Low intensity transcranial electric stimulation: Safety, ethical, legal regulatory and application guidelines.

Low intensity transcranial electrical stimulation (TES) in humans, encompassing transcranial direct current (tDCS), transcutaneous spinal Direct Current Stimulation (tsDCS), transcranial alternating current (tACS), and transcranial random noise (tRNS) stimulation or their combinations, appears to be safe. No serious adverse events (SAEs) have been reported so far in over 18,000 sessions administered to healthy subjects, neurological and psychiatric patients, as summarized here. Moderate adverse events (AEs), as defined by the necessity to intervene, are rare, and include skin burns with tDCS due to suboptimal electrode-skin contact. Very rarely mania or hypomania was induced in patients with depression (11 documented cases), yet a causal relationship is difficult to prove because of the low incidence rate and limited numbers of subjects in controlled trials. Mild AEs (MAEs) include headache and fatigue following stimulation as well as prickling and burning sensations occurring during tDCS at peak-to-baseline intensities of 1-2mA and during tACS at higher peak-to-peak intensities above 2mA. The prevalence of published AEs is different in studies specifically assessing AEs vs. those not assessing them, being higher in the former. AEs are frequently reported by individuals receiving placebo stimulation. The profile of AEs in terms of frequency, magnitude and type is comparable in healthy and clinical populations, and this is also the case for more vulnerable populations, such as children, elderly persons, or pregnant women. Combined interventions (e.g., co-application of drugs, electrophysiological measurements, neuroimaging) were not associated with further safety issues. Safety is established for low-intensity 'conventional' TES defined as <4mA, up to 60min duration per day. Animal studies and modeling evidence indicate that brain injury could occur at predicted current densities in the brain of 6.3-13A/m2 that are over an order of magnitude above those produced by tDCS in humans. Using AC stimulation fewer AEs were reported compared to DC. In specific paradigms with amplitudes of up to 10mA, frequencies in the kHz range appear to be safe. In this paper we provide structured interviews and recommend their use in future controlled studies, in particular when trying to extend the parameters applied. We also discuss recent regulatory issues, reporting practices and ethical issues. These recommendations achieved consensus in a meeting, which took place in Göttingen, Germany, on September 6-7, 2016 and were refined thereafter by email correspondence.

The development of synovial joints.

During vertebrate evolution, successful adaptation of animal limbs to a variety of ecological niches depended largely on the formation and positioning of synovial joints. The function of a joint is to allow smooth articulation between opposing skeletal elements and to transmit biomechanical loads through the structure, and this is achieved through covering the ends of bones with articular cartilage, lubricating the joint with synovial fluid, using ligaments to bind the skeletal elements together, and encapsulating the joint in a protective fibrous layer of tissue. The diversity of limb generation has been proposed to occur through sequential branching and segmentation of precartilaginous skeletal elements along the proximodistal axis of the limb. The position of future joints is first delimited by areas of higher cell density called interzones initially through an as yet unidentified inductive signal, subsequently specification of these regions is controlled hierarchically by wnt14 and gdf5, respectively. Joint-forming cell fate although specified is not fixed, and joints will fuse if growth factor signaling is perturbed. Cavitation, the separation of the two opposing skeletal elements, and joint morphogenesis, the process whereby the joint cells organize and mature to establish a functional interlocking and reciprocally shaped joint, are slowly being unraveled through studying the plethora of molecules that make up the unique extracellular matrix of the forming structure. The joint lining tissue, articular cartilage, is avascular, and this limits its reparative capacity such that arthritis and associated joint pathologies are the single largest cause of disability in the adult population. Recent discoveries of adult stem cells and more specifically the isolation of chondroprogenitor cells from articular cartilage are extending available therapeutic options, though only with a more complete understanding of synovial joint development can such options have greater chances of success.

The cellular responses of articular cartilage to sharp and blunt trauma.

