Cartilage Regeneration in Preannealed Silk Elastin-Like Co-Recombinamers Injectable Hydrogel Embedded with Mature Chondrocytes in an Ex Vivo Culture Platform.

Tissue engineering for cartilage repair requires biomaterials that show rapid gelation and adequate mechanical properties. Although the use of hydrogel is the most promising biomaterial, it often lacks in rigidity and anchorage of cells when they are surrounded by synovial fluid while they are subjected to heavy loads. We developed and produced the Silk Elastin-Like co-Recombinamer (SELR), which contains both the physical interaction from elastin motifs and from silk motifs. In the first part of this work, we set up and optimized a preannealing treatment based on the evolution of silk motifs into ß-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new preannealed SELRs (pA(EIS)2-(I5R)6) were characterized with the combination of several experimental techniques (CD, TEM, SEM, and rheology) to provide a deep insight into the material features. Finally, the regeneration properties of the pA(EIS)2-(I5R)6 hydrogel embedded with chondrocytes were evaluated. After 4 weeks of culturing in a standardized and representative ex vivo model, the biochemical and histological analysis revealed the production of glycosaminglycans and collagen. Moreover, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage. Hence, we conclude that the pA(EIS)2-(I5R)6 hydrogel presents improved mechanical properties while conserving the injectability, which leads to successful regeneration of hyaline cartilage in an ex vivo model.

Age estimation from canine volumes.

Techniques for estimation of biological age are constantly evolving and are finding daily application in the forensic radiology field in cases concerning the estimation of the chronological age of a corpse in order to reconstruct the biological profile, or of a living subject, for example in cases of immigration of people without identity papers from a civil registry. The deposition of teeth secondary dentine and consequent decrease of pulp chamber in size are well known as aging phenomena, and they have been applied to the forensic context by the development of age estimation procedures, such as Kvaal-Solheim and Cameriere methods. The present study takes into consideration canines pulp chamber volume related to the entire teeth volume, with the aim of proposing new regression formulae for age estimation using 91 cone beam computerized scans and a freeware open-source software, in order to permit affordable reproducibility of volumes calculation.

Sevelamer is cost-saving vs. calcium carbonate in non-dialysis-dependent CKD patients in italy: a patient-level cost-effectiveness analysis of the INDEPENDENT study.

OBJECTIVES: To conduct a cost-effectiveness analysis of sevelamer versus calcium carbonate in patients with non-dialysis-dependent CKD (NDD-CKD) from the Italian NHS perspective using patient-level data from the INDEPENDENT-CKD study. METHODS: Patient-level data on all-cause mortality, dialysis inception and phosphate binder dose were obtained for all 107 sevelamer and 105 calcium carbonate patients from the INDEPENDENT-CKD study. Hospitalization and frequency of dialysis data were collected post hoc for all patients via a retrospective chart review. Phosphate binder, hospitalization, and dialysis costs were expressed in 2012 euros using hospital pharmacy, Italian diagnosis-related group and ambulatory tariffs, respectively. Total life years (LYs) and costs per treatment group were calculated for the 3-year period of the study. Bootstrapping was used to estimate confidence intervals around outcomes, costs, and cost-effectiveness and to calculate the cost-effectiveness acceptability curve. A subgroup analysis of patients who did not initiate dialysis during the INDEPENDENT-CKD study was also conducted. RESULTS: Sevelamer was associated with 0.06 additional LYs (95% CI -0.04 to 0.16) and cost savings of EUR -5,615 (95% CI -10,066 to -1,164) per patient compared with calcium carbonate. On the basis of the bootstrap analysis, sevelamer was dominant compared to calcium carbonate in 87.1% of 10,000 bootstrap replicates. Similar results were observed in the subgroup analysis. RESULTS were driven by a significant reduction in all-cause mortality and significantly fewer hospitalizations in the sevelamer group, which offset the higher acquisition cost for sevelamer. CONCLUSIONS: Sevelamer provides more LYs and is less costly than calcium carbonate in patients with NDD-CKD in Italy.

Implications of Atopic Dermatitis on the Quality of Life of 6-11 Years Old Children and Caregivers (PEDI-BURDEN).

