An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

A sciatic nerve gap-injury model in the rabbit.

There has been an increased number of studies of nerve transection injuries with the sciatic nerve gap-injury model in the rabbit in the past 2 years. We wanted to define in greater detail what is needed to test artificial nerve guides in a sciatic nerve gap-injury model in the rabbit. We hope that this will help investigators to fully exploit the robust translational potential of the rabbit sciatic nerve gap-injury model in its capacity to test devices whose diameter and length are in the range of those commonly applied in hand and wrist surgery (diameter ranging between 2 and 4 mm; length up to 30 mm). We suggest that the rabbit model should replace the less translational rat model in nerve regeneration research. The rabbit sciatic model, however, requires an effective strategy to prevent and control self-mutilation of the foot in the postoperative period, and to prevent pressure ulcers.

Hoechst 33342 as a marker for imaging neurites of Dorsal Root Ganglion in vitro.

Fluorescent markers, generally targeting neurotubules, are used to visualize the radiating crown of growing neurites that is produced by dorsal root ganglion cells in vitro. Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate) is a widely used fluorescent DNA marker that stain both live and fixed nuclei. We have recently found that H33342 can visualize the neurites of DRG too, but only when they are fixed in formalin. Images have a good signal-to-noise ratio. We noticed that besides H33342 being a specific marker for DNA, it also stains the transmembrane P-glycoprotein (P-gp) which is involved in the active pump-out of alien molecules from the cytoplasm; so, H33342 remains associated with P-gp after fixation. P-gp is quite ubiquitous in healthy cells and, notably, P-gp has been detected in DRG of several species as well as in human DRG. The use of H33342 as a staining for neurites of DRG in fixed samples could have a practical value due to its widespread use and its better affordability compared to other fluorescent markers for neurites.

A method to deliver patterned electrical impulses to Schwann cells cultured on an artificial axon.

Information from the brain travels back and forth along peripheral nerves in the form of electrical impulses generated by neurons and these impulses have repetitive patterns. Schwann cells in peripheral nerves receive molecular signals from axons to coordinate the process of myelination. There is evidence, however, that non-molecular signals play an important role in myelination in the form of patterned electrical impulses generated by neuronal activity. The role of patterned electrical impulses has been investigated in the literature using co-cultures of neurons and myelinating cells. The co-culturing method, however, prevents the uncoupling of the direct effect of patterned electrical impulses on myelinating cells from the indirect effect mediated by neurons. To uncouple these effects and focus on the direct response of Schwann cells, we developed an in vitro model where an electroconductive carbon fiber acts as an artificial axon. The fiber provides only the biophysical characteristics of an axon but does not contribute any molecular signaling. In our "suspended wire model", the carbon fiber is suspended in a liquid media supported by a 3D printed scaffold. Patterned electrical impulses are generated by an Arduino 101 microcontroller. In this study, we describe the technology needed to set-up and eventually replicate this model. We also report on our initial in vitro tests where we were able to document the adherence and ensheath of human Schwann cells to the carbon fiber in the presence of patterned electrical impulses (hSCs were purchased from ScienCell Research Laboratories, Carlsbad, CA, USA; ScienCell fulfills the ethic requirements, including donor's consent). This technology will likely make feasible to investigate the response of Schwann cells to patterned electrical impulses in the future.

Open Surgery for Trigger Finger Required Combined a1-a2 Pulley Release. A Retrospective Study on 1305 Case.

OBJECTIVES: We retrospectively reviewed 1305 open-surgery for idiopathic trigger finger performed by 4 senior hand surgeons between 2014 and 2016. MATERIAL AND METHODS: Medical records and a telephone interview made with a minimum follow-up of 1 year were used to identify the recurrent rate of triggering and other complications. RESULTS: This retrospective study let us note that 169 fingers (13%) required simultaneous release of the A1-A2 pulleys because the sectioning of the A1 pulley alone did not lead to complete free sliding of the tendons. We did not record any bowstring complication and we ascribe this to both surgery and bandaging technique. Overall rate of complication was 11.8% and relapse triggering or permanent proximal interphalangeal joint flexion (PPIJF) were among them; notably, however, they occurred only in patients where the A2 pulley was not sectioned. CONCLUSIONS: Is possible to reduce the percentage of relapse triggering or PPIJF after trigger finger surgery, by performing that combined A1-A2 pulley release. LEVEL OF EVIDENCE: Level III.

