Myofascial junction: Emerging insights into the connection between deep/muscular fascia and muscle.

Muscles and fasciae are mutually connected and are influenced by force transmission. However, the anatomical connectivity and histological features of these structures remain unclear. The aim of this study was to assess the evidence for connection between muscles and deep/muscular fasciae. We assessed this relationship in different topographical regions of human cadavers and in mice. The results showed that myofascial junctions (MFJ) were made up of collagen I immune-positive structures occupying an average area of 5.11 ± 0.81 µm2, distributed in discrete regions at the interface between muscle and fascia with an average density of 9.7 ± 2.51 MFJ/mm and an average inclination angle of 35.25 ± 1.52°. These specialized structures also showed collagen III and HA immunopositivity and the presence of elastic fibers. The human myofascial junction can be visualized, opening emerging insights into the connection between deep/muscular fascia and muscle.

Blood supply to the superficial fascia of the abdomen: An anatomical study.

The aim of this study was to examine data demonstrating that Scarpa's fascia, a superficial fascia of the anterior abdominal wall, is a vascularized tissue. Specimens of the fascia of seven volunteers undergoing abdominoplasty surgical procedures at the Plastic Surgery Unit of the University of Padova Medical Center were collected. Fractal analysis and quantitative assessment of the vascular network of the fascia was carried out, exploiting the presence of blood in the vessels. Each sample was divided and processed for histological/immunohistochemical analysis (into 5 micron-paraffin embedded sections and cryo-sectioned free-floating samples) as well as for electron microscopy study. A rich vascular pattern forming a fine, dense meshwork with an area percentage of 6.20% ± 2.10% von Willebrand factor stained vessels was noted in all the specimens of the fascia examined; the area percentage of the αSMA-stained vessels was 2.93% ± 1.80%. The diameters of the vessels fell between the 13 and 65 µm range; the network was composed of arteries, veins, capillaries and lymphatic segments. Topological results showed that the vascular network within Scarpa's fascia is well branched (segments: 6615 ± 3070 and 8.40 ± 3.40 per mm2 ; crossing points: 3092 ± 1490 and 3.40 ± 1.90 per mm2 ). Fractal analysis (fractal dimension = 1.063 ± 0.10; lacunarity = 0.60 ± 0.10) revealed that this particular vascular network has an optimal spatial distribution and homogeneity occupying the entire space of the superficial fascia. These findings could undoubtedly be useful to plastic surgeons as well as to pain management specialists.

From Muscle to the Myofascial Unit: Current Evidence and Future Perspectives.

The "motor unit" or the "muscle" has long been considered the quantal element in the control of movement. However, in recent years new research has proved the strong interaction between muscle fibers and intramuscular connective tissue, and between muscles and fasciae, suggesting that the muscles can no longer be considered the only elements that organize movement. In addition, innervation and vascularization of muscle is strongly connected with intramuscular connective tissue. This awareness induced Luigi Stecco, in 2002, to create a new term, the "myofascial unit", to describe the bilateral dependent relationship, both anatomical and functional, that occurs between fascia, muscle and accessory elements. The aim of this narrative review is to understand the scientific support for this new term, and whether it is actually correct to consider the myofascial unit the physiological basic element for peripheral motor control.

Detection of Mast Cells in Human Superficial Fascia.

The recent findings showed that the superficial fascia is a fibrous layer in the middle of hypodermis, richly innervated and vascularized, and more complex than so far demonstrated. This study showed the presence of mast cells in the superficial fascia of the human abdomen wall of three adult volunteer patients (mean age 42 ± 4 years; 2 females, 1 male), by Toluidine Blue and Safranin-O stains and Transmission Electron Microscopy. Mast cells are distributed among the collagen bundles and the elastic fibers, near the vessels and close to the nerves supplying the tissue, with an average density of 20.4 ± 9.4/mm2. The demonstration of the presence of mast cells in the human superficial fascia highlights the possible involvement of the tissue in the inflammatory process, and in tissue healing and regeneration processes. A clear knowledge of this anatomical structure of the hypodermis is fundamental for a good comprehension of some fascial dysfunctions and for a better-targeted clinical practice.

Elastic Fibres in the subcutaneous tissue: Is there a difference between superficial and muscular fascia? A cadaver study.

BACKGROUND: In last years the role of fascia in proprioception and pain has been confirmed in numerous papers, but the real structure of fasciae is not still entirely known. To date, many studies have evaluated the elastic fibres in arteries, ligaments, lungs, epidermis and dermis, but only two studies exist about the elastic fibres in the fasciae, and they did not distinguish between superficial (in the subcutaneous tissue) and deep/muscular fasciae. The aim of the study was to assess the percentage of elastic fibres between superficial and deep fascia. MATERIALS AND METHODS: Three full thickness specimens (proximal, middle and distal respectively) were taken from each of four regions of the thigh of three non-embalmed cadavers: the anterior (Ant), the lateral (Lat), the posterior (Post) and the medial (Med) aspect. Thus, a total of 12 specimens were collected from each analysed thigh and histological Weigert Van Gieson stains was performed. Three sections per specimen were considered for the morphometric analysis. RESULTS: In all the specimens the superficial and deep fasciae were clearly recognizable. The difference in percentage of elastic fibres between superficial and deep fasciae in same region for all four was highly significant (p < 0.001). They are abundant in the superficial fascia than deep fascia. CONCLUSIONS: In the light of these findings is evident that the superficial (in the subcutaneous tissue) and deep fasciae have different elasticity. This difference may improve grading of fascial dysfunction in dermatological diseases as burns, scars and lymphedema to better plan treatments.

Fascial Innervation: A Systematic Review of the Literature.

Currently, myofascial pain has become one of the main problems in healthcare systems. Research into its causes and the structures related to it may help to improve its management. Until some years ago, all the studies were focused on muscle alterations, as trigger points, but recently, fasciae are starting to be considered a new, possible source of pain. This systematic review has been conducted for the purpose of analyze the current evidence of the muscular/deep fasciae innervation from a histological and/or immunohistochemical point of view. A literature search published between 2000 and 2021 was made in PubMed and Google Scholar. Search terms included a combination of fascia, innervation, immunohistochemical, and different immunohistochemical markers. Of the 23 total studies included in the review, five studies were performed in rats, four in mice, two in horses, ten in humans, and two in both humans and rats. There were a great variety of immunohistochemical markers used to detect the innervation of the fasciae; the most used were Protein Gene Marker 9.5 (used in twelve studies), Calcitonin Gene-Related Peptide (ten studies), S100 (ten studies), substance P (seven studies), and tyrosine hydroxylase (six studies). Various areas have been studied, with the thoracolumbar fascia being the most observed. Besides, the papers highlighted diversity in the density and type of innervation in the various fasciae, going from free nerve endings to Pacini and Ruffini corpuscles. Finally, it has been observed that the innervation is increased in the pathological fasciae. From this review, it is evident that fasciae are well innerved, their innervation have a particular distribution and precise localization and is composed especially by proprioceptors and nociceptors, the latter being more numerous in pathological situations. This could contribute to a better comprehension and management of pain.

The Effects of Aging on the Intramuscular Connective Tissue.

The intramuscular connective tissue plays a critical role in maintaining the structural integrity of the muscle and in providing mechanical support. The current study investigates age-related changes that may contribute to passive stiffness and functional impairment of skeletal muscles. Variations in the extracellular matrix in human quadriceps femoris muscles in 10 young men, 12 elderly males and 16 elderly females, and in the hindlimb muscles of 6 week old, 8 month old and 2 year old C57BL/6J male mice, were evaluated. Picrosirius red, Alcian blue and Weigert Van Gieson stainings were performed to evaluate collagen, glycosamynoglycans and elastic fibers. Immunohistochemistry analyses were carried out to assess collagen I, collagen III and hyaluronan. The percentage area of collagen was significantly higher with aging (p < 0.01 in humans, p < 0.001 in mice), mainly due to an increase in collagen I, with no differences in collagen III (p > 0.05). The percentage area of elastic fibers in the perimysium was significantly lower (p < 0.01) in elderly men, together with a significant decrease in hyaluronan content both in humans and in mice. No significant differences were detected according to gender. The accumulation of collagen I and the lower levels of hyaluronan and elastic fibers with aging could cause a stiffening of the muscles and a reduction of their adaptability.

An anatomical comparison of the fasciae of the thigh: A macroscopic, microscopic and ultrasound imaging study.

