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Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Some of these unsolved issues may involve the grantees of teaching and training activities, the role of academic anatomical institutes, the role of family members in the donation process, the universal time limit indicated for any donation, the handling of corpses, and the limited body donation and its subordination to the donation of organs and tissues. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Decellularized extracellular matrix is one of the most promising biological scaffold supporting in vitro tissue growth and in vivo tissue regeneration in both preclinical research and clinical practice. In case of thick tissues or even organs, conventional static decellularization methods based on chemical or enzymatic treatments are not effective in removing the native cellular material without affecting the extracellular matrix. To overcome this limitation, dynamic decellularization methods, mostly based on perfusion and agitation, have been proposed. In this study, we developed a low-cost scalable 3D-printed sample-holder for agitation-based decellularization purposes, designed for treating multiple specimens simultaneously and for improving efficiency, homogeneity and reproducibility of the decellularization treatment with respect to conventional agitation-based approaches. In detail, the proposed sample-holder is able to house up to four specimens and, immersed in the decellularizing solution within a beaker placed on a magnetic stirrer, to expose them to convective flow, enhancing the solution transport through the specimens while protecting them. Computational fluid dynamics analyses were performed to investigate the fluid phenomena establishing within the beaker and to support the sample-holder design. Exploratory biological tests performed on human skin specimens demonstrated that the sample-holder reduces process duration and increases treatment homogeneity and reproducibility.

Introduction: Age, gender, body mass index percentiles and the adherence to Mediterranean diet were investigated as potential predictor factors in this assessment. Aim: To assess the parental perception of children's weight status. Methods: This cross-sectional observational study was carried out during a corporate wellness program (Ferrari Formula Benessere) implemented by Ferrari S.p.A. and managed by Med-Ex s.r.l. The children’s real weight status was assessed through Body Mass Index percentiles (ArthroPlus software—WHO). Results: 328 children (66.4%) were normal-weight, 10 were underweight (2%), 66 were overweight (13.4%) and 90 were obese (18.2%). 289 children (59%) were classified correctly by parents, while 205 children (41%) were not. 64 of 66 overweight children and 90 of 90 children with obesity have been completely underestimated (53 of 90 children with obesity were judged normal weight). The parents’ probability to estimate children’s weight status correctly decreased increasing body mass index percentiles paradoxically [OR = 0.96 (0.95–0.97)] and was lower in boys [OR = 0.65 (0.44–0.98)]. Although not statistically significant, children with higher adherence to Mediterranean diet seems to have higher probability to be correctly estimated [low adherence: reference, medium adherence: OR = 1.06 (0.61–1.85), high adherence: OR = 1.48 (0.81–2.75)]. Conclusions: A high percentage of children is overweight or obese and almost half of parents classified their weight status incorrectly. Increasing children’s body mass index percentiles decreases the probability to be correctly classified.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-μm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE.

Background. Recent findings indicate that the connective tissue of tendons hosts cells that can self-renew and are multipotent. Even if these cells seemingly fail to accomplish tendon regeneration in pathological conditions, their identification and characterization represents a milestone in the research and development of new biological therapies for tendinopathies. Methods. We isolated the adult human tendon-derived stem cells (hTDSCs) from the fragments of patellar or calcaneal tendons and characterized these cells in vitro by immunochemistry and histochemistry. Subsequently, the MTT test and Trypan Blue were used for the evaluation of cytotoxicity of the supplements/drugs commonly used for the treatment of musculoskeletal disorders: Curcumin, Hyaluronic Acid, Palmi-toylethanolamide, Diclofenac sodium, Triamcinolone acetonide and Thiocolchicoside. Results. Cells obtained by outgrowth expressed mesenchymal markers, were clono-genic and differentiated into chondroblasts, osteoblasts and adipocytes. High concen-trations of the anti-inflammatory glucocorticoid Triamcinolone and the analge-sic fatty-acid amide Palmitoylethanolamide significantly reduced cell viability. Only curcumin had a positive effect on cell survival, both in the normal and oxidative stress conditions. Conclusions. Adult human tendons posses stem cells. The influence of several drugs or supplements used for the treatment of musculoskeletal disorders should be taken into consideration in order to take the full advantage of the healing properties of stem cells within tendons.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of &alpha, smooth muscle actin (&alpha, SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