Reciprocal nerve staining (RNS) for the concurrent detection of choline acetyltransferase and myelin basic protein on paraffin-embedded sections.

BACKGROUND: Objective of our work was to develop a sequential double nonfluorescent immunostaining method which allows the selective identification of myelinated motor fibers in paraffin-embedded samples of peripheral nerves. Motor recovery after a nerve gap-lesion repaired by artificial nerve-guides ("conduits") is often less complete and slower than sensory recovery. The mechanism for this is not fully understood. NEW METHOD: Incubation in sheep polyclonal choline acetyltransferase antibody (Abcam 18,736) at dilution of 1:150 was followed by incubation in mouse monoclonal anti-myelin basic protein antibody (Abcam 62,631) at a dilution of 1:5000. Counterstaining was performed with hematoxylin QS (Vector Labs H-3404). RESULTS: Immunostaining of choline acetyltransferase and myelin basic protein can be combined together and results show a good contrast between the light brown of the choline acetyltransferase reaction product and the green of myelin basic protein reaction product. Cell nuclei are stained blue. This new protocol retains the advantages of paraffin embedded sections such as (i) having a relatively simple methodology, (ii) years-long storage life, and (iii) easy sharing among laboratories. Comparison with existing method. This specific combinatorial protocol has never been used before on paraffin embedded sections. It has been named "reciprocal nerve staining" (RNS). CONCLUSIONS: Routine combination of choline acetyltransferase and myelin basic protein immunostaining provides a highly specific, highly contrasted paraffin-embedded sections where optical differentiation of myelinated motor fibers is easy and straightforward. This method will likely simplify and speed-up the routine histological study of nerve regeneration and will contribute a better identification of the nerve motor component.

"Ruffled border" formation on a CaP-free substrate: A first step towards osteoclast-recruiting bone-grafts materials able to re-establish bone turn-over.

Osteoclasts are large multinucleated giant cells that actively resorb bone during the physiological bone turnover (BTO), which is the continuous cycle of bone resorption (by osteoclasts) followed by new bone formation (by osteoblasts). Osteoclasts secrete chemotactic signals to recruit cells for regeneration of vasculature and bone. We hypothesize that a biomaterial that attracts osteoclasts and re-establishes BTO will induce a better healing response than currently used bone graft materials. While the majority of bone regeneration efforts have focused on maximizing bone deposition, the novelty in this approach is the focus on stimulating osteoclastic resorption as the starter for BTO and its concurrent new vascularized bone formation. A biodegradable tyrosine-derived polycarbonate, E1001(1k), was chosen as the polymer base due to its ability to support bone regeneration in vivo. The polymer was functionalized with a RGD peptide or collagen I, or blended with ß-tricalcium phosphate. Osteoclast attachment and early stages of active resorption were observed on all substrates. The transparency of E1001(1k) in combination with high resolution confocal imaging enabled visualization of morphological features of osteoclast activation such as the formation of the "actin ring" and the "ruffled border", which previously required destructive forms of imaging such as transmission electron microscopy. The significance of these results is twofold: (1) E1001(1k) is suitable for osteoclast attachment and supports osteoclast maturation, making it a base polymer that can be further modified to optimize stimulation of BTO and (2) the transparency of this polymer makes it a suitable analytical tool for studying osteoclast behavior.

Development of a Device-Assisted Nerve-Regeneration Procedure in Disruptive Lesions of the Brachial Plexus.

BACKGROUND: We describe the development of a new surgical procedure to be used in the treatment of disruptive brachial plexus (BP) lesions. It is centered on an artificial device designed to assist nerve regeneration by providing a confined and protected environment. Nerve fibers can repair inside the device, while the adverse massive scar-tissue formation is limited to the outside of the device. METHODS: Steps in the development of the procedure were (1) definition of the rationale, (2) design of the device, (3) choice of an in vivo translational model, (4)refinement of the surgical procedure, and (5) performance of an in vivo pilot study as a proof of concept. An interdisciplinary team from several laboratories was involved in this work over a period of 6 years. RESULTS: Results showed the absence of significant scar tissue in the regenerate and the presence of myelinated fibers aligned proximodistally between the stumps. This surgical approach can be seen not only as a definitive treatment but also as an early examination and stabilization before some different surgery will be later performed. It may also be used as additional protection for traditional surgery like end-to-end coaptation. CONCLUSIONS: We conclude that the availability of a suitable device-assisted early treatment, even if not to be considered definitive, could help in addressing the BP lesions at an earlier stage and this may improve the final outcome. Our evidence justifies further experimentation on this approach.

