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In the field of bone tissue engineering, particular interest is devoted to the development of 3D cultures to study bone cell proliferation under conditions similar to in vivo ones, e.g. by artificially producing mechanical stresses promoting a biological response (mechanotransduction). Of particular relevance in this context are the effects generated by the flow shear stress, which governs the nutrients delivery rate to the growing cells and which can be controlled in perfusion reactors. However, the introduction of 3D scaffolds complicates the direct measurement of the generated shear stress on the adhered cells inside the matrix, thus jeopardizing the potential of using multi-dimensional matrices. In this study, an anisotropic hydroxyapatite-based set of scaffolds is considered as a 3D biomimetic support for bone cells deposition and growth. Measurements of sample-specific flow resistance are carried out using a perfusion system, accompanied by a visual characterization of the material structure. From the obtained results, a subset of three samples is reproduced using 3D-Computational Fluid Dynamics (CFD) techniques and the models are validated by virtually replicating the flow resistance measurement. Once a good agreement is found, the analysis of flow-induced shear stress on the inner B-HA structure is carried out based on simulation results. Finally, a statistical analysis leads to a simplified expression to correlate the flow resistance with the entity and extensions of wall shear stress inside the scaffold. The study applies CFD to overcome the limitations of experiments, allowing for an advancement in multi-dimensional cell cultures by elucidating the flow conditions in 3D reactors.

Bone substitute biomaterials (BSBs) represent a promising alternative to bone autografts, due to their biocompatibility, osteoconduction, slow resorption rates, and the ability to define and maintain volume for bone gain in dentistry. Many biomaterials are tailored to provide structural and biological support for bone regeneration, and allow the migration of bone-forming cells into the bone defect. Neural crest-derived stem cells isolated from human dental pulp (hDPSCs) represent a suitable stem cell source to study the biological effects of BSBs on osteoprogenitor cells involved in the physiological bone regenerative processes. This study aimed to evaluate how three different BSBs affect the stem cell properties, osteogenic differentiation, and inflammatory properties of hDPSCs. Our data highlight that BSBs do not alter cell proliferation and stemness markers expression, nor induce any inflammatory responses. Bone metabolism data show that hDPSCs exposed to the three BSBs distinctively secrete the factors supporting osteoblast activity and osteoclast activity. Our data indicate that (i) hDPSCs are a suitable stem cell source to study the effects of BSBs, and that (ii) the formulation of BSBs may condition the biological properties of stem cells, suggesting their versatile suitability to different dentistry applications.

Endometriosis is a gynecological disease characterized by pain and infertility. The diagnosis is very often made during the infertility work-up, together with other reproductive diseases and uterine disorders. A retrospective cohort study was conducted on infertile women with clinical or ultrasound suspect of endometriosis, undergoing an ultrasound (US) evaluation by a team of expert sonographers (

This study investigates the biological effects on a 3D scaffold based on hydroxyapatite cultured with MC3T3 osteoblasts in response to flow-induced shear stress (FSS). The scaffold adopted here (B-HA) derives from the biomorphic transformation of natural wood and its peculiar channel geometry mimics the porous structure of the bone. From the point of view of fluid dynamics, B-HA can be considered a network of micro-channels, intrinsically offering the advantages of a microfluidic system. This work, for the first time, offers a description of the fluid dynamic properties of the B-HA scaffold, which are strongly connected to its morphology. These features are necessary to determine the FSS ranges to be applied during in vitro studies to get physiologically relevant conditions. The selected ranges of FSS promoted the elongation of the attached cells along the flow direction and early osteogenic cell differentiation. These data confirmed the ability of B-HA to promote the differentiation process along osteogenic lineage. Hence, such a bioactive and naturally derived scaffold can be considered as a promising tool for bone regeneration applications.

Background: The last two decades have seen a growing number of pregnancies in women who needed the donation of oocytes. With oocyte donation pregnancies, studies on obstetric outcomes among these w...

