In vitro neurotoxicity testing: lessons from chemotherapy-induced peripheral neurotoxicity.

INTRODUCTION: Chemotherapy induced peripheral neurotoxicity (CIPN) is a long-lasting, or even permanent, late toxicity caused by largely used anticancer drugs. CIPN affects a growing population of cancer survivors and diminishes their quality of life since there is no curative/preventive treatment. Among several reasons for this unmet clinical need, there is an incomplete knowledge on mechanisms leading to CIPN. Therefore, bench side research is still greatly needed: in vitro studies are pivotal to both evaluate neurotoxicity mechanisms and potential neuroprotection strategies. AREAS COVERED: Advantages and disadvantages of in vitro approaches are addressed with respect to their applicability to the CIPN field. Different cell cultures and techniques to assess neurotoxicity/neuroprotection are described. PubMed search-string: (chemotherapy-induced) AND (((neuropathy) OR neurotoxicity) OR neuropathic pain) AND (in vitro) AND (((((model) OR SH-SY5Y) OR PC12) OR iPSC) OR DRG neurons); (chemotherapy-induced) AND (((neuropathy) OR neurotoxicity) OR neuropathic pain) AND (model) AND (((neurite elongation) OR cell viability) OR morphology). No articles published before 1990 were selected. EXPERT OPINION: CIPN is an ideal experimental setting to test axonal damage and, in general, peripheral nervous system mechanisms of disease and neuroprotection. Therefore, starting from robust preclinical data in this field, potentially, relevant biological rationale can be transferred to other human spontaneous diseases of the peripheral nervous system.

A novel "cells-on-particles" cytotoxicity testing platform in vitro: design, characterization, and validation against engineered nanoparticle aerosol.

The presented research introduces the "Cells-on-Particles" integrated aerosol sampling and cytotoxicity testing in vitro platform, which allows for the direct assessment of the biological effects of captured aerosol particles on a selected cell type without the need for extraction or resuspension steps. By utilizing particles with unaltered chemical and physical properties, the method enables simple and fast screening of biological effects on specific cell types, making it a promising tool for assessing the cytotoxicity of particulate matter in ambient and occupational air. Platforms fabricated from cellulose acetate (CA) and poly[ε]caprolactone (PCL) were proven to be biocompatible and promoted the attachment and growth of the human bronchial epithelial cell line BEAS-2B. The PCL platforms were exposed to simulated occupational aerosols of silver, copper, and graphene oxide nanoparticles. Each nanoparticle type exhibited different and dose-dependent cytotoxic effects on cells, evidenced by reduced cell viability and distinct, particle type-dependent gene expression patterns. Notably, copper nanoparticles were identified as the most cytotoxic, and graphene oxide the least. Comparing the "Cells-on-Particles" and submerged exposure ("Particles-on-Cells") testing strategies, BEAS-2B cells responded to selected nanoparticles in a comparable manner, suggesting the developed testing system could be proposed for further evaluation with more complex environmental aerosols. Despite limitations, including particle agglomeration and the need for more replicates to address variability, the "Cells-on-Particles" platform enables effective detection of toxicity induced by relatively low levels of nanoparticles, demonstrating good sensitivity and a relatively simpler procedure compared to standard 2D cell exposure methods.

Toward a more realistic estimate of exposure to chromium and nickel in soils of geogenic and/or anthropogenic origin: importance of oral bioaccessibility.

To enhance risk assessment for contaminated sites, incorporating bioavailability through bioaccessibility as a corrective factor to total concentration is essential to provide a more realistic estimate of exposure. While the main in vitro tests have been validated for As, Cd, and/or Pb, their potential for assessing the bioaccessibility of additional elements remains underexplored. In this study, the physicochemical parameters, pseudototal Cr and Ni concentrations, soil phase distribution, and oral bioaccessibility of twenty-seven soil samples were analysed using both the ISO 17924 standard and a simplified test based on hydrochloric acid. The results showed wide variability in terms of the concentrations (from 31 to 21,079 mg kg-1 for Cr, and from 26 to 11,663 mg kg-1 for Ni) and generally low bioaccessibility for Cr and Ni, with levels below 20% and 30%, respectively. Bioaccessibility variability was greater for anthropogenic soils, while geogenic enriched soils exhibited low bioaccessibility. The soil parameters had an influence on bioaccessibility, but the effects depended on the soils of interest. Sequential extractions provided the most comprehensive explanation for bioaccessibility. Cr and Ni were mostly associated with the residual fraction, indicating limited bioaccessibility. Ni was distributed in all phases, whereas Cr was absent from the most mobile phase, which may explain the lower bioaccessibility of Cr compared to that of Ni. The study showed promising results for the use of the simplified test to predict Cr and Ni bioaccessibility, and its importance for more accurate human exposure evaluation and effective soil management practices.

Identification of two novel chemical classes of Autotaxin (ATX) inhibitors using Enalos Asclepios KNIME nodes.

Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.