Cutaneous melanoma (CM) represents one of the most metastasizing and drug resistant solid tumors. CM is characterized by a remarkable metabolic plasticity and an important connection between oncogenic activation and energetic metabolism. In fact, melanoma cells can use both cytosolic and mitochondrial compartments to produce adenosine triphosphate (ATP) during cancer progression. However, the CM energetic demand mainly depends on glycolysis, whose upregulation is strictly linked to constitutive activation of BRAF/MAPK pathway affected by BRAFV600E kinase mutant. Furthermore, the impressive metabolic plasticity of melanoma allows the development of resistance mechanisms to BRAF/MEK inhibitors (BRAFi/MEKi) and the adaptation to microenvironmental changes. The metabolic interaction between melanoma cells and tumor microenvironment affects the immune response and CM growth. In this review article, we describe the regulation of melanoma metabolic alterations and the metabolic interactions between cancer cells and microenvironment that influence melanoma progression and immune response. Finally, we summarize the hallmarks of melanoma therapies and we report BRAF/MEK pathway targeted therapy and mechanisms of metabolic resistance.

Induced pluripotent stem cells (iPSCs) are adult somatic cells genetically reprogrammed to an embryonic stem cell‐like state. Notwithstanding their autologous origin and their potential to differentiate towards cells of all three germ layers, iPSC reprogramming is still affected by low efficiency. As dermal fibroblast is the most used human cell for reprogramming, we hypothesize that the variability in reprogramming is, at least partially, because of the skin fibroblasts used. Human dermal fibroblasts harvested from five different anatomical sites (neck, breast, arm, abdomen and thigh) were cultured and their morphology, proliferation, apoptotic rate, ability to migrate, expression of mesenchymal or epithelial markers, differentiation potential and production of growth factors were evaluated in vitro. Additionally, gene expression analysis was performed by real‐time PCR including genes typically expressed by mesenchymal cells. Finally, fibroblasts isolated from different anatomic sites were reprogrammed to iPSCs by integration‐free method. Intriguingly, while the morphology of fibroblasts derived from different anatomic sites differed only slightly, other features, known to affect cell reprogramming, varied greatly and in accordance with anatomic site of origin. Accordingly, difference also emerged in fibroblasts readiness to respond to reprogramming and ability to form colonies. Therefore, as fibroblasts derived from different anatomic sites preserve positional memory, it is of great importance to accurately evaluate and select dermal fibroblast population prior to induce reprogramming.

Aims The aim of our study was to assess the clinical significance of the exercise stress testing endpoints, namely 85% of maximal theoretical heart rate (MTHR), metabolic equivalent of task, and rating of perceived exertion (RPE), and their relation to electrocardiographic (ECG) changes in a healthy adult population. Methods A cross-sectional study was conducted on 408 males and 52 females (mean age 39.4 ± 8.6 years) who performed the maximal cycle ergometer exercise stress test until volitional exhaustion, reporting the RPE score at 85% of MTHR and at peak exercise. Metabolic equivalents of task were indirectly calculated from the maximum workload and compared with the predicted values. Sitting torso-lead ECG and blood pressure were recorded at rest, during exercise and during recovery. Results Of 460 participants, 73% exceeded 85% of MTHR. The RPE score represented the overall most significant endpoint of exercise stress testing, with the median value of 17 at peak exercise. ECG events were detected in 23/124 (18.5%) who reached ≤ 85% of MTHR and in 61/336 (18.2%) who achieved >85% of MTHR ( p = 0.92). In the latter group, 54% of ECG changes occurred at 85% of MTHR ( p = 0.51). If the exercise stress testing had been interrupted at ≤ 85% of MTHR, almost half of the ECG events would have remained undetected and 35% of the cardiovascular abnormalities observed at the diagnostic follow-up would have remained undiagnosed. Conclusion Terminating exercise stress testing before volitional exhaustion and an RPE score of 17 limits the test accuracy and reduces the possibility to detect cardiovascular abnormalities in apparently healthy adult populations.