The excellent biocompatibility, controllable biodegradability, non-immunogenicity and intrinsic biological activity, make silk sericin an ideal candidate for the formulation of drug delivery systems. Unfortunately, nanoparticles, nanocapsules or nanofibers based on silk sericin “alone” cannot be produced due to its physicochemical instability, influenced by the high water solubility. For this reason, cross-linking, blending, or combination with other polymers/compounds is required. This chapter provides an overview of the main preparation methods of sericin-based nanoparticles and nanofibers, as reported in the literature. Their applications, both in vitro and in vivo, are also described.

Mesenchymal stem/stromal cells (MSCs) are a therapeutic target to promote tissue regeneration, mainly when oxidative stress-mediated damage is involved in disease pathogenesis. Here, slow-release silk sericin nanoparticles (SNPs) loaded with natural antioxidant polyphenols were developed to sustain regeneration by tissue-resident MSCs. SNPs were prepared by exploiting a self-assembly method with poloxamer and were loaded with proanthocyanidins (P), quercetin (Q) or epigallocatechin gallate (E). SNPs, with a diameter less than 150 nm, were able to encapsulate both hydrophilic (P and E) and hydrophobic (Q) drugs. A slow and controlled release was obtained from SNPs for all the actives in PBS, while in EtOH, Q and E showed a burst release but P did not. Kinetic models revealed lower diffusion of P than other biomolecules, probably due to the higher steric hindrance of P. The in vitro anti-oxidant, anti-elastase and anti-tyrosinase properties of SNPs were assessed: loading the P and E into SNPs preserved the in vitro biological activities whereas for Q, the anti-elastase activity was strongly improved. Moreover, all formulations promoted MSC metabolic activity over 72 h. Finally, SNPs exhibited a strong ability to protect MSCs from oxidative stress, which supports their potential use for regenerative purposes mediated by tissue-resident MSCs.

Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnological applications, as anticancer drug, anticoagulant, cell culture additive, wound healing agent and drug delivery carrier. Reasons for SS use in biomedical applications derive from its physical-chemical composition. As a consequence, a detailed characterization of SS in terms of average molecular weight, molecular weight distribution and hydro/lipophilic character is crucial to properly address and assess its quality, cosmetic or pharmacological use. In this study, the application of different and complementary chromatographic modes allows a detailed investigation of SS protein isolated from wastewater using two diverse extraction methods. Hydrophilic interaction liquid chromatography (HILIC using an AdvanceBio Glycan Map column) and reverse phase (RP using Symmetry300 C18 column) were applied to intact protein characterization to derive data on protein hydrophilicity and on hydrophobic components of the two SS preparations (SS#1 and SS#2). A higher hydrophilic character of SS#1 was observed by HILIC trace, coherently with the preparation method used, while no significant differences in hydrophobicity were detectable in the RPLC separations. Size distribution was also defined by using a SEC-UV-MS method (using TSKgel SuperSW2000 column) properly optimized to maximize both the size selectivity and the method sensitivity. Taken together, the chromatographic data allowed to better characterize the SS samples obtained by different extraction methods, and the structural properties were correlated to their biological activities.

Background: The introduction of the immunomodulatory drug lenalidomide has revolutionized the treatment of patients with myelodysplastic syndromes (MDS) and deletion of the long arm of chromosome 5. Treatment with lenalidomide results in transfusion independence in the majority of patients, but some questions remain unresolved, among them the duration of treatment. Moreover, a number of unexpected long-term remissions in patients who stopped lenalidomide for various reasons have been observed. Case Report: We report the case of a 60-year-old Caucasian male with deletion of the long arm of chromosome 5 and International Prognostic Scoring System (IPSS)-defined low-risk MDS who was treated with lenalidomide, achieving complete cytogenetic remission and erythroid response. After tapering off and interrupting the treatment, the patient relapsed and showed a new response by lenalidomide retreatment. Six years after the initial treatment, we registered a durable erythroid long-term response and good tolerance, but there was no evidence of a very profound cytogenetic response compared to using lenalidomide as a first-line treatment. Cytogenetic and fluorescence in situ hybridization together with hemoglobin level, mean corpuscular volume (MCV) and vitamin B12 level helped us to monitor the patient response; during the various phases of lenalidomide treatment, MCV and vitamin B12 normalization correlated with good response. Conclusion: Lenalidomide interruption and rechallenge in some 5q- MDS patients, with low risk according to the IPSS, is safe and feasible but does not result in a profound cytogenetic response.

