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Fatal Adverse Events (FADEs) are a major public health problem, and some FADEs could be preventable. The aim of the present study is to describe the frequency, the drugs involved and the preventability in the FADEs collected through the MEREAFaPS Study between 2012 and 2018. All cases including the outcome “death” have been examined. We excluded cases with vaccine-related ADEs, overdose or suicide, and ADEs occurred during the hospitalisation. Two trained assessors evaluated all cases fulfilling the inclusion criteria. ADEs’ preventability was evaluated applying the Schumock and Thornton algorithm. During the study period, we observed 429 cases of death, 92 of which were excluded. The remaining 337 cases involved 187 women and 150 men, with a mean age of 79 and of 77years, respectively. For each report, the suspected drugs and concomitant ones were 1.26 and 4.20, respectively. Anticoagulants and antiplatelet agents account for more than 40% of FADE cases and the most frequent reactions are haemorrhages (37.5%). The 25% of the FADEs were preventable. This study confirms that FADEs are still a relevant clinical occurrence, and are often caused by widely used old drugs associated with adverse events. The death of one in four patients was preventable. Further efforts should be done to improve the appropriateness of the therapy, especially in older patients who are treated with anticoagulants.

The aetiology of impaired growth hormone (GH) secretion in Prader-Willi syndrome (PWS) remains controversial due to the common occurrence of obesity. To further clarify whether suboptimal GH secretion in PWS is an artefact of excess weight, we evaluated both GH immunological activity and GH bioactivity after arginine administration in 23 non-obese PWS patients [seven females, aged 6.9 +/- 0.9 years, body mass index (BMI) SDS 0.63 +/- 0.26], in comparison with a control group of 32 healthy subjects, matched for age, gender and BMI (10 females, aged 7.9 +/- 0.3 years, BMI SDS 0.21 +/- 0.20). Serum GH concentration was measured with a time-resolved immunofluorometric assay (IFMA), while GH bioactivity was evaluated by the Nb2 cell bioassay. Serum IGF-I concentrations were measured by double-antibody RIA. GH mean peak after pharmacological stimulation was significantly lower in PWS individuals compared with controls when measured either by IFMA (6.05 +/- 1.23 microg/L vs. 23.7 +/- 1.06 microg/L, p < 0.0001) or by Nb2 (6.87 +/- 0.55 microg/L vs. 12.88 +/- 0.19 microg/L, p < 0.0001). Analysis of integrated GH secretion (AUC) confirmed that the PWS group differed significantly from the control subjects (387.9 +/- 76.1 microg/L/h vs. 1498.1 +/- 56.2 microg/L/h, p < 0.0001); the same result was obtained when the GH rise after arginine administration was expressed as nAUC (278.2 +/- 53.3 microg/L/h vs. 1443.6 +/- 52.5 microg/L/h, p < 0.0001). PWS patients had an IGF-I SDS significantly lower than those found in control subjects (p < 0.0001). Subnormal IGF-I values were present in 19 PWS individuals (82.6%) and two healthy controls (6.2%). These findings are in agreement with the hypothesis that a complex derangement of hypothalamus-pituitary axis occurs in PWS.

Background Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in ‘real-world’ services. Methods/Design The Psychosis early Intervention and Assessment of Needs and Outcome (PIANO) trial is part of a larger research program (Genetics, Endophenotypes and Treatment: Understanding early Psychosis - GET UP) which aims to compare, at 9 months, the effectiveness of a multi-component psychosocial intervention versus treatment as usual (TAU) in a large epidemiologically based cohort of patients with FEP and their family members recruited from all public community mental health centers (CMHCs) located in two entire regions of Italy (Veneto and Emilia Romagna), and in the cities of Florence, Milan and Bolzano. The GET UP PIANO trial has a pragmatic cluster randomized controlled design. The randomized units (clusters) are the CMHCs, and the units of observation are the centers’ patients and their family members. Patients in the experimental group will receive TAU plus: 1) cognitive behavioral therapy sessions, 2) psycho-educational sessions for family members, and 3) case management. Patient enrolment will take place over a 1-year period. Several psychopathological, psychological, functioning, and service use variables will be assessed at baseline and follow-up. The primary outcomes are: 1) change from baseline to follow-up in positive and negative symptoms’ severity and subjective appraisal; 2) relapse occurrences between baseline and follow-up, that is, episodes resulting in admission and/or any case-note records of re-emergence of positive psychotic symptoms. The expected number of recruited patients is about 400, and that of relatives about 300. Owing to the implementation of the intervention at the CMHC level, the blinding of patients, clinicians, and raters is not possible, but every effort will be made to preserve the independency of the raters. We expect that this study will generate evidence on the best treatments for FEP, and will identify barriers that may hinder its feasibility in ‘real-world’ clinical settings, patient/family conditions that may render this intervention ineffective or inappropriate, and clinical, psychological, environmental, and service organization predictors of treatment effectiveness, compliance, and service satisfaction. Trial registration ClinicalTrials.gov Identifier NCT01436331