Although the number of Ultrasound (US) imaging studies investigating the fascial layers are becoming more numerous, the majority tend to use different reference points and terminology to describe their findings. The current work set out to compare macroscopic and microscopic data of specimens of the fascial layers of the thigh with US imaging findings. Specimens of the different fascial layers of various regions of the thigh were collected for macroscopic and histological analyses from three fresh cadavers and compared with in vivo US images of the thighs of 20 healthy volunteers. The specimens showed that the subcutaneous tissue of the thigh is made up of three layers: a superficial adipose layer, a membranous layer/superficial fascia, and a deep adipose layer. The deep fascia is composed of an aponeurotic fascia, which envelops all the thigh muscles and is laterally reinforced by the iliotibial tract and an epimysial fascia, which is specific for each muscle. The morphometric measurements of the thickness of the superficial fascia were different (anterior: 153.2 ± 39.3 µm; medial: 128.4 ± 24.7 µm; lateral: 154 ± 28.9 µm; and posterior: 148.8 ± 33.2 µm) as were those of the deep fascia (anterior: 556.8 ± 176.2 µm; medial: 820.4 ± 201 µm; lateral: 1112 ± 237.9 µm; and posterior: 730.4 ± 186.5 µm). The US scans showed a clear picture of the superficial adipose tissue, the superficial fascia, and the deep adipose tissue, as well as the deep fasciae. The epimysial and aponeurotic fasciae of only some topographic areas could be independently identified. The US imaging findings confirmed that the superficial and deep fascia have different thicknesses, and they showed that the US measurements were always larger with respect to those produced by histological analysis (p < 0.001) probably due to shrinkage during the processing. The posterior region (level 1) of the superficial fascia had, for example, a mean thickness of 0.56 ± 0.12 mm at US, while the histological analysis showed that it was 148.8 ± 33.2 µm. Showing a similar pattern, the thickness of the deep fascia was as follows: 1.64 ± 0.85 mm versus 730.4 ± 186.5 µm. Study results have confirmed that US can be considered a valid, non-invasive instrument to evaluate the fascial layers. In any event, there is a clear need for a set of standardised protocols since the thickness of the fascial layers of different parts of the human body varies and the data obtained using inaccurate reference points are not reproducible or comparable. Given the inconsistent terminology used to describe the fascial system, it would also be important to standardise the terminology used to define its parts. The difficulty in distinguishing between the epimysial and aponeurotic/deep fascia can also impede data interpretation.

A Closer Look at the Cellular and Molecular Components of the Deep/Muscular Fasciae.

The fascia can be defined as a dynamic highly complex connective tissue network composed of different types of cells embedded in the extracellular matrix and nervous fibers: each component plays a specific role in the fascial system changing and responding to stimuli in different ways. This review intends to discuss the various components of the fascia and their specific roles; this will be carried out in the effort to shed light on the mechanisms by which they affect the entire network and all body systems. A clear understanding of fascial anatomy from a microscopic viewpoint can further elucidate its physiological and pathological characteristics and facilitate the identification of appropriate treatment strategies.

Pilot Study of Sacroiliac Joint Dysfunction Treated with a Single Session of Fascial Manipulation® Method: Clinical Implications for Effective Pain Reduction.

Background and Objectives: Sacroiliac joint dysfunction (SIJD) generally refers to pain in the lower back due to abnormal sacroiliac joint movement, either from hypomobility or hypermobility. It is considered to be the principal cause in up to 40% of low back pain cases. In literature, it emerges that the "fascia", by its anatomical continuity, if altered or densified in different regions of the body with respect to the sacroiliac joint and its surroundings, may have a fundamental role in the genesis of SIJD and low back pain. The purpose of the present study is to evaluate the effectiveness of incorporating a single session of Fascial Manipulation®-Stecco method®, treating the muscular fasciae at distance from the painful region. Materials and Methods: Twenty patients with acute and chronic sacroiliac joint dysfunction (SIJD) were recruited (16 males and 4 females, mean age of 46.6 ± 12.98 years). Patients underwent a predefined assessment protocol, followed by an evaluation of myofascial pain and subsequent manipulation of the fascia at points at least 20 cm away from the posterior inferior iliac spines (PIIS). Each patient underwent three pain evaluations: pre-treatment (t0), post-treatment (t1), and at a 1-month follow-up (t2). For the evaluation in t0, t1 the numerical rating scale (NRS) for the intensity of pain and the algometer for the pain threshold at the PIIS were used; in t2 only the NRS scale. Results: The results obtained by comparing the algometer measurements with the NRS values between t0 and t1 were in both cases statistically significant (p < 0.0001), whereas the comparison between the NRS values at t1 and at t2 was not statistically significant (p > 0.05). Conclusions: A single Fascial Manipulation treatment, even when applied at least 20 cm from the PIIS, can potentially decrease pain around the SIJ. The inclusion of this type of approach in SIJD can allow for improved patient management, better tolerance for other treatments and a more rapid application of pain-free exercise programs.

