ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

A glycan-based approach to cell characterization and isolation: Hematopoiesis as a paradigm.

Cell surfaces display a wide array of molecules that confer identity. While flow cytometry and cluster of differentiation (CD) markers have revolutionized cell characterization and purification, functionally heterogeneous cellular subtypes remain unresolvable by the CD marker system alone. Using hematopoietic lineages as a paradigm, we leverage the extraordinary molecular diversity of heparan sulfate (HS) glycans to establish cellular "glycotypes" by utilizing a panel of anti-HS single-chain variable fragment antibodies (scFvs). Prospective sorting with anti-HS scFvs identifies functionally distinct glycotypes within heterogeneous pools of mouse and human hematopoietic progenitor cells and enables further stratification of immunophenotypically pure megakaryocyte-erythrocyte progenitors. This stratification correlates with expression of a heptad of HS-related genes that is reflective of the HS epitope recognized by specific anti-HS scFvs. While we show that HS glycotyping provides an orthogonal set of tools for resolution of hematopoietic lineages, we anticipate broad utility of this approach in defining and isolating novel, viable cell types across diverse tissues and species.

Anastomoses (Superficial Cervical Ansa) Between the Cervical Plexus and Peripheral Facial Nerve Branches: Implications for Regional Anesthesia in Carotid Endarterectomies - Anatomical Study.

PURPOSE: Sensory innervation in the carotid triangle involves the cervical plexus, cranial nerves, and the sympathetic trunk. This innervation also applies to skin incision, including various anatomical structures with potentially different innervation, such as the skin (dermatomes), the platysma (myotomes), and the superficial layer of the cervical fascia (fasciotomes), as well as retromandibular retractor insertion (co-innervation: V, VII). The aim of this anatomical study was to develop an injection technique for carotid endarterectomies to additionally block anastomoses between the transverse cervical nerve (TCN), the cervical branch VII (CB VII), and the marginal mandibular branch VII (MMB VII). These anastomoses are also termed superficial cervical ansa (SCA). MATERIALS AND METHODS: Preparations (n=16) were performed on unembalmed donor cadavers (n=8). Subplatysmal injections (each using 5 mL of Alcian blue) were performed cranially within the carotid triangle between the anterior margin of the sternocleidomastoid muscle and the submandibular gland. RESULTS: Anastomoses between the TCN, CB VII, and MMB VII were stained in all preparations (n=16). CONCLUSION: This anatomical study presents an ultrasound-guided subplatysmal SCA block to optimize, in addition to a cervical plexus block, the quality of anesthesia for carotid endarterectomies.

T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice.

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

Serious Complications After Epidural Catheter Placement: Two Case Reports.

Thoracic epidural analgesia (TEA) is a standard procedure in multimodal analgesia applied in major thoracic and abdominal surgeries. Two cases are presented with serious complications related to TEA. In both cases, earlier reaction of the treating physicians to patient-reported sensory symptoms could have prevented the complicated course. The first case was a 73-year-old patient with bronchial carcinoma who underwent right lower lobe resection. In this case, dabigatran 150 mg/d (indication: permanent atrial fibrillation) had been discontinued 72 hours before surgery, and enoxaparin 80 mg (every 12 hours) had been started 11 hours after surgery. An epidural hematoma developed postoperatively. Magnetic resonance imaging (MRI) was performed only after paraplegia had developed the next day. Unfortunately, delayed hematoma evacuation could not prevent persistent paraplegia in this case, which was complicated by hospital-acquired pneumonia with sepsis and acute renal failure. The second case was a 39-year-old patient with ulcerative colitis and an initially undetected malposition of the epidural catheter. Immediately after test bolus injection, the patient reported paresthesia and overall discomfort, which however could not be safely attributed to either the test dose or the already started general anesthesia. The patient could only be extubated after stopping the epidural infusion. Accidental re-start of epidural infusion led to coma, conjugate eye deviation, and respiratory arrest, necessitating re-intubation. Computed tomography (CT) ruled out intracerebral pathology and showed a catheter position centrally in the spinal canal. Fortunately, no neurological deficits were detected after catheter removal.