Introduction : Every governmental health institution in Italy implements its strategic annual plan though a balance score card system (BSC). The ASL (Azienda Sanitaria Locale) of Viterbo that coordinates the entire Viterbo province health system, including several hospitals, primary care districts, home care, veterinary activity, hygiene, vaccination, prevention and food and water control, is using a BSC tool to implement its strategic plan. The BSCs with specific targets and objectives are set at the beginning of the year. They are discussed and negotiated with each director of the 102 Operative Units (O.U.) within the ASL of Viterbo. Each director of the O.U after signing the commitment contract will try to reach or realize the single targets and objective. Most of them up today are used to work in an individual way and they want to be judged at the end of the year as “single” players instead on how they are performing as “team” players. Objective : The aim of our study was to plan an integrated care system using the existing BSC system and monitor the integration level between the shared objectives and O.U. through a social network analysis to validate and estimate the integrated care level within the ASL system before signing the BSC. The final goal was indeed the improvement of healthcare performance at regional and national level. Methods : Instead giving “single” player objectives to the directors of the O.U. we set shared goals that will require a “team play” integrated approach to reach the final goal. The same objectives were indeed given to all the O.U. involved in the process. We focus the measure on the results of a good team work between the players. We extracted data and goals from governmental national and regional performance evaluation system for regional healthcare system. Subsequently we choose critical areas of intervention where integrated care was highly needed through a need assessment analysis. The same goals were sets to different O.U. with the recommendation to collaborate with the other linked O.U. in the process. To obtain a big picture of integration of care between the different O.U. before releasing the final BSCs we used the Social network analysis (SNA). The SNA was set as a bimodal network between O.U. and objectives, that were linking the O.U. together. We used the “island method” and the statistical metrics to evaluate the integration and the clustering effect. Results : The clustering analysis of the SNA permitted us to understand the real functional teams that were different form the formal organizational structure. The cluster indeed reflect a functional structure that was invisible before. This allowed to redistribute the resources between the O.U. in a different way using the functional clusters. The SNA statistical metrics permitted to understand which O.U. were the crossroads between functional clusters using the betweenness centrality but also which O.U. had a more controlling activity of the process using the Eigenvector centrality and the closeness centrality. Conclusions : The SNA was an effective support tool to draw an effective integrated care plan but it was also useful to plan a strategic deployment of objectives and resources. The shared goals on the balance score forced the directors to meet and prepare integrated strategic plans to respond to the issue. The final integrated care effects were visible through the improvements on several regional and national indicators.

Background The achievement of complete response (CR) significantly correlates with a better clinical outcome in multiple myeloma (MM) patients treated with autologous stem cell transplant (ASCT). The depth of response is one of the most relevant factors to predict patient’s outcome, however the definition of CR through standard criteria has shown several limitations. Methods In this study we evaluated the minimal residual disease (MRD) in 50 consecutive MM patients who underwent an up-front tandem ASCT in our center, using a single-tube six-colors flow cytometry assay (FC) based on intra-cytoplasmic immunoglobulin (cy-Ig) light chains ratio evaluated on patient-specific plasma cells (PC) immune profile, in a real-life setting. Results With a sensitivity up to 10−5, clonal-PC were documented by FC in 36.4 % (12/33) of patients in conventional CR after second transplant. The number of flow MRD-negative patients significantly increased after induction and first ASCT, but not between first and second transplant. The 5-years progression-free survival (5ys-PFS) of flow MRD-negative patients after second transplant was significantly better than patients who remained MRD-positive considering both all patients (5ys-PFS: 70 % vs 5 %) and patients in CR according to standard criteria (5ys-PFS: 67 % vs 0 %). Conclusions FC remission through cy-Ig light ratio on PC sub-populations is a sensitive, highly informative, low-cost and routinely applicable MRD assay, a powerful tool in treatment response evaluation and a crucial marker of outcome in MM.