A Dynamic Elbow Testing Apparatus for Simulating Elbow Joint Motion in Varying Shoulder Positions.

Purpose: To develop and evaluate the capabilities of a dynamic elbow testing apparatus that simulates unconstrained elbow motion throughout the range of humerothoracic (HTA) abduction. Methods: Elbow flexion was generated by six computer-controlled electromechanical actuators that simulated muscle action, while six degree-of-freedom joint motion was measured using an optical tracking device. Repeatability of joint kinematics was assessed at four HTA angles (0°, 45°, 90°, 135°) and with two muscle force combinations (A1-biceps brachialis, brachioradialis and A2-biceps, brachioradialis). Repeatability was determined by comparing kinematics at every 10° of flexion over five flexion-extension cycles (0° to 100°). Results: Multiple muscle force combinations can be used at each HTA angle to generate elbow flexion. Trials showed that the testing apparatus produced highly repeatable joint motion at each HTA angle and with varying muscle force combinations. The intraclass correlation coefficient was greater than 0.95 for all conditions. Conclusions: Repeatable smooth cadaveric elbow motion was created that mimicked the in vivo situation. Clinical relevance: These results suggest that the dynamic elbow testing apparatus can be used to characterize elbow biomechanics in cadaver upper extremities.

Mining the ZINC database of natural products for specific, testosterone-like, OXER1 antagonists.

OXER1, the receptor for the oxidized arachidonic acid metabolite 5-oxo-ETE has been reported to play a significant role in inflammatory responses, being responsible for leucocyte chemotactic responses. Recently, we have identified OXER1 (GPR170) as a membrane receptor for androgens in prostate and breast cancer cells. Testosterone action via OXER1 induces specific Ca2+ release from intracellular organelles, modifies polymerized actin distribution induces apoptosis and decreases cancer cell migration. These actions are antagonized by 5-oxo-ETE. In addition, 5-oxo-ETE through a Gαi protein decreases cAMP, an action antagonized by testosterone. In this work, we mined the ZINC15 database, using QSAR, for natural compounds able to signal through Gαi and Gßγ simultaneously, mimicking testosterone actions, as well as for specific Gßγ interactors, inhibiting 5-oxo-ETE tumor promoting actions. We were able to identify four druggable Gαßγ and seven Gßγ specific OXER1 interactors. We further confirmed by bio-informatic methods their binding to the 5-oxo-ETE/testosterone binding groove of the receptor, their ADME properties and their possible interaction with other receptor and/or enzyme targets. Two compounds, ZINC04017374 (Naphthofluorescein) and ZINC08589130 (Puertogaline A) were purchased, tested in vitro and confirmed their OXER1 Gßγ and Gαßγ activity, respectively. The methodology followed is useful for a better understanding of the mechanism by which OXER1 mediates its actions, it has the potential to provide structural insights, in order to design small molecular specific interactors and ultimately design new anti-inflammatory and anti-cancer agents. Finally, the methodology may also be useful for identifying specific agonists/antagonists of other GPCRs.

Advanced in vitro hemocompatibility assessment of biomaterials using a new flow incubation system.

Physiologically relevant in vitro hemocompatibility assessment of biomaterials remains challenging. We present a new setup that enables standardized whole blood incubation of biomedical materials under flow. A blood volume of 2 mL is recirculated over test surfaces in a custom-made parallel plate incubation system to determine the activation of hemostasis and inflammation. Controlled physiological shear rates between 125 s-1 and 1250 s-1 and minimized contact to air are combined with a natural-like pumping process. A unique feature of this setup allows tracing adhesion of blood cells to test surfaces microscopically in situ. Validation testing was performed in comparison to previously applied whole blood incubation methodologies. Experiments with the newly developed setup showed that even small obstacles to blood flow activate blood (independent of materials-induced blood activation levels); that adhesion of blood cells to biomaterials equilibrates within 5 to 10 min; that high shear rates (1250 compared to 375 s-1) induce platelet activation; and that hemolysis, platelet factor 4 (PF4) release and platelet loss - but not thrombin formation - depend on shear rate (within the range investigated, 125 to 1250 s-1).

Significance of cell culture phases for utilizing 3D cultures of A549 cells for in vitro studies.

Three-dimensional (3D) culture systems of human cancer cell lines have become popular experimental models for a variety of applications including drug screening. It is understood that the 2D and 3D cultures of the same cell line behave differently in several aspects. One such difference is in the duration of cell culture phases (the lag, log, plateau and the decline stages). We obtained 3D cultures of A549 cells on agarose hydrogels. We observed and compared the morphological differences in the progression of 2D and 3D cultures of A549 cells in a time-dependent manner. The morphological features along with the cell counts and viabilities obtained for the 2D and 3D cultures at different time intervals clearly indicate that the cell culture phases occurred as more extended one for the 3D cultures compared to that of the 2D counterparts. The plateau stage for the 2D and 3D cultures occurred at 48 and 69 h, respectively. Such cell culture phase durations can be different for different cell lines as a function of their doubling times. We propose that the cell culture phase durations for any cell line should be first established before using them for drug testing or for studies involving toxicity to obtain useful results from 3D cell cultures. Also, we propose that the late-exponential (lag) phase of 3D cultures of cancer cell lines is the most ideal one for drug testing owing to the various optimal features of the aggregates in this cell culture phase.