Role of fasciae around the median nerve in pathogenesis of carpal tunnel syndrome: microscopic and ultrasound study.

This study investigated the connections between the median nerve paraneural sheath and myofascial structures near it, from both macroscopic and microscopic points of view. Four samples of median nerve and surrounding tissues were excised from nine non-embalmed upper limbs for microscopic analysis. Ultrasound images were analysed in 21 healthy subjects and 16 carpal tunnel syndrome patients to evaluate median nerve transversal displacement during finger motion at carpal tunnel and forearm levels. An anatomical continuity between epimysium and paraneural sheath and a reduction of paraneural fat tissue from proximal to distal was found in all samples. Median nerve displacements at both levels were significantly reduced in carpal tunnel syndrome subjects (P < 0.001). It was observed that the median nerve is not an isolated structure but is entirely connected to myofascial structures. Therefore, unbalanced tension of epimysial fasciae can affect the paraneural sheath, limiting nerve displacement, and consequently this must be included in carpal tunnel syndrome pathogenesis.

Sensitivity of the Fasciae to the Endocannabinoid System: Production of Hyaluronan-Rich Vesicles and Potential Peripheral Effects of Cannabinoids in Fascial Tissue.

The demonstrated expression of endocannabinoid receptors in myofascial tissue suggested the role of fascia as a source and modulator of pain. Fibroblasts can modulate the production of the various components of the extracellular matrix, according to type of stimuli: physical, mechanical, hormonal, and pharmacological. In this work, fascial fibroblasts were isolated from small samples of human fascia lata of the thigh, collected from three volunteer patients (two men, one woman) during orthopedic surgery. This text demonstrates for the first time that the agonist of cannabinoid receptor 2, HU-308, can lead to in vitro production of hyaluronan-rich vesicles only 3-4 h after treatment, being rapidly released into the extracellular environment. We demonstrated that these vesicles are rich in hyaluronan after Alcian blue and Toluidine blue stainings, immunocytochemistry, and transmission electron microscopy. In addition, incubation with the antagonist AM630 blocked vesicles production by cells, confirming that release of hyaluronan is a cannabinoid-mediated effect. These results may show how fascial cells respond to the endocannabinoid system by regulating and remodeling the formation of the extracellular matrix. This is a first step in our understanding of how therapeutic applications of cannabinoids to treat pain may also have a peripheral effect, altering the biosynthesis of the extracellular matrix in fasciae and, consequently, remodeling the tissue and its properties.

Effects of Cesarean Section and Vaginal Delivery on Abdominal Muscles and Fasciae.

Background and objectives: Possible disorders after delivery may interfere with the quality of life. The aim of this study was to ascertain whether abdominal muscles and fasciae differ in women depending on whether they experienced transverse cesarean section (CS) or vaginal delivery (VA) in comparison with healthy nulliparous (NU). Materials and methods: The thicknesses of abdominal muscles and fasciae were evaluated by ultrasound in 13 CS, 10 VA, and 13 NU women (we examined rectus abdominis (RA); external oblique (EO); internal oblique (IO); transversus abdominis (TrA); total abdominal muscles (TAM = EO + IO + TrA); inter-rectus distance (IRD); thickness of linea alba (TLA); rectus sheath (RS), which includes anterior fascia of RS and posterior fascia of RS (P-RS); loose connective tissue between sublayers of P-RS (LCT); abdominal perimuscular fasciae (APF), which includes anterior fascia of EO, fasciae between EO, IO, and TrA, and posterior fascia of TrA). Data on pain intensity, duration, and location were collected. Results: Compared with NU women, CS women had wider IRD (p = 0.004), thinner left RA (p = 0.020), thicker right RS (p = 0.035) and APF (left: p = 0.001; right: p = 0.001), and IO dissymmetry (p = 0.009). VA women had thinner RA (left: p = 0.008, right: p = 0.043) and left TAM (p = 0.024), mainly due to left IO (p = 0.027) and RA dissymmetry (p = 0.035). However, CS women had thicker LCT (left: p = 0.036, right: p < 0.001), APF (left: p = 0.014; right: p = 0.007), and right IO (p = 0.028) than VA women. There were significant correlations between pain duration and the affected fasciae/muscles in CS women. Conclusions: CS women showed significant alterations in both abdominal fasciae and muscle thicknesses, whereas VA women showed alterations mainly in muscles. Thinner RA and/or dissymmetric IO, wider IRD, and thicker LCT and APF after CS may cause muscle deficits and alteration of fascial gliding, which may induce scar, abdominal, low back, and/or pelvic pain.

Inter-rater reliability and variability of ultrasound measurements of abdominal muscles and fasciae thickness.

Ultrasound (US) imaging is being increasingly used by Physical and Rehabilitation Medicine (PRM) specialists to measure the thickness of abdominal muscles. The current study set out to assess the inter-rater reliability of US measurements of the thickness of the abdominal muscles/fasciae. Three raters (1 = orthopedic specialist, expert on fasciae; 2 = PRM resident; 3 = PRM specialist) with different levels of US training examined the abdominal muscles and fasciae of a healthy volunteer under supine resting and dynamic conditions following a standard US protocol. The probe was positioned along the right lateral abdominal wall at the height of the 12th rib: (1) above the umbilicus at the linea alba, (2) to the side of and approximately 2 cm from the umbilicus, (3) along the mammillary line, and (4) along the anterior axillary line. Each rater measured 17 anatomical structures six times during two sessions. The relative error of the measurements (intra-rater variability) was slightly higher for the fasciae than for the muscles, and during the dynamic condition than the resting condition. Inter-rater reliability was good under both conditions for the fasciae (Intraclass Correlation Coefficient = ICC = 0.83) and excellent for the muscles (ICC = 0.99). Knowledge of the fascial anatomy of the abdominal wall is essential for accurate ultrasound examinations and for improving reliability. These findings confirm that US imaging is a reliable, non-invasive, cost-effective instrument for evaluating the abdominal muscles/fasciae. Clin. Anat. 32:948-960, 2019. © 2019 Wiley Periodicals, Inc.

Dermatome and fasciatome.

Increased knowledge of the rich innervation of the deep fascia and its anatomical organization indicates the need to reevaluate maps of the dermatome according to the new findings. The authors present a distinction between dermatome and fasciatome, basing their approach to the literature on nerve root stimulation and comparing dermatomeric and myomeric maps. The former represents the portion of tissue composed of skin, hypodermis, and superficial fascia supplied by all the cutaneous branches of an individual spinal nerve; the latter includes the portion of deep fascia supplied by the same nerve root and organized according to force lines to emphasize the main directions of movement. The dermatome is important for esteroception, whereas the fasciatome is important for proprioception. If they are altered, the dermatome shows clearly localized pain and the fasciatome irradiating pain according to the organization of the fascial anatomy. Clin. Anat. 32:896-902, 2019. © 2019 Wiley Periodicals, Inc.

The fasciacytes: A new cell devoted to fascial gliding regulation.

Hyaluronan occurs between deep fascia and muscle, facilitating gliding between these two structures, and also within the loose connective tissue of the fascia, guaranteeing the smooth sliding of adjacent fibrous fascial layers. It also promotes the functions of the deep fascia. In this study a new class of cells in fasciae is identified, which we have termed fasciacytes, devoted to producing the hyaluronan-rich extracellular matrix. Synthesis of the hyaluronan-rich matrix by these new cells was demonstrated by Alcian Blue staining, anti-HABP (hyaluronic acid binding protein) immunohistochemistry, and transmission electron microscopy. Expression of HAS2 (hyaluronan synthase 2) mRNA by these cells was detected and quantified using real time RT-PCR. This new cell type has some features similar to fibroblasts: they are positive for the fibroblast marker vimentin and negative for CD68, a marker for the monocyte-macrophage lineage. However, they have morphological features distinct from classical fibroblasts and they express the marker for chondroid metaplasia, S-100A4. The authors suggest that these cells represent a new cell type devoted to the production of hyaluronan. Since hyaluronan is essential for fascial gliding, regulation of these cells could affect the functions of fasciae so they could be implicated in myofascial pain. Clin. Anat. 31:667-676, 2018. © 2018 Wiley Periodicals, Inc.