We aimed at developing a more detailed understanding of cyclin D1 in early stage human breast cancer and defining the biologic profiles with different prognostic value correlating cyclin D1 gene amplification and chromosome 11 aneusomy with bio-pathologic variables of known clinical importance. Cyclin D1 gene amplification and chromosome 11 aneusomy were investigated using fluorescence in situ hybridization whereas cyclin D1, PgR, HER-2, Bcl2, p53, and Ki-67 expressions were analyzed by immunohistochemistry in 121 stage I or II breast cancer patients uniformly treated with cyclophosphamide/metotrexate/5-fluorouracil-based chemotherapy. Cyclin D1 was amplified in 6.6% and overexpressed in 32.2% of cases. Amplification was not associated with any selected bio-pathologic variables, whereas the chromosome 11 aneusomy level significantly increased in tumors with higher histologic grade (P0.01), higher tumor size (P0.003), p53 nuclear accumulation (P0.04), and ERalpha negativity (P0.049). Multiple correspondence analysis showed 4 different biologic tumor profiles. The first, characterized by high Ki-67 score, p53+, cyclin D1+, HER-2+, aneusomy level30%, ratio (cyclin D1 gene/CEP11)2, was associated with tumor relapse defining the most unfavorable biologic profile. Kaplan-Meier's method showed significantly shorter disease-free survival in patients with at least 3 variables positive out of the 6 detected by multiple correspondence analysis. In multivariate analysis, the identified biologic profile emerged as the only significant prognostic indicator. Our findings are of particular clinical interest for early stage breast cancer patients, because the assessment of biologic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.

Background Prostate cancer (PCa) is the most common male cancer in Europe and the US. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits. Among the new tests currently under study, one of the most promising is the prostate cancer gene 3 (PCA3), a non-coding mRNA whose level increases up to 100 times in PCa tissues when compared to normal tissues. With the present study we contribute to the validation of the clinical utility of the PCA3 test and to the evaluation of its prognostic potential. Methods 407 Italian men, with two or more PCa risk factors and at least a previous negative biopsy, entering the Urology Unit of Regina Elena National Cancer Institute, were tested for PCA3, total PSA (tPSA) and free PSA (fPSA and f/tPSA) tests. Out of the 407 men enrolled, 195 were positive for PCa and 114 of them received an accurate staging with evaluation of the Gleason score (Gs). Then, the PCA3 score was correlated to biopsy outcome, and the diagnostic and prognostic utility were evaluated. Results Out of the 407 biopsies performed after the PCA3 test, 195 (48%) resulted positive for PCa; the PCA3 score was significantly higher in this population (p

Background Renal tumor subtypes are associated with distinct, recurring cytogenetic abnormalities and hereditary cancer syndromes. In papillary renal carcinoma, trisomy 7 and 17 and loss of the Y chromosome are the most common chromosomal defects. Case presentation The present paper analyzes the chromosomes 7, 17, and Y alterations found in familial papillary carcinoma and in the normal tissue of two brothers. The evaluation, performed by fluorescence in situ hybridization (FISH) and cytogenetic conventional methods, was carried out on blood samples, normal kidney tissue, and tumor samples of the two brothers. Patient 1 showed gains of chromosomes 7 and 17 both in normal and tumor tissue. Chromosome Y status was normal. Patient 2 showed chromosome 7 and 17 gains, chromosome Y loss in tumor and chromosome 17 and Y alterations in normal kidney tissue. The constitutional karyotype was normal in both brothers. Conclusions Of particular relevance were the chromosome aberrations found in normal kidney parenchyma. In fact, the progressive alterations of 7, 17, and Y chromosomes could provide evidence of early genetic instability of tissue and may even precede the development of macroscopically identifiable lesions.

Fluorescence in situ hybridization analysis was performed to evaluate P16 (9p21), P53 (17p13.1), RB1 (13q14), HER2 (17q11.2) genes and chromosomes 3, 7, 9 and 17 in 62 bladder cancer cystectomies. We found chromosome aneusomy ranged between 74.2% and 91.9%. The highest percentage of homozygous deletion was found in the P16 gene (48.4%), while the highest percentage of heterozygous deletion was in the RB1 gene (51.6%). Chromosome 17 aneusomy significantly increased in tumors with higher stage, and RB heterozygous deletion showed a higher percentage of tumors with positive lymph node. Multiple correspondence analysis (MCA) showed four different biological tumor profiles, but only one could be associated with survival, defining the most unfavorable biological profile, characterized by P53 and P16 as homozygous and heterozygous and RB as homozygous deletion. Overall survival was significantly shorter in patients with at least two positive variables out of the five detected by MCA using the Kaplan-Meier's method. The biological stratification of advanced bladder cancer patients is of particular clinical interest, because the assessment of genetic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.

TO THE EDITOR: In a previously published issue of the Journal, we reported an unusual case of chronic myelomonocytic leukemia (CMML) type 2 (CMML-2) that evolved from refractory anemia (RA) [1]. The complete development of CMML was preceded by the loss of response to epoetin alpha (EPO). EPO had allowed the patient to maintain the transfusion independence (TI) for 7 years until then. The patient received azacitidine, achieving complete remission (CR) of CMML-2 after 6 treatment courses. However, the TI was observed after 2 cycles of hypomethylating therapy despite the persistence of myelodysplastic features. The optimal response to azacitidine in this patient was in line with that reported by our group earlier [2]. Moreover, the limited clinical efficacy of azacitidine in transfusion-dependent and erythropoiesis-stimulating agent (ESA)-resistant, low and intermediate-1 risk myelodysplastic syndrome (MDS) has been recently outlined [3]. Therefore, we could speculate that several and different biological components and pathogenic mechanisms, stemming from different patterns of response to distinct therapeutic agents, may be responsible for the myelodysplasia and the resulting clinical phenotype. These findings might be observed more easily during treatment with hypomethylating agents as these agents induce downstaging of high-risk MDS to low-risk MDS. Here, we report the updated follow-up of the clinical course of our patient, wherein we observed a dynamic evolution of MDS clones that showed different responses to azacitidine and EPO. The response to EPO was likely related to the persistence of RA clones that re-emerged despite azacitidine. After the achievement of the TI and the CR following the second and the sixth azacitidine courses, respectively, the patient continued to receive hypomethylating therapy as maintenance. However, after the fourteenth course, preceded by several weeks in which the patient complained of profound weakness and fatigue [4], progressive anemia (hemoglobin ~7.0 g/dL) requiring red blood cell (RBC) transfusions was noted. Therefore, the patient was reevaluated in the light of the suspicion of a loss of response to azacitidine and evolution to acute myeloid leukemia. However, examination of the bone marrow (BM) showed the disappearance of blast cells and a marked reduction of monocytes in a MDS framework, which was characterized by trilineage dysplasia and erythroid predominance with the features of ineffective erythropoiesis, as observed at the onset of MDS when the patient was diagnosed with RA [1]. According to its disease-modifying effects in MDS, azacitidine had "down-staged" the patient's malignancy from the transformed form of CMML-2 to refractory cytopenia with multilineage dysplasia (RCMD), resulting in a clinical phenotype similar to primary RA. On the basis of these findings, the patient continued to receive azacitidine as maintenance hypomethylating therapy and EPO was reintroduced. A rapid response with the achievement of TI was soon recorded. Therefore, the patient displaying near normal peripheral blood counts, continued to receive azacitidine concomitant with EPO, in order to maintain CR and TI. Our case presents some interesting observations. Initially, our patient was responsive to EPO for many years until the occurrence of an important transfusion requirement corresponding to transformation towards CMML-2. The treatment with azacitidine resulted in the clearance of this transformed, aggressive leukemic component probably by restoring a previous framework consistent with a low MDS risk, such as RCMD, as observed at the onset of primary MDS diagnosis. Therefore, it is likely that the clinical picture of the patient reflected maintained response to azacitidine, with good control of the CMML-2 and re-emergence of ineffective erythropoiesis typically associated with RA/RCMD MDS, which was again responsive to EPO. The patient was 83 years old and had a MDS history for 9 years; he was well after 21 courses of azacitidine and 6 months of TI with EPO. Although both the use of ESA [5] (to reduce the high burden of transfusion requirement in MDS patients [6]) and azacitidine [7,8] (to contain or reverse the natural progression of disease) are well established, very few studies have explored the therapeutic role exerted by the association of azacitidine with EPO in the setting of higher-risk MDS. A retrospective French study that included 32 higher-risk MDS patients, who concomitantly received ESA and azacitidine found that the addition of ESA to the hypomethylating therapy significantly improved overall survival independent of azacitidine schedule and duration [9]. These findings may reflect synergistic effects exerted by two agents or their separate actions on distinct mechanisms and/or components of MDS. However, no prospective studies have confirmed these findings and there is a need for prospective studies for confirmation [10]. However, we have not been able to thoroughly investigate this case with molecular techniques, owing to which we cannot clarify the biological and pathogenic bases for what we observed. Our case showed that MDS over its course can display different clinical manifestations, combining aspects associated with both high- and low-risk MDS forms. These different manifestations could correspond to underlying biological heterogeneity of different neoplastic clones or their transformation over time in the context of a continuum clonal evolution that may be influenced by the pattern of response to disease-modifying treatments.

Fluorescence in situ hybridization analysis for evaluation of 7, 8, X chromosomes and EGFR, LPL, MYC, AR genes in 79 neoplastic foci from 56 patients with clinically localized prostate cancer was performed. We found aneusomy for chromosome 7, 8 and X in 74/77 (96.1%), 56/76 (73.7%), 26/70 (37.1%) of examined foci respectively. No specimen was amplified for EGFR and AR genes, only 2/71 (2.8%) specimens showed MYC gene amplified. LPL deletion was present in 52/76 (68.4%) specimens. Statistically association between Gleason score and both chromosome 7 aneusomy and 8p21 deletion was present. The frequency of chromosome 7 aneusomy was statistically higher in T3-4 cases than T2c and T2a-T2b ones. We considered as unfavorable a genetic set if aneusomy for at least two chromosomes and one altered gene were present. The percentage of tumors, with unfavorable genetic pattern, increased from 36.4 to 75.0% in those with Gleason >7 and from 40.0 to 73.7% in those with stage T3 or more. These alterations could be considered potent genetic markers adjunctive to conventional prognostic parameters. Our objective was to establish specific genetic profiles which may discriminate favorable and unfavorable genetic prognosis tumors.

Here we describe how we detected the BCR/ABL fusion gene on the short arm of der(9) combining classical GTG banding and Fluorescence In Situ Hybridization (FISH) in a case of chronic myeloid leukemia (CML). To our knowledge, variant translocations involving the short arm of chromosome 9, in literature, are almost rare in chronic myeloid leukemia. It is not clear if this complex genetic translocation represents clonal evolution or a unique, initial presentation variant of the Philadelphia chromosome (Ph).