Synchrotron Radiation-based Micro-computed Tomography Research Articles

First flower inclusion and fossil evidence of Cryptocarya (Laurales, Lauraceae) from Miocene amber of Zhangpu (China)

Lauraceae have one of the oldest fossil records of angiosperms with the earliest known evidence from the mid-Cretaceous. However, most of these records are based on leaves, especially from the Cenozoic of Asia, which are often challenging to assign to extinct or extant genera or species. In contrast, fossils of reproductive organs are more informative, but remain scarce. We here described the first Cenozoic Lauraceae flower of Asia and confirmed the presence of Cryptocarya in the Miocene Zhangpu flora (Fujian Province, south-eastern China) based on an amber inclusion. We scanned the specimen using synchrotron radiation-based micro-computed tomography (SRμCT) and then compared the fossil with extant flowers of the genus. The present fossil flower is small, bisexual, and polysymmetric, with a whorled and trimerous perianth and androecium along with a hypanthium around the gynoecium. The perianth comprises six undifferentiated tepals, the androecium consists of nine stamens and three innermost staminodes, and the gynoecium of a single carpel with a superior, unilocular (and uniovulate) ovary. Our study also shows that the fossil shares an unusual position of the typical staminal glands and a short androecial tube on the rim of the hypanthium with at least one extant Australian species of Cryptocarya, which have not been reported before. Nowadays, Lauraceae are still present in tropical to subtropical regions, mostly in American and Asian rainforests. The discovery of many Lauraceae leaf fossils in Zhangpu, as well as the amber flower of this study, is consistent with the current reconstruction of the amber source environment as a megathermal seasonal rainforest during the Mid-Miocene.

Large-scale morphometry of the subarachnoid space of the optic nerve

BackgroundThe meninges, formed by dura, arachnoid and pia mater, cover the central nervous system and provide important barrier functions. Located between arachnoid and pia mater, the cerebrospinal fluid (CSF)-filled subarachnoid space (SAS) features a variety of trabeculae, septae and pillars. Like the arachnoid and the pia mater, these structures are covered with leptomeningeal or meningothelial cells (MECs) that form a barrier between CSF and the parenchyma of the optic nerve (ON). MECs contribute to the CSF proteome through extensive protein secretion. In vitro, they were shown to phagocytose potentially toxic proteins, such as α-synuclein and amyloid beta, as well as apoptotic cell bodies. They therefore may contribute to CSF homeostasis in the SAS as a functional exchange surface. Determining the total area of the SAS covered by these cells that are in direct contact with CSF is thus important for estimating their potential contribution to CSF homeostasis.MethodsUsing synchrotron radiation-based micro-computed tomography (SRµCT), two 0.75 mm-thick sections of a human optic nerve were acquired at a resolution of 0.325 µm/pixel, producing images of multiple terabytes capturing the geometrical details of the CSF space. Special-purpose supercomputing techniques were employed to obtain a pixel-accurate morphometric description of the trabeculae and estimate internal volume and surface area of the ON SAS.ResultsIn the bulbar segment, the ON SAS microstructure is shown to amplify the MECs surface area up to 4.85-fold compared to an “empty” ON SAS, while just occupying 35% of the volume. In the intraorbital segment, the microstructure occupies 35% of the volume and amplifies the ON SAS area 3.24-fold.ConclusionsWe provided for the first time an estimation of the interface area between CSF and MECs. This area is of importance for estimating a potential contribution of MECs on CSF homeostasis.

A multimodal 3D imaging approach of pore networks in the human femur to assess age-associated vascular expansion and Lacuno-Canalicular reduction.

Cellular communication in the mechanosensory osteocyte Lacuno-Canalicular Network (LCN) regulates bone tissue remodeling throughout life. Age-associated declines in LCN size and connectivity dysregulate mechanosensitivity to localized remodeling needs of aging or damaged tissue, compromising bone quality. Synchrotron radiation-based micro-Computed Tomography (SRμCT) and Confocal Laser Scanning Microscopy (CLSM) were employed to visualize LCN and vascular canal morphometry in an age series of the anterior femur (males n=14, females n=11, age range=19-101, mean age=55). Age-associated increases in vascular porosity were driven by pore coalescence, including a significant expansion in pore diameter and a significant decline in pore density. In contrast, the LCN showed significant age-associated reductions in lacunar volume fraction, mean diameter, and density, and in canalicular volume fraction and connectivity density. Lacunar density was significantly lower in females across the lifespan, exacerbating their age-associated decline. Canalicular connectivity density was also significantly lower in females but approached comparable declining male values in older age. Our data illuminate the trajectory and potential morphometric sources of age-associated bone loss. Increased vascular porosity contributes to bone fragility with aging, while an increasingly reduced and disconnected LCN undermines the mechanosensitivity required to repair and reinforce bone. Understanding why and how this degradation occurs is essential for improving the diagnosis and treatment of age-related changes in bone quality and fragility.