Insight into Antibiotic Synergy Combinations for Eliminating Colistin Heteroresistant Klebsiella pneumoniae.

Colistin heteroresistance has been identified in several bacterial species, including Escherichia coli and Klebsiella pneumoniae, and may underlie antibiotic therapy failures since it most often goes undetected by conventional antimicrobial susceptibility tests. This study utilizes population analysis profiling (PAP) and time-kill assay for the detection of heteroresistance in K. pneumoniae and for evaluating the association between in vitro regrowth and heteroresistance. The mechanisms of colistin resistance and the ability of combination therapies to suppress resistance selection were also analysed. In total, 3 (18%) of the 16 colistin-susceptible strains (MIC ≤ 2 mg/L) were confirmed to be heteroresistant to colistin by PAP assay. In contrast to the colistin-susceptible control strains, all three heteroresistant strains showed regrowth when exposed to colistin after 24 h following a rapid bactericidal action. Colistin resistance in all the resistant subpopulations was due to the disruption of the mgrB gene by various insertion elements such as ISKpn14 of the IS1 family and IS903B of the IS5 family. Colistin combined with carbapenems (imipenem, meropenem), aminoglycosides (amikacin, gentamicin) or tigecycline was found to elicit in vitro synergistic effects against these colistin heteroresistant strains. Our experimental results showcase the potential of combination therapies for treatment of K. pneumoniae infections associated with colistin heteroresistance.

Protein extracts from regenerating lizard tail show an inhibitory effect on human cancer cells cultivated in-vitro.

BACKGROUND: accumulating evidence indicates that during tail regeneration in lizards the initial stage of regenerative blastema is a tumor-like proliferative outgrowth that rapidly elongates into a new tail composed of fully differentiated tissues. Both oncogenes and tumor-suppressors are expressed during regeneration, and it has been hypothesized that an efficient control of cell proliferation avoids that the blastema is turned into a tumor outgrowth. METHODS: in order to determine whether functional tumor-suppressors are present in the growing blastema we have utilized protein extracts collected from early regenerating tails of 3-5 mm that have been tested for a potential anti-tumor effect on in-vitro culture by using cancer cell lines from human mammary gland (MDA-MB-231) and prostate cancer (DU145). RESULTS: at specific dilutions, the extract determines a reduction of viability in cancer cells after 2-4 days of culture, as supported by statistical and morphological analyses. While control cells appear viable, treated cells result damaged and produce an intense cytoplasmic granulation and degeneration. CONCLUSIONS: this negative effect on cell viability and proliferation is absent using tissues from the original tail supporting the hypothesis that only regenerating tissues synthesize tumor-suppressor molecules. The study suggests that the regenerating tail of lizard at the stages here selected contains some molecules that determine inhibition of cell viability on the cancer cells analyzed.

Cross-link augmentation enhances CFR-PEEK short fixation in lumbar metastasis stabilization.

Introduction: Spinal stability plays a crucial role in the success of the surgical treatment of lumbar vertebral metastasis and, in current practice, less invasive approaches such as short constructs have been considered. Concurrently, carbon fiber-reinforced (CFR) poly-ether-ether-ketone (PEEK) fixation devices are expanding in oncologic spinal surgery thanks to their radiotransparency and valid mechanical properties. This study attempts to provide an exhaustive biomechanical comparison of different CFR-PEEK surgical stabilizations through a highly reproducible experimental setup. Methods: A Sawbones biomimetic phantom (T12-S1) was tested in flexion, extension, lateral bending, and axial rotation. An hemisome lesion on L3 vertebral body was mimicked and different pedicle screw posterior fixations were realized with implants from CarboFix Orthopedics Ltd: a long construct involving two spinal levels above and below the lesion, and a short construct involving only the levels adjacent to L3, with and without the addition of a transverse rod-rod cross-link; to provide additional insights on its long-term applicability, the event of a pedicle screw loosening was also accounted. Results: Short construct reduced the overloading onset caused by long stabilization. Particularly, the segmental motion contribution less deviated from the physiologic pattern and also the long-chain stiffness was reduced with respect to the prevalent long construct. The use of the cross-link enhanced the short stabilization by making it significantly stiffer in lateral bending and axial rotation, and by limiting mobiliza-tion in case of pedicle screw loosening. Discussion: The present study proved in vitro the biomechanical benefits of cross-link augmentation in short CFR-PEEK fixation, demonstrating it to be a potential alternative to standard long fixation in the surgical management of lumbar metastasis.

The In Vitro Inhibitory Effect of Selected Asteