Extrusion 3D printing of keratin protein hydrogels free of exogenous chemical agents.

Keratins are a class of intermediate filament proteins that can be obtained from numerous sources including human hair. Materials fabricated from keratins offer desirable characteristics as scaffolds for tissue engineering, including intrinsic cell adhesion sequences and tunable degradation kinetics. The capacity to create 3D printed constructs from keratin-based bio-inks generates unique opportunities for spatial control of scaffold physicochemical properties to direct scaffold functions in ways not readily achieved through other means. The aim of this study was to leverage the controllable rheological properties of keratin hydrogels to create a strategy for extrusion 3D printing of keratin bio-inks without the use of exogenous rheological modifiers, crosslinking agents, or photocurable resins. The rheological properties of keratin hydrogels were tuned by varying two parameters: (a) the ratio of keratose (obtained by oxidative extraction of keratin) to kerateine (obtained by reductive extraction of keratin); and (b) the weight percentage of total keratin protein in the gel. A computational model of the dispensing nozzle for a commercially available extrusion 3D printer was developed to calculate the needed pneumatic printing pressures based on the known rheological properties of the gels. Keratin hydrogel constructs, of varying keratose/kerateine ratios and total keratin weight percentages, were 3D printed in cylindrical geometries via extrusion 3D printing. Rheology and degradation studies showed that gels with greater relative kerateine content exhibited greater flow resistance and slower degradation kinetics when submerged in phosphate buffered saline solution at 37 °C, owing to the presence of cysteine residues in kerateine and the capability of forming disulfide bonds. Total keratin weight percentage was found to influence gel yield stress, with possible implications for tuning filament fidelity. Findings from this work support the use of keratose/kerateine ratio and total keratin weight percentage as handles for modulating rheological characteristics of keratin hydrogels to enhance printability and control scaffold properties.

Fallow time determination in dentistry using aerosol measurement in mechanically and non-mechanically ventilated environments.

Aim To calculate fallow time (FT) required following dental aerosol generating procedures (AGPs) in both a dental hospital (mechanically ventilated) and primary care (non-mechanically ventilated). Secondary outcomes were to identify spread and persistence of aerosol in open clinics compared to closed surgeries (mechanically ventilated environment), and identify if extraoral scavenging (EOS) reduces FT and production of aerosol.Methods In vitro simulation of fast handpiece cavity preparations using a manikin was conducted in a mechanically and non-mechanically ventilated environment using Optical Particle Sizer and NanoScan at baseline, during the procedure and fallow period.Results AGPs carried out in the non-mechanically, non-ventilated environment failed to achieve baseline particle levels after one hour. In contrast, when windows were opened after AGPs, there was an immediate reduction in all particle sizes. In mechanically ventilated environments, the baseline levels of particles were very low and particle count returned to baseline within ten minutes following the AGP. There was no detectable difference between particles in mechanically ventilated open bays and closed surgeries. The effect of the EOS on reducing the particle count was greater in the non-mechanically ventilated environment; additionally, it also reduced the spikes in particle counts in mechanically ventilated environments.Conclusion High-efficiency particulate, air-filtered mechanical ventilation, along with mitigation (high-volume suction), resulted in reduction of fallow time (ten minutes). Non-ventilated rooms failed to reach baseline level even after one hour of fallow time. There was no difference in particle counts in open bays or closed surgeries in mechanically ventilated settings with an extraoral suction device reducing particulate spikes. This study confirms that AGPs are not recommended in dental surgeries where no ventilation is possible.

Cultivation of hierarchical 3D scaffolds inside a perfusion bioreactor: scaffold design and finite-element analysis of fluid flow.

The use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6-12% w/v and 10-40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%-93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% ß-tricalcium phosphate and 60% hydroxyapatite (ß-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering.

Quantitative analysis of particulate matter release during orthodontic procedures: a pilot study.