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based μCT—A comparison with histology

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRμCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRμCT and histology. Phase-contrast SRμCT was performed with two different magnifications (voxel sizes 6.5 μm and 0.65 μm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 μm high-resolution SRμCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 μm low-resolution SRμCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRμCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method’s non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

Intraskeletal consistency in patterns of vascularity within bat limb bones.

Bats are the only mammals to have achieved powered flight. A key innovation allowing for bats to conquer the skies was a forelimb modified into a flexible wing. The wing bones of bats are exceptionally long and dynamically bend with wingbeats. Bone microarchitectural features supporting these novel performance attributes are still largely unknown. The humeri and femora of bats are typically avascular, except for large-bodied taxa (e.g., pteropodid flying foxes). No thorough investigation of vascular canal regionalization and morphology has been undertaken as historically it has been difficult to reconstruct the 3D architecture of these canals. This study augments our understanding of the vascular networks supporting the bone matrix of a sample of bats (n=24) of variable body mass, representing three families (Pteropodidae [large-bodied, species=6], Phyllostomidae [medium-bodied, species=2], and Molossidae [medium-bodied, species=1]). We employed Synchrotron Radiation-based micro-Computed Tomography (SRμCT) to allow for a detailed comparison of canal morphology within humeri and femora. Results indicate that across selected bats, canal number per unit volume is similar independent of body size. Differences in canal morphometry based on body size and bone type appear primarily related to a broader distribution of the canal network as cortical volume increases. Heavier bats display a relatively rich vascular network of mostly longitudinally-oriented canals that are localized mainly to the mid-cortical and endosteal bone envelopes. Taken together, our results suggest that relative vascularity of the limb bones of heavier bats forms support for nutrient exchange in a regional pattern.

Evaluating the effect of Avastin on breast cancer angiogenesis using synchrotron radiation.

The visualization of microvasculature is an essential step in understanding the mechanisms underlying early vessel disorders involved in breast cancer and for developing effective therapeutic strategies. However, generating detailed and reproducible data using immunohistochemistry analysis of breast cancer angiogenesis has been difficult. To analyze the diversification of angiogenesis in the development of tumor growth and evaluate the anti-vascular effects of Avastin (bevacizumab), we used new X-ray microangiography and third-generation synchrotron radiation-based micro-computed tomography (SR micro-CT) technology. With these techniques, we were able to investigate the structures and density of microvessels in xenograft mouse models (n=24). Barium sulfate nanoparticles were injected into the left cardiac ventricle of the mice to allow the visualization of blood vessels. Three-dimensional structures of microvessels were displayed with a high spatial image resolution of 20-30 µm. The density of angiogenesis and the incidence of lung metastasis were significantly reduced in xenograft mouse models of breast cancer treated with Avastin compared with control groups. Also, the density of smaller vessels (diameter <50 µm) was significantly decreased in the Avastin-treated mice, while the density of larger vessels (diameter >100 µm) was not significantly changed. Avastin inhibited tumor growth and lung metastasis by reducing microvessels. Additionally, synchrotron radiation (SR) techniques are useful as an additional tool for more precise quantification of angiogenesis.

Synchrotron micro-CT scanning leads to the discovery of a new genus of morphologically conserved echinoid (Echinodermata: Cassiduloida).

Here we use synchrotron radiation-based micro-computed tomography (SRµCT) images of type specimens to confidently place Cassidulus malayanus in a new genus (Kassandrina gen. nov.) that would not have been discovered with traditional techniques, and to describe a new species of Cassidulus (Cassidulus briareus sp. nov.) from Australia and designate a neotype for Cassidulus caribaearum. We also provide remarks describing the taxonomic history of each taxon and a diagnostic table of all living cassidulid species, and extend the known geographic and bathymetric range of C. caribaearum and C. malayanus. Besides rendering novel morphological data, the SRµCT images provide significant insights in the evolution of bourrelets of these cassiduloid echinoids.