Human bones are biological examples of functionally graded lattice capable to withstand large in vivo loading and allowing optimal stress distribution. Disruption of bone integrity may require biocompatible implants capable to restore the original bone structure and properties. This study aimed at comparing mechanical properties and biological behavior in vitro of uniform (POR-FIX) and graded (POR-VAR) Cobalt-chrome alloy lattice structures manufactured via Selective Laser Melting. In compression, the POR-VAR equivalent maximum stress was about 2.5 times lower than that of the POR-FIX. According to the DIC analysis, the graded lattice structures showed a stratified deformation associated to unit cells variation. At each timepoint, osteoblast cells were observed to colonize the surface and the first layer of both scaffolds. Cell activity was always significantly higher in the POR-VAR (p< 0.0005). In terms of gene expression, the OPG/RANKL ratio increased significantly over time (p< 0.0005) whereas IL1β and COX2 significantly decreased (7 day vs 1 day; p< 0.0005) in both scaffolds. Both uniform- and graded-porosity scaffolds provided a suitable environment for osteoblasts colonization and proliferation, but graded structures seem to represent a better solution to improve stress distribution between implant and bone of orthopedic implants.

Highly porous composites made up of biodegradable poly-e-caprolactone (PCL) and stoichiometric hydroxyapatite (HA) particles have been developed as substrate for bone-tissue regeneration. The processing technique consists of phase inversion and particulate (salt crystals) leaching. Three different HA contents (13, 20 and 26 vol %) in PCL-based composite were considered in this study. Pore microstructure with fully interconnected network and pore sizes ranging around a few hundred of μm (macroporosity) was obtained as a result of salt particles removal by leaching process. Several microns (microporosity) porosity was also created through phase inversion of polymer solution. Total porosity up to 95% was achieved. Human marrow stromal cells (MSC) were seeded onto porous PCL-based composites for 1–5 weeks and cultured in osteogenic medium. MSC were able to adhere and grow on PCL-based substrates with a plateau at 3–4 weeks. However, the small effect of bioactive signals on the biological response evaluated in MSC cell culture suggests a prior role of topography on the biological response. Importantly, the presence of HA as a bioactive solid signal determines an increase of mechanical properties. On the overall, the results indicated that porous PCL-based composites are potential candidate for bone substitution with beneficial influence on structural characteristics by solid signal addition. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2008

Polycaprolactone (PCL), a semicrystalline linear resorbable aliphatic polyester, is a good candidate as a scaffold for bone tissue engineering, due to its biocompatibility and biodegradability. However, the poor mechanical properties of PCL impair its use as scaffold for hard tissue regeneration, unless mechanical reinforcement is provided. To enhance mechanical properties and promote osteoconductivity, hydroxyapatite (HA) particles were added to the PCL matrix: three PCL-based composites with different volume ratio of HA (13%, 20%, and 32%) were studied. Mechanical properties and structure were analysed, along with biocompatibility and osteoconductivity. The addition of HA particles (in particular in the range of 20% and 32%) led to a significant improvement in mechanical performance (e.g., elastic modulus) of scaffold. Saos-2 cells and osteoblasts from human trabecular bone (hOB) retrieved during total hip replacement surgery were seeded onto 3D PCL samples for 1-4 weeks. Following the assessment of cell viability, proliferation, morphology, and ALP release, HA-loaded PCL was found to improve osteoconduction compared to the PCL alone. The results indicated that PCL represents a potential candidate as an efficient substrate for bone substitution through an accurate balance between structural/ mechanical properties of polymer and biological activities. (c) 2005 Wiley Periodicals, Inc