OBJECTIVE: To determine the response of immature articular cartilage to both sharp and blunt trauma in terms of cell death, cell proliferation and matrix synthesis. DESIGN: Blunt wounds were made with a trephine in full depth immature bovine articular cartilage explants which were cut in half through the center of the trephine wound with a sharp scalpel to produce blunt and sharp trauma on the same explant. Explants were maintained in culture for up to 10 days. Prior to fixation at days 2, 5 and 10, medium was supplemented with 10 microCi ml-1 35S-sulphate, [3H]-proline or [3H]-thymidine for 24h to assess matrix synthesis and cell proliferation. Cell death was assessed using a Live/Dead label. RESULTS: In the case of blunt wounds, a band of cell death was observed adjacent to the lesion edge. Microautoradiography demonstrated little radiolabel incorporation and, therefore, no new matrix synthesis or cell proliferation within this region. In contrast, wounds made with a sharp scalpel showed restricted cell death, with radiolabel incorporation adjacent to the lesion edge at all time points. This demonstrated not only chondrocyte proliferation and new matrix synthesis at the wound margin, but also an up-regulation of matrix synthesis adjacent to the lesion edge. CONCLUSIONS: In terms of clinical relevance, the use of sharp precise instruments during the surgical management of cartilage defects may be necessary to reduce cell death and promote matrix elaboration at the lesion edge in order to facilitate successful integration.

A mechanism underlying the movement requirement for synovial joint cavitation.

Many studies have highlighted the importance of movement-induced mechanical stimuli in the development of functional synovial joints. However, such phenomenological results have failed to provide a full explanation of the mechanism essential for the morphogenesis of fluid-filled joint cavities. We have previously demonstrated that the large glycosaminoglycan hyaluronan (HA), in association with its principal cell surface receptor CD44, plays a major role during the morphogenesis of chick joints. We have taken cells from the surface of recently cavitated joints and subjected them to a brief period of dynamic mechanical strain (3800 microE for 10 min) and measured changes in HA synthesis/release, CD44 expression and HA synthase gene expression. In addition, we subjected cells to matrix depletion prior to the application of mechanical strain in order to examine any potential modulatory function of the ECM during the cell response to strain. Removal of the cell-associated HA-containing matrix with hyaluronidase significantly increased the release of HA into tissue culture media over 24 h and is associated with increased CD44 expression, alterations in HA synthase gene expression and enhanced binding of HA to the cell surface. Such changes in HA release were shown to be blocked by addition of exogenous HA and synergistically enhanced by the application of dynamic mechanical strain. These results show that cell-matrix interactions modify the response of embryonic cells to mechanical strain and provide further insight into the mechano-dependent mechanism of joint cavity morphogenesis.

The distribution of Notch receptors and their ligands during articular cartilage development.

We examined the distribution of Notch family members and their ligands during the development of articular cartilage and the growth plate. Notch 1 was expressed by the chondrocytes of the developing articular surface but became increasingly restricted to the deeper layers after birth whilst expression of this family member was restricted to hypertrophic chondrocytes in the growth plate. Notch 2 and 4, Delta and Jagged 2 showed a broadly similar distribution, being present throughout the articular cartilage during development and becoming increasingly restricted to deeper layers with age. Hypertrophic chondrocytes within the growth plate also expressed Notch 2 and 4, Delta and Jagged 2 (which was also expressed in prehypertrophs). Notch 3 and Jagged 1 were absent from developing articular cartilage but were present in deeper layers at later time points (> 1 month) and both receptor and ligand were expressed in hypertrophic chondrocytes at all ages examined. These results highlight the complex Notch signalling interactions that result in the formation of the heterogeneous articular cartilage and allow for the co-ordinated ossification and elongation of the growth plate. Mechanisms by which these processes are controlled are discussed in light of recent advances in the understanding of Notch signalling pathways.

Short-term rigid and flaccid paralyses diminish growth of embryonic chick limbs and abrogate joint cavity formation but differentially preserve pre-cavitated joints.

The influence of movement on joint space formation during limb development has been the subject of much interest. Our aim was to investigate the short-term influence of movement upon cavitation by immobilizing chick embryos in ovo, both in a rigid manner where dynamic stimulation is removed, and a flaccid manner where both dynamic and static stimulation are absent. Induction of rigid immobilization with decamethonium bromide (DMB) or the novel induction of flaccid immobilization with pancuronium bromide (PB) for 3 days, during the normal cavitation of joints resulted in the loss of cavity formation. Immobilization after the formation of an overt cavity demonstrated that static stimulation (during rigid paralysis) was able to maintain joint cavities and preserve some of the hyaluronan (HA) content of articular surfaces, whereas flaccid paralysis resulted in the loss of cavities and a marked depletion of HA content. Assessments of the growth and deposition of cartilage and bone in the limbs of embryos immobilised during cavitation showed that the length of limb elements was greatly reduced and that decreases in epiphyseal widths were most marked and more pronounced distally. The volume of bone in these elements remained unchanged whereas the cartilage volume decreased significantly, suggesting that chondrogenic but not osteogenic events in the embryo are particularly sensitive to mechanical stimulation. In addition to describing a novel method of inducing flaccid immobility in ovo, these data point towards the important role of both static and dynamic stimuli in the growth of embryonic limbs and the development of a functional joint space.