Post-breakage changes in particle properties using synchrotron tomography

Granular recycled Construction and Demolition (C&D) materials reused in pavements, roads, and embankments experience particle breakage causing serious issues, such as settlement, during their service life. Particle breakage is of paramount importance for understanding the behaviour of particulate materials used not only in pavements, roads, and rail tracks, but also in the oil and gas industry and mineral processing. The engineering characteristics of a granular assembly is closely dependent on properties altered by breakage. Changes in particle properties of uniformly graded and spectrum of C&D specimens due to breakage under compressive loads were studied using three-dimensional Synchrotron Radiation-based Micro-Computed Tomography. The high-resolution 4D imaging was used to scan the inner body of the granular assemblies under compression. The resulting images were subsequently processed using a variety of techniques, including image thresholding, filtering, and segmentation, to identify and label each fragment in the assemblies. The fractal distribution of granular assemblies demonstrated that breakage becomes dominant in smaller particles rather than larger particles, where an increase in newly generated fine fragments brings about high coordination number surrounding the larger particles. More prominently, the results of particle morphology evolution showed a reversal trend as the stress increased. The C&D particles tended to generate more spherical fragments with higher aspect ratio, although by increasing the stress this tendency totally reversed. In addition, it has been found that the general trend of changes in particle shape obeys universality. In other words, for the materials tested; same generic evolution by increasing stress was observed irrespective of the material types or sizes.

Occurrence of osteon banding in adult human cortical bone.

Differentiating human from nonhuman fragmented bone is often accomplished using histological methods if the observation of gross morphology proves insufficient. Linearly oriented primary and/or secondary osteonal systems, commonly referred to as osteon bands, are described as a strong indicator of nonhuman bone, particularly the occurrence of multiple bands. This phenomenon has been conventionally documented using two-dimensional (2D) histology, but such analyses are destructive and typically limited to a single cross-section. Progressive developments in high-resolution X-ray imaging, however, allow for the nondestructive three-dimensional (3D) visualization of bone microarchitecture. The primary objective of the current research was to visualize and document the occurrence of osteon banding in adult human cortical bone using high-resolution synchrotron radiation-based micro-Computed Tomography (SR micro-CT). Synchrotron radiation-based micro-CT scanning was carried out at the Canadian Light Source (CLS) national synchrotron facility. The presence or absence of osteon banding was visualized in human skeletal elements from three adult males with representative samples from all regions of the skeleton (n = 129). If present, osteon banding was described and quantified. Results indicated that 23 of 129 human cortical bone specimens exhibited osteon banding, representing 18% of the sample. Linear arrangements of primary and/or secondary osteons were observed in the following skeletal elements: temporal, parietal, frontal, occipital, clavicle, mandible, femur, tibia, ulna, second metatarsal, and sacrum. The present work represents the first 3D examination of inter-element variation in osteon banding in adult human cortical bone. Findings indicate that the presence of multiple osteon bands in a single specimen is not diagnostic of nonhuman bone. As such, osteon banding categorically should not be taken as evidence of nonhuman bone in forensic and archaeological contexts.

Macro-mesoporous composites containing PEEK and mesoporous diopside as bone implants: characterization, in vitro mineralization, cytocompatibility, and vascularization potential and osteogenesis in vivo.

Bone implants of polyetheretherketone (PEEK) have become increasingly popular in the orthopedic field but its bioinertness and poor osteogenic properties significantly limit its applications for bone regeneration and osseointegration with host bone. In this study, by incorporation of mesoporous diopside (MD) into the PEEK matrix, macro-mesoporous PEEK/MD (PM) composites as bone implants with interconnected macropores of 300-400 μm were fabricated using the method of cold press-sintering and salt-leaching. The results showed that the compressive strength, porosity and water absorption of the porous PM composites significantly increased with an increase in the MD content. In addition, the incorporation of MD into PEEK obviously enhanced the mineralization ability of the PM composites, indicating good bioactivity. Moreover, the in vitro cell experiments indicated that the macro-mesoporous PM composites significantly promoted the adhesion and proliferation as well as osteogenic differentiation of MC3T3-E1 cells, which depended on the MD content. The results of synchrotron radiation-based micro-computed tomography (SRmCT) and histological analysis revealed that the porous PM composites possessed excellent osteogenic properties in vivo, which were obviously enhanced with an increase in the MD content. Moreover, the immunohistochemistry evaluation of type I collagen (COL I) and vascular endothelial growth factor (VEGF) confirmed that addition of MD into PEEK obviously enhanced the osteogenesis and vascularization potential of the macro-mesoporous PM composites in vivo. The results suggested that the PM composites containing macropores and mesopores as bone implants had great potential for bone repair/substitution.

