Contrast-enhanced Micro-computed Tomography Research Articles

Revealing Detailed Cartilage Function Through Nanoparticle Diffusion Imaging: A Computed Tomography & Finite Element Study

The ability of articular cartilage to withstand significant mechanical stresses during activities, such as walking or running, relies on its distinctive structure. Integrating detailed tissue properties into subject-specific biomechanical models is challenging due to the complexity of analyzing these characteristics. This limitation compromises the accuracy of models in replicating cartilage function and impacts predictive capabilities. To address this, methods revealing cartilage function at the constituent-specific level are essential. In this study, we demonstrated that computational modeling derived individual constituent-specific biomechanical properties could be predicted by a novel nanoparticle contrast-enhanced computer tomography (CECT) method. We imaged articular cartilage samples collected from the equine stifle joint (n = 60) using contrast-enhanced micro-computed tomography (µCECT) to determine contrast agents’ intake within the samples, and compared those to cartilage functional properties, derived from a fibril-reinforced poroelastic finite element model. Two distinct imaging techniques were investigated: conventional energy-integrating µCECT employing a cationic tantalum oxide nanoparticle (Ta2O5-cNP) contrast agent and novel photon-counting µCECT utilizing a dual-contrast agent, comprising Ta2O5-cNP and neutral iodixanol. The results demonstrate the capacity to evaluate fibrillar and non-fibrillar functionality of cartilage, along with permeability-affected fluid flow in cartilage. This finding indicates the feasibility of incorporating these specific functional properties into biomechanical computational models, holding potential for personalized approaches to cartilage diagnostics and treatment.

Postnatal changes in thyroid cartilage shape and cartilage matrix composition are not synchronized in Mus musculus.

The study was conducted to quantify laryngeal cartilage matrix composition and to investigate its relationship with cartilage shape in a mouse model. A sample of 30 mice (CD-1 mouse, Mus musculus) from five age groups (postnatal Days 2, 21, 90, 365, and 720) were used. Three-dimensional mouse laryngeal thyroid cartilage reconstructions were generated from contrast-enhanced micro-computed tomography (CT) image stacks. Cartilage matrix composition was estimated as Hounsfield units (HU). HU were determined by overlaying 3D reconstructions as masks on micro-CT image stacks and then measuring the attenuation. Cartilage shape was quantified with landmarks placed on the surface of the thyroid cartilage. Shape differences between the five age groups were analyzed using geometric morphometrics and multiparametric analysis of landmarks. The relationship between HU and shape was investigated with correlational analyses. Among five age groups, HU became higher in older animals. The shape of the thyroid cartilage changes with age throughout the entire life of a mouse. The changes in shape were not synchronized with changes in cartilage matrix composition. The thyroid cartilage of young and old M. musculus larynx showed a homogenous mineralization pattern. High-resolution contrast-enhanced micro-CT imaging makes the mouse larynx accessible for analysis of genetic and environmental factors affecting shape and matrix composition.

Abstract 14594: Dual Contrast-Enhanced Micro-Computed Tomography for Simultaneous Characterization of Macrocalcification and Tri-Layered Structural Remodeling in Explanted Human Aortic Valves

Background: Micro-computed tomography (microCT) is excellent for detecting macrocalcification but ineffective at revealing other pathologically relevant structural details in the valves affected by aortic stenosis (AS). Here, we aimed to develop a dual contrast-enhanced microCT technique to simultaneously assess macrocalcification and layer-specific remodeling in diseased human aortic valves. Methods: Diseased (n=15) and normal (n=9) aortic valve leaflets were obtained from human subjects undergoing aortic valve replacement for AS and heart transplantation/autopsy, respectively. Leaflets were fixed (4% paraformaldehyde and 2% glutaraldehyde), stained with dual contrast (1% osmium tetroxide and 1% uranyl acetate), and embedded in epoxy resin. Additional unstained leaflets (n=3 diseased, n=3 normal), similarly processed otherwise, served as staining controls. MicroCT of embedded leaflets was performed using a ZEISS Xradia 520 Versa scanner, with low energy beam (80 kVp, 5 W, LE4 filter) and high-resolution acquisitions (15 μm x 15 μm, 5 sec exposure x 1601 projections). Reconstructed images were analyzed for leaflet/layer thicknesses at midline and macrocalcification volume using Dragonfly. Results: The tri-layered tissue structure was only visualized in stained but not unstained leaflets ( p =0.002). With staining, each layer was found to be thicker in the diseased than the normal leaflets (0.28±0.13 mm vs 0.18±0.07 mm, p =0.0002 for fibrosa; 0.64±0.68 mm vs 0.29±0.19 mm, p =0.012 for spongiosa; 0.32±0.16 mm vs 0.16±0.07 mm, p <0.0001 for ventricularis). The macrocalcification volume in the diseased leaflets was greater than that in the normal leaflets (84.7±84.4 mm 3 vs 0 mm 3 , p <0.0001) and correlated only with the thickness of spongiosa ( r =0.79, p <0.001) but not other layers ( p =0.67, p =0.26). Macrocalcification was as frequently observed in spongiosa as in fibrosa (66.7% vs 66.7% of diseased leaflets, p =0.3) but never in ventricularis. Conclusions: Dual contrast-enhanced microCT enables high-quality imaging of both macrocalcification and layer-specific remodeling in diseased human aortic valves. The results here suggest an intimate relationship between macrocalcification and spongiosa that warrants further investigation.