Breast cancer is the most common cancer in women, which incidence has increased in recent years. It is constituted by very heterogeneous tissue characterized by an abnormal microenvironment regulating tumor progression and providing evasion from cancer therapies. Breast cancer-associated fibroblasts (BCAFs) are the main cell type of breast cancer microenvironment and can represent up to 80% of the tumor mass. In particular, BCAFs induce cancer initiation, proliferation, invasion and metastasis by undergoing an activation process associated with the secretion of growth factors, cytokines, and paracrine interactions. Therapy resistance is the main cause of poor therapeutic results or even failure in breast cancer patients. Despite recent advances in breast cancer management, there is a need for new prognostic markers and novel agents for targeting key signalling pathways to either improve the efficacy of the current therapies, or reduce toxicity. In this view, BCAFs represent markers useful to clinical diagnosis, therapy, and prognosis of breast cancer. This review focuses on the role of BCAFs in cancer, and describes the processes of endocrine/chemotherapy resistance linked to BCAFs action. Moreover, it points to molecules and pathways regulating therapy resistance induced by BCAFs. Finally, potential therapeutic strategies targeting BCAFs and offering new tools in breast cancer therapy are highlighted.

The increasing availability of food supplements, aggressive media advertising, and common beliefs that these substances have only positive effects on health and sport performance indicate a need for continuous monitoring of this phenomenon. The aim of this study was to investigate the habits and beliefs related to diet supplementation among medical, health professional, and other university/high school students by means of a cross-sectional anonymous survey online. Among the respondents aware of supplements, 37.4% were taking or had taken them in the past. Food supplement use was more common among university students (in particular, those in health professional graduate courses) than high school students. Individual sport practice, rather than team sport, was associated with higher likelihood of food supplement use. Multivitamins were most commonly used, while weight-loss formulations were the least popular. Strikingly, filling nutrient gaps was statistically not considered the main reason for taking food supplements. Instead, they were used to enhance mental performance or enhance well-being. There was statistical evidence that students not enrolled in health or medical professional studies strongly agreed more often than medical students that taking food supplements prevents illness. These results indicate a striking difference between the evidence-based and personal reasons for food supplement use. Arguably, it calls for an improvement in education about diet supplementation and a change in attitude of health care providers to its implementation.

Objective Only a few anatomic studies of the entire course of the hypoglossal nerve (cranial nerve XII) have been reported. We analyzed all relationships of the 12th nerve with surrounding structures from the brainstem to the tongue through a microscopic perspective. A comprehensive anatomically and clinically oriented classification of its different segments is proposed. Methods Ten formalin-fixed adult human cadaveric heads (20 sides) were dissected with the aim to explore the entire course of cranial nerve XII via lateral suboccipital, far lateral partial, or total transcondylar routes. Different segments of the nerve were identified based on the hypoglossal course and its relationship with surrounding structures. Measurements of every portion of the nerve were taken in all specimens during dissection. Results The hypoglossal nerve was divided into 5 segments: cisternal, intracanalar, descending, horizontal, and ascending. Detailed and comprehensive examination of basic anatomic relationships through the view of different transcranial and endoscope-assisted approaches was performed. A new perspective of the hypoglossal canal is proposed, and the venous plexus surrounding the intracanalar segment of the nerve is described in detail. Conclusions Classification of 5 segments for the hypoglossal nerve seems anatomically valid, and it is surgically oriented with respect to all surgical approaches. Precise knowledge of the relationships with the surrounding structures may help to prevent some complications during surgery, and it is useful to explain, segment by segment, the pathogenic mechanisms for nerve injuries that are evidenced by lesions that exist along the entire intracranial and extracranial course.