Changes to Grain Properties due to Breakage in a Sand Assembly using Synchrotron Tomography

Grain breakage is of paramount importance for understanding the behaviour of granular materials used in various engineering applications, such as pavements, roads, rail tracks, the oil and gas industry, and mineral processing. Changes to grain properties of a uniformly graded sand specimen stemming from breakage during compression were studied with the aid of three-dimensional Synchrotron Radiation-based Micro-Computed Tomography. The fast scanning and high-resolution 4D imaging were utilised to capture images from the interior body of the granular assembly during loading. The fractal distribution of the sand assembly showed that breakage becomes dominant in smaller grains rather than larger ones, where an increase in the amount of newly generated fine fragments leads to high coordination number surrounding the larger grains. More importantly, the results of morphological changes in the particulate assembly revealed that there is a reversal trend in the grain morphology evolution with increasing stress. The sand grains tended to create more spherical fragments with higher aspect ratio whereas by increasing the stress this trend completely shifted.

Quantification of soil aggregate microstructure on abandoned cropland during vegetative succession using synchrotron radiation-based micro-computed tomography

Information for the microstructure of soil aggregates is crucial for understanding the mechanisms of various soil processes. The quantification of complex aggregate microstructure and its relationship to vegetative restoration, however, remains elusive. The objective of this study was to evaluate the impact of natural revegetation on aggregate microstructure using synchrotron-based X-ray micro-computed tomography and image analysis. Soil samples were collected from an active cropland and from four former croplands that have been abandoned for 6, 12, 23, and 32 years on the Loess Plateau, China. Soil aggregates (3–5mm) were scanned at a voxel resolution of 3.25μm, and the aggregate pore structure was visualized and quantified with ImageJ software. The stability of wet aggregates and other soil properties were also evaluated. The amount of soil organic carbon increased significantly and soil bulk density decreased significantly with abandonment age. Aggregate water stability was higher after revegetation but did not differ significantly among the abandoned sites. Total porosity, percentage of pores >75μm, and the fraction of elongated pores increased, but the number of pores, percentage of pores <75μm, and fractions of regular and irregular pores decreased after the croplands were abandoned. The fractal dimension, degree of anisotropy and the Euler number all indicated that aggregate microstructures were more connected and developed at all abandoned sites than in the active cropland, but the fractal dimension was more sensitive for monitoring the quality of the soil structure. The results from this study can help to improve our understanding of the soil processes during natural vegetative succession and of the importance of pore morphology in monitoring the quality of soil structure.

Three-dimensional reconstruction of Haversian systems in human cortical bone using synchrotron radiation-based micro-CT: morphology and quantification of branching and transverse connections across age.

This study uses synchrotron radiation-based micro-computed tomography (CT) scans to reconstruct three-dimensional networks of Haversian systems in human cortical bone in order to observe and analyse interconnectivity of Haversian systems and the development of total Haversian networks across different ages. A better knowledge of how Haversian systems interact with each other is essential to improve understanding of remodeling mechanisms and bone maintenance; however, previous methodological approaches (e.g. serial sections) did not reveal enough detail to follow the specific morphology of Haversian branching, for example. Accordingly, the aim of the present study was to identify the morphological diversity of branching patterns and transverse connections, and to understand how they change with age. Two types of branching morphologies were identified: lateral branching, resulting in small osteon branches bifurcating off of larger Haversian canals; and dichotomous branching, the formation of two new osteonal branches from one. The reconstructions in this study also suggest that Haversian systems frequently target previously existing systems as a path for their course, resulting in a cross-sectional morphology frequently referred to as 'type II osteons'. Transverse connections were diverse in their course from linear to oblique to curvy. Quantitative assessment of age-related trends indicates that while in younger human individuals transverse connections were most common, in older individuals more evidence of connections resulting from Haversian systems growing inside previously existing systems was found. Despite these changes in morphological characteristics, a relatively constant degree of overall interconnectivity is maintained throughout life. Altogether, the present study reveals important details about Haversian systems and their relation to each other that can be used towards a better understanding of cortical bone remodeling as well as a more accurate interpretation of morphological variants of osteons in cross-sectional microscopy. Permitting visibility of reversal lines, synchrotron radiation-based micro-CT is a valuable tool for the reconstruction of Haversian systems, and future analyses have the potential to further improve understanding of various important aspects of bone growth, maintenance and health.

