Comparison of HepaRG and HepG2 cell lines to model mitochondrial respiratory adaptations in non­alcoholic fatty liver disease.

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non­alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non­alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for in vitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA­treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II­driven respiration and ß­oxidation were linked to increased complex I and II activities in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA­treated HepaRG cells than in FFA­treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early­stage NASH.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a founder variant in the 5'-untranslated region of UNC45A.

BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

Solvent-assisted dispersive micro-solid phase extraction of bisphenols using iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a new nanostructured sorbent: a proof of concept.

In this work, we describe for the first time the use of iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a novel sorbent for solvent-assisted dispersive micro-solid phase extraction (SA-dµSPE) of bisphenols from water samples. The extraction procedure is based on the formation of nanoparticles in situ following the rapid injection of a methanolic solution of Fe(TTA)3 into the stirred aqueous sample. Herein, the synthesis of Fe(TTA)3 and study of the essential parameters of the preparative procedure are described. The optimized procedure allowed for efficient enrichment of bisphenols from various water samples, chosen as model contaminants and matrix, within 2.5 min. The sorbent was collected by centrifugation, dissolved in methanol, and injected to perform HPLC with spectrophotometric detection. The limits of detection and quantification obtained ranged from 1.0-3.1 and 3.1-7.5 µg L-1, respectively. Intraday and interday precisions of <7% relative standard deviation (RSD) and <8% RSD with analyte recoveries ranging between 70-117% (103.8% on average) were obtained for the analysis of river water, wastewater treatment plant effluent, and bottled water.

High-Pressure Inactivation of Bacillus cereus in Human Breast Milk.

Although Holder pasteurization is the recommended method for processing breast milk, it does affect some of its nutritional and biological properties and is ineffective at inactivating spores. The aim of this study was to find and validate an alternative methodology for processing breast milk to increase its availability for newborn babies and reduce the financial loss associated with discarding milk that has become microbiologically positive. We prepared two series of breast milk samples inoculated with the Bacillus cereus (B. cereus) strain to verify the effectiveness of two high-pressure treatments: (1) 350 MPa/5 min/38 °C in four cycles and (2) cumulative pressure of 350 MPa/20 min/38 °C. We found that the use of pressure in cycles was statistically more effective than cumulative pressure. It reduced the number of spores by three to four orders of magnitude. We verified that the method was reproducible. The routine use of this method could lead to an increased availability of milk for newborn babies, and at the same time, reduce the amount of wasted milk. In addition, high-pressure treatment preserves the nutritional quality of milk.

An Investigation of the Accuracy and Reproducibility of 3D Printed Transparent Endodontic Blocks.

Due to a broad spectrum of endodontic rotary instruments on the market and no standardised protocol for comparing their mechanical properties, it can be challenging for clinician to choose proper instruments. In vitro studies using resin blocks with artificial canals can offer many valuable information because of their uniformity compared to studies performed on extracted teeth. To improve precision and reproducibility of artificial canals, 3D printing was used in this study to manufacture endodontic test block samples. 20 commercially available endodontic blocks Endo-Training-Bloc-J by Dentsply Sirona were tested. The mean values of the measured parameters were used for a 3D CAD model of their replicas. 20 copies of the endodontic training blocks were printed from acrylic resin (VeroClear-RGD810, Stratasys, Eden Prairie, USA) using the 3D printer Objet30 Pro (Stratasys, Eden Prairie, USA). The key dimensions of the commercial blocks and the 3D printed blocks were measured under and compared using t - test and Levene's test for equality of variances. The profiles of the 3D printed artificial canals showed significantly lower dimensional variability when compared with the commercial blocks. 3D polyjet printing proved to be a precise and reproducible method for production of blocks for testing endodontic rotary instruments.

Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells.

Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO2 P25. We used four techniques for TiO2 P25 dispersion. We estimated the colloid stability of TiO2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO2 P25 (0-100 µg.mL-1) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO2 P25. Under these conditions, TiO2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO2 P25 in A549 cells were met, similar results on the biological effects of TiO2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO2 P25 as a reference material.

Contactless Measurement of Integrity of Silicone Coating on Self-Expandable Esophageal Nitinol Stents.