Surgical treatment of proximal femoral fractures in high-risk geriatric patients under peripheral regional anesthesia : A prospective feasibility study.

BACKGROUND AND OBJECTIVE: Due to changing demographics geriatric patients with multiple comorbidities and proximal femoral fractures are an increasing patient population. In these patient groups, peripheral regional anesthesia could become increasingly more important besides established procedures, such as neuraxial or general anesthesia. The aim of this single center feasibility study was to evaluate a combined blockade technique of the lumbosacral plexus for three predefined subgroups depending on the type of hip fracture. METHODS: We used a unilateral double injection three-step technique to block the sacral (parasacral block) and lumbar plexus (anterior quadratus lumborum and psoas compartment block, n = 78). The blockade was performed both under ultrasound guidance and simultaneous nerve stimulation and 20 ml ropivacaine 0.375% was injected at each of the 3 injection sites (total dose 225 mg). RESULTS: In 42% of cases the surgery was opioid-free (n = 33). In 5 patients a conversion to general anesthesia (insertion of a laryngeal mask and pressure-controlled or pressure-supported ventilation) was necessary (6%). The overall success rate of combination anesthesia (peripheral nerve blocks with supplemental sedative (propofol 1-2 mg/kg*h) or analgesic (incremental doses of 5 µg sufentanil) medication) was 94%, regardless of fracture type and surgical treatment. CONCLUSION: The combined anesthetic technique presented in this study enables surgical treatment of proximal femoral fractures. The associated effort and requirement for expert knowledge in regional anesthesia indicates that this method should be considered especially in cases with high anesthetic risk, suitable sonoanatomy, and non-compromised coagulation.

The multiscale architecture of tessellated cartilage and its relation to function.

When describing the architecture and ultrastructure of animal skeletons, introductory biology, anatomy and histology textbooks typically focus on the few bone and cartilage types prevalent in humans. In reality, cartilage and bone are far more diverse in the animal kingdom, particularly within fishes (Chondrichthyes and Actinopterygii), where cartilage and bone types are characterized by features that are anomalous or even pathological in human skeletons. This review discusses the curious and complex architectures of shark and ray tessellated cartilage, highlighting similarities and differences with their mammalian skeletal tissue counterparts. By synthesizing older anatomical literature with recent high-resolution structural and materials characterization work, this review frames emerging pictures of form-function relationships in this tissue and of the evolution and true diversity of cartilage and bone.

Shape-preserving erosion controlled by the graded microarchitecture of shark tooth enameloid.

The teeth of all vertebrates predominantly comprise the same materials, but their lifespans vary widely: in stark contrast to mammals, shark teeth are functional only for weeks, rather than decades, making lifelong durability largely irrelevant. However, their diets are diverse and often mechanically demanding, and as such, their teeth should maintain a functional morphology, even in the face of extremely high and potentially damaging contact stresses. Here, we reconcile the dilemma between the need for an operative tooth geometry and the unavoidable damage inherent to feeding on hard foods, demonstrating that the tooth cusps of Port Jackson sharks, hard-shelled prey specialists, possess unusual microarchitecture that controls tooth erosion in a way that maintains functional cusp shape. The graded architecture in the enameloid provokes a location-specific damage response, combining chipping of outer enameloid and smooth wear of inner enameloid to preserve an efficient shape for grasping hard prey. Our discovery provides experimental support for the dominant theory that multi-layered tooth enameloid facilitated evolutionary diversification of shark ecologies.

Natural hybrid silica/protein superstructure at atomic resolution.

Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

Endoskeletal mineralization in chimaera and a comparative guide to tessellated cartilage in chondrichthyan fishes (sharks, rays and chimaera).

