM and comparable increases in bone volume were observed, indicating that avian cells were responsible for the bone mineralisation process. Finally, CAM-implantation of acellular collagen sponges, containing bone morphogenetic protein 2, resulted in the deposition of extracellular matrix and tissue mineralisation. These studies indicate that the CAM can respond to osteogenic stimuli and support formation or resorption of implanted human bone, providing a humanised CAM model for regenerative medicine research and a novel short-term in vivo model for tissue engineering and biomaterial testing.

* The Chorioallantoic Membrane Assay for Biomaterial Testing in Tissue Engineering: A Short-Term In Vivo Preclinical Model.

The fields of regenerative medicine and tissue engineering offer significant promise to address the urgent unmet need for therapeutic strategies in a number of debilitating conditions, diseases, and tissue needs of an aging population. Critically, the safety and efficacy of these pioneering strategies need to be assessed before clinical application, often necessitating animal research as a prerequisite. The growing number of newly developed potential treatments, together with the ethical concerns involved in the application of in vivo studies, requires the implementation of alternative models to facilitate such screening of new treatments. The present review examines the current in vitro and in vivo models of preclinical research with particular emphasis on the chorioallantoic membrane (CAM) assay as a minimally invasive, short-term in vivo alternative. Traditionally used as an angiogenic assay, the CAM of the developing chick embryo provides a noninnervated rapidly growing vascular bed, which can serve as a surrogate blood supply for organ culture, and hence a platform for biomaterial testing. This review offers an overview of the CAM assay and its applications in biomedicine as an in vivo model for organ culture and angiogenesis. Moreover, the application of imaging techniques (magnetic resonance imaging, microcomputed tomography, fluorescence labeling for tracking) will be discussed for the evaluation of biomaterials cultured on the CAM. Finally, an overview of the CAM assay methodology will be provided to facilitate the adoption of this technique across laboratories and the regenerative medicine community, and thus aid the reduction, replacement, and refinement of animal experiments in research.

The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering.

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (µCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by µCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

Transient Canonical Wnt Stimulation Enriches Human Bone Marrow Mononuclear Cell Isolates for Osteoprogenitors.

Activation of the canonical Wnt signaling pathway is an attractive anabolic therapeutic strategy for bone. Emerging data suggest that activation of the Wnt signaling pathway promotes bone mineral accrual in osteoporotic patients. The effect of Wnt stimulation in fracture healing is less clear as Wnt signaling has both stimulatory and inhibitory effects on osteogenesis. Here, we tested the hypothesis that transient Wnt stimulation promotes the expansion and osteogenesis of a Wnt-responsive stem cell population present in human bone marrow. Bone marrow mononuclear cells (BMMNCs) were isolated from patients undergoing hip arthroplasty and exposed to Wnt3A protein. The effect of Wnt pathway stimulation was determined by measuring the frequency of stem cells within the BMMNC populations by fluorescence-activated cell sorting and colony forming unit fibroblast (CFU-F) assays, before determining their osteogenic capacity in in vitro differentiation experiments. We found that putative skeletal stem cells in BMMNC isolates exhibited elevated Wnt pathway activity compared with the population as whole. Wnt stimulation resulted in an increase in the frequency of skeletal stem cells marked by the STRO-1(bright) /Glycophorin A(-) phenotype. Osteogenesis was elevated in stromal cell populations arising from BMMNCs transiently stimulated by Wnt3A protein, but sustained stimulation inhibited osteogenesis in a concentration-dependent manner. These results demonstrate that Wnt stimulation could be used as a therapeutic approach by transient targeting of stem cell populations during early fracture healing, but that inappropriate stimulation may prevent osteogenesis.