OBJECTIVES: A stent is a mesh tube inserted into a natural passage in the body to prevent disease induction. Self-expandable esophageal nitinol stents such as SX-ELLA Stent Esophageal HV (HV Stent Plus) can be indicated for palliation of malignant esophageal strictures, for the treatment of benign esophageal strictures that are refractory to standard therapy and for the treatment of esophago-respiratory fistulas. A silicone-stent coating is used for tumor in-growth prevention and esophago-respiratory fistula occlusion. The thickness of the stent and the overall integrity of the silicone coating of all wires indicate the overall mechanical properties of the esophageal stent and the resistance to external adverse events such as corrosion and mechanical and chemical resistance. METHODS: The polymer multicomponent epoxy resin - a mixture of Epon and Durcupan - was used as a method for robust sample stabilization. A cutting system using a thin water beam with a powder (Blue Line) was chosen as the best variant to obtain 6 samples for both-sided measurement (10 measuring sides). The optical microscopic reflective light method was used to examine wire crossing points in the sections. Fifty values were measured on either sample side for the internal, external and mesh thickness of the silicone stent layer. The wire crossing points were selected so that the silicone layer structure could be clearly seen, and the wires approached each other most closely. Only approximately 4 to 8 crossing points in each section could be measured when applying this approach. The resolution of the microscope and calibration (based on the camera used) was 0.677 µm/pixel. RESULTS: Additional data could be obtained on 8 planes. Two boundary samples were destroyed by the cutting process. Whole coating of the stent was around all mesh wires, especially in areas with higher mechanical stress (wire crossing). The minimum detectable and admissible value determined for all 3 measuring areas (internal, external, mesh) on the wire crossings was 6.77 µm, i.e., 10 pixels, based on the microscope resolution and manufacturer's methodology. The results were characterized by p < 0.001 for all 3 parameters. We tested opposite samples in each section to verify the section quality and data consistency. For the 4 areas, the data were significantly different, but the thickness differences were only on the order of units percent, so the measurements were not appreciably affected. We assume that the material cutting loss, making up 1-2 mm, contributed to the differences in the sections. CONCLUSION: We examined the overall integrity of the silicone coating of the esophageal stent. The method of HV stent anchoring in a polymeric bath followed by cutting with a waterjet and sample measurement under an optical microscope proved to be very simple and reliable. Sufficient thicknesses of the silicone layer on the wire cross sections were verified. The coated silicone layer thickness appeared to be significantly different along the stent from the proximal part to the distant part, presumably due to the manufacturing technology.

The Effect of Chronic Exposure of Graphene Nanoplates on the Viability and Motility of A549 Cells.

Graphene and its derivatives are popular nanomaterials used worldwide in many technical fields and biomedical applications. Due to such massive use, their anticipated accumulation in the environment is inevitable, with a largely unknown chronic influence on living organisms. Although repeatedly tested in chronic in vivo studies, long-term cell culture experiments that explain the biological response to these nanomaterials are still scarce. In this study, we sought to evaluate the biological responses of established model A549 tumor cells exposed to a non-toxic dose of pristine graphene for eight weeks. Our results demonstrate that the viability of the A549 cells exposed to the tested graphene did not change as well as the rate of their growth and proliferation despite nanoplatelet accumulation inside the cells. In addition, while the enzymatic activity of mitochondrial dehydrogenases moderately increased in exposed cells, their overall mitochondrial damage along with energy production changes was also not detected. Conversely, chronic accumulation of graphene nanoplates in exposed cells was detected, as evidenced by electron microscopy associated with impaired cellular motility.

A Complex In Vitro Degradation Study on Polydioxanone Biliary Stents during a Clinically Relevant Period with the Focus on Raman Spectroscopy Validation.