An accepted uniting character of modern cartilaginous fishes (sharks, rays, chimaera) is the presence of a mineralized, skeletal crust, tiled by numerous minute plates called tesserae. Tesserae have, however, never been demonstrated in modern chimaera and it is debated whether the skeleton mineralizes at all. We show for the first time that tessellated cartilage was not lost in chimaera, as has been previously postulated, and is in many ways similar to that of sharks and rays. Tesserae in Chimaera monstrosa are less regular in shape and size in comparison to the general scheme of polygonal tesserae in sharks and rays, yet share several features with them. For example, Chimaera tesserae, like those of elasmobranchs, possess both intertesseral joints (unmineralized regions, where fibrous tissue links adjacent tesserae) and recurring patterns of local mineral density variation (e.g. Liesegang lines, hypermineralized 'spokes'), reflecting periodic accretion of mineral at tesseral edges as tesserae grow. Chimaera monstrosa's tesserae, however, appear to lack the internal cell networks that characterize tesserae in elasmobranchs, indicating fundamental differences among chondrichthyan groups in how calcification is controlled. By compiling and comparing recent ultrastructure data on tesserae, we also provide a synthesized, up-to-date and comparative glossary on tessellated cartilage, as well as a perspective on the current state of research into the topic, offering benchmark context for future research into modern and extinct vertebrate skeletal tissues.

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Mechanistic dissection of the PD-L1:B7-1 co-inhibitory immune complex.

The B7 family represents one of the best-studied subgroups within the Ig superfamily, yet new interactions continue to be discovered. However, this binding promiscuity represents a major challenge for defining the biological contribution of each specific interaction. We developed a strategy for addressing these challenges by combining cell microarray and high-throughput FACS methods to screen for promiscuous binding events, map binding interfaces, and generate functionally selective reagents. Applying this approach to the interactions of mPD-L1 with its receptor mPD-1 and its ligand mB7-1, we identified the binding interface of mB7-1 on mPD-L1 and as a result generated mPD-L1 mutants with binding selectivity for mB7-1 or mPD-1. Next, using a panel of mB7-1 mutants, we mapped the binding sites of mCTLA-4, mCD28 and mPD-L1. Surprisingly, the mPD-L1 binding site mapped to the dimer interface surface of mB7-1, placing it distal from the CTLA-4/CD28 recognition surface. Using two independent approaches, we demonstrated that mPD-L1 and mB7-1 bind in cis, consistent with recent reports from Chaudhri A et al. and Sugiura D et al. We further provide evidence that while CTLA-4 and CD28 do not directly compete with PD-L1 for binding to B7-1, they can disrupt the cis PD-L1:B7-1 complex by reorganizing B7-1 on the cell surface. These observations offer new functional insights into the regulatory mechanisms associated with this group of B7 family proteins and provide new tools to elucidate their function in vitro and in vivo.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Thoracic-paravertebral blocks: comparative anatomical study with different injection techniques and volumes.

BACKGROUND AND OBJECTIVES: We hypothesized that different injection techniques and volumes in thoracic-paravertebral blocks (TPVB) lead to different patterns of dye spread. In particular, we investigated whether an alternating injection technique leads to complete staining of all adjacent intercostal nerves. METHODS: This comparative anatomical investigation was performed using 10 or 20 mL of dye (Alcian Blue) in 10 unfixed donor cadavers (54 injections) that were designated for education or research purposes. RESULTS: In landmark-guided TPVB, the thoracic-paravertebral space (TPVS) was either not stained at all (spread of dye in the paraspinal muscles, n=3) or the dye was predominantly found in the epidural space (n=3). In ultrasound-guided TPVB, the TPVS was correctly identified in all cases (n=48). The sympathetic trunk was stained in 84.6% of injections (multi-injection technique: 100%), independent of injection technique and volume. The epidural space was stained more frequently (p≤0.001) if both the puncture site (sagittal transducer position) and guidance of the needle were more medial (77.8%). Finally, a higher injection volume (20 vs 10 mL) resulted in a higher number of stained intercostal nerves (p=0.04). CONCLUSION: For ultrasound-guided techniques, a higher injection volume resulted in a larger number of stained intercostal nerves. Staining of the sympathetic trunk was independent of the injection technique. Epidural spread was observed significantly less frequently if the injection was lateral (transducer transversal) or with a strictly cranial injection direction (transducer sagittal). Landmark-guided injections reliably achieved the TPVS (and the epidural space) only after a needle advance of 2.5 cm after initial contact with the transverse process.