Differences in repair responses between immature and mature cartilage.

Cartilage has a poor reparative capacity although it is unclear as to what extent this may be dependent on age or maturation. In the current study, the cellular responses of chondrocytes to experimental wounding in vitro using embryonic, immature, and mature cartilage have been compared. In all cases, the response was consistent (a combination of cell death that included apoptosis and proliferation). The speed of response varied in terms of cell death with embryonic cartilage showing the most rapid response and mature cartilage showing the slowest response. Intrinsic repair as assessed by the ability to heal the lesion was not detected in any of the culture systems used. It was concluded that the poor repair potential of cartilage is not maturation dependent in the systems studied.

The development of articular cartilage: evidence for an appositional growth mechanism.

It is well-established that cartilage grows by a combination of matrix secretion, cell hypertrophy and cell proliferation. The extent to which this growth is by appositional, as opposed to interstitial mechanisms, however, remains unclear. Using the knee joints of the marsupial Monodelphis domestica to study cartilage growth, we have combined an immunohistochemical study of the TGF-beta family of cartilage growth and differentiation factors between 30 days postpartum to 8 months, together with a stereological analysis of cartilage morphology during growth. Furthermore, to gain an insight into the generation of the characteristic zones within cartilage, we have examined the effects of intra-articular administration of bromodeoxyuridine, an agent that is incorporated into DNA during cell division and blocks further cell cycling. During early growth, TGF-beta2 and -beta3 were widely expressed but TGF-beta1 was less so. After the formation of the secondary centre of ossification, all isoforms became more restricted to the upper half of the tissue depth and their distribution was similar to that previously described for IGFs, and PCNA-positive cells. Stereological analysis of tissue sections from the femoral condylar cartilage at 3 and 6 months showed that there was a 17% increase in total cartilage volume but a 31% decrease in cell density on a unit volume basis. Finally, cell-cycle perturbation with BrDU, which was injected into the knee joints of 3-month-old animals and analysed 1 and 4 months post-injection, revealed that the chondrocytes occupying the transitional zone were depleted 1 month post-injection, resulting in thinning of the articular cartilage. This effect was reversed 4 months post-injection. Immunohistochemical analysis revealed that BrDU-treatment altered the expression patterns of all TGF-beta isoforms, with a marked reduction in labelling of TGF-beta1 and -beta3 isoforms in the upper half of the cartilage depth. Overall, the data lends further support to the notion of articular cartilage growing by apposition from the articular surface rather than by interstitial mechanisms.

The effect of mechanical strain on hyaluronan metabolism in embryonic fibrocartilage cells.

The development of the synovial joint cavity between the cartilage anlagen of the long bones is thought to be mediated by differential matrix synthesis at the developing articular surfaces. In addition, many studies have shown that removal of movement-induced mechanical stimuli from developing diarthrodial joints prevents cavity formation or produces a secondary fusion of previously cavitated joints. Herein, we describe an inductive influence of mechanical strain on hyaluronan metabolism and the expression of hyaluronan-binding proteins in cultured cells isolated from the articular surface of the distal tibial condyles of 18-day chick embryos. The effect of 10 min of mechanical strain on hyaluronan release into culture media, intracellular uridine diphospho-glucose dehydrogenase activity (an enzyme required for hyaluronan saccharide precursor production), cell surface hyaluronan-binding protein expression and HA synthase mRNA expression were analysed up to 24 h later. Six hours after the application of strain, there was a significant increase in the accumulation of hyaluronan released into tissue culture media by strained fibrocartilage cells compared with controls, an effect still detectable after 24 h. Strained cells also showed increased activity for uridine diphospho-glucose dehydrogenase and expressed higher levels of the hyaluronan-binding protein CD44 at 24 h. In addition, at 24 h mRNA for HA synthase 2 was expressed in all samples whereas mRNA for HA synthase 3 was only expressed in strained cells. These results further highlight the role for movement-induced stimuli in differential extracellular matrix metabolism during joint development and also show that strain may facilitate differential HA synthase gene expression.

An essential role for the interaction between hyaluronan and hyaluronan binding proteins during joint development.

We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HA-HABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HA-HABP interactions play a major role in the initial separation events occurring during joint formation.