Nicholas S. Kalson

Using transmission electron microscopy and 3View to determine collagen fibril size and three-dimensional organization

Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face–scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ∼8 d from isolating the tissue to having a 3D image stack.

Nonmuscle myosin II powered transport of newly formed collagen fibrils at the plasma membrane

Significance Collagen is the most abundant protein in vertebrates and is the building block of strong tissues such as tendons, skin, and bones. The fibrils can be millimeters long and occur in the extracellular matrix as a scaffold for tissue growth. Important questions remain unanswered about how cells assemble and transport the fibrils. We show here that collagen fibril assembly can occur at the plasma membrane in structures called fibripositors. We show that fibripositors are a nonmuscle myosin II (NMII)-dependent mechanical interface between the actinomyosin machinery and the extracellular matrix; thus, we propose a new function for NMII. A unique mechanism of fibril transport is presented as a basis for studies of tissue morphogenesis and conditions including wound healing and fibrosis. Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1–10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures.

Determinants of Summiting Success and Acute Mountain Sickness on Mt Kilimanjaro (5895 m)

Abstract Objective.—To determine the incidence of acute mountain sickness (AMS), the frequency of summiting success, and the factors that affect these in trekkers on Kilimanjaro, one of the worlds most summitted high-altitude peaks. Methods.—The study group comprised 312 trekkers attempting Mt Kilimanjaro summit by the Marango Route. Trekkers ascended over 4 or 5 days along a fixed ascent profile, stopping at 3 huts on ascent (2700 m, 3700 m, and 4700 m) before attempting the summit. Researchers were stationed at each hut for 16 days. Each night we measured heart rate, respiratory rate, blood pressure, oxygen saturation, and Lake Louise Score. We recorded the highest altitude that trekkers reached on the mountain. Results.—Of 181 complete sets of data, 111 (61%) trekkers reached the summit, and 139 (77%) developed AMS. Physiological results were not related to summit success. The incidence of AMS and summiting success were similar in those on the 4- or 5-day route. Trekkers on the 5-day route who used acetazolamide were less likely to develop AMS and more likely to summit than were those not taking acetazolamide (P  =  <.05); this difference was not present with trekkers on the 4-day route. Conclusions.—The risk of developing AMS is high on Mt Kilimanjaro. Although taking an extra day to acclimatize with the use of acetazolamide did provide some protection against AMS, ideally trekkers need a more gradual route profile for climbing this mountain.

A structure-based extracellular matrix expansion mechanism of fibrous tissue growth.

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis. DOI: http://dx.doi.org/10.7554/eLife.05958.001

An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe–linear–fail stress–strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and 14C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

Tenocyte contraction induces crimp formation in tendon-like tissue

Tendons are composed of longitudinally aligned collagen fibrils arranged in bundles with an undulating pattern, called crimp. The crimp structure is established during embryonic development and plays a vital role in the mechanical behaviour of tendon, acting as a shock-absorber during loading. However, the mechanism of crimp formation is unknown, partly because of the difficulties of studying tendon development in vivo. Here, we used a 3D cell culture system in which embryonic tendon fibroblasts synthesise a tendon-like construct comprised of collagen fibrils arranged in parallel bundles. Investigations using polarised light microscopy, scanning electron microscopy and fluorescence microscopy showed that tendon constructs contained a regular pattern of wavy collagen fibrils. Tensile testing indicated that this superstructure was a form of embryonic crimp producing a characteristic toe region in the stress–strain curves. Furthermore, contraction of tendon fibroblasts was the critical factor in the buckling of collagen fibrils during the formation of the crimp structure. Using these biological data, a finite element model was built that mimics the contraction of the tendon fibroblasts and monitors the response of the Extracellular matrix. The results show that the contraction of the fibroblasts is a sufficient mechanical impulse to build a planar wavy pattern. Furthermore, the value of crimp wavelength was determined by the mechanical properties of the collagen fibrils and inter-fibrillar matrix. Increasing fibril stiffness combined with constant matrix stiffness led to an increase in crimp wavelength. The data suggest a novel mechanism of crimp formation, and the finite element model indicates the minimum requirements to generate a crimp structure in embryonic tendon.