Ultra-high-field MRI of postmortem human fetal wrist joints: initial experience

BackgroundThis study aimed to assess the feasibility of postmortem ultra-high-field magnetic resonance imaging (UHF-MRI) to study fetal musculoskeletal anatomy and explore the contribution of variation in iodine and formaldehyde (paraformaldehyde, PFA) treatment of tissue.MethodsSeven upper extremities from human fetuses with gestational ages of 19 to 24 weeks were included in this experimental study, approved by the Medical Research Ethics Committee. The specimens were treated with various storage (0.2–4% PFA) and staining (Lugol’s solution) protocols and the wrist joint was subsequently imaged with 7.0 T UHF-MRI. Soft-tissue contrast was quantified by determining regions of interest within a chondrified carpal bone (CCB) from the proximal row, the triangular fibrocartilage (TFC), and the pronator quadratus muscle (PQM) and calculating the contrast ratios (CRs) between mean signal intensities of CCB to TFC and CCB to PQM.ResultsUHF-MRI showed excellent soft-tissue contrast in different musculoskeletal tissues. Increasing storage time in 4% PFA, CRs decreased, resulting in a shift from relatively hyperintense to hypointense identification of the CCB. Storage in 0.2% PFA barely influenced the CRs over time. Lugol’s solution caused an increase in CRs and might have even contributed to the inversion of the CRs.ConclusionsUHF-MRI is a feasible technique to image musculoskeletal structures in fetal upper extremities and most successful after short storage in 4% PFA or prolonged storage in 0.2% PFA. The use of Lugol’s solution is not detrimental on soft-tissue MRI contrast and therefore enables effectively combining UHF-MRI with contrast-enhanced micro-computed tomography using a single preparation of the specimen.Relevance statementUHF-MRI can be performed after CE-micro-CT to take advantage of both techniques.Key points• UHF-MRI is feasible to study human fetal cartilaginous and ligamentous anatomy.• Storage in low PFA concentrations (i.e., 0.2%) improves soft-tissue contrast in UHF-MRI.• Limited preservation time in high concentrations of PFA improves soft-tissue contrast in UHF-MRI.• Prior staining with Lugol’s solution does not reduce soft-tissue contrast in UHF-MRI.Graphical

Ontogeny of cranial musculoskeletal anatomy and its relationship to allometric increase in bite force in an insectivorous bat (Eptesicus fuscus).

Bite force is a performance metric commonly used to link cranial morphology with dietary ecology, as the strength of forces produced by the feeding apparatus largely constrains the foods an individual can consume. At a macroevolutionary scale, there is evidence that evolutionary changes in the anatomical elements involved in producing bite force have contributed to dietary diversification in mammals. Much less is known about how these elements change over postnatal ontogeny. Mammalian diets drastically shift over ontogeny-from drinking mother's milk to feeding on adult foods-presumably with equally drastic changes in the morphology of the feeding apparatus and bite performance. Here, we investigate ontogenetic morphological changes in the insectivorous big brown bat (Eptesicus fuscus), which has an extreme, positive allometric increase in bite force during development. Using contrast-enhanced micro-computed tomography scans of a developmental series from birth to adult morphology, we quantified skull shape and measured skeletal and muscular parameters directly related to bite force production. We found pronounced changes in the skull over ontogeny, including a large increase in the volume of the temporalis and masseter muscles, and an expansion of the skull dome and sagittal crest that would serve to increase the temporalis attachment area. These changes indicate that development of the jaw adductors play an important role in the development of biting performance of these bats. Notably, static bite force increases with positive allometry with respect to all anatomical measures examined, suggesting that modifications in biting dynamics and/or improved motor coordination also contribute to increases in biting performance.