Purpose: Atopic dermatitis (AD) is a chronic, relapsing and remitting inflammatory skin disease characterized by intense itch. The disease burden includes physical limitations, psychosocial discomfort, and a reduced quality of life (HRQoL). This study presents the results of a parent-reported survey on the psychosocial impact of AD on Italian pre-adolescent children (6-11 years old), with a specific focus on bullying, self-isolation, absenteeism, and presenteeism. Methods: An online questionnaire was sent to 3067 random recipients and 160 matched the inclusion criteria for age, self-reported AD diagnosis, localizations (according to ISAAC), and disease severity (POEM ≥8). 100 children, with comparable ages, not matching the inclusion criteria for AD, were recruited as a control group. Results: Children with AD and their caregivers had a significantly lower quality of sleep (QoS) compared to the control group. The presence of AD was directly responsible for many restless nights, both in children and caregivers (58.9 and 55.4 respectively). Children with AD and their parents also experienced significantly more daytime drowsiness (43.6 and 54.6 days, respectively). Children with AD were more frequently victims of bullying at school (20.0% vs 9.0%; p≤0.05) or in other social environments (16.9% vs 3.0%; p≤0.05). AD caused 17.7 days of absenteeism and 20.1 days of presenteeism per student over the previous 12 months, accounting for 37.8 days of study impairment overall. Severe/very severe AD had a significantly greater impact on presenteeism than moderate AD (25.1 vs 17.5 days; p≤0.05). Presenteeism, which was more pronounced among bullied students, was positively correlated with absenteeism only in the AD cohort. Conclusion: AD has a detrimental impact on the HRQoL of pediatric patients, causing stigmatization and social isolation. Functional distress was also reported by caregivers. Our study might inform the public and policymakers about the disease burden of AD at a young age.

Dupilumab treatment induced similar improvements in signs, symptoms, and quality of life in adults with moderate-to-severe atopic dermatitis with baseline Eczema Area and Severity Index Score <24 or ≥24.

BACKGROUND: In multiple phase 3 trials, dupilumab improved signs, symptoms (including pruritus), and quality-of-life (QoL) in adults with moderate-to-severe atopic dermatitis (AD). In Italy, dupilumab received innovation status but is currently only reimbursed by the National Health Service for adults with Eczema Area Severity Index (EASI) scores ≥24. This analysis assesses disease burden and dupilumab efficacy in adults with EASI scores above and below this threshold. METHODS: This post-hoc analysis included 299 adults pooled from two, randomized, placebo-controlled, phase 3 trials, LIBERTY AD CAFÉ (NCT02755649) and LIBERTY AD CHRONOS (NCT02260986), who received the approved dupilumab regimen (300 mg every 2 weeks) or placebo, with concomitant topical corticosteroids. EASI, Peak Pruritus Numerical Rating Scale (PP-NRS), and Dermatology Life Quality Index (DLQI) were assessed in patients with EASI scores ≥20 to <24 and ≥24 at week 16. RESULTS: At baseline, EASI was weakly correlated with PP-NRS and DLQI (Spearman's correlation coefficient: 0.22 and 0.29, respectively). At week 16, in both the EASI<24 and EASI≥24 populations, respectively, significantly more patients vs. control achieved: ≥50% improvement in EASI (95.5% vs. 55.6%; 80.6% vs. 33.1%); ≥3-point improvement in PP-NRS (68.4% vs. 35.3%; 55.3% vs. 17.7%); and ≥4-point improvement in DLQI (83.3% vs. 43.8%; 84.2% vs. 41.9%); from baseline. Dupilumab was generally well tolerated with an acceptable safety profile. CONCLUSIONS: Dupilumab treatment improves signs, symptoms, and QoL in moderate-to-severe AD adults with EASI<24, who can present with high disease burden. Opportunity may exist to use additional parameters to define disease severity and access to new therapies.

Modeling the cost-effectiveness for cement-less and hybrid prosthesis in total hip replacement in Emilia Romagna, Italy.

BACKGROUND: The aim of the present study was to assess the cost-effectiveness of cement-less versus hybrid prostheses in total hip replacement (THR) in patients diagnosed with primary osteoarthritis. METHODS: Effectiveness data were obtained from the Emilia-Romagna Regional Registry on Orthopaedic Prosthesis (RIPO), which collects information on all orthopaedic intervention performed in Emilia-Romagna (41,199 total hip replacements performed from 2000 to 2007), and from which we obtained survival curves and transition probabilities for the cement-less and hybrid prostheses, respectively. Conversely, costs were derived from regional databases through a specific procedure, which allowed us to register individual component's costs for both primary and subsequent revision interventions. A specific Markov transition model was constructed in order to consider the 3 types of revisions that an implant could possibly undergo through its life-span: total, cup or stem, head insert or neck. The cost-effectiveness was expressed in terms of cost per "revision-free" life year. RESULTS AND CONCLUSIONS: Considering a 70-y old patient undergoing THR, the cementless strategy resulted more effective but more costly than the hybrid solution, with an incremental cost effectiveness ratio of 2401.63 € per revision-free life year. Following a deterministic sensitivity analysis, hybrid and cementless fixation showed, respectively, a dominance profile for patients older than 83 y and younger than 43 y, whereas for all ages in between, we report a progressive increase in the ICER of cementless prostheses. Our results proved to be robust, as underlined by the probabilistic sensitivity analysis performed using cost distributions.

Atopic Dermatitis and Patient Perspectives: Insights of Bullying at School and Career Discrimination at Work.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by recurrent eczematous lesions and intense pruritus. AD patients are known to face a considerable disease burden, including physical and emotional limitations. There is still limited knowledge about daily implications in education and occupation. We describe disease social stigmatization by measuring bullying and self-isolation in students and professional discrimination in workers. Overall loss of productivity, either at school and at the workplace, was quantified as the sum of absenteeism (number of days AD sick leave) and presenteeism (number of days with decreased focus and functionality). METHODS: An on-line web survey was sent to 3235 random recipients and 401 met the inclusion criteria (self-reporting AD and ≥12 yo). The survey domains included daily limitations, QoL, feelings and relationships, together with specific questions about bullying, discrimination and loss of productivity. RESULTS: AD negatively affected QoL in 51.6% of respondents, whereas 68.8% considered AD as a real limit to daily routine. More in detail, 39.3% of students were victims of bullying and 33.9% of workers felt discriminated because of AD. On average, absenteeism in students was for 17.1 days/year (presenteeism: 19.5 days/year), whereas in workers, the estimate was 10.9 days/year (presenteeism: 13.1 days/year). Absenteeism and presenteeism were more pronounced in bullied/discriminated subjects. CONCLUSION: AD multidimensional implications deeply affect and undermine personal and professional fulfillments. Our results contribute to a better understanding of what living with AD means.

Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae.

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

Hydrophobic Cholesteryl Moieties Trigger Substrate Cell-Membrane Interaction of Elastin-Mimetic Protein Coatings in Vitro.

A cellular coating based on hydrophobic interactions of an elastin-like recombinamer (ELR) with the cell membrane is presented. It is well-documented that biophysical properties such as net charge, hydrophobicity, and protein-driven cell-ligand (integrin binding) interactions influence the interaction of polymers, proteins or peptides with model membranes and biological cells. Most studies to enhance membrane-substrate interactions have focused on the introduction of positively charged groups to foster electrostatic interactions with the negatively charged membrane. Herein, we present an antagonistic approach based on ELRs with varying amounts of hydrophobic cholesteryl groups (ELRCTAs). The ability of the membranes to stabilize cholesteryl groups is hypothesized to assist the coordination of hydrophobic ELRs with the membrane. The main objective was to generate a defined cellular coating of a recombinant protein that allows for total sequence control and less host, or batch-to-batch, variation as a substitute for the existing coatings like alginate, polyelectrolytes, collagens, and fibronectin. We used an in vitro cell-binding assay to quantify cell-substrate interactions, showing enhanced cellular recognition and matrix distribution with an increasing number of cholesteryl groups incorporated. These novel materials and the versatile nature of their protein sequence have great potential as cellular markers, drug carriers, or hydrophobic cell-binding domains.

Bicyclic RGD peptides with high integrin α v ß 3 and α 5 ß 1 affinity promote cell adhesion on elastin-like recombinamers.

Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In contrast, the few biological alternatives employed as biomaterials lack the versatility that chemical synthesis can offer. Herein, we studied the HUVEC adhesion and proliferation properties of elastin-like recombinamers (ELRs) that were covalently functionalized with each three high-affinity and selectivity α v ß 3- and α 5 ß 1-binding bicyclic RGD peptides. Next to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent functionalization with the RGD peptides, as validated by MALDI-TOF analysis, guarantees flexibility and minimal steric hindrance for interactions with cellular integrins. In addition to the covalently modified RGD-ELRs, we also synthesized another benchmark ELR comprising RGD as part of the backbone. HUVEC adhesion and proliferation analysis using the PicoGreen® assay revealed a higher short-term adhesion and proliferative capacity of cells on ELR surfaces functionalized with high affinity, integrin-binding bicyclic RGD-peptides compared with the ELRs containing RGD in the backbone.

An elastin-like recombinamer-based bioactive hydrogel embedded with mesenchymal stromal cells as an injectable scaffold for osteochondral repair.

The aim of this study was to evaluate injectable, in situ cross-linkable elastin-like recombinamers (ELRs) for osteochondral repair. Both the ELR-based hydrogel alone and the ELR-based hydrogel embedded with rabbit mesenchymal stromal cells (rMSCs) were tested for the regeneration of critical subchondral defects in 10 New Zealand rabbits. Thus, cylindrical osteochondral defects were filled with an aqueous solution of ELRs and the animals sacrificed at 4 months for histological and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality and the new matrix in the defects. Although both approaches helped cartilage regeneration, the results suggest that the specific composition of the rMSC-containing hydrogel permitted adequate bone regeneration, whereas the ELR-based hydrogel alone led to an excellent regeneration of hyaline cartilage. In conclusion, the ELR cross-linker solution can be easily delivered and forms a stable well-integrated hydrogel that supports infiltration and de novo matrix synthesis.

Sevelamer is cost effective versus calcium carbonate for the first-line treatment of hyperphosphatemia in new patients to hemodialysis: a patient-level economic evaluation of the INDEPENDENT-HD study.

BACKGROUND: The recent multicenter, randomized, open-label INDEPENDENT study demonstrated that sevelamer improves survival in new to hemodialysis (HD) patients compared with calcium carbonate. The objective of this study was to determine the cost-effectiveness of sevelamer versus calcium carbonate for patients new to HD, using patient-level data from the INDEPENDENT study. STUDY DESIGN: Cost-effectiveness analysis. SETTING AND POPULATION: Adult patients new to HD in Italy. MODEL, PERSPECTIVE, TIMEFRAME: A patient-level cost-effectiveness analysis was conducted from the perspective of the Servizio Sanitario Nazionale, Italy's national health service. The analysis was conducted for a 3-year time horizon. The cost of dialysis was excluded from the base case analysis. INTERVENTION: Sevelamer was compared to calcium carbonate. OUTCOMES: Total life years (LYs), total costs, and the incremental cost per LY gained were calculated. Bootstrapping was used to estimate confidence intervals around LYs, costs, and cost-effectiveness and to calculate the cost-effectiveness acceptability curve. RESULTS: Sevelamer was associated with a gain of 0.26 in LYs compared to calcium carbonate, over the 3-year time horizon. Total drug costs were €3,282 higher for sevelamer versus calcium carbonate, while total hospitalization costs were €2,020 lower for sevelamer versus calcium carbonate. The total incremental cost of sevelamer versus calcium carbonate was €1,262, resulting in a cost per LY gained of €4,897. The bootstrap analysis demonstrated that sevelamer was cost effective compared with calcium carbonate in 99.4 % of 10,000 bootstrap replicates, assuming a willingness-to-pay threshold of €20,000 per LY gained. LIMITATIONS: Data on hospitalizations was taken from a post hoc retrospective chart review of the patients included in the INDEPENDENT study. Patient quality of life or health utility was not included in the analysis. CONCLUSIONS: Sevelamer is a cost-effective alternative to calcium carbonate for the first-line treatment of hyperphosphatemia in new to HD patients in Italy.

Identification of a co-activator that links growth factor signalling to c-Jun/AP-1 activation.

The AP-1 transcription factor c-Jun is essential for cellular proliferation in many cell types, but the molecular link between growth factors and c-Jun activation has been enigmatic. In this study we identify a previously uncharacterized RING-domain-containing protein, RACO-1 (RING domain AP-1 co-activator-1), as a c-Jun co-activator that is regulated by growth factor signalling. RACO-1 interacted with c-Jun independently of amino-terminal phosphorylation, and was both necessary and sufficient for c-Jun/AP-1 activation. Growth factor-mediated stimulation of AP-1 was attributable to MEK/ERK-dependent stabilization of RACO-1 protein. Stimulation of the MEK/ERK pathway strongly promoted Lys 63-linked ubiquitylation of RACO-1, which antagonized Lys 48-linked degradative auto-ubiquitylation of the same Lys residues. RACO-1 depletion reduced cellular proliferation and decreased expression of several growth-associated AP-1 target genes, such as cdc2, cyclinD1 and hb-egf. Moreover, transgenic overexpression of RACO-1 augmented intestinal tumour formation triggered by aberrant Wnt signalling and cooperated with oncogenic Ras in colonic hyperproliferation. Thus RACO-1 is a co-activator that links c-Jun to growth factor signalling and is essential for AP-1 function in proliferation.