Fibrin glue as a stabilization strategy in peripheral nerve repair when using porous nerve guidance conduits.

Porous conduits provide a protected pathway for nerve regeneration, while still allowing exchange of nutrients and wastes. However, pore sizes >30 µm may permit fibrous tissue infiltration into the conduit, which may impede axonal regeneration. Coating the conduit with Fibrin Glue (FG) is one option for controlling the conduit's porosity. FG is extensively used in clinical peripheral nerve repair, as a tissue sealant, filler and drug-delivery matrix. Here, we compared the performance of FG to an alternative, hyaluronic acid (HA) as a coating for porous conduits, using uncoated porous conduits and reverse autografts as control groups. The uncoated conduit walls had pores with a diameter of 60 to 70 µm that were uniformly covered by either FG or HA coatings. In vitro, FG coatings degraded twice as fast as HA coatings. In vivo studies in a 1 cm rat sciatic nerve model showed FG coating resulted in poor axonal density (993 ± 854 #/mm2), negligible fascicular area (0.03 ± 0.04 mm2), minimal percent wet muscle mass recovery (16 ± 1 in gastrocnemius and 15 ± 5 in tibialis anterior) and G-ratio (0.73 ± 0.01). Histology of FG-coated conduits showed excessive fibrous tissue infiltration inside the lumen, and fibrin capsule formation around the conduit. Although FG has been shown to promote nerve regeneration in non-porous conduits, we found that as a coating for porous conduits in vivo, FG encourages scar tissue infiltration that impedes nerve regeneration. This is a significant finding considering the widespread use of FG in peripheral nerve repair.

A suspended carbon fiber culture to model myelination by human Schwann cells.

Understanding of myelination/remyelination process is essential to guide tissue engineering for nerve regeneration. In vitro models currently used are limited to cell population studies and cannot easily identify individual cell contribution to the process. We established a novel model to study the contribution of human Schwann cells to the myelination process. The model avoids the presence of neurons in culture; Schwann cells respond solely to the biophysical properties of an artificial axon. The model uses a single carbon fiber suspended in culture media far from the floor of the well. The fiber provides an elongated structure of defined diameter with 360-degree of surface available for human Schwann cells to wrap around. This model enabled us to spatially and temporally track the myelination by individual Schwann cells along the fiber. We observed cell attachment, elongation and wrapping over a period of 9 days. Cells remained alive and expressed Myelin Basic Protein and Myelin Associated Glycoprotein as expected. Natural and artificial molecules, and external physical factors (e.g., p atterned electrical impulses), may be tested with this model as possible regulators of myelination.

Trapeziometacarpal joint osteoarthritis: a prospective trial on two widespread conservative therapies.

INTRODUCTION: The trapeziometacarpal (TMC) joint osteoarthritis (OA) is at the origin of important secondary functional disability to pinch as well as a painful grip. Several conservative therapies are often considered in the early stages of TMC OA to decrease pain, recover function and slow the evolution of OA. They include massage therapy, heat applications, stretching of the first web span and assisted mobilization of the TMC joint. However, as with other arthritic joints, many physicians often suggest administering intra-articular corticosteroids. The aim of this study was to assess the effect of 10 sessions of physiotherapy versus a single corticoid intra-articular injection. Both treatments were associated with TMC splinting. METHODS: Two groups of twenty-five patients received either physiotherapy or a corticoid injection. They were followed over a one-year period. All of them were assessed for pain, function, strength at 2, 6 and 12 months and overall satisfaction at the end of the study. RESULTS: With the infiltrative therapy, the parameters improve more quickly, whereas patients treated with physiotherapy show longer persistence regarding remission of pain. The final functional evaluation scores at one year are similar to pretreatment scores. CONCLUSION: In early stages of TMC OA, corticosteroids articular injections and physiotherapy treatments can improve the painful symptoms but treatment with corticosteroids is faster. Although hand functions return to average scores similar to those of pre-treatment, physiotherapy program is associated with a longer remission of pain.