Head anatomy of a lantern shark wet-collection specimen (Chondrichthyes: Etmopteridae).

In this study, we apply a two-step (untreated and soft tissue stained) diffusible iodine-based contrast-enhanced micro-computed tomography array to a wet-collection Lantern Shark specimen of Etmopterus lucifer. The focus of our scanning approach is the head anatomy. The unstained CT data allow the imaging of mineralized (skeletal) tissue, while results for soft tissue were achieved after staining for 120 h in a 1% ethanolic iodine solution. Three-dimensional visualization after the segmentation of hard as well as soft tissue reveals new details of tissue organization and allows us to draw conclusions on the significance of organs in their function. Outstanding are the ampullae of Lorenzini for electroreception, which appear as the dominant sense along with the olfactory system. Corresponding brain areas of these sensory organs are significantly enlarged as well and likely reflect adaptations to the lantern sharks' deep-sea habitat. While electroreception supports the capture of living prey, the enlarged olfactory system can guide the scavenging of these opportunistic feeders. Compared to other approaches based on the manual dissection of similar species, CT scanning is superior in some but not all aspects. For example, fenestrae of the cranial nerves within the chondrocranium cannot be identified reflecting the limitations of the method, however, CT scanning is less invasive, and the staining is mostly reversible and can be rinsed out.

Early patellofemoral cartilage and bone pathology in a rat model of noninvasive anterior cruciate ligament rupture

ABSTRACT Objective Anterior cruciate ligament rupture (ACLR) is a risk factor for the development of post-traumatic osteoarthritis (PTOA). While PTOA in the tibiofemoral joint compartment is well-characterized, very little is known about pathology in the patellofemoral compartment after ACL injury. Here, we evaluated the extent to which ACLR induces early patellofemoral joint damage in a rat model. Methods Adult female Lewis rats were randomized to noninvasive ACLR or Sham. Two weeks post-injury, contrast-enhanced micro-computed tomography (µCT) of femoral and patellar cartilage was performed using 20% v/v ioxaglate. Morphometric parameters of femoral trochlear and patellar cartilage, subchondral bone, and trabecular bone were derived from µCT. Sagittal Safranin-O/Fast-Green-stained histologic sections were graded using the OARSI score in a blinded fashion. Results Cartilage and bone remodelling consistent with an early PTOA phenotype were observed in both femoral trochleas and patellae. ACLR caused osteophyte formation of the patella and pathology in the superficial zone of articular cartilage, including surface fibrillation, fissures, increased cellularity, and abnormal chondrocyte clustering. There were significant increases in thickness of patellar and trochlear cartilage. Loss of subchondral bone thickness, bone volume fraction, and tissue mineral density, as well as changes to patellar and trochlear trabecular microarchitecture, were indicative of catabolic bone remodelling. Several injury-induced changes, including increased cartilage thickness and trabecular spacing and decreased trabecular number were more severe in the patella compared to the trochlea. Conclusion The patellofemoral joint develops mild but evident pathology in the early period following ACL rupture, extending the utility of this model to the study of patellofemoral PTOA.

Contrast-enhanced microCT evaluation of degeneration following partial and full width injuries to the mouse lumbar intervertebral disc