Biodegradable biliary stents are promising treatments for biliary benign stenoses. One of the materials considered for their production is polydioxanone (PPDX), which could exhibit a suitable degradation time for use in biodegradable stents. Proper material degradation characteristics, such as sufficient stiffness and disintegration resistance maintained for a clinically relevant period, are necessary to ensure stent safety and efficacy. The hydrolytic degradation of commercially available polydioxanone biliary stents (ELLA-CS, Hradec Králové, Czech Republic) in phosphate-buffered saline (PBS) was studied. During 9 weeks of degradation, structural, physical, and surface changes were monitored using Raman spectroscopy, differential scanning calorimetry, scanning electron microscopy, and tensile and torsion tests. It was found that the changes in mechanical properties are related to the increase in the ratio of amorphous to crystalline phase, the so-called amorphicity. Monitoring the amorphicity using Raman spectroscopy has proven to be an appropriate method to assess polydioxanone biliary stent degradation. At the 1732 cm-1 Raman peak, the normalized shoulder area is less than 9 cm-1 which indicates stent disintegration. The stent disintegration started after 9 weeks of degradation in PBS, which agrees with previous in vitro studies on polydioxanone materials as well as with in vivo studies on polydioxanone biliary stents.

The Outcomes of Cemented Femoral Revisions for Periprosthetic Femoral Fractures in the Elderly: Comparison with Cementless Stems.

INTRODUCTION: The present study compares the outcome of the long cemented stem and the revision uncemented stem used in periprosthetic femoral fractures. We propose that the revision with a long stem cemented prosthesis does not compromise fracture healing. PATIENTS AND METHODS: A consecutive series of 37 patients, operated between 2010 and 2017, were enrolled in a retrospective analysis. A long cemented stem was implanted in 21 patients (study group; age at operation: 63 to 89 years). A distally tapered fluted uncemented stem was used in 16 patients (control group; age at operation: 35 to 77 years). The clinical outcome was evaluated with Merle d'Aubigné and Postel scoring system. Standard radiographs were taken before surgery, at 3, 6, and 12 months postoperatively, and last follow-up. Any and all complications during the follow-up period were recorded. RESULTS: Although a significant difference (P = 0.006) was observed in the post-operative Merle d´Aubigné score over the 12-month follow-up period, no significant difference (P = 0.066) was found in the post-operative pain score between the study and control groups. Periodic radiographic assessments showed the disappearance of radiolucent lines and the diaphyseal part of the fracture was healed in all 34 followed-up cases during the first annual follow-up. Early surgical complications were seen in both groups, the medical complications were observed only in the study group. CONCLUSION: Based on our results, periprosthetic fractures of the femur after a total hip arthroplasty were associated with significant morbidity and increased mortality in elderly patients. Revision with a long-stem cemented prosthesis provided early pain-free weight-bearing without compromising the healing of femoral fractures in elderly patients with osteoporotic bone, altered mobility, poor balance, and reduced cognitive capacity.

Raman Spectroscopy as a Novel Method for the Characterization of Polydioxanone Medical Stents Biodegradation.

Polydioxanone (PPDX), as an FDA approved polymer in tissue engineering, is an important component of some promising medical devices, e.g., biodegradable stents. The hydrolytic degradation of polydioxanone stents plays a key role in the safety and efficacy of treatment. A new fast and convenient method to quantitatively evaluate the hydrolytic degradation of PPDX stent material was developed. PPDX esophageal stents were degraded in phosphate-buffered saline for 24 weeks. For the first time, the changes in Raman spectra during PPDX biodegradation have been investigated here. The level of PPDX hydrolytic degradation was determined from the Raman spectra by calculating the area under the 1732 cm-1 peak shoulder. Raman spectroscopy, unlike Fourier transform infrared (FT-IR) spectroscopy, is also sensitive enough to monitor the decrease in the dye content in the stents during the degradation. Observation by a scanning electron microscope showed gradually growing cracks, eventually leading to the stent disintegration. The material crystallinity was increasing during the first 16 weeks, suggesting preferential degradation of the amorphous phase. Our results show a new easy and reliable way to evaluate the progression of PPDX hydrolytic degradation. The proposed approach can be useful for further studies on the behavior of PPDX materials, and for clinical practice.

Clinicopathological Characteristics and Prognostic Factors in Ovarian Metastases from Right- and Left-Sided Colorectal Cancer.