Introduction Transmission of SARS-CoV-2 through aerosol has been suggested, particularly in the presence of highly concentrated aerosols in enclosed environments. It is accepted that aerosols are produced during a range of dental procedures, posing potential risks to both dental practitioners and patients. There has been little agreement concerning aerosol transmission associated with orthodontics and associated mitigation.Methods Orthodontic procedures were simulated in a closed side-surgery using a dental manikin on an acrylic model using composite-based adhesive. Adhesive removal representing debonding was undertaken using a 1:1 contra-angle handpiece (W&H Synea Vision WK-56 LT, Bürmoos, Austria) and fast handpiece with variation in air and water flow. The removal of acid etch was also simulated with the use of combined 3-in-1 air-water syringe. An optical particle sizer (OPS 3330, TSI Inc., Minnesota, USA) and a portable scanning mobility particle sizer (NanoScan SMPS Nanoparticle Sizer 3910, TSI Inc., Minnesota, USA) were both used to assess particulate matter ranging in dimension from 0.08 to 10 µm.Results Standard debonding procedure (involving air but no water) was associated with clear increase in the 'very small' and 'small' (0.26-0.9 µm) particles but only for a short period. Debonding procedures without supplementary air coolant appeared to produce similar levels of aerosol to standard debonding. Debonding in association with water tended to produce large increases in aerosol levels, producing particles of all sizes throughout the experiment. The use of water and a fast handpiece led to the most significant increase in particles. Combined use of the 3-in-1 air-water syringe did not result in any detectable increase in the aerosol levels.Conclusions Particulate matter was released during orthodontic debonding, although the concentration and volume was markedly less than that associated with the use of a fast handpiece. No increase in particulates was associated with prolonged use of a 3-in-1 air-water syringe. Particulate levels reduced to baseline levels over a short period (approximately five minutes). Further research within alternative, open environments and without air exchange systems is required.

The efficacy of an extraoral scavenging device on reduction of splatter contamination during dental aerosol generating procedures: an exploratory study.

Introduction This study was conducted in light of the SARS-CoV-2 pandemic, which brought UK dentistry to a standstill. The market has seen a recent influx of unproven extraoral scavengers (EOSs), which claim to reduce the risk of particulate spread.Aims To investigate the efficacy of a commercially available EOS device on contamination reduction during dental aerosol generating procedures (AGPs). The secondary aim was to investigate differences between open and closed dental operatories.Method Dental procedures were simulated on a dental manikin using citric acid (10%) added to the water lines with universal indicating paper (UIP) placed in strategic locations in the operatory, on the clinician and assistant. Chromatic change related to settling of splatter containing citric acid on the UIP was analysed to calculate percentage intensity of splatter contamination.Results EOSs resulted in 20% reduction in frequency and 75% reduction in mean intensity of contamination of operatory sites. There was a 33% and 76% reduction in mean intensity contamination for clinician and assistant, respectively. Use of rubber dam and four-handed dentistry resulted in further reduction.Discussion This exploratory study demonstrates contamination by splatter in a simulated dental setting. The concern in dentistry regarding aerosol requires further quantitative investigation of smaller particles.Conclusions The routine use of four-handed dentistry and rubber dam should continue where possible to maximise risk mitigation during AGPs. However, on the basis of our findings, the use of an EOS device can further mitigate the magnitude and concentration of splatter.

Validation of scaffold design optimization in bone tissue engineering: finite element modeling versus designed experiments.

This study reports the development of biological/synthetic scaffolds for bone tissue engineering (TE) via 3D bioplotting. These scaffolds were composed of poly(L-lactic-co-glycolic acid) (PLGA), type I collagen, and nano-hydroxyapatite (nHA) in an attempt to mimic the extracellular matrix of bone. The solvent used for processing the scaffolds was 1,1,1,3,3,3-hexafluoro-2-propanol. The produced scaffolds were characterized by scanning electron microscopy, microcomputed tomography, thermogravimetric analysis, and unconfined compression test. This study also sought to validate the use of finite-element optimization in COMSOL Multiphysics for scaffold design. Scaffold topology was simplified to three factors: nHA content, strand diameter, and strand spacing. These factors affect the ability of the scaffold to bear mechanical loads and how porous the structure can be. Twenty four scaffolds were constructed according to an I-optimal, split-plot designed experiment (DE) in order to generate experimental models of the factor-response relationships. Within the design region, the DE and COMSOL models agreed in their recommended optimal nHA (30%) and strand diameter (460 µm). However, the two methods disagreed by more than 30% in strand spacing (908 µm for DE; 601 µm for COMSOL). Seven scaffolds were 3D-bioplotted to validate the predictions of DE and COMSOL models (4.5-9.9 MPa measured moduli). The predictions for these scaffolds showed relative agreement for scaffold porosity (mean absolute percentage error of 4% for DE and 13% for COMSOL), but were substantially poorer for scaffold modulus (51% for DE; 21% for COMSOL), partly due to some simplifying assumptions made by the models. Expanding the design region in future experiments (e.g., higher nHA content and strand diameter), developing an efficient solvent evaporation method, and exerting a greater control over layer overlap could allow developing PLGA-nHA-collagen scaffolds to meet the mechanical requirements for bone TE.

Long-Lasting WNT-TCF Response Blocking and Epigenetic Modifying Activities of Withanolide F in Human Cancer Cells.

The WNT-TCF signaling pathway participates in adult tissue homeostasis and repair, and is hyperactive in a number of human diseases including cancers of the colon. Whereas to date there are no antagonists approved for patient use, a potential problem for their sustained use is the blockade of WNT signaling in healthy tissues, thus provoking potentially serious co-lateral damage. Here we have screened a library of plant and microorganism small molecules for novel WNT signaling antagonists and describe withanolide F as a potent WNT-TCF response blocker. This steroidal lactone inhibits TCF-dependent colon cancer xenograft growth and mimics the effects of genetic blockade of TCF and of ivermectin, a previously reported WNT-TCF blocker. However, withanolide F is unique in that it imposes a long-lasting repression of tumor growth, WNT-TCF targets and cancer stem cell clonogenicity after drug treatment. These findings are paralleled by its modulation of chromatin regulators and its alteration of overall H3K4me1 levels. Our results open up the possibility to permanently repress essential signaling responses in cancer cells through limited treatments with small molecules.

Thrombin inhibition by dabigatran attenuates atherosclerosis in ApoE deficient mice.

INTRODUCTION: Atherosclerosis is a chronic inflammatory disease characterized by endothelial cell damage, infiltration, proliferation and accumulation of macrophages, lymphocytes and transformed vascular smooth muscle cells within the vascular wall and procoagulation processes involving activation of plasmatic coagulation events and platelets. Numerous studies suggested a close interaction between thrombin action and atherogenesis, but possibly underlying mechanisms are multiple and specific treatment options were missing until now. MATERIAL AND METHODS: Atherosclerosis prone 12 weeks old ApoE(-/-) mice were fed a cholesterol rich diet for 4 weeks and were concomitantly treated orally with placebo or the thrombin inhibitor dabigatran (1.2 g/kg/day). RESULTS: The thrombin time (HEMOCLOT(®)) was significant extended in dabigatran treated animals. Vascular oxidative stress was significantly reduced during thrombin inhibition, as assessed by L012 chemiluminescence in aortic segments (212 ±84 vs. 69 ±21 RLU/s/mg dry weight, p = 0.048). Organ chamber experiments of isolated aortic rings showed that dabigatran treatment significantly improved endothelium-derived vasorelaxation (p < 0.001). Dabigatran treated mice developed less atherosclerotic lesions (6.2 ±0.2% vs. 9 ±1.1%, p = 0.037) and showed less infiltration of atherosclerotic lesions with macrophages (2.59 ±0.3% vs. 5.14 ±0.7%, p = 0.0046), as determined by systematic histological and immunohistological analyses of the aortic root. Blood pressure, body weight and food intake were not altered by the treatment. CONCLUSIONS: The thrombin inhibitor dabigatran reduces vascular oxidative stress and inflammation, improves endothelial function and decreases atherosclerosis in mice.

Producing homogeneous cryogel phantoms for medical imaging: a finite-element approach.