A targeted injury to the mouse intervertebral disc (IVD) is often used to recapitulate the degenerative cascade of the human pathology. Since injuries can vary in magnitude and localization, it is critical to examine the effects of different injuries on IVD degeneration. We thus evaluated the degenerative progression resulting from either a partial- or full-width injury to the mouse lumbar IVD using contrast-enhanced micro-computed tomography and histological analyses. A lateral-retroperitoneal surgical approach was used to access the lumbar IVD, and the injuries to the IVD were produced by either incising one side of the annulus fibrosus or puncturing both sides of the annulus fibrosus. Female C57BL/6J mice of 3–4 months age were used in this study. They were divided into three groups to undergo partial-width, full-width, or sham injuries. The L5/6 and L6/S1 lumbar IVDs were surgically exposed, and then the L6/S1 IVDs were injured using either a surgical scalpel (partial-width) or a 33G needle (full-width), with the L5/6 serving as an internal control. These animals recovered and then euthanized at either 2-, 4-, or 8-weeks after surgery for evaluation. The IVDs were assessed for degeneration using contrast-enhanced microCT (CEµCT) and histological analysis. The high-resolution 3D CEµCT evaluation of the IVD confirmed that the respective injuries were localized within one side of the annulus fibrosus or spanned the full width of the IVD. The full-width injury caused significant deteriorations in the nucleus pulposus, annulus fibrous and at the interfaces after 2 weeks, which was sustained through the 8 weeks, while the partial width injury caused localized disruptions that remained limited to the annulus fibrosus. The use of CEµCT revealed distinct IVD degeneration profiles resulting from partial- and full-width injuries. The partial width injury may serve as an alternative model for IVD degeneration resulting from localized annulus fibrosus injuries.

The anatomy of the head muscles in caecilians (Amphibia: Gymnophiona): Variation in relation to phylogeny and ecology?

In limbless fossorial vertebrates such as caecilians (Gymnophiona), head-first burrowing imposes severe constraints on the morphology and overall size of the head. As such, caecilians developed a unique jaw-closing system involving the large and well-developed m. interhyoideus posterior, which is positioned in such a way that it does not significantly increase head diameter. Caecilians also possess unique muscles among amphibians. Understanding the diversity in the architecture and size of the cranial muscles may provide insights into how a typical amphibian system was adapted for a head-first burrowing lifestyle. In this study, we use dissection and non-destructive contrast-enhanced micro-computed tomography (μCT) scanning to describe and compare the cranial musculature of 13 species of caecilians. Our results show that the general organization of the head musculature is rather constant across extant caecilians. However, the early-diverging Rhinatrema bivittatum mainly relies on the 'ancestral' amphibian jaw-closing mechanism dominated by the m. adductores mandibulae, whereas other caecilians switched to the use of the derived dual jaw-closing mechanism involving the additional recruitment of the m. interhyoideus posterior. Additionally, the aquatic Typhlonectes show a greater investment in hyoid musculature than terrestrial caecilians, which is likely related to greater demands for ventilating their large lungs, and perhaps also an increased use of suction feeding. In addition to three-dimensional interactive models, our study provides the required quantitative data to permit the generation of accurate biomechanical models allowing the testing of further functional hypotheses.

Contrast-enhanced micro-computed tomography of compartment and time-dependent changes in femoral cartilage and subchondral plate in a murine model of osteoarthritis.

A lack of understanding of the mechanisms underlying osteoarthritis (OA) progression limits the development of effective long-term treatments. Quantitatively tracking spatiotemporal patterns of cartilage and bone degeneration is critical for assessment of more appropriately targeted OA therapies. In this study, we use contrast-enhanced micro-computed tomography (μCT) to establish a timeline of subchondral plate (SCP) and cartilage changes in the murine femur after destabilization of the medial meniscus (DMM). We performed DMM or sham surgery in 10-12-week-old male C57Bl/6J mice. Femora were imaged using μCT after 0, 2, 4, or 8 weeks. Cartilage-optimized scans were performed after immersion in contrast agent CA4+. Bone mineral density distribution (BMDD), cartilage attenuation, SCP, and cartilage thickness and volume were measured, including lateral and medial femoral condyle and patellar groove compartments. As early as 2 weeks post-DMM, cartilage thickness significantly increased and cartilage attenuation, SCP volume, and BMDD mean significantly decreased. Trends in cartilage and SCP metrics within each joint compartment reflected those seen in global measurements, and both BMDD and SCP thickness were consistently greater in the lateral and medial condyles than the patellar groove. Sham surgery also resulted in significant changes to SCP and cartilage metrics, highlighting a potential limitation of using surgical models to study tissue morphology or composition changes during OA progression. Contrast-enhanced μCT analysis is an effective tool to monitor changes in morphology and composition of cartilage, and when combined with bone-optimized μCT, can be used to assess the progression of degenerative changes after joint injury.

Subchondral involvement in osteonecrosis of the femoral head: insight on local composition, microstructure and vascularization

To determine changes of subchondral bone composition, micro-structure, bone marrow adiposity and micro-vascular perfusion in end-stage osteonecrosis of the femoral head (ONFH) compared to osteoarthritis (OA) using a combined in vivo and ex vivo approach. Male patients up to 70 years old referred for total hip replacement surgery for end-stage ONFH were included (n=14). Fifteen patients with OA were controls. Pre-operative MRI was used to assess bone perfusion (dynamic contrast-enhanced (DCE) sequences) and marrow fat content (chemical shift imaging). Three distinct zones of femoral head subchondral bone - necrotic, sclerotic, distant - were compared between groups. After surgery, plugs were sampled in these zones and Raman spectroscopy was applied to characterize bone mineral and organic components (old and newly-formed), and contrast-enhanced micro-computed tomography (CE-μCT) to determine bone micro-structural parameters and volume of bone marrow adipocytes, using conventional 2D histology as a reference. In the necrotic zone of ONFH patients compared to OA patients: 1) the subchondral plate did not exhibit significant changes in composition nor structure; 2) the volume fraction of subchondral trabecular bone was significantly lower; 3) type-B carbonate substitution was less pronounced, 4) collagen maturity was more pronounced; and 5) bone marrow adipocytes were significantly depleted. The sclerotic zone from the ONFH group showed greater trabecular thickness, and higher DCE-MRI AUC and Ktrans. Volume fraction of subchondral bone, trabecular number, and Kep were significantly lower in the distant zone of the ONFH group. This study demonstrated alterations of subchondral bone microstructure, composition, perfusion and/or adipose content in all zones of the femoral head.

Hind foot drumming: Volumetric micro-computed tomography investigation of the hind limb musculature of three African mole-rat species (Bathyergidae).

Several species of African mole-rats use seismic signalling by means of hind foot drumming for communication. The present study aimed to create three-dimensional reconstructions and compare volumetric measurements of 27muscles of the hind limb of two drumming (Georychus capensis and Bathyergus suillus) and one non-drumming (Cryptomys hottentotus natalensis) species of African mole-rats. Diffusible iodine contrast-enhanced micro-computed tomography (diceCT) scans were performed on six specimens per species. Manual segmentation of the scans using VGMAX Studio imaging software allowed for individual muscles to be separated while automatically determining the volume of each muscle. The volume of the individual muscles was expressed as a percentage of the total hind limb volume and statistically compared between species. Subsequently, three-dimensional reconstructions of these muscles were created. Musculus gracilis anticus had a significantly larger percentage of the total hind limb muscle volume in both drumming species compared to the non-drumming C. h. natalensis. Furthermore, several hip and knee extensors, namely mm. gluteus superficialis, semimembranosus, gluteofemoralis, rectus femoris and vastus lateralis, had significantly larger muscle volume percentages in the two drumming species (G. capensis and B. suillus) compared to the non-drumming species. While not statistically significant, G. capensis had larger muscle volume percentages in several key hip and knee extensors compared to B. suillus. Additionally, G capensis had the largest summed percentage of the total hind limb volume in the hip flexor, hip extensor, knee extensor and ankle plantar flexor muscle groups in all the three species. This could be indicative of whole muscle hypertrophy in these muscles due to fast eccentric contractions that occur during hind foot drumming. However, significantly larger muscle volume percentages were observed in the scratch digging B. suillus compared to the other two chisel tooth digging species. Moreover, while not statistically significant, B. suillus had larger muscle volume percentages in several hip extensor and knee flexor muscles compared to G. capensis (except for m. vastus lateralis). These differences could be due to the large relative size of this species but could also be influenced by the scratch digging strategy employed by B. suillus. Therefore, while the action of hind foot drumming seems to influence certain key muscle volumes, digging strategy and body size may also play a role.

Characterization of Eocene flint

Eocene flint 48–56.0 million years old (mya) from the Negev desert (Israel) was characterized using a suite of analytical techniques. High-resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) of the inorganic component showed the texture, morphology, size, and distribution of two silica polymorphs: α–quartz and moganite. While euhedral forms were attributed to α-quartz, moganite crystals were comprised of spherulitic grains. An electron less-dense amorphous material (no scattering under SAED) was found between the siliceous crystallites. Energy dispersive X-rays (EDS) and electron energy loss spectroscopy (EELS) demonstrated that this electron less-dense amorphous material is composed solely of carbon. Low vacuum, low energy backscattered environmental scanning electron microscopy (BSE-eSEM) imaging of flint surfaces showed the presence of micrometer-sized organic inclusions randomly distributed throughout the siliceous matrix. Energy-dispersive X-ray studies (EDS) demonstrated that these organic micro-inclusions were composed of carbon, sulfur, and nitrogen with a C/N ratio attributed to marine sources. These micro-inclusions were not directly associated with hard-shell fossils. BSE-eSEM imaging conditions allowed the identification of entrapped carbon-rich organic material, which is not possible when applying commonly used electron microscopy conditions that require carbon coating and high acceleration voltages, rendering carbon-rich features electron-transparent. Phase contrast-enhanced micro-computed tomography (PC-μCT) showed that these organic micro-inclusions were randomly distributed throughout the siliceous matrix.Time-of-flight secondary ion mass spectrometry (ToF-SIMS), nano-Fourier transform infrared spectroscopy (nano-FTIR), and scanning probe microscopy (SPM) were used to further characterize these organic micro-inclusions. These three in situ analytical techniques with nanometer resolution provided complementary information on the chemical composition and structure of the organic material. Specifically, ToF-SIMS analysis revealed amino acid and hydrocarbon mass spectra fingerprints inside the organic micro-inclusions. While the former were exclusively found in the organic micro-inclusions, the mass spectral fingerprints for hydrocarbons were also found in the siliceous matrix in agreement with the HR-TEM/EDS/EELS results, where pure carbon was found between the siliceous nanocrystals. While ToF-SIMS provides chemical information, it does not provide structural information. Nano-FTIR analysis showed the presence of amide I and II infrared vibrations exclusively on the organic micro-inclusions. The scanning probe microscopy (SPM) techniques Peak Force Quantitative Nanomechanics (PF-QNM) and Contact Resonance Atomic Force Microscopy (CR-AFM) were used to assess the mechanical properties. PF-QNM measurements on the organic micro-inclusions, under dry and liquid conditions, demonstrated that the organic micro-inclusions swell upon hydration and soften, pointing toward the presence of hydrophilic molecules in agreement with nano-FTIR and ToF-SIMS results. CR-AFM allows in situ determination of the mechanical properties of materials with high stiffness at nanometer resolution. This technique, rarely used in a geological context, revealed that the organic micro-inclusions had an unusually high stiffness atypical for modern organic material, which was attributed to molecular cross-linking promoted by diagenesis.This work provided a comprehensive view of the inorganic and organic components of Eocene flint from the Negev desert with implications for paleontology and archaeology. It offers a roadmap of novel complementary techniques that can be used in the exploration of entrapped organic material in flint.

Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model

Articular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

Ontogenetic development of the shoulder joint muscles in frogs (Amphibia: Anura) assessed by digital dissection with implications for interspecific muscle homologies and nomenclature

Previous myological studies show inconsistencies with regard to the identification and naming of the shoulder joint muscles in frogs and toads (Amphibia: Anura). Those inconsistencies were revealed and resolved by assessing the ontogenetic development, innervation, and adult morphology of selected anuran species representing ancient lineages and two major neobatrachian groups. To do so, digital dissections of volumes acquired by histological serial sectioning, episcopic microtomy, and contrast-enhanced micro-computed tomography scanning were performed and three-dimensional reconstructions were derived. Muscle units crossing the shoulder joint were defined, their ontogenetic development was described, their homology across species was established, and a consistent nomenclature was suggested. The mm. anconaeus, dorsalis scapulae, latissimus dorsi, and the group of scapulohumeralis muscles were ontogenetically derived from the dorsal pre-muscle mass present in all tetrapods. The ventral pre-muscle mass gave rise to the mm. cleidohumeralis, episternohumeralis, supracoracoideus, coracoradialis, subcoracoscapularis, coracobrachialis, and pectoralis. The results indicate that the mm. anconaeus, dorsalis scapulae, latissimus dorsi, coracoradialis, and the portionis sternalis and abdominalis of the m. pectoralis have consistently been recognized and denoted in previous studies, whereas the names for the muscle units commonly denoted as m. coraco-brachialis longus and as parts of the m. deltoideus are misleading with regard to the ontogenetic origin of these muscles. The mm. scapulohumeralis profundus anterior and posterior, although present, have been overlooked in some studies. The mm. cleidohumeralis, supracoracoideus, and coracobrachialis are present with two parts or portions in some species, these portions have previously not always been recognized and assigned correctly.