Platinum ions and nanoparticles applications in the biomedical field include a number of pathologies. In particular, platinum ions are useful in the preparation of antitumoral drugs, whereas Pt nanoparticles (PtNPs) are known to reduce cellular oxidative stress. Herein, we developed composite materials using poly(ethylenimine) functionalized hydroxyapatite (HAPEI) as support for platinum. The presence of polyelectrolyte promotes interaction with platinum ion, which is loaded in greater amount than on pure hydroxyapatite. The dimensions of PtNPs are very small (about 1 nm), but increase up to about 20–30 nm on heat treatment. Loading Pt(II) ion on the apatitic supports provide a material with cytotoxic properties towards osteosarcoma derived cell lines (SaOS2 and MG63). On the contrary, PtNPs loaded apatitic supports display good anti-oxidant properties, in agreement with their quenching activity towards H2O2. Moreover, they promote differentiation of both primary osteoblast and osteoblast cell line, as shown by the increase of the levels of Alkaline Phosphatase and Collagen type 1, and reduce the level of reactive oxygen species with a consequent decrease of cellular oxidative stress.

Background Lombardy was the epicenter in Italy of the first wave of COVID-19 pandemic. To face the contagion growth, from March 8 to May 8 2020, a regional law re-designed the hub-and-spoke system for time-dependent diseases to better allocate resources for COVID-19 patients. Methods We report the reorganization of the major hospital in Lombardy during COVID-19 pandemic, including the rearrangement of its ICU beds to face COVID-19 pandemic and fulfill its role as extended hub for time-dependent diseases while preserving transplant activity. To highlight the impact of the emergently planned hub-and-spoke system, all patients admitted to a COVID-19-free ICU hub for trauma, neurosurgical emergencies and stroke during the two-month period were retrospectively collected and compared to 2019 cohort. Regional data on organ procurement was retrieved. Observed-to-expected (OE) in-ICU mortality ratios were computed to test the impact of the pandemic on patients affected by time-dependent diseases. Results Dynamic changes in ICU resource allocation occurred according to local COVID-19 epidemiology/trends of patients referred for time-dependent diseases. The absolute increase of admissions for trauma, neurosurgical emergencies and stroke was roughly two-fold. Patients referred to the hub were older and characterized by more severe conditions. An increase in crude mortality was observed, though OE ratios for in-ICU mortality were not statistically different when comparing 2020 vs. 2019. An increase in local organ procurement was observed, limiting the debacle of regional transplant activity. Conclusions We described the effects of a regional emergently planned hub-and-spoke system for time-dependent diseases settled in the epicenter of COVID-19 pandemic in Italy.

Multi-functionalization of calcium phosphates to get delivery systems of therapeutic agents is gaining increasing relevance for the development of functional biomaterials aimed to solve problems related to disorders of the muscolo-skeletal system. In this regard, we functionalized Strontium substituted hydroxyapatite (SrHA) with some β-lactam integrin agonists to develop materials with enhanced properties in promoting cell adhesion and activation of intracellular signaling as well as in counteracting abnormal bone resorption. For this purpose, we selected two monocyclic β-lactams on the basis of their activities towards specific integrins on promoting cell adhesion and signalling. The amount of β-lactams loaded on SrHA could be modulated on changing the polarity of the loading solution, from 3.5-24 wt% for compound 1 and from 3.2-8.4 wt% for compound 2. Studies on the release of the β-lactams from the functionalized SrHA in aqueous medium showed an initial burst followed by a steady-release that ensures a small but constant amount of the compounds over time. The new composites were fully characterized. Co-culture of human primary mesenchymal stem cells (hMSC) and human primary osteoclast (OC) demonstrated that the presence of β-lactams on SrHA favors hMSC adhesion and viability, as well as differentiation towards osteoblastic lineage. Moreover, the β-lactams were found to enhance the inhibitory role of Strontium on osteoclast viability and differentiation.

One of the main biomechanical causes for aseptic failure of orthopaedic implants is the stress shielding. This is caused by an uneven load distribution across the bone normally due to a stiff metal prosthesis component, leading to periprosthetic bone resorption and to implant loosening. To reduce the stress shielding and to improve osseointegration, biocompatible porous structures suitable for orthopaedic applications have been developed. Aim of this study was to propose a novel in-vitro model of the mechanical interaction between metal lattice structures and bovine cortical bone in compression. Analysis of the strain distribution between metal structure and bone provides useful information on the potential stress shielding of orthopaedic implants with the same geometry of the porous scaffold. Full density and lattice structures obtained by the repetition of 1.5 mm edge cubic elements via Laser Powder Bed Fusion of CoCrMo powder were characterized for mechanical properties using standard compressive testing. The two porous geometries were characterized by 750 μm and 1000 μm pores resulting in a nominal porosity of 43.5% and 63.2% respectively. Local deformation and strains of metal samples coupled with fresh bovine cortical bone samples were evaluated via Digital Image Correlation analysis up to failure in compression. Visualization and quantification of the local strain gradient across the metal-bone interface was used to assess differences in mechanical behaviour between structures which could be associated to stress-shielding. Overall stiffness and local mechanical properties of lattice and bone were consistent across samples. Full-density metal samples appeared to rigidly transfer the compression force to the bone which was subjected to large deformations (2.2 ± 0.3% at 15 kN). Larger porosity lattice was associated to lower stiffness and compressive modulus, and to a smoother load transfer to the bone. While tested on a limited sample size, the proposed in-vitro model appears robust and repeatable to assess the local mechanical interaction of metal samples suitable for orthopaedic applications with the bone tissue. CoCrMo scaffolds made of 1000 μm pores cubic cells may allow for a smoother load transfer to the bone when used as constitutive material of orthopaedic implants.

Low-intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm(2) LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7d, 14d, 21d). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21d CD73 + /CD90 + : 6%; OCT-3/4 + /NANOG + /SOX2 + : 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation. This article is protected by copyright. All rights reserved.

Background Adverse drug event (ADEs) are a significant cause of emergency department (ED) visits and consequent hospitalization. Preventing ADEs and their related ED visits in outpatients remains a public health safety challenge. In this context, the aims of the present study were to describe the frequency, seriousness and preventability of outpatients' ADE-related ED visits and hospitalizations in the Italian general population, and to identify the presence of potential predictors of ADE-related hospitalization. Methods We performed a nationwide, multicentre, observational, retrospective study based on reports of suspected ADEs collected between January 1, 2007 and December 31, 2018 in 94 EDs involved in the MEREAFaPS project. Patients' demographic characteristics, their clinical status, suspected and concomitant drugs, ADE description, and its degree of seriousness, were collected. Causality and preventability were assessed using validated algorithms, and logistic regression analyses were used to estimate the reporting odds ratios (RORs) with 95% confidence intervals (CIs) of ADE-related hospitalization, considering the following covariates: age, sex, ethnicity, number of implicated medications, parenteral administration, presence of interaction, therapeutic error, and/or complementary and alternative medicines (CAM). Results Within 12 years, 61,855 reports of suspected ADE were collected, of which 18,918 (30.6%) resulted in hospitalization (ADE defined as serious). Patients were mostly female (56.6%) and Caucasians (87.7%), with a mean age of 57.5 ± 25.0 years. 58% of patients were treated with more than two drugs, and 47% of ADEs leading to hospitalization were preventable. Anticoagulants, antibiotics, and nonsteroidal anti-inflammatory drugs (NSAIDs) were the most frequently implicated agents for ED visits and/or hospitalization, which included clinically significant ADEs, such as haemorrhage for anticoagulants, moderate to severe allergic reactions for antibiotics, and dermatologic reactions and gastrointestinal disturbances for NSAIDs. Older age (1.54 [1.48-1.60]), higher number of concomitantly taken drugs (2.22 [2.14-2.31]), the presence of drug-drug interactions (1.52 [1.28-1.81]), and therapeutic error (1.54 [1.34-1.78]), were significantly associated with an increased risk of hospitalization. Conclusion Our long-term active pharmacovigilance study in ED provided a valid estimation of ADE-related hospitalization in a representative sample of the Italian general population and can suggest further focus on medication safety in outpatients, in order to early recognise and prevent ADEs.

Rheumatoid Arthritis (RA) is the most common chronic inflammatory autoimmune disease involving joints. Among several pathogenic mechanisms, the impairment of homeostatic regulators of inflammation seems to be critically important to sustain persistent infiltration and activation of immune and stromal cells within the diseased synovium. Tyrosine kinase receptors Tyro3, Axl, and Mer are members of the TAM family. Upon binding their ligands Growth Arrest-Specific gene 6 (Gas6) and Protein S (ProS1), TAM receptors (TAMs) exert numerous and diverse biologic functions. Activated Axl and Mer, for instance, can negatively regulate the inflammatory cascade and mediate phagocytosis of apoptotic cells, contributing to prevent the development of autoimmunity. Thus, a role for TAMs has been hypothesized in RA. In this review, we will summarise unmet clinical needs in RA, depict the biology of TAMs and TAM ligands, focussing on their ability to regulate the immune system and inflammation cascade, and finally offer an overview of the state-of-the-art literature about the putative role of TAM axis in RA.