Anatomical and functional relationships between external abdominal oblique muscle and posterior layer of thoracolumbar fascia.

The abdominal muscles are important for the stability of the lumbar region through the thoracolumbar fascia (TLF). However, there is not full agreement regarding the posterior transversal continuity of the external abdominal oblique muscle (EO) with the TLF. To clarify this point, 10 cadavers and computed tomography (CT) images from 27 subjects were used to evaluate the transversal continuity of the TLF with the abdominal muscles. The width of the fascial continuity of the EO with the posterior layer of TLF along the posterior border of the EO was also measured (40.70 ±3.92 mm). The epimysial fascia of the EO was in direct continuity with the posterior layer of TLF in eight cadavers and 23 CT images, whereas in two cadavers and four CT images, the epimysial fascia of the EO first fused with the fascia covering the latissimus dorsi, and then, both fasciae were in continuity with the posterior layer of TLF. Therefore, the transversal fascial continuity of the EO could explain the transmission of tension from the EO to the posterior layer of TLF and its importance in maintaining the stability of the lumbar spine through a hydraulic effect. Regarding fascial continuity in the trunk, and taking the EO into consideration, the TLF is formed by the fascia of all the abdominal muscles as the rectus sheath. In this manner, myofascial continuity between the TLF and the abdominal muscles is achieved through the aponeurosis and fascia, which ensures synchronization between the erector spinae and the rectus abdominis. Clin. Anat. 31:1092-1098, 2018. © 2018 Wiley Periodicals, Inc.

Morphometric and dynamic measurements of muscular fascia in healthy individuals using ultrasound imaging: a summary of the discrepancies and gaps in the current literature.

PURPOSE: The objectives of this work was to conduct a comprehensive state-of-the art review of the current literature to identify any gaps or discrepancies and summarize the main challenges for obtaining a homogeneous evaluation of muscular fascia in healthy individuals. METHODS: An electronic document search using key words and MeSH terms was performed with various databases. Two independent investigators were tasked with the screening of articles and data extraction. A critical appraisal of what is known was then conducted. RESULTS: The literature search identified 65 articles related to healthy facia in the various databases consulted and 20 articles were kept for the review. The thickest portion of the fascia lata (the iliotibial tract) and the plantar fascia are the most often studied muscular fasciae whereas there is paucity of studies on fascia related to other muscles in the body. CONCLUSION: US imaging is suitable to complement physical examination and for evaluating treatment outcomes. However, the small number of studies and the heterogeneity of the methods did not allow us to establish normal reference values for muscular fascia thickness and to provide strong recommendations about measurement protocols.

Evaluation of gold nanoparticles toxicity towards human endothelial cells under static and flow conditions.

A new in vitro model system, adding advection and shear stress associated with a flowing medium, is proposed for the investigation of nanoparticles uptake and toxicity towards endothelial cells, since these processes are normally present when nanoparticles formulations are intravenously administered. In this model system, mechanical forces normally present in vivo, such as advection and shear stress were applied and carefully controlled by growing human umbilical vein endothelial cells inside a microfluidic device and continuously infusing gold nanoparticle (Au NPs) solution in the device. The tests performed in the microfluidic device were also run in multiwells, where no flow is present, so as to compare the two model systems and evaluate if gold nanoparticles toxicity differs under static and flow culture conditions. Full characterization of Au NPs in water and in culture medium was accomplished by standard methods. Two-photon fluorescence correlation spectroscopy was also employed to map the flow speed of Au NPs in the microfluidic device and characterize Au NPs before and after interactions with the cells. Au NPs uptake in both in vitro systems was investigated through electron and fluorescence microscopy and ICP-AES, and NPs toxicity measured through standard bio-analytical tests. Comparison between experiments run in multiwells and in microfluidic device plays a pivotal role for the investigation of nanoparticle-cell interaction and toxicity assessment: our work showed that administration of equal concentrations of Au NPs under flow conditions resulted in a reduced sedimentation of nanoparticle aggregates onto the cells and lower cytotoxicity with respect to experiments run in ordinary static conditions (multiwells).