Myofibroblasts are activated fibroblasts, involved in tissue repair and cancer, characterized by de novo expression of alpha smooth muscle actin (α-SMA), increased secretion of growth factors and immunoregulatory phenotype [1]. At the end of wound healing myofibroblasts undergo apoptotic cell death, whereas in vitro they are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response [1,2]. Moreover, fibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this investigation, we produced and analysed clusters such as spheroids from human primary cutaneous myofibroblasts to evaluate apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation does not induce apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α-SMA in protein extracts of spheroids, the anti- migratory effect of spheroid-conditioned medium on normal cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts undergo a deactivation process within spheroids. The cells of spheroids, reverted to adhesion growth, preserve their proliferation capability and are able to reacquire a myofibroblastic phenotype. Furthermore, the spontaneous formation of clusters and spheroids on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and indicates that fibroblast clusters could be a cell reservoir regulating tissue turnover., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Introduction Lateral epicondylitis is a common painful elbow disorder. Several approaches to treatment have been proposed, with a local injection of corticosteroids being the most frequently used. Recent insights into the pathophysiology encouraged the introduction of autologous blood injections as an alternative treatment method. The aim of this meta-analysis is to summarize quantitatively the evidence regarding the efficacy of corticosteroids and autologous blood injections for treatment of pain in lateral epicondylitis. Evidence acquisition Studies were considered eligible based on the following inclusion criteria: adult human, diagnosis of lateral epicondylitis, randomized controlled trials comparing corticosteroids versus autologous blood injections, pain assessment. Exclusion criteria were previous surgery for lateral epicondylitis or for other elbow disorders, concurrent treatment with drugs or physiotherapy, diagnosis of musculoskeletal systemic disorder. A systematic search of literature was performed according to the PRISMA statement. Effect size of each included study was calculated and analyzed in a random-effects model. Evidence synthesis Four studies, enrolling total of 218 patients (139 females and 79 males), were included in quantitative analysis. At 2 weeks, there was a trend towards a reduction of VAS score in the corticosteroid group (WMD=2.12 [95% CI: 4.38 to 0.14], P=0.07). No significant differences were recorded in the medium-term (4-12 weeks; WMD=0.85 [95% CI: -0.44 to 2.15], P=0.19) and long-term (24 weeks; WMD=0.63 [95% CI: -2.40 to 3.66], P=0.68) follow-up. Conclusions Few high-quality trials compare the efficacy of corticosteroid and autologous blood injections in the control of pain related to lateral epicondylitis. Available data indicate that corticosteroids tend to reduce VAS score in short-term follow-up, although these data are not statistically significant. No differences were recorded in the medium and long term. Contrary to popular opinion among medical professionals, and despite pathophysiological cues, the currently available data offer no support for the effectiveness of autologous blood injections in medium- and long-term follow-up. Further studies are necessary to establish which treatment has more impact on pain in lateral epicondylitis. These data could be then used as a basis for practical guidelines and new protocols of treatment.

Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts.

Among biomaterials tested so far in cardiac tissue engineering, the decellularized native ECM (d-ECM) appears by far the most promising and appealing[1]. In order to obtain intact scaffold of d-ECM while preserving its composition, we compared the human cardiac d-ECM produced through four different protocols (named Pr1, Pr2, Pr3 and Pr4) in terms of efficiency of decellularization, composition of scaffolds and their suitability for repopulation with cardiac stem cells to develop myocardial patches. As regards efficiency of decellularization, DNA measurement revealed ineffectiveness of Pr1 and Pr3 in producing thoroughly acellular d-ECM, as they yielded scaffolds containing 46,33+5,3 and 96,7+12,2 ng of DNA/mg of tissue, respectively, while DNA content was as low as 9+2,1 in d-ECMs produced with Pr2, which includes DNase treatment, and 17,33+2.22 in the ones obtained with Pr4. Hematoxylin and Eosin staining confirmed the effectiveness of the decellularization procedure for Pr2 and Pr4, while Masson's staining revealed that Pr4 produced a d-ECM with more preserved architecture. Sirius Red, PAS and Gomori stainings documented the retention of collagen, non-collagenous proteins and elastic fibers in d-ECM from all protocols. Immunodetection of fibronectin, tenascin and laminin confirmed the retention of such ECM proteins in d-ECM, while assay for insoluble collagen and sulphated glycosaminoglycan confirmed the presence of collagen and GAGs in all protocols. Mostly due to ineffective decellularization and, as such, to mild treatment, ECM obtained through Pr3 retained more collagen and GAGs. Nevertheless, collagen content was only 1,5-fold higher than in d-ECM obtained with Pr4, which retained the highest amount of GAGs as well. Notably, GAGs protect growth factors against proteolysis, and protein array to investigate the retention of growth factors by d-ECM proved the highest efficiency of Pr4 in retaining growth factors as HGF, VEGF, TGF and IGF-1 in d-ECM. Finally, to assess suitability of d-ECM scaffolds for cell repopulation, we cultured cardiac stem cells (CSCs) on d-ECM to evaluate their viability and differentiation trend. Among all, Pr2 and Pr4 better supported cellular viability, but Pr4 provided the best environment to promote terminal differentiation towards myocardium, endothelium and smooth muscle cell lineages. Interestingly, we previously reported the inability of human cardiac stem cells to acquire fully differentiated state in vitro[2], but d-ECM supports stem cell terminal differentiation prompting progenitor cells to further proceed in differentiation towards precursor and mature phenotype. In conclusion, the composition and architecture of scaffolds of human cardiac d-ECM depend on decellularization protocol. We report here an adaptation of previously described protocol which yields highly preserved d-ECM, in terms of composition and architectures, that might be successfully be employed in regenerative medicine as biological scaffold.