Debris of carbon-fibers originated from a CFRP (pEEK) wrist-plate triggered a destruent synovitis in human.

Application of carbon-fiber-reinforced-polymer (CFRP) artifacts in humans has been promoted in Orthopedic and Trauma Surgery. Literature documents the biocompatibility of materials used, namely carbon fibers (CF) and poly-ether thermoplastics, like poly-ether-ether-ketone (PEEK). A properly designed and accurately implanted composite artifact should not expose its fibers during or after surgery: however this may happen. A white Caucasian woman came to our attention 11 months after surgery for a wrist fracture. She had a severe impairment, being unable to flex the thumb; index finger and distal phalanx of third finger. We retrieved a correctly positioned plate and documented an aggressive erosive flexor tendons synovitis with eroded stumps of flexor tendons. The plate and soft tissues were analyzed by Visible Light and Scanning Electron Microscopy. Histopathology showed granulomatous fibrogenic process with CF engulfed inside multinucleated giant cells. Fibers were unmasked and disrupted inside the holes where screws were tightened and corrugation of the polymer coating led to further unmasking. The mechanism of foreign-body reaction to CF has not been studied in depth yet, particularly at the ultrastructural level and in Humans. This case documents a damage occurred in a clinical application and which was theoretically possible. Our opinion is that a proper way to promote the use of CRFP in the Clinic in the short term is to direct Research towards finding a better way to prevent CF debris to be exposed and released. In the longer term, the biological response to CF deserves a deeper understanding.

Peripheral nerve regeneration inside collagen-based artificial nerve guides in humans.

PURPOSE: Nerve gap injuries may be associated with lesions in other structures, like tendons or bones; in these cases, it is common to plan a second surgery to improve functional recovery. Since macroscopic observations of nerve regeneration in humans are rare, we exploited these second surgeries for the purpose of studying nerve regeneration in humans. METHODS: We assessed the clinical outcomes of 50 implants of collagen-based nerve guides in the upper limb. We performed a second look at 20, assessing macroscopically both nerve regeneration and collagen degradation. RESULTS AND CONCLUSIONS: Pain was never recorded in these patients. An adequate sensory recovery took place whenever nerve regeneration was found inside the guide. Motor recovery seemed to occur only when the gap lesion was shorter than 10 mm. The degree of degradation appeared to be variable and was not directly correlated with time; we hypothesize that it could be associated with the site of implantation. Such a large number of second looks in humans has never been previously reported in the literature.

Can we regrow a human arm? A negative perspective from an upper-limb surgeon.

If we would like to devote time and money to the task of regrowing a human arm, we should feel free to do it, in principle. However, if we recognize a purpose in biomedical research, we must scrutinize this task in the light of a possible clinical application. We will then discover that regrowing a human arm is not only likely to be not possible, but also not required in the clinic. Bionic arms and better reconstructive surgery already provide a different, simpler and easier solution to the loss of a human arm, and should be promoted. Probably, ''can we regrow a human arm?'' is not the right question. Instead, we should ask, ''can we restore the function of a lost human arm?''.

Persistence of abnormal electrophysiological findings after carpal tunnel release.

Practitioners may refer to experienced hand surgeons to differentiate a recurrence in carpal tunnel syndrome (CTS) from a failed carpal tunnel release. The patient may complain about the reappearance of symptoms, whatever is the cause. Nerve conduction studies (NCS) are often required by the practitioner to assist the final diagnosis. We observed abnormal values in NCS in patients who were clinically healed from CTS. We evaluated the changes preoperatively and, then, at 1, 3, 6, 9, and 12 month postoperatively. At the same time, we performed a retrospective study on a group of 37 clinically healed patients. Follow-up ranged from 2 to 20 years. Surgical treatment let the electrophysiological parameters to improve toward physiological values; however, normality is hardly ever reached. This sort of ''electrophysiological scar'' is true for all the parameters measured. In presence of CTS, the latency difference between the radial and median sensory nerve action potentials, recorded following thumb stimulation, produces a double peak shift. The ''double peak shift'' best described this ''electrophysiological scar,'' being a parameter that should measure about zero in the normal population. In conclusion, abnormal postoperative electrophysiological findings cannot substantiate the diagnosis of a poor outcome of a carpal tunnel release nor a recurrence of CTS.

