Figure - available from: Contrast Media & Molecular Imaging
This content is subject to copyright. Terms and conditions apply.
Bone architecture and contrast-enhanced vessel visualisation in rat long bones. (a) Bone architecture of rat tibia (3D rendering) with compact bone (black arrows), trabecular structure (star), and epiphyseal gap (red arrow). (b) 3D rendering of contrast-enhanced scan showing diaphysis of a rat femur with contrasted central vessel (red arrows). (c) 2D slice of the data set shown in (b). Red pixels represent grey values typical for vessels. Vessel course (red arrow) and branching (white arrows) are clearly visible. (d) Length of contrasted central vessel. (e) Haematoxylin and eosin staining of mid-diaphysis of a rat femur showing bone (star), bone marrow (plus), ink-gelatin-filled (triangles), and empty (arrow) vessels. (f) Diameter of central vessel measured in histology and microtomography. (g) Histogram of tibial vessel diameters (n=10) showing a unimodal distribution, which is skewed left and wider for data from histology. Femoral vessel diameters display a similar distribution. (h) Closest distance of vessel center and bone measured in histology and microtomography.
Source publication
Objectives
Bone ischemia and necrosis are challenging to treat, requiring investigation of native and engineered bone revascularisation processes through advanced imaging techniques. This study demonstrates an experimental two-step method for precise bone and vessel analysis in native bones or vascularised bone grafts using X-ray microtomography (μ...
Citations
... Cerebral vessel density was measured by ink-gelatin perfusion (Hasan et al., 2013;Xue et al., 2014;Sutter et al., 2017). Mice were anesthetized with 4% chloral hydrate 7 days after the administration of nmFGF1. ...
Diabetes increases the risk of stroke, exacerbates neurological deficits, and increases mortality. Non-mitogenic fibroblast growth factor 1 (nmFGF1) is a powerful neuroprotective factor that is also regarded as a metabolic regulator. The present study aimed to investigate the effect of nmFGF1 on the improvement of functional recovery in a mouse model of type 2 diabetic (T2D) stroke. We established a mouse model of T2D stroke by photothrombosis in mice that were fed a high-fat diet and injected with streptozotocin (STZ). We found that nmFGF1 reduced the size of the infarct and attenuated neurobehavioral deficits in our mouse model of T2D stroke. Angiogenesis plays an important role in neuronal survival and functional recovery post-stroke. NmFGF1 promoted angiogenesis in the mouse model of T2D stroke. Furthermore, nmFGF1 reversed the reduction of tube formation and migration in human brain microvascular endothelial cells (HBMECs) cultured in high glucose conditions and treated with oxygen glucose deprivation/re-oxygenation (OGD). Amp-activated protein kinase (AMPK) plays a critical role in the regulation of angiogenesis. Interestingly, we found that nmFGF1 increased the protein expression of phosphorylated AMPK ( p -AMPK) both in vivo and in vitro . We found that nmFGF1 promoted tube formation and migration and that this effect was further enhanced by an AMPK agonist (A-769662). In contrast, these processes were inhibited by the application of an AMPK inhibitor (compound C) or siRNA targeting AMPK. Furthermore, nmFGF1 ameliorated neuronal loss in diabetic stroke mice via AMPK-mediated angiogenesis. In addition, nmFGF1 ameliorated glucose and lipid metabolic disorders in our mouse model of T2D stroke without causing significant changes in body weight. These results revealed that nmFGF1-regulated glucolipid metabolism and angiogenesis play a key role in the improvement of functional recovery in a mouse model of T2D stroke and that these effects are mediated by the AMPK signaling pathway.
... The damaged vasculature and bone marrow create a hypoxic microenvironment that recruits MSCs, fibroblasts, and endothelial cells to the fracture site. 21 The reparative phase consists of two subphases: the soft callus and the hard callus phases. 20 In the soft callus phase, the recruited MSCs start differentiating in two different ways according to their microenvironment. ...