The choice of the appropriate material having suitable compositional and morphological surface characteristics, is a crucial step in the development of orthopedic implants. The purpose of this paper is to elucidate, on this regard, the influence of two important hits, i.e., biogenic apatite with bone-like composition and nanostructured morphology, providing the evidence of the efficacy of nanostructured biogenic apatite coatings in favoring adhesion, growth, proliferation, and in vitro osteogenic differentiation of human mesenchymal stromal cells (hMSCs) isolated from the bone marrow. The specific features of this coating in terms of topographical and biochemical cues, obtained by Ionized Jet Deposition, are perceived by hMSCs, as suggested by changes in different morphologic parameters as Aspect Ratio or Elongation index, suggesting the impact exerted by the nanostructure on early adhesion events, cytoskeleton organization, and cells fate. In addition, the nanostructured CaP coating sustained the metabolic activity of the cells and facilitated the osteogenic differentiation of MSC by supporting the osteogenesis-related gene expression. These findings support the use of a combined approach between technological advancement and instructive surfaces, both from the topographical and the biochemical point of view, in order to manufacture smart biomaterials able to respond to different needs of the orthopedic practice.

ObjectivesMast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). However, their contribution remains controversial. To establish their role in RA, we analysed their presence in the synovium of treatment-naïve patients with early RA and their association and functional relationship with histological features of synovitis.MethodsSynovial tissue was obtained by ultrasound-guided biopsy from treatment-naïve patients with early RA (n=99). Immune cells (CD3/CD20/CD138/CD68) and their relationship with CD117+MCs in synovial tissue were analysed by immunohistochemistry (IHC) and immunofluorescence (IF). The functional involvement of MCs in ectopic lymphoid structures (ELS) was investigated in vitro, by coculturing MCs with naïve B cells and anticitrullinated protein antibodies (ACPA)-producing B cell clones, and in vivo in interleukin-27 receptor alpha (IL27ra)-deficient and control mice during antigen-induced arthritis (AIA).ResultsHigh synovial MC counts are associated with local and systemic inflammation, autoantibody positivity and high disease activity. IHC/IF showed that MCs reside at the outer border of lymphoid aggregates. Furthermore, human MCs promote the activation and differentiation of naïve B cells and induce the production of ACPA, mainly via contact-dependent interactions. In AIA, synovial MC numbers increase in IL27ra deficient mice, in association with ELS and worse disease activity.ConclusionsSynovial MCs identify early RA patients with a severe clinical form of synovitis characterised by the presence of ELS.

Objective Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKα and IKKβ are essential kinases that activate NF-κB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy. Methods Expression of IKKα or IKKβ was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed. Results The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKα or IKKβ. Silencing of IKKβ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKα dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKα-deficient cells retained the phenotype of cells in a pre–hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses. Conclusion IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKα were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKα activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenoctyes. Twenty-four male Sprague-Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1β, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1β was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.Abstract The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenoctyes. Twenty-four male Sprague-Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1β, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1β was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.

Osteoporosis due to estrogen deficiency is an increasing bone health issue worldwide: new strategies are being studied for regenerative medicine of bone pathologies in these patients. The most commonly used cells for tissue engineering therapy are the bone marrow mesenchymal stem cells (BMSCs), but they might be negatively affected by aging and estrogen deficiency. Besides the general advantages of adipose-derived mesenchymal stem cells (ADSCs) over BMSCs, ADSCs also seem to be less affected by aging than BMSCs, but in the literature, little is known about ADSCs in estrogen deficiency. The present study investigated the in vitro behavior of ADSCs, isolated from healthy (SHAM) and estrogen-deficient (OVX) rats. Phenotype, clonogenicity, viability, and osteogenic differentiation, at both cellular and molecular levels, were evaluated with or without osteogenic stimuli. Pro-inflammatory cytokines, growth factors, and adipogenic differentiation markers were also analyzed. There were no significant differences between OVX and SHAM ADSCs in some analyzed parameters. In addition, clonogenicity, osteopontin (Spp1) gene expression, alkaline phosphatase (ALP) activity at 2 weeks of culture, total collagen (COLL), osteocalcin (Bglap) gene expression and production, and matrix mineralization were significantly higher in OVX than in SHAM ADSCs. Besides the increase in some osteogenic markers, peroxisome proliferator-activated receptor gamma (Pparg) gene was also more expressed in OVX in osteogenic medium, with a concomitant estrogen receptor 1 (Esr1) gene expression decrease. These results underlined that ADSCs were not affected by estrogen deficiency in an osteogenic microenvironment.