A more detailed mechanism to explain the "bands of Fontana" in peripheral nerves.

INTRODUCTION: In 1779, Fontana identified transverse and oblique bands along peripheral nerves. Subsequent studies pointed alternatively to endoneural or perineural components as the cause. Our aim was to clarify these conflicting findings. METHODS: Recoiling of the bands of Fontana was video-recorded in the rat sciatic nerve. Computer-assisted design (CAD) software was used to model the nerve by interference figures. RESULTS: In vivo microdissection showed distinctive, black-and-white, closely packed bands in the perineurium, which differed from the widely spaced, translucent, dark/pale gray, staggered bands in the endoneurium. CAD merging of these 2 patterns produced images resembling the bands observed in vivo. CONCLUSIONS: Two repetitive structures with different characteristics, 1 in the perineurium and the other in the endoneurium, merge to give the appearance of these bands.

Combining an external fixator and an artificial nerve-guide for the treatment of a complex digital injury.

A complex digital injury, with tendon, nerve and bone losses, may pose the problem of which structure deserves the highest priority. Authors were able to treat tendon, nerve and bone lesions with the same level of priority thanks to the combined use of an external fixator and a nerve-guide.

A degradable soybean-based biomaterial used effectively as a bone filler in vivo in a rabbit.

The 'gold standard' for bone filling is currently the bone autograft, but its use is limited by material availability and by the possible risks of infection or other donor site morbidity. Materials proposed so far as bone fillers do not show all the characteristics which are desirable. These are (a) osteoconductivity, (b) controlled biodegradation and (c) ease of adaptation to the implantation site. Recently, a new class of biodegradable material based on soybeans has been presented which shows good mechanical properties and an intrinsic bioactivity on inflammatory and tissue cells in vitro. The authors investigated the morphology in vivo of bone response in repairing a surgical lesion in the presence of granules of a novel soybean-based biomaterial (SB), comparing it with a sham-operated contralateral lesion of critical size (non-healing model); 26 operations were performed in New Zealand White rabbits, with back scattered electron microscopy as the analysis technique of choice. Implantation of SB granules over 8 weeks produced bone repair with features distinct from those obtained by healing in a non-treated defect. New and progressively maturing trabeculae appeared in the animal group where SB granules were implanted, while sham operation produced only a rim of pseudo-cortical bone still featuring a large defect. The trabeculae forming in the presence of SB granules had features typical of reticular bone. These findings suggest that the bone regeneration potential of SB granules and their intrinsic bioactivity, combined with their relatively easy and cost-effective preparation procedures, make them suitable candidates as a bone filler in clinical applications.

Role of phosphatidyl-serine in bone repair and its technological exploitation.

In the 1970s, morphological evidence collected by electron microscopy linked mineral deposition ("calcification" or "mineralization") in newly-forming bone to membrane-encapsulated particles of a diameter of approximately 100 nm (50-200 nm) that were called "matrix vesicles". As the characterisation of these vesicles progressed towards their biochemical composition, the role of lipids in the biomineralization process appeared to be crucial. In particular, a group of cell-membrane phospholipids were identified as major players in the crystal formation process. Indeed, in the 1980s it became clear that phosphatidylserine, together with proteins of the annexin family, was among the most important molecules in binding calcium ions and that this phospholipid was involved in the regulation of the early stages of mineralization in vivo. During the same period of time, the number of surgical implantations of orthopaedic, dental and maxilo-facial devices requiring full integration with the treated bone prompted the study of new functionalization molecules able to establish a stable bonding with the mineral phase of the host tissue. In the late 1990 s studies started that aimed at exploiting the potential of calcium-binding phospholipids and, in particular, of the phosphatidylserine as functionalization molecules to improve the osteointegration of artificial implants. Later, papers have been published that show the potential of the phophatidylserine and phosphatidylserine-mimicking coating technology to promote calcification both in vitro and in vivo. The promising results support the future clinical application of these novel osteointegrative biomaterials.