Traditional bone tissue engineering (BTE) strategies induce direct bone-like matrix formation by mimicking the embryological process of intramembranous ossification. However, the clinical translation of these clinical strategies for bone repair is hampered by limited vascularization and poor bone regeneration after implantation in vivo. An alternative strategy for overcoming these drawbacks is engineering cartilaginous constructs by recapitulating the embryonic processes of endochondral ossification (ECO); these constructs have shown a unique ability to survive under hypoxic conditions as well as induce neovascularization and ossification. Such developmentally engineered constructs can act as transient biomimetic templates to facilitate bone regeneration in critical-sized defects. This review introduces the concept and mechanism of developmental BTE, explores the routes of endochondral bone graft engineering, highlights the current state of the art in large bone defect reconstruction via ECO-based strategies, and offers perspectives on the challenges and future directions of translating current knowledge from the bench to the bedside.
... After explantation, micro-computed tomography (CT) scans were performed with a tungsten X-ray source at 70 kV/260 mA and a 0.5-mm aluminum filter (Nanotom M, GE, USA) in order to image vessels, after complete decalcification, as previously described (Sutter et al., 2017). ...
Introduction: Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. We have previously shown that axially-vascularized osteogenic constructs, engineered by combining human stromal vascular fraction (SVF) cells and a ceramic scaffold, can revitalize necrotic bone of clinically relevant size in a rat model of AVN. For a clinical translation, the fetal bovine serum (FBS) used to generate such grafts should be substituted by a non-xenogeneic culture supplement. Human thrombin-activated platelet-rich plasma (tPRP) was evaluated in this context.
Material and methods: SVF cells were cultured inside porous hydroxyapatite scaffolds with a perfusion-based bioreactor system for 5 days. The culture medium was supplemented with either 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bovine bone cylinders to mimic the treatment of a necrotic bone. A ligated vascular bundle was inserted into the constructs upon subcutaneous implantation in the groin of nude rats. After 1 and 8 weeks, constructs were harvested and vascularization, host cell recruitment and bone formation were analyzed.
Results: After 1 week in vivo, constructs were densely vascularized, with no difference between tPRP- and FBS-based ones. After 8 weeks, bone formation and vascularisation was found in both tPRP- and FBS-pre-cultured constructs. However, the amount of bone and the vessel density were respectively 2.2- and 1.8-fold higher in the tPRP group. Interestingly, the density of M2, pro-regenerative macrophages was also significantly higher (6.9-fold) following graft preparation with tPRP than with FBS.
Discussion: Our findings indicate that tPRP is a suitable substitute for FBS to generate vascularized, osteogenic grafts from SVF cells and could thus be implemented in protocols for clinical translation of this strategy towards the treatment of bone loss and AVN.
... The damaged vasculature and bone marrow create a hypoxic microenvironment that recruits MSCs, fibroblasts, and endothelial cells to the fracture site. 21 The reparative phase consists of two subphases: the soft callus and the hard callus phases. 20 In the soft callus phase, the recruited MSCs start differentiating in two different ways according to their microenvironment. ...
... many) and a heparinized saline solution (100 IU/ml, 0.9% NaCl, B. Braun AG, Switzerland). Based on a previously developed protocol allowing for analysis of native bone and vasculature by microtomography (μCT), as well as histology [15], we then injected a dark-blue contrasting agent (5% w/v Gelatine-Gold, Carl Roth GmbH, Karlsruhe, Germany; 50% v/v Indian Ink, Lefranc & Bourgeois, Le Mans, France; 4% w/v D-Mannitol, Carl Roth GmbH; distilled water) until the skin of the lower extremities turned black, followed by immediate euthanasia of the rat by exsanguination. The cadavers were refrigerated at 4°C overnight to allow for polymerization of the contrast agent-containing gelatin. ...
... All samples were scanned twice: first without any contrast agent (for imaging of mineralization/bone) and a second time after decalcification with a 15% w/v EDTA (Sigma-Aldrich) solution in distilled water at 37°C for one week and consecutive radio-dense contrasting of the vessels. The latter was performed with 5% w/v phosphotungstic acid (PTA, Sigma-Aldrich) in distilled water at room temperature for 24 h, to image the vascular bed, as previously described [15]. This sequence allowed us to first obtain native scans of the radio-dense pre-bone and bone material without any signal overlap that a radio-dense vessel contrasting agent would produce if already applied at the time of explantation. ...