Mechanical properties of stingray tesserae: High-resolution correlative analysis of mineral density and indentation moduli in tessellated cartilage.

Skeletal tissues are built and shaped through complex, interacting active and passive processes. These spatial and temporal variabilities make interpreting growth mechanisms from morphology difficult, particularly in bone, where the remodeling process erases and rewrites local structural records of growth throughout life. In contrast to the majority of bony vertebrates, the elasmobranch fishes (sharks, rays, and their relatives) have skeletons made of cartilage, reinforced by an outer layer of mineralized tiles (tesserae), which are believed to grow only by deposition, without remodeling. We exploit this structural permanence, performing the first fine-scale correlation of structure and material properties in an elasmobranch skeleton. Our characterization across an age series of stingray tesserae allows unique insight into the growth processes and mechanical influences shaping the skeleton. Correlated quantitative backscattered electron imaging (qBEI) and nanoindentation measurements show a positive relationship between mineral density and tissue stiffness/hardness. Although tessellated cartilage as a whole (tesserae plus unmineralized cartilage) is considerably less dense than bone, we demonstrate that tesserae have exceptional local material properties, exceeding those of (mammal) bone and calcified cartilage. We show that the finescale ultrastructures recently described in tesserae have characteristic material properties suggesting distinct mechanical roles and that regions of high mineral density/stiffness in tesserae are confined predominantly to regions expected to bear high loads. In particular, tesseral spokes (laminated structures flanking joints) exhibit particularly high mineral densities and tissue material properties, more akin to teeth than bone or calcified cartilage. We conclude that these spokes toughen tesserae and reinforce points of contact between them. These toughening and reinforcing functions are supported by finite element simulations incorporating our material data. The high stresses predicted for spokes, and evidence we provide that new spoke laminae are deposited according to their local mechanical environment, suggest tessellated cartilage is both mutable and responsive, despite lacking remodeling capability. STATEMENT OF SIGNIFICANCE: The study of vertebrate skeletal materials is heavily biased toward mammal bone, despite evidence that bone and cartilage are extremely diverse. We broaden the perspective on vertebrate skeleton materials and evolution in an investigation of stingray tessellated cartilage, a curious type of unmineralized cartilage with a shell of mineralized tiles (tesserae). Combining high-resolution imaging and material testing, we demonstrate that tesserae have impressive local material properties for a vertebrate skeletal tissue, arguing for unique tissue organization relative to mammalian calcified cartilage and bone. Incorporating our materials data into mechanical models, we show that finescale material arrangements allow this cartilage to act as a functional and responsive alternative to bone, despite lacking bone's ability to remodel. These results are relevant to a diversity of researchers, from skeletal, developmental, and evolutionary biologists, to materials scientists interested in high-performance, low-density composites.

The femoral vein diameter and its correlation with sex, age and body mass index - An anatomical parameter with clinical relevance.

BACKGROUND: The femoral vein diameter is a critical factor when assessing endoprosthetic valve size for the treatment of chronic venous insufficiency. To examine the previously stated correlation between body mass index and femoral vein diameter and to re-assess the anatomical and physiological demands for a valve implant for chronic venous insufficiency treatment, we measured the femoral vein diameter in 82 subjects. METHOD: Femoral vein diameters (164 legs) were measured with B-mode sonography both in supine position at rest and in upright position during Valsalva maneuver. RESULT: The mean femoral vein diameter differed significantly between supine position (13.6 ± 3.0 mm) and upright position (16.4 ± 2.6 mm). Males possessed a significant bigger diameter than females. A significant positive correlation between femoral vein diameter and body mass index was observed. CONCLUSION: Assuming an increased femoral vein diameter due to obesity would further impair valve functionality by increasing distance between both valve cusps. For the development of artificial venous valves, it is crucial to consider patient- and condition-dependent vein dilation.