Slow stretching that mimics embryonic growth rate stimulates structural and mechanical development of tendon‐like tissue in vitro

A distinctive feature of embryonic tendon development is the steady increase in collagen fibril diameter and associated improvement of tissue mechanical properties. A potential mechanical stimulus for these changes is slow stretching of the tendon during limb growth. Testing this hypothesis in vivo is complicated by the presence of other developmental processes including muscle development and innervation. Here we used a cell culture tendon‐like construct to determine if slow stretch can explain the increases in fibril diameter and mechanical properties that are observed in vivo. Non‐stretched constructs had an ultrastructural appearance and mechanical properties similar to those of early embryonic tendon. However, slowly stretching during 4 days in culture increased collagen fibril diameter, fibril packing volume, and mechanical stiffness, and thereby mimicked embryonic development. 3D EM showed cells with improved longitudinal alignment and elongated nuclei, which raises the hypothesis that nuclear deformation could be a novel mechanism during tendon development. Developmental Dynamics 240:2520–2528, 2011.

Fibre bundles in the human extensor carpi ulnaris tendon are arranged in a spiral

The extensor carpi ulnaris musculotendinous unit has important agonist and antagonist action in wrist motion, including the dart-throwing action, and is a dynamic stabilizer of the distal radioulnar joint during forearm rotation. Despite its functional and clinical importance, little is known about its internal structure. Investigation of the ultrastructure of the human extensor carpi ulnaris (ECU) tendon was undertaken using plane polarized light microscopy and microcomputer tomography with 3D reconstruction. The study demonstrates that the tendon comprises fibre bundles (fascicles) approximately 0.1 mm in diameter that are arranged in a gradual spiral. The spiralling fibres make an angle of 8º to the longitudinal axis of the tendon. The spiral structure of the human ECU tendon has important biomechanical implications, allowing fascicular sliding during forearm rotation. The observed features may prevent injury.

International consensus on the definition and classification of fibrosis of the knee joint.

AIMS The aim of this consensus was to develop a definition of post-operative fibrosis of the knee. PATIENTS AND METHODS An international panel of experts took part in a formal consensus process composed of a discussion phase and three Delphi rounds. RESULTS Post-operative fibrosis of the knee was defined as a limited range of movement (ROM) in flexion and/or extension, that is not attributable to an osseous or prosthetic block to movement from malaligned, malpositioned or incorrectly sized components, metal hardware, ligament reconstruction, infection (septic arthritis), pain, chronic regional pain syndrome (CRPS) or other specific causes, but due to soft-tissue fibrosis that was not present pre-operatively. Limitation of movement was graded as mild, moderate or severe according to the range of flexion (90° to 100°, 70° to 89°, < 70°) or extension deficit (5° to 10°, 11° to 20°, > 20°). Recommended investigations to support the diagnosis and a strategy for its management were also agreed. CONCLUSION The development of standardised, accepted criteria for the diagnosis, classification and grading of the severity of post-operative fibrosis of the knee will facilitate the identification of patients for inclusion in clinical trials, the development of clinical guidelines, and eventually help to inform the management of this difficult condition. Cite this article: Bone Joint J 2016;98-B:1479-88.

Post-radiation sciatic neuropathy: a case report and review of the literature

BackgroundPost-radiation peripheral neuropathy has been reported in brachial and cervical plexuses and the femoral nerve.Case presentationWe describe a patient who developed post-radiation sciatic neuropathy after approximately 3 years and discuss the pathophysiology, clinical course and treatment options available for the deleterious effects of radiation to peripheral nerves.ConclusionThis is the first case of post-radiation involvement of the sciatic nerve reported in the literature.