BACKGROUND: Secondary tumors of the ovary (STOs) account for 10-25% of all ovarian malignancies, including metastases from primary gynecological tumors. Colorectal cancer (CRC) has been recognized as one of the most common causes of STOs in Western countries. Despite it being well-known that CRC originating from the right versus left side of the colon/rectum differ substantially, there is a paucity of information regarding the effect of the primary tumor sidedness on the clinicopathological characteristics of STOs. METHODS: This retrospective, observational chart review study included patients with histologically confirmed STOs of CRC origin diagnosed between January 2000 and December 2019. The clinicopathological characteristics of STOs originating from left-sided and right-sided CRC were compared. Univariable and multivariable analyses employing elastic net Cox proportional hazard models were used to evaluate potential prognostic factors. Further, the role of imaging methods in STOs diagnostics was evaluated. RESULTS: Fifty-one patients with STOs of colorectal origin were identified. The primary tumor originated in the right and left colon/rectum in 39% and 61% of the cases, respectively. STOs originating from right-sided primary tumors were more frequently bilateral, associated with peritoneal carcinomatosis, had the ovarian surface affected by the tumor, and contained a mucinous component. The independent prognostic factors for overall survival in the whole cohort included: the presence of macroscopic residual disease after cytoreductive surgery, menopausal status, the application of systemic therapy, and the application of targeted therapy. In 54% of cases, the imaging methods failed to determine the laterality of the STOs correctly as compared to pathological reports and/or intraoperative findings. CONCLUSION: STOs originating from left-sided and right-sided CRC show distinct clinicopathological characteristics. Moreover, different metastatic pathways might be employed according to the primary tumor sidedness. Considering the discrepancies between radiological assessment and histopathological findings regarding the laterality of STOs, bilateral adnexectomy should be advised whenever feasible.

Mechanical properties of a biodegradable self-expandable polydioxanone monofilament stent: In vitro force relaxation and its clinical relevance.

Biodegradable stents are promising treatments for many diseases, e.g., coronary artery disease, urethral diseases, tracheal diseases, and esophageal strictures. The mechanical properties of biodegradable stent materials play a key role in the safety and efficacy of treatment. In particular, insufficient creep resistance of the stent material could result in premature stent collapse or narrowing. Commercially available biodegradable self-expandable SX-ELLA stents made of polydioxanone monofilament were tested. A new, simple, and affordable method to measure the shear modulus of tiny viscoelastic wires is presented. The important mechanical parameters of the polydioxanone filament were obtained: the median Young's modulus was [Formula: see text] = 958 (922, 974) MPa and the shear modulus was [Formula: see text] = 357 (185, 387) MPa, resulting in a Poisson's ratio of ν = 0.34. The SX-ELLA stents exhibited significant force relaxation due to the stress relaxation of the polydioxanone monofilament, approximately 19% and 36% 10 min and 48 h after stent application, respectively. However, these results were expected, and the manufacturer and implanting clinician should be aware of the known behavior of these biodegradable materials. If possible, a biodegradable stent should be designed considering therapeutic force rather than initial force. Additionally, new and more advanced biodegradable shape-memory polymers should be considered for future study and use.

Digital orbitoplethysmograph: A new device to study the regional cerebral circulation using extraorbital plethysmography.

BACKGROUND: Noninvasive diagnostic methods utilizing pulse wave measurements on the surface of the head are an important tool in diagnosing various types of cerebrovascular disease. The measurement of extraorbital pressure fluctuations reflects intraocular and intracranial pressure changes and can be used to estimate pressure changes in intracranial arteries and the collateral circulation. NEW METHOD: In this paper, we describe our patented (CZ 305757) digital device for noninvasive measuring and monitoring of orbital movements using pressure detection. We conducted preclinical tests (126 measurements on 42 volunteers) to evaluate the practical capabilities of our device. Two human experts visually assessed the quality of the pressure pulsation and discriminability among various test conditions (specifically, subject lying, sitting, and the Matas carotid occlusion test). RESULTS: The results showed that our device provided clinically relevant outcomes with a sufficient level of detail of the pulse wave and a high reliability (not less than 85%) in all clinically relevant situations. It was possible to record the effect of the Matas carotid occlusion test. COMPARISON WITH EXISTING METHOD(S): Our fully noninvasive, lightweight (185 g), portable, and wireless device provides a considerably cheaper alternative to the current diagnostic methods (e.g., transcranial ultrasound, X-ray, or MRI angiography) for specific assessment of cerebral circulation. Within a minute, it can detect the Willis circle integrity and thus eliminate the potential risks associated with the Matas test using standard EEG. CONCLUSIONS: Our device represents an improvement and a valid alternative to the current methods diagnosing regional cerebral circulation.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

The toxic effect of cytostatics on primary cilia frequency and multiciliation.