Automated segmentation of complex patterns in biological tissues: Lessons from stingray tessellated cartilage.

INTRODUCTION: Many biological structures show recurring tiling patterns on one structural level or the other. Current image acquisition techniques are able to resolve those tiling patterns to allow quantitative analyses. The resulting image data, however, may contain an enormous number of elements. This renders manual image analysis infeasible, in particular when statistical analysis is to be conducted, requiring a larger number of image data to be analyzed. As a consequence, the analysis process needs to be automated to a large degree. In this paper, we describe a multi-step image segmentation pipeline for the automated segmentation of the calcified cartilage into individual tesserae from computed tomography images of skeletal elements of stingrays. METHODS: Besides applying state-of-the-art algorithms like anisotropic diffusion smoothing, local thresholding for foreground segmentation, distance map calculation, and hierarchical watershed, we exploit a graph-based representation for fast correction of the segmentation. In addition, we propose a new distance map that is computed only in the plane that locally best approximates the calcified cartilage. This distance map drastically improves the separation of individual tesserae. We apply our segmentation pipeline to hyomandibulae from three individuals of the round stingray (Urobatis halleri), varying both in age and size. RESULTS: Each of the hyomandibula datasets contains approximately 3000 tesserae. To evaluate the quality of the automated segmentation, four expert users manually generated ground truth segmentations of small parts of one hyomandibula. These ground truth segmentations allowed us to compare the segmentation quality w.r.t. individual tesserae. Additionally, to investigate the segmentation quality of whole skeletal elements, landmarks were manually placed on all tesserae and their positions were then compared to the segmented tesserae. With the proposed segmentation pipeline, we sped up the processing of a single skeletal element from days or weeks to a few hours.

[Cervical Plexus Blocks]. / Zervikale Plexusblockaden.

Blockades of the cervical plexus are established anesthesia procedures, not only in the context of operative carotid revascularizations. Recent investigations define inner sonoanatomic landmarks as well as the importance of brain nerves for innervation in the neck region. The present practice-oriented article discusses current study results, alternative techniques (ultrasound- versus landmark-guided) and fields of application (carotid desobliteration, surgery of the shoulder, ear and infraclavicular region).

Calcified cartilage or bone? Collagens in the tessellated endoskeletons of cartilaginous fish (sharks and rays).

The primary skeletal tissue in elasmobranchs -sharks, rays and relatives- is cartilage, forming both embryonic and adult endoskeletons. Only the skeletal surface calcifies, exhibiting mineralized tiles (tesserae) sandwiched between a cartilage core and overlying fibrous perichondrium. These two tissues are based on different collagens (Coll II and I, respectively), fueling a long-standing debate as to whether tesserae are more like calcified cartilage or bone (Coll 1-based) in their matrix composition. We demonstrate that stingray (Urobatis halleri) tesserae are bipartite, having an upper Coll I-based 'cap' that merges into a lower Coll II-based 'body' zone, although tesserae are surrounded by cartilage. We identify a 'supratesseral' unmineralized cartilage layer, between tesserae and perichondrium, distinguished from the cartilage core in containing Coll I and X (a common marker for mammalian mineralization), in addition to Coll II. Chondrocytes within tesserae appear intact and sit in lacunae filled with Coll II-based matrix, suggesting tesserae originate in cartilage, despite comprising a diversity of collagens. Intertesseral joints are also complex in their collagenous composition, being similar to supratesseral cartilage closer to the perichondrium, but containing unidentified fibrils nearer the cartilage core. Our results indicate a unique potential for tessellated cartilage in skeletal biology research, since it lacks features believed diagnostic for vertebrate cartilage mineralization (e.g. hypertrophic and apoptotic chondrocytes), while offering morphologies amenable for investigating the regulation of complex mineralized ultrastructure and tissues patterned on multiple collagens.