Background Although progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures. Methods Normal human articular chondrocytes (NHAC-kn) and human mesenchymal stromal cells (hMSC) from healthy donors were seeded in polystyrene wells as controls (CTR), and on decellularized human dermis batches (HDM_derm) for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure. Results Both cell cultures had a similar proliferation rate that increased significantly (p < 0.0005) at 14 days. In comparison with CTR, at 14 days NHAC-kn enhanced procollagen type II (CPII, p < 0.05) and aggrecan synthesis (p < 0.0005), whereas hMSC significantly enhanced aggrecan synthesis (p < 0.0005) and transforming growth factor-beta1 release (TGF-β1, p < 0.0005) at both experimental times. Neither inflammatory stimulus nor catabolic activity induction was observed. By comparing data of the two primary cells, NHAC-kn synthesized significantly more CPII than did hMSC at both experimental times (p < 0.005), whereas hMSC synthesized more aggrecan at 7 days (p < 0.005) and TGF-β1 at both experimental times than did NHAC-kn (p < 0.005). Conclusions The results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

Hyaline cartilage lesions represent an important global health problem. Several approaches have been developed in the last decades to resolve this disability cause, including tissue engineering, but to date, there is not a definitive procedure that is able to promote a repair tissue with the same mechanical and functional characteristics of native cartilage, and to obtain its integration in the subchondral bone. The need of resolutive technologies to obtain a "more effective" tissue substitutes has led Butler to propose the "Functional Tissue Engineering" (FTE) paradigm, whose principles are outlined in a so-called FTE road map. It consists of a two-phase strategy: in vitro tissue engineering and clinically surgery evaluation. The first phase, based on construct development, should take into account not only the chondrocyte biology, as their sensitivity to biochemical and physical stimuli, the risk of dedifferentiation in culture, and the ability to produce extracellular matrix, but also the features of suitable scaffolds. The in vivo phase analyzes the inflammatory microenvironment where the construct will be placed, because the cytokines released by synoviocytes and chondrocytes could affect the construct integrity, and, in particular, cause matrix degradation. The use of pulsed electromagnetic fields (PEMFs) represents an innovative therapeutic approach, because it is demonstrated that this physical stimulus increases the anabolic activity of chondrocytes and cartilage explants with consequent increase of matrix synthesis, but, at the same time, PEMFs limit the catabolic effects of inflammatory cytokines, reducing the construct degradation inside the surgical microenvironment. PEMFs mediate an up-regulation of A2A adenosine receptors and a potentiation of their anti-inflammatory effects.

Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

OBJECTIVE: To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. METHODS: MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. RESULTS: Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. CONCLUSION: MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

Silicate-based filling materials were designed to obtain new endodontic sealers and root-end filling materials with adequate workability and consistency. Four different formulations (TC, TC 1%, TCf 1%, and TCf) were prepared incorporating calcium chloride as accelerant agent. A plasticizing compound (phyllosilicate) was added to TC 1% and TCf 1%. TC and TC 1% were prepared with water, whereas TCf and TCf 1% were mixed with a latex polymer as fluidizing agent. The aim of this study was to assess the in vitro biological compatibility of designed materials. White-MTA and AH Plus were tested as reference materials. Human osteoblast-like Saos-2 cells were challenged in short-term cultures (72 h) with solid materials and with material extracts in culture medium, and cell viability and number, cellular adhesion, and morphology were assessed. The new cements exerted no acute toxicity in the assay systems. Saos-2 like cells adhered and proliferated on solid samples of the experimental cements and MTA whilst AH Plus did not allowed cell growth. The extracts from the latex-containing cements showed some toxicity. By SEM analysis, osteoblast-like cells appeared adherent and spread on the new materials, and showed the maintenance of polygonal osteoblastic phenotype. Similar morphology was observed for cells on MTA, whereas only few cells were noted on the AH Plus surface. In conclusion, the new materials proved non toxic and supported the growth of bone-like cells, and resulted suitable to be used as endodontic sealers and root-end filling materials.

This study was aimed to investigate whether the activation of poly-(epsilon-caprolactone) (PCL) surface by low-energy irradiation and/or the biofunctionalization by absorption of arginine-glycine-aspartic sequences (RGD), can modify the expression of integrins closely related to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization, the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5 x 10(15)He(+)/cm(2) (10 keV energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover, irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was observed only on irr-PCL (surface coverage: 7.0 pmol/cm(2)). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed alpha1, alpha2, alpha3, alpha5, beta1, and alphaVbeta3; after 24h, cells cultured on tissue-plastic expressed high levels of beta1 and alphaVbeta3, while alpha1 showed a low intensity and alpha2, alpha3, and alpha5 were negative. beta1 and alphaVbeta3 were selected to evaluate the interaction between cells and PCL samples. The beta1 expression was higher in hOB cultured on irr-PCL than on the other samples. A significant increase in alphaVbeta3 expression was observed only in irr-PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to improve the osteoconductivity of selected materials.

Background: Platelet alpha granules contain growth factors released into the surrounding environment during activation. This property has been used in clinical medicine to accelerate the repair process by activating in vitro autologous platelets with thrombin and has also been proposed to promote the proliferation of bone cells. The aim of this research was to assess the effect of platelet concentrates activated with thrombin on human gingival fibroblasts and human osteoblasts from trabecular bone. Methods: Platelet concentrates, activated with bovine thrombin, were added to the cells in serum-free medium. The cultures were assessed for proliferation by vital stain and cell count after 72-hour incubation. Alkaline phosphatase activity was tested after 72-hour incubation on the osteoblast lysates by a colorimetric assay. After 21 days the formation of mineral nodules was tested in the osteoblast cultures by alizarin red staining. The effects of the activated platelet concentrates (APC) were compared with the serum-free medium (SF), or with platelet-poor plasma added medium (PPP). Results: The fibroblast growth in the presence of APC was higher, though not significantly, than SF. APC resulted in a nonsignificant decrease in proliferation and alkaline phosphatase expression in osteoblasts, compared both to serum free medium, and PPP. Mineralization was only modestly increased after incubation with APC in comparison with serum-free medium. Conclusions: There were no statistical differences in fibroblast proliferation, or in osteoblast growth and functions between serum-free conditions and the platelet gel treatment. Therefore, neither fibroblast proliferation nor osteoblast growth and functions were affected by the activated platelet concentrates in vitro. J Periodontol 2005;76:323-328.

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al(2)O(3)) or 'ultra-high molecular weight polyethylene' (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 microg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al(2)O(3) HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al(2)O(3) wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.

Objective Studies of memory T cells transferred with the graft are relevant to better understand the early immune reconstitution of patients given autologous bone marrow transplantation (A-BMT). A critical question is whether memory T cells resident in bone marrow (BM) of patients with hematological malignancies are resistant to either pretransplant chemotherapy or ex vivo pharmacological purging. Patients and methods To address these issues, we evaluated the frequency of tetanus-toxoid (TT)-specific proliferating T-cell precursors (TT-PTCp) in BM and peripheral blood (PB) of eight patients with acute myeloid leukemia (AML) given A-BMT after in vitro purging of BM with mafosfamide. Patients were studied at the time of BM harvesting and five of them also after A-BMT. Results The range of TT-PTCp frequencies found after A-BMT were comparable with those observed in PB and in BM at the time of harvesting and did not differ significantly from those of eight age-matched healthy subjects who donated BM for a human leukocyte antigen-identical sibling. TT-PTCp frequencies in BM, studied before and after ex vivo purging, appeared not to be affected by incubation with mafosfamide. We also compared the T-cell receptor (TCR)-Vβ-repertoire usage of TT-specific T-cell lines (TT-TCL) in BM of patients at the time of harvesting and in their PB 2 months after transplantation. The same TCR-clonotypes were detected in TT-TCL at time of harvesting and after A-BMT. Conclusion These data indicate that BM-resident memory T cells of patients with AML are resistant to both pretransplant chemotherapy and ex vivo pharmacological purging and may contribute to immune reconstitution after A-BMT.

Successful ex-vivo priming and long-term maintenance of anti-tumor cytotoxic T-cell (CTL) lines are preliminary conditions for their use in approaches of adoptive immunotherapy for patients with cancer. We describe the results of a novel procedure for generating in vitro anti-tumor CTL using CD8-enriched peripheral blood mononuclear cells (PBMC) and dendritic cells (DC), pulsed with irradiated tumor cells (TC) as source of tumor antigen. Eight patients were enrolled in our study: 4 sarcoma, 2 renal cell carcinoma, 1 ovarian carcinoma and 1 breast carcinoma. Ten anti-tumor CTL-lines cytotoxic towards patient TC were generated. Five CTL-lines were obtained using both DC and PBMC from the patients (autologous setting). For 5 CTL-lines, DC derived from an HLA-identical sibling were employed (allogeneic setting): patients or siblings PBMC were used to generate CTL-lines in 2 and 3 cases, respectively,. After tumor-specific rounds of stimulation, followed by antigen-independent cycle of expansion, CTL-lines obtained in both autologous and allogeneic setting showed an expansion of the absolute number of cultured cells. In 6 of 10 CTL-lines, the majority of effector cells (>70%) were CD3+/CD8+, while in the remaining 4, 40-70% of effector cells were CD3+/CD4+. Both CD8+ and CD4+ T cells displayed anti-tumor cytotoxic activity. Spectratyping analysis of the TCR-Vbeta subfamilies revealed a preferential expansion of oligoclonal populations in 18 of 24Vbeta subfamily. Altogether these results demonstrate that our experimental approach is suitable for efficiently generating and expanding anti-solid tumor CTL to be used for adoptive immunotherapy. Copyright 2004 Wiley-Liss, Inc.

In the present study, the effect of mechanically alloyed particles of new FeAlCr alloys developed for potential applications as surgical implants has been tested on osteoblast- and macrophage-like cells and compared to particles of the Ti6Al4V alloy, for which there is a good clinical experience. After microstructural characterisation of the particles, cells were cultured with particles for 24-48h using three different concentrations of particles, and the response of cells was quantified by assessment of viability, proliferation, and morphology. Mineralisation by osteoblasts was verified after 21 days. The amount of aluminium and chromium ions in the culture medium of macrophages was measured by graphite furnace atomic absorption and phagocytosis of particles assessed by light microscopy. Viability and proliferation of osteoblast- and macrophage-like cells were substantially unaffected by the presence of particles of the new alloys, which were phagocytosed according to their size. Aluminium and chromium ions were released in the culture medium, but no direct correlation with the cell behaviour was found. In vitro mineralisation was achieved by osteoblasts in due time. The new alloys are well tolerated in in vitro systems, and, due to their chemical and mechanical characteristics, they are under development for surgical implants. © 2004 Elsevier Ltd. All rights reserved.

Particle-induced macrophage activation, mainly by UHMWPE wear, has been recognized as the biological mechanism leading to periprosthetic bone resorption, which is responsible for the loosening of the total hip replacements (THR). Ceramic-on-ceramic implants have been advocated as a means of reducing wear products. Many studies investigated the effect of alumina (Al(2)O(3)) particles on monocytes/macrophages, but only limited information are available on their participation to bone turnover. An in vitro model was performed to investigate how Al(2)O(3) and UHMWPE particles may influence the osteoblast-osteoclast interaction: human osteoblasts (HOB) were obtained from trabecular bone, while osteoclasts were derived from peripheral blood mononuclear cells (PBMC) of healthy donors. The amount of IL6, TNF alpha, GM-CSF, and other factors acting on the bone turnover, i.e. the 'receptor activator of NF kappa B' ligand (RANKL) and osteoprotegerin (OPG), was detected in culture medium of particle-challenged HOB (HOB-CM). The Al(2)O(3) and UHMWPE particles did not affect either cell viability or TNF and GM-CSF release, while the increase in IL6 release seemed to be dependent on the particle concentration. UHMWPE increased the release of RANKL from HOB, while OPG and OPG-to-RANKL ratio were significantly inhibited. The ability of HOB-CM to promote osteoclastogenesis was tested via osteoblast/monocyte cooperation: after seven days of culture UHMWPE HOB-CM induced a large amount of multinucleated TRAP-positive giant cells, as well as significantly reduced the amount of IL6, GM-CSF and RANKL in the supernatant. With regard to the inductive effect on the osteoclastogenesis, our results show that the Al(2)O(3) wear debris are less active.