The pulmonary artery autograft (PA) is the ideal substitute for aortic valve disease in children and young adult. However, it is harnessed by the issue of long-term dilation and regurgitation, often requiring surgery. PA implanted in aortic position during the growth phase in children undergoes a process of mechanical remodeling. We previously developed a semiresorbable armored prosthesis able to mechanically sustain the neoaorta preventing dilation and to gradually integrate with the PA wall inducing a progressive arterial-like tissue positive remodeling. We also described the mechanisms of growth, remodeling and stress shielding of the reinforced PA through a mathematical model. We sought to demonstrate the biological counterpart and the potential molecular mechanisms underlying this histological and mechanical remodeling. A specific mathematical model was developed to describe mechanical behavior of the PA. Mallory trichrome red staining and immunohistochemistry for MMP-9 were performed to elucidate extracellular matrix remodeling phenomena. Apoptosis and cell proliferation were determined by TUNEL assay and immunohistochemistry for Ki67, respectively. An histological remodeling phenomenon sustained by increased level of MMP-9, augmented cell proliferation and reduced apoptosis in the reinforced PA was demonstrated. The mathematical model predicted the biomechanical behavior subtended by the histological changes of the PA in these settings. Changes in metalloproteinases (MMP-9), cell proliferation and apoptosis are the main actors in the remodeling process occurring after transposition of the PA into systemic regimens. Use of semiresorbable reinforcements might induce a positive remodeling of the PA in the context of Ross operation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2785-2793, 2016.

Bi-layered scaffolds with a 0°/90° lay-down pattern were prepared by melt-extrusion additive manufacturing (AM) using a poly(ester urethane) (PU) synthesized from poly(ε-caprolactone) diol, 1,4-butandiisocyanate and l -lysine ethyl ester dihydrochloride chain extender. Rheological analysis and differential scanning calorimetry of the starting material showed that compression moulded PU films were in the molten state at a higher temperature than 155°C. The AM processing temperature was set at 155°C after verifying the absence of PU thermal degradation phenomena by isothermal thermogravimetry analysis and rheological characterization performed at 165°C. Scaffolds highly reproduced computer-aided design geometry and showed an elastomeric-like behaviour which is promising for applications in myocardial regeneration. PU scaffolds supported the adhesion and spreading of human cardiac progenitor cells (CPCs), whereas they did not stimulate CPC proliferation after 1–14 days culture time. In the future, scaffold surface functionalization with bioactive peptides/proteins will be performed to specifically guide CPC behaviour.

Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM forin vitrostudies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

Background Nitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca2+ entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca2+ inflow is, however, unknown. Methods In this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca2+-sensitive fluorescent dye, Fura-2/AM. Results We demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca2+. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca2+ inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca2+ response. Conclusions The data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization.

Adult human heart hosts a population of cardiac primitive CD117-positive cells (CPCs), which are responsible for physiological tissue homeostasis and regeneration. While the bona fide stem cells express telomerase, their progenies are no longer able to preserve telomeric DNA; hence the balance between their proliferation and differentiation has to be tightly controlled in order to prevent cellular senescence and apoptosis of CPCs before their maturation can be accomplished. We have examined at cellular and molecular level the proliferation, apoptosis and commitment of CPCs isolated from normal (CPC-N) and age-matched pathological adult human hearts (CPC-P) with ischemic heart disease. In the CPC-P, genes related to early stages of developmental processes, nervous system development and neurogenesis, skeletal development, bone and cartilage development were downregulated, while those involved in mesenchymal cell differentiation and heart development were upregulated, together with the transcriptional activation of TGFβ/BMP signaling pathway. In the pathological heart, asymmetric division was the prevalent type of cardiac stem cell division. The population of CPC-P consisted mainly of progenitors of cardiac cell lineages and less precursors; these cells proliferated more, but were also more susceptible to apoptosis with respect to CPC-N. These results indicate that CPCs fail to reach terminal differentiation and functional competence in pathological conditions. Adverse effects of underlying pathology, which disrupts cardiac tissue structure and composition, and cellular senescence, resulting from cardiac stem cell activation in telomere dysfunctional environment, can be responsible for such outcome.