Intra-arterial Contrast-enhanced Micro-computed Tomography Can Evaluate Intracranial Status in the Ultra-early Phase of Experimental Subarachnoid Hemorrhage in Rats

The endovascular perforation (EP) model is a common technique for experimental subarachnoid hemorrhage (SAH) in rats, simulating the pathophysiological features observed in the acute phase of SAH. Due to the drawbacks of large variations in the amount of bleeding, the results obtained from this model require severity evaluation. However, no less-invasive procedure could confirm the precise intracranial conditions immediately after establishing the rat EP model. We created a novel method for evaluating SAH immediately after establishing the rat EP model using intra-arterial contrast-enhanced micro-computed tomography (CT). We administered contrast agents continuously via the carotid artery during surgery and performed CT examination immediately after SAH induction. First, bleeding severity was classified by establishing a scoring system based on the CT findings (cSAH scoring system). Subsequently, we determined the actual SAH distribution macroscopically and histologically and compared it with the cSAH scores. Second, we investigated the contrast agent’s neurotoxicity in rats. Finally, we confirmed the correlation between cSAH scores and SAH severity, including neurological status, cerebral vasospasm, and hematoma volume 24 hr after SAH. Intra-arterial contrast-enhanced micro-CT could visualize the distribution of SAH proportionally to the bleeding severity immediately after establishing the EP model. Moreover, the contrast agent administration was determined not to be neurotoxic to rats. The cSAH scoring revealed a significant correlation with the SAH severity in the rat EP model (P <0.01). Thus, our minimally invasive method provided precise information on intracranial status in the ultra-early phase of SAH in rats EP model.

Quantitative Imaging Parameters of Contrast-Enhanced Micro-Computed Tomography Correlate with Angiogenesis and Necrosis in a Subcutaneous C6 Glioma Model.

Simple SummaryContrast-enhanced (CE) X-ray imaging techniques have been used to assess angiogenesis in patients and animal models of cancer in order to overcome the limitations of histological quantification of angiogenesis, such as spatial and temporal heterogeneity of tumors. Some studies have compared the quantitative imaging parameters obtained with static and dynamic CE X-ray imaging techniques, but their association with histological biomarkers of angiogenesis has never been directly compared. This study aimed to provide such a comparison in a suitable animal model for the study of angiogenesis, namely, the subcutaneous C6 glioma model. We found an agreement among the quantitative imaging parameters obtained with these techniques, and we also found an association between a set of them with angiogenesis and necrosis descriptors. This set of quantitative imaging parameters demonstrated a high potential to describe angiogenesis and could be used to assess treatment response in further studies with this animal model.The aim of this work was to systematically obtain quantitative imaging parameters with static and dynamic contrast-enhanced (CE) X-ray imaging techniques and to evaluate their correlation with histological biomarkers of angiogenesis in a subcutaneous C6 glioma model. Enhancement (E), iodine concentration (CI), and relative blood volume (rBV) were quantified from single- and dual-energy (SE and DE, respectively) micro-computed tomography (micro-CT) images, while rBV and volume transfer constant (Ktrans) were quantified from dynamic contrast-enhanced (DCE) planar images. CI and rBV allowed a better discernment of tumor regions from muscle than E in SE and DE images, while no significant differences were found for rBV and Ktrans in DCE images. An agreement was found in rBV for muscle quantified with the different imaging protocols, and in CI and E quantified with SE and DE protocols. Significant strong correlations (Pearson r > 0.7, p < 0.05) were found between a set of imaging parameters in SE images and histological biomarkers: E and CI in tumor periphery were associated with microvessel density (MVD) and necrosis, E and CI in the complete tumor with MVD, and rBV in the tumor periphery with MVD. In conclusion, quantitative imaging parameters obtained in SE micro-CT images could be used to characterize angiogenesis and necrosis in the subcutaneous C6 glioma model.