The micro-vascular morphological changes of the rat spinal cord after injury by Absorb-contrast imaging of synchrotron radiation

Objective Using the synchrotron radiation-based micro-computed tomography (SRμCT) to observe the changes of three-dimensional micro-vascular morphology structure after acute spinal cord injury in rat.Methods Eight male Sprague-Dawley rats were randomly divided into control group and injury group (n =4).Injury group were deal with improved Allen' s weight dropping to establish the acute spinal cord injury model.All rats were deal with ascending aorta angiography in the next day.Freezing the specimens for 24 hours and then scanned them in Shanghai Institute of Applied Physics,Chinese Academy of Sciences.Processed and reconstructed the Data to three-dimensional images.Results The SRμCT can show the micro-vascular structure of spinal cord in rat completely and clearly.Combining with control group,we can see the lack and fracture of the micro-vessels of spinal cord in injury group significantly.The number and diameter of the micro-vessels were decreased obviously.The average number of vascular in injury group was 77.25 ± 16.80,and the control group was 168.30 ± 12.79 ; The average diameter of vascular in injury group was (16.270 ± 1.201) μm,but the control group was (20.210 ± 1.094) μm.Significant differences were existed between control group and injury group (P ＜ 0.05).Conclusion The SRμCT can get the three-dimensional and high resolution imaging of micro-vessels in small animal' s spinal cord.The micro-circulation network structure of spinal cord was prominently destroyed after acute spinal cord injury.The blood circulation of spinal cord in injury areas and its vicinity was serious damaged. Key words: Synchrotron radiation; Spinal cord injury ; Micro-vessels ; Morphology

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.

CHARACTERIZATION OF POROUS Mg–Zn ALLOY USING X-RAY TOMOGRAPHY AND ITS DRUG-LOADING BEHAVIOR

Porous Mg alloys will be promising orthopedic implants materials. In this study, porous Mg – Zn alloy with a porosity of 30% was prepared by powder metallurgy process. XRD was used to examine the composition of the sample and synchrotron radiation-based X-ray micro-computed tomography (SR-μCT) was used to investigate the pore properties of the sample. The results showed that the sample comprised a single Mg phase and a Mg 2 Zn 11 phase. The SR-μCT results showed that the porosity of the sample is about 30% which is approximate to the theoretical porosity. The pore morphology was irregular and the largest pore was about 200 μm in diameter. And a large pore cluster was identified meaning that most of the pores in the sample were interconnected with each other. This is beneficial for the materials transformation after the implantation of the sample in human body, which will boost the bone regeneration. Drug-loading experiment showed that Ibuprofen can be successfully loaded in porous Mg – Zn alloy, which means that porous Mg – Zn alloy could be an orthopedic drug delivery system (DDS).

The importance of the intracortical canal network for murine bone mechanics

As shown by recent data bone strength estimation can greatly be improved by including microarchitectural parameters in the analysis. Our previous results showed that intracortical canals (the living space of the vasculature and/or remodeling units) are a major contributor to cortical tissue porosity, and therefore, can be linked to mechanical bone properties. Consequently, the goal of this study was to investigate the importance of the intracortical canal network for murine bone mechanics. To study intracortical canals within murine femoral bone, we used a mouse model, including two mouse strains, C57BL/6J-Ghrhrlit/J (B6-lit/+) and C3.B6-Ghrhrlit/J (C3.B6-lit/+) representing low and high bone mass, respectively. The intracortical canal network was assessed by synchrotron radiation-based micro-computed tomography and the mechanical bone properties were derived from three-point bending experiments. Multiple linear regression models were built to explain the variation in ultimate force, work to fracture, and stiffness in terms of the morphometric parameters. The power to explain the variation in bone mechanics was increased significantly for most mechanical measures when including morphometric parameters of intracortical canals in addition to macroscopic morphometric measures. Specifically, we could derive generalized (mouse strain-independent) models for ultimate force, where the incorporation of intracortical canals in addition to macroscopic bone measures improved the explained variation in ultimate force considerably, which was confirmed by an increase in adjusted R2 of 73% and 8% for B6-lit/+ and C3.B6-lit/+, respectively. Further, we observed that the heterogeneity of the morphometric measures for the individual canal branches play an important role for explaining the variation in ultimate force. Finally, the current study provides strong evidence that work to fracture of murine bone, which is triggered critically by microcracks, is affected by intracortical canals. In summary, the study suggests that the intracortical canal network is important for bone mechanics.