Endothelial dysfunction or loss is the early event that leads to a host of severe cardiovascular diseases, such as atherosclerosis, hypertension, brain stroke, myocardial infarction, and peripheral artery disease. Ageing is regarded among the most detrimental risk factor for vascular endothelium and predisposes the subject to atheroscleorosis and inflammatory states even in absence of traditional comorbid conditions. Standard treatment to restore blood perfusion through stenotic arteries are surgical or endovascular revascularization. Unfortunately, ageing patients are not the most amenable candidates for such interventions, due to high operative risk or unfavourable vascular involvement. It has recently been suggested that the transplantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) might constitute an alternative and viable therapeutic option for these individuals. Albeit pre-clinical studies demonstrated the feasibility of EPC-based therapy to recapitulate the diseased vasculature of young and healthy animals, clinical studies provided less impressive results in old ischemic human patients. One hurdle associated to this kind of approach is the senescence of autologous EPCs, which are less abundant in peripheral blood and display a reduced pro-angiogenic activity. Conversely, umbilical cord blood (UCB)-derived EPCs are more suitable for cellular therapeutics due to their higher frequency and sensitivity to growth factors, such as vascular endothelial growth factor (VEGF). An increase in intracellular Ca(2+) concentration is central to EPC activation by VEGF. We have recently demonstrated that the Ca(2+) signalling machinery driving the oscillatory Ca(2+) response to this important growth factor is different in UCB-derived EPCs as compared to their peripheral counterparts. In particular, we focussed on the so-called endothelial colony forming cells (ECFCs), which are the only EPC population belonging to the endothelial lineage and able to form capillary-like structures in vitro and stably integrate with host vasculature in vivo. The present review provides a brief description of how exploiting the Ca(2+) toolkit of juvenile EPCs to restore the repairative phenotype of senescent EPCs to enhance their regenerative outcome in therapeutic settings.

Although low-energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non-invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four-month-old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin-I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c-kit–positive, Ki67-positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65-fold more numerous in the treated myocardium. In conclusion, non-invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.

Epithelial-mesenchymal transition (EMT) involves genotypic and phenotypic changes leading to the conversion of polarized epithelial cells into motile mesenchymal cells. This process occurs typically in mesothelial cells of developing cardiac tissue, but recent discoveries indicate the preservation or reactivation of embryonic potential of epicardial cells in the adult heart. Moreover, cells with cardiac stem cell phenotype and properties have been identified among epicardially derived cell (EPDC) population, indicating EMT and EPDCs as yet another source of cells for adult cardiac tissue regeneration in cardiovascular diseases. Understanding how and when EPDCs arise from epicardial cells by EMT in the adult human heart could have a strong impact in clarifying the mechanisms of cardiac tissue self-renewal and regeneration. The chapter includes a protocol for adult human epicardial cell culture and EMT induction leading to a formation of cardiac progenitor cells in vitro.

Scientific views of heart tissue biology in normal and pathological conditions have changed over recent years. This chapter describes the current concept of cardiac tissue homeostasis and summarizes the latest hypotheses and findings regarding cardiac stem/ progenitor cell origin, phenotype, and contribution to myocardial regeneration. It then discusses the possible cardiac stem cell-based methods of myocardial repair applicable in the therapy of heart failure and highlights the latest clinical trials.

The goal of the present study was to develop a three-dimensional (3D) geometrical model based on pre- and post-dissection Digital Imaging and Communication in Medicine (DICOM) images of both transcranial and endonasal skull base approaches. Such model was structured for either teaching surgical anatomy and to evaluate the amount of bone removal over the skull base surface through a 3D digital perspective. Twenty-five human cadaveric heads were dissected at the Laboratory of Surgical NeuroAnatomy (LSNA) of the University of Barcelona (Spain) between 2007 and 2009. Before and after each dissection, a computed tomography-scan (CT-scan) was obtained in order to create a 3D geometrical model of the same approach performed in the dissection laboratory. The model protocol was designed as follows: (1) preoperative CT-scan of the specimens; (2) creation of a computer-generated 3D model of the specimen using specific imaging software for visualization and manipulation of biomedical data; (3) dissection of the specimens; (4) development of a 3D CT-based model of the approach as a result of the overlapping of the DICOM data of the specimens before and after the dissection. The fusion of the pre- and post-dissection 3D models allowed evaluation of the amount of bone removal over the skull base surface. Measurements of the bony landmarks as well as the visual feedback of the drilled bone over the skull base provided by our 3D model gives the opportunity to improve the tailoring of each approach to the different skull base areas.