Large and complex bone defects represent challenging clinical scenarios, typically requiring autologous vascularized bone transplants. In order to bypass the numerous associated limitations, here we aimed at ectopically prefabricating a bone graft surrogate with vascular pedicle. A hollow cylinder of devitalized cancellous bone was used to define the space of a large bone substitute. This space was filled with devitalized pellets of engineered hypertrophic cartilage as bone-inducing material, in combination or not with stromal vascular fraction (SVF) of adipose tissue as source of osteoprogenitors and endothelial cells. Vascularization of the space was targeted through axial insertion of an arterio-venous (AV) bundle. Constructs were subcutaneously implanted in nude rats for 12 weeks and analyzed for bone formation and vascularization by histology and microtomography. Retrieved constructs were extensively vascularized in all conditions, with vessels sprouting from the AV bundle and reaching a higher density in the axially central volume. Bone tissue was formed through remodeling of hypertrophic cartilage, and quantitatively correlated with de novo vascularization. Our study demonstrates feasibility to prefabricate large, pedicled bone grafts in predefined shapes. The combination of an AV bundle with engineered hypertrophic cartilage provided a germ for the coupled processes of vascularization and bone formation. The demonstrated osteoinductivity of devitalized hypertrophic cartilage offers the opportunity of implementing the proposed regenerative surgery strategy through off-the-shelf materials.
... Ex vivo, contrastenhanced μCT involves the incubation of harvested samples in contrast agent prior to imaging, while in vivo it requires the nonhazardous agent to be locally or systemically delivered to the animal. Successful applications of contrast-enhanced μCT in the mouse include 3D visualization of articular cartilage (Das Neves Borges et al., 2014;Lakin et al., 2016), vasculature (Gayetskyy et al., 2014;Sutter et al., 2017), kidney (Missbach-Guentner et al., 2018), and heart (Dunmore-Buyze et al., 2014) morphology. ...
With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome‐wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development.
Current Protocols in Mouse Biology
... For bone and vessel analysis, 8-week samples were scanned twice: once for bone analysis and a second time after increasing vessel-to-bone contrast as previously described [19]. For this, samples were completely decalcified with 15% w/v EDTA pH 7.0 in distilled water at 37°C and then incubated overnight in 5% w/v phosphotungstic acid (Sigma-Aldrich) in distilled water before being scanned again. ...
Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses...
Background:
Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. The standard of care, vascularized bone grafts, is limited by donor site morbidity and restricted availability. The aim of this study was to generate and test engineered, axially vascularized SVF cells-based bone substitutes in a rat model of AVN.
Methods:
SVF cells were isolated from lipoaspirates and cultured onto porous hydroxyapatite scaffolds within a perfusion-based bioreactor system for 5 days. The resulting constructs were inserted into devitalized bone cylinders mimicking AVN-affected bone. A ligated vascular bundle was inserted upon subcutaneous implantation of constructs in nude rats. After 1 and 8 weeks in vivo, bone formation and vascularization were analyzed.
Results:
Newly-formed bone was found in 80% of SVF-seeded scaffolds after 8 weeks but not in unseeded controls. Human ALU+ cells in the bone structures evidenced a direct contribution of SVF cells to bone formation. A higher density of regenerative, M2 macrophages was observed in SVF-seeded constructs. In both experimental groups, devitalized bone was revitalized by vascularized tissue after 8 weeks.
Conclusion:
SVF cells-based osteogenic constructs revitalized fully necrotic bone in a challenging AVN rat model of clinically-relevant size. SVF cells contributed to accelerated initial vascularization, to bone formation and to recruitment of pro-regenerative endogenous cells.
Statement of significance:
Avascular necrosis (AVN) of bone often requires surgical treatment with autologous bone grafts, which is surgically demanding and restricted by significant donor site morbidity and limited availability. This paper describes a de novo engineered axially-vascularized bone graft substitute and tests the potential to revitalize dead bone and provide efficient new bone formation in a rat model. The engineering of an osteogenic/vasculogenic construct of clinically-relevant size with stromal vascular fraction of human adipose, combined to an arteriovenous bundle is described. This construct revitalized and generated new bone tissue. This successful approach proposes a novel paradigm in the treatment of AVN, in which an engineered, vascularized osteogenic graft would be used as a germ to revitalize large volumes of necrotic bone.