Anatomy of the cardiac conduction system

The specialized cardiomyocytes that constitute the conduction system in the human heart, initiate the electric impulse and result in rhythmic and synchronized contraction of the atria and ventricles. Although the atrioventricular (AV) conduction axis was described more than a century ago by Sunao Tawara, the anatomic pathway for propagation of impulse from atria to the ventricles has been a topic of debate for years. Over the past 2 decades, there has been a resurgence of conduction system pacing (CSP) by implanting pacing leads in the His bundle region in lieu of chronic right ventricular pacing that is associated with worse clinical outcomes. The inherent limitations of implanting the leads in the His bundle region has led to the emergence of left bundle branch area pacing in the past 3years as an alternative strategy for CSP. The clinical experience from performing CSP has helped electrophysiologists gain deeper insight into the anatomy and physiology of cardiac conduction system. This review details the anatomy of the cardiac conduction system, and highlights some of the recently published articles that aid in better understanding of the AV conduction axis and its variations, the knowledge of which is critical for CSP. The remarkable evolution in technology has led to visualization of the cardiac conduction system using noninvasive, nondestructive high-resolution contrast-enhanced micro-computed tomography imaging that may aid in future CSP. We also discuss from anatomical perspective, the differences seen clinically with His bundle pacing and left bundle branch area pacing.

3D reconstruction of coronary artery bifurcations from coronary angiography and optical coherence tomography: feasibility, validation, and reproducibility

The three-dimensional (3D) representation of the bifurcation anatomy and disease burden is essential for better understanding of the anatomical complexity of bifurcation disease and planning of stenting strategies. We propose a novel methodology for 3D reconstruction of coronary artery bifurcations based on the integration of angiography, which provides the backbone of the bifurcation, with optical coherence tomography (OCT), which provides the vessel shape. Our methodology introduces several technical novelties to tackle the OCT frame misalignment, correct positioning of the OCT frames at the carina, lumen surface reconstruction, and merging of bifurcation lumens. The accuracy and reproducibility of the methodology were tested in n = 5 patient-specific silicone bifurcations compared to contrast-enhanced micro-computed tomography (µCT), which was used as reference. The feasibility and time-efficiency of the method were explored in n = 7 diseased patient bifurcations of varying anatomical complexity. The OCT-based reconstructed bifurcation models were found to have remarkably high agreement compared to the µCT reference models, yielding r2 values between 0.91 and 0.98 for the normalized lumen areas, and mean differences of 0.005 for lumen shape and 0.004 degrees for bifurcation angles. Likewise, the reproducibility of our methodology was remarkably high. Our methodology successfully reconstructed all the patient bifurcations yielding favorable processing times (average lumen reconstruction time < 60 min). Overall, our method is an easily applicable, time-efficient, and user-friendly tool that allows accurate and reproducible 3D reconstruction of coronary bifurcations. Our technique can be used in the clinical setting to provide information about the bifurcation anatomy and plaque burden, thereby enabling planning, education, and decision making on bifurcation stenting.

Me2SO perfusion time for whole-organ cryopreservation can be shortened: Results of micro-computed tomography monitoring during Me2SO perfusion of rat hearts.

Cryopreservation of whole organs and specific tissues is an important and continually expanding field of medicine. The protocols currently used for organ preservation do not ensure survivability and functionality; the protocols for ovarian tissue lead to acceptable outcomes, but these are still capable of further improvement. In general, cryopreservation protocols need to be optimized. One important approach to improving cryopreservation protocols in general involves reducing exposure to cytotoxic cryoprotective agents prior to freezing. This study, therefore, evaluated the real-time tissue penetration of dimethyl sulfoxide, a cryoprotective agent that is widely used in cryopreservation. Dimethyl sulfoxide penetration in rat hearts perfused with a 15% (v/v) dimethyl sulfoxide solution was examined in real-time using dynamic contrast-enhanced micro-computed tomography imaging. Viability of cardiomyocytes was not significantly affected by the dimethyl sulfoxide perfusion procedure. Two different perfusion rates were evaluated and compared with perfusion using a common iodine-based contrast agent (iomeprol). The dynamic contrast-enhanced micro-computed tomography imaging data showed that dimethyl sulfoxide flushes both the extracellular and intracellular spaces in rat heart tissue to 95% equilibration after ≈ 35 s via perfusion. Subsequent wash-out via perfusion is completed to 95% within ≈ 49 s. The equilibration duration routinely used in dimethyl sulfoxide–based protocols for cryopreservation should therefore be questioned. Shorter incubation duration would perhaps be sufficient, as well as being beneficial in relation to cell survivability. It would be helpful to have techniques for non-invasive real-time monitoring of the penetration of cryoprotective agents and such techniques should be used to revise cryopreservation protocols. Switching to perfusion-based equilibration procedures might be beneficial, if feasible.