The search for ideal cell candidate for heart regeneration, as well as for putative cardiac stem cell responsible for cardiac tissue homeostasis, is occupying both basic scientists and clinicians. Growing number of studies and publications indicate epicardium-derived cells as cardiac stem cells. While it is beyond doubt that these cells contribute to normal development of the heart during organogenesis, it remains an open question whether mesothelial epicardial cells can preserve their embryonic potential and if they can undergo epithelial–mesenchymal transition, giving origin to cardiac cell lineages, also in the adult human heart. Recent observations in vitro confirm this hypothesis, but direct evidence from the adult human heart is difficult to obtain. We report the absence of epicardial cells from the surface of adult human hearts with ischemic cardiomyopathy and the accumulation of cells with epithelial and mesenchymal markers in the subepicardium. We argue that these findings may correspond to the activation of the epithelial–mesenchymal transition in the chronic pathological conditions requiring cardiac cell regeneration, followed by epicardial cell pool exhaustion. Hence, observation of the epicardium of patients with cardiovascular disease, although not offering immediate diagnostic advantage, could provide some urging answers concerning the self-renewal potential of the adult heart.

We investigated the effects of human granulocyte macrophage- colony stimulating factor (GM-CSF) on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I) and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix) mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II), and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by “laddering analysis”, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

CD117-positive cells contributing to cardiac cell turnover in normal and pathological conditions have recently been described in adult human heart. Since the precise spatial and temporal expression of extracellular matrix proteins and their receptors is critical for organ formation, we compared the distribution of cardiac primitive CD117-positive cells in the human adult normal and pathological hearts with ischemic cardiomyopathy, with respect to localization and expression of laminin and integrin isoforms. In the pathological hearts, CD117-positive cells were significantly more numerous than in the normal hearts. They were localized mainly in the atria and were up to 38-fold more numerous in the subepicardium than in the myocardium. Compared with normal hearts, most CD117-positive cells in the subepicardium of pathological hearts were α6 integrin-positive. Laminin-1, typical of developing heart, was found predominantly in the subepicardium of adult heart. Immunoblotting revealed its highest expression in the normal atrium and pathological left ventricle. Both laminin isoforms reduced apoptosis and increased proliferation and migration of CD117-positive cells in vitro with respect to control, but the effects of laminin-1 significantly outweighed those of laminin-2. Signaling mediated by α6 integrin was implicated in the migration and protection from apoptosis, as documented by transfection with specific small interfering RNA. These data reveal that the increase in the number of cardiac CD117-positive cells and the expression of laminin-1 are observed in ischemic cardiomyopathy. Subepicardial localization of CD117-positive cells and expression of laminin-1 and α6 integrin subunits may all correspond to the activation of regeneration involving an epithelial-mesenchymal transition recently described in adult heart. Disclosure of potential conflicts of interest is found at the end of this article.

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

Surface properties may affect the clinical outcome of titanium dental implants. The aim of the present study was to investigate the effects of 3 different titanium surfaces—smooth (S), sandblasted (SB), and titanium plasma-sprayed (TPS)—on proliferation, differentiation, and apoptosis of human osteoblast-like cells, SaOS-2. Cell proliferation was significantly (p < 0.05) higher on the S surface, and synthesis of extracellular matrix proteins was more abundant on TPS and SB than on S surfaces. Analysis of integrin receptors showed a higher expression of α2, α5, αVβ3, and ß1 on TPS as compared with SB and S surfaces. An increase in alkaline phosphatase activity was detected only on SB and TPS surfaces. Analysis of cell apoptosis did not demonstrate any significant difference among the 3 different surfaces. The results indicate that titanium surface topography affects proliferation and differentiation of osteoblast-like SaOS-2 cells, suggesting that surface properties might be important for bone response around dental implants in vivo.