Bone regeneration using cell-mediated responsive degradable PEG-based scaffolds incorporating with rhBMP-2

The treatment of large osseous defects remains a challenging clinical problem in orthopedic surgery. Particularly, strategies to control the appropriate degradation rate adapting to the tissue reconstruction are of essential for tissue regeneration. Here we report on a strategy to achieve adaptive degradation rate using cell-secreted protease as a switch. Disulfide-containing PEG-based scaffolds have been synthesized, and demonstrated to be responsive to the cell-secreted redox microenvironment. Thus, the cell-triggered degradation and liberation of growth factor are achieved. The osteoinductive growth factor, recombinant human bone morphogenetic protein-2 (rhBMP-2), is incorporated into the scaffold for bioactivity promotion. Degradations under the stimuli of reduced glutathione (GSH) at intracellular and extracellular concentrations was studied with the results of duration time ranging from 0.5 h to 22 days regulated by both concentrations of redox medium and polymer precursors. The rhBMP-2 loaded scaffolds evidently induced the ectopic bone formation in the mouse thigh muscles. In addition, we further investigated the in vivo effects of rhBMP-2-loaded scaffolds in a rabbit radius critical defect by radiography, three dimensional micro-computed tomographic (μCT) and synchrotron radiation-based micro-computed tomography (SRμCT) imaging, histological analysis, and biomechanical measurement. Scaffolds underwent gradual resorption and replacement by new bone and induced reunion of bone marrow cavity at 12 weeks, much better than the effect of self-repairing group. The results indicated that both osteoinduction and appropriate degradation played a crucial role in accelerating and promoting bone augmentation, as well as effective proangiogenesis. Such a strategy appears promising as 3D temporal scaffolds for potential orthopedic applications.

Detection of microvasculature in rat hind limb using synchrotron radiation

To detect deep-level microvascular structure in rat hind limb by synchrotron radiation and microangiographic technique. Microangiography in vivo and ex vivo was performed by synchrotron radiation based absorption imaging and phase contrast imaging, with omnipaque and barium sulfate solution as contrast media, respectively, and synchrotron radiation-based micro-computed tomography (SRmCT) was also performed to reveal three-dimensional morphology of the blood vessel in rat hind limb. Using microangiographic technique in vivo and in vitro (with barium sulfate), blood vessels in the rat limb muscle could be visualized with high resolution, and the fourth branches of iliac artery in rat hind limb could be detected with the minimum visualized blood vessels about 40 µm and 9 µm in diameter, respectively. In addition, the vascular network could be defined and analyzed at the micrometer scale from the 3D renderings of limb vessel as shown by SRmCT. Synchrotron radiation-based microangiography and SRmCT thus provided a practical and effective means to observe the microvasculature of rat hindlimb, which might be useful in assessment of angiogenesis in lower limbs.

P2-09-14: Evaluation of Angiogenesis Using Synchrotron Radiation in Xenograft Mouse Models of Breast Cancer.

Abstract Visualization of microvasculatures is an important step in understanding the mechanism of early vessel disorders in breast cancer and developing effective therapeutic strategies. However, quantitative evaluation of breast cancer angiogenesis using immunohistochemistry has been limited in generating detailed and reproducible data. To analyze the diversification of angiogenesis in the development of tumor growth and evaluate anti-vascular effects of Avastin, a new method of microvascular imaging was introduced in this paper. We used New X-ray microangiography and third-generation synchrotron radiation-based micro-computed tomography (SR micro-CT) to investigate the structures and density of microvessels at each stage of xenograft mouse models that were created by inoculation with MDA-MB-231HM cells (one breast cancer cell line with high metastasis potential). Barium Sulfate Nanoparticles, was used as a blood vessels contrast agent through left cardiac ventricle. Three dimensional structures of microvessels were displayed with a high spatial image resolution at around 20–30 μm. The density of microvessles was significantly reduced from the third week in mouse xenograft models. This observation with SR micro-CT was consistent with the results analyzed by immunohistochemical techniques. In addition, human breast cancer-bearing mice were treated site-specifically with humanized monoclonal antibody (Avastin) that targets all isoforms of VEGF-A. Tumor volume and the density of angiogenesis were significantly reduced in xenograft mouse models of breast cancer treated with avastin when compared with untreated mice as assessed by SR micro-CT. Specifically, the density of smaller vessels (di&amp;lt;50μm) was decreased significantly while the density of larger vessels was little changed in the Avastin-treated mice. Therefore, synchrotron radiation technique is a novel method for investigating breast cancer angiogenesis, and this might be considered as an additional complementary tool for more precise quantification of angiogenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-09-14.