The primary cilium is considered as a key component of morphological cellular stability. However, cancer cells are notorious for lacking primary cilia in most cases, depending upon the tumour type. Previous reports have shown the effect of starvation and cytostatics on ciliogenesis in normal and cancer cells although with limited success, especially when concerning the latter. In this study, we evaluated the presence and frequency of primary cilia in breast fibroblasts and in triple-negative breast cancer cells after treatment with cytostatics finding that, in the case of breast fibroblasts, primary cilia were detected at their highest incidence 72 hours after treatment with 120 nM doxorubicin. Further, multiciliated cells were also detected after treatment with 80 nM doxorubicin. On the other hand, treatment with taxol increased the number of ciliated cells only at low concentrations (1.25 and 3.25 nM) and did not induce multiciliation. Interestingly, triple-negative breast cancer cells did not present primary cilia after treatment with either doxorubicin or taxol. This is the first study reporting the presence of multiple primary cilia in breast fibroblasts induced by doxorubicin. However, the null effect of these cytostatics on primary cilia incidence in the evaluated triple negative breast carcinomas cell lines requires further research.

Comparative Study on the Application of Mesenchymal Stromal Cells Combined with Tricalcium Phosphate Scaffold into Femoral Bone Defects.

This prospective study sought to evaluate the healing quality of implanted ultraporous ß-tricalcium phosphate sown with expanded autologous mesenchymal stromal cells (MSCs) into femoral defects during revision hip arthroplasty. A total of 37 osseous defects in 37 patients were treated and evaluated concerning bone regeneration. Nineteen subjects received ß-tricalcium phosphate graft material serving as a carrier of expanded autologous MSCs (the trial group A), nine subjects received ß-tricalcium phosphate graft material only (the study group B) and nine subjects received cancellous allografts only (the control group C). Clinical and radiographic evaluations were scheduled at 6 weeks, 3, 6, and 12 months post-operatively, and performed at the most recent visit as well. All observed complications were recorded during follow-up to assess the use of an ultraporous ß-tricalcium phosphate synthetic graft material combined with expanded MSCs in bone defect repair. The resulting data from participants with accomplished follow-up were processed and statistically evaluated with a Freeman-Halton modification of the Fischer's exact test, a P < 0.05 value was considered to be significant. Whereas no significant difference was observed between the trial group A with ß-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs and control group C with cancellous impaction allografting in terms of the bone defect healing, significant differences were documented between the study group B with ß-tricalcium phosphate graft material only and control group C. Regarding adverse effects, six serious events were recorded during the clinical trial with no causal relationship to the cell product. ß-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs appears safe and promotes the healing of bone defects in a jeopardized and/or impaired microenvironment. This clinical trial was registered at the EU Clinical Trials Register before patient recruitment (Registration number: EudraCT number 2012-005599-33; Date of registration: 2013-02-04).

Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth.

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

The Outcomes of Total Hip Replacement in Patients with Parkinson's Disease: Comparison of the Elective and Hip Fracture Groups.

INTRODUCTION: The aim of the study was to compare the clinical outcomes following elective and traumatic total hip arthroplasty in Parkinson's disease patients. MATERIALS AND METHODS: Ten patients with osteoarthritis comprise the elective group (mean age at operation 74 years; mean follow-up 82 months). Thirteen patients with femoral fracture comprise the hip fracture group (mean age 76 years; mean follow-up 54 months). All patients were followed up at 6 and 36 months postoperatively and at the time of the latest follow-up. RESULTS: Despite the significant improvement in Merle d'Aubigné-Postel and pain scores, disability related to Parkinson's disease increased during the follow-up. Whereas more than 1/3 of hip fracture patients and all elective patients walked independently at 36 months after total hip arthroplasty, 43% of living patients from both groups were able to walk independently at the time of the latest follow-up. The medical complications were seen mainly in patients with hip fracture. CONCLUSIONS: Excellent pain relief with preserved walking ability without support of another person and acceptable complication profile was observed in Parkinson's disease patients at 36 months after elective total hip arthroplasty. This procedure may be indicated in Parkinson's disease patients after careful and individualized planning.