In the present study the effects of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on fibroblast growth and activity have been studied. In this regard the AA have evaluated in primary cultures of human gengival normal fibroblasts (PG1 cells): a)-the expression of GM-CSF receptor (GM-CSFR) (alfa unit) on the cell surface; b)-the in vitro effects of different doses of GM-CSF on the GM-CSFR expression and on the proliferation and activity of fibroblasts. PG1 cells have been stimulated in vitro with different concentrations of GM-CSF (10, 50, 80, 100 and 150 ng/ml) using promonocytic cell line U937 as positive control for GM-CSFR expression. GM-CSFR was investigated by flow cytometry, with mouse monoclonal antibody (mAb) against the alfa chain of the human GM-CSFR and fluorescein-conjugated goat antimouse immunoglobulin G (IgG). At high GM-CSF concentration (80 ng/ml) the AA observed: 1)-A marked increase of GM-CSFR expression evaluated as fluorescence intensity (about three fold in respect to the controls); 2)-Maximal increase of PG1 cells proliferation. Moreover immunofluorescence on fibroblasts obtained from culture plates showed increased actin stress fibers and fibronectin production with low stimulation by GM-CSF, while higher concentration of this cytokine determined increased proliferation of cells, but a decreased formation of actine fibers and vinculin plaques. These results demonstrate: 1)-The presence of GM-CSFR on the surface of fibroblasts; 2)-The proliferation and the synthesis activity of these cells (in vitro) are modulated by different concentration of GM-CSF. We hypothesize that GM-CSF until 80 ng/ml can upregulate the expression of the receptor. Therefore, on the basis of previous findings of high serum levels of GM-CSF in course of scleroderma, a disease characterized by fibroblast hyperactivity, a possible role of this cytokine in the pathogenic process of this disease can be hypothesized.

Fifty patients with intracranial meningiomas, verified histologically, are retrospectively analyzed to correlate the peritumoral brain edema with the positivity of estrogen receptors and progesterone receptors. The extent of edema was quantified on CT scan and/or MR. Monoclonal antibodies were used to test the estrogen receptors and the dextran-coated charcoal method was used to test the progesterone receptors. Significant levels of both receptors were found in 41 (82%) specimens. 80% of the cases with positive receptors had brain edema, in contrast to only 2 among 9 cases with negative receptors. Thus, the presence of brain edema has resulted to be correlated to the positivity of sex hormone receptors, although we did not find significant correlation between the amount of receptors and the amount of edema. The suggested mechanisms responsible for the brain edema surrounding intracranial meningiomas are discussed. We can suggest that progesterone may induce the secretion of some substances from meningioma cells, such as prostaglandins and biogenic amines, which may result in vagogenic edema.

Estradiol and progesterone receptors were studied in 14 patients with meningiomas. Estrogen receptors were detected by specific monoclonal antibodies, whereas progesterone receptors were assayed by the dextran-coated charcoal method. In 9 cases the estrogen receptors were also investigated in cultured tumor cells. Positive estrogen and progesterone receptors were found in 86% of patients. The results have been compared with 11 published series of sex steroid assays in meningiomas. The different rate of positive results in most series can be explained by preoperative glucocorticoid therapy. There is no correlation between the estrogen and progesterone receptor activity, the sex and age of the patients, and the location and histological features of the meningioma. The authors suggest that assays of antiestrogen and antiprogestin drugs in cultured cells can indicate whether this estrogen and progesterone receptor activity may be of therapeutic use.

Purpose To evaluate the feasibility, efficacy and safety of strict prone posturing taken for 2 hours after operation in preventing the occurrence of unintentional retinal displacement in elderly patients operated on for retinal detachment (RD). Methods Twenty patients aged 60 or more with diagnosis of macula-off RD were asked to keep a strict face-down posturing for 2 hours after vitrectomy and 20% sulfur hexafluoride tamponade. IOP was measured immediately before and after surgery and after the 2-hour posturing. A questionnaire was administered to each patient to evaluate the rate of discomfort experienced because of the face-down posturing. Unintentional displacement of the retina was assessed by evaluating the presence of retinal vessel printings on fundus autofluorescence images taken 4 weeks after operation. Results The 2-hour posturing was generally well-tolerated. A mild neck pain was the most common reported symptom. Only a few patients experienced moderate breath shortness while posturing and none had to break the posturing because of respiratory problems. Intraocular pressure (IOP) measured before operation (11.7 ± 2.6 mmHg) was significantly different from IOP recorded at the end of surgery (18.9 ± 4.9 mmHg) and from IOP measured 2 hours after surgery (16.8 ± 4.7 mmHg, P