Tissue-mimicking phantoms with well-defined properties can help in identifying the potential weaknesses in medical imaging systems. Among the imaging systems, magnetic resonance elastography is a new noninvasive technique used to quantify the shear modulus of biological tissues, and therefore has shown promise in studying liver and brain pathologies. Polyvinyl alcohol (PVA) cryogel prepared by the freeze-thaw technique is a potential candidate for mimicking the mechanical properties of soft tissues and has been extensively used as a phantom material. However, large PVA cryogels suffer from variations in properties, partly due to the low thermal conductivity of PVA solution. The loss of homogeneity in cryogel phantoms is also attributed to inhomogeneous thawing rates during the freeze-thaw cycle. We have used a modified freeze-thaw process that imposes multiple isotherms so as to enhance the homogeneity of the produced cryogels. In addition, we have developed a finite-element modeling tool (a virtual controller) to optimize the temperature profile during the freeze-thaw cycle. Our experimental validations demonstrated the potential of the virtual controller in predicting the optimal temperature profile for the freeze-thaw process (phantom diameters: 60 and 100 mm). A robust simulation framework can fill the gap in the scientific literature with regard to phantom design for medical imaging and will help to reduce phantom development time and cost.

"When doing good is just the start to being good": a possible tool to improve the organizational effectiveness of non-profit health care organizations.

Health care is an area where many non-profit organizations do good work, relieving governments and for-profit providers of workload. They contribute often specific knowledge and competence or provide better access to patients through their cultural and ethnic affiliations. Non-profit organizations, NPOs, are often dependent on one or more significant donors, sometimes governments, sometimes for-profit health providers and mostly private/corporate donors with an interest in specific medical areas, such as Alzheimers, or in unique communities, such as the Laotian neighborhoods in Southern California. In the media, high profile scandals involving financial irresponsibility have caused shock waves around the world. Concerns among some donors over the lack of a transparent performance measurement of non-profit entities have increased with the greater call for transparency and good governance in the corporate world. Not all the scandals have been in the commercial sector; some Not for Profit Organizations (NPOs) have been identified as having less than credible governance structures (Thomson, 2003). The watchdog organization SustainAbility reported that accountability and transparency are issues on which several NPOs are found wanting (Anon., 2003) and the New York Times reports that until recently NPOs were thought to be exempt from traditional oversight; their do-good nature and the commitment of their participants were thought to be sufficient to produce positive results (Christensen, 2004).

Recurrent intracardiac thrombosis as an unusual manifestation of inherited thrombophilia.

Patients with inherited thrombophilia are at high risk for the development of venous thrombosis that manifests mainly as deep venous thrombosis of the legs and/or pulmonary embolism. We report spontaneous right-sided intracardiac thrombosis in a young man as an unusual manifestation of inherited thrombophilia. The diagnosis of thrombophilia was confirmed by demonstration of the prothrombin-G20210A-mutation in the homozygous state. A second spontaneous intracardiac thrombosis occurred 3 years after discontinuation of oral anticoagulant treatment. This indicates the high risk for recurrence in patients developing intracardiac thrombosis in the absence of an underlying cardiac disease and warrants long-term oral anticoagulant treatment.

Development, validation and evaluation of a homogenous one-step reverse transcriptase-initiated PCR assay with competitive internal control for the detection of hepatitis C virus RNA.

Nucleic acid amplification testing for hepatitis C virus (HCV) RNA has become an essential tool for the prevention and clinical management of hepatitis C. We describe the development, validation and evaluation of a homogenous reverse transcriptase-initiated HCV-PCR assay with competitive internal control that is applicable to both the quantitative detection of HCV genomes in single patient samples and the screening of blood donations by mini-pool testing. For the implementation of a positive run control, a HCV RNA-positive plasma sample was calibrated against an international HCV RNA standard preparation. For quantification purposes, an in vitro-transcribed RNA calibrator sequence was used. The detection limit of the assay (95% positive cut-off) was determined by probit analysis and was calculated as 114 IU/mL. Comparable sensitivity to different HCV template sequences was verified for HCV genotypes 1-5. Quantitative test results correlated well with viral loads that had been previously determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay (n=53, R=0.943, p<0.001). During more than 5 years of blood donation testing, the specificity of the assay was found to be 99.51%. All assay components showed constant performance during this time period. In conclusion, we introduce a well-proven method that allows fast and reliable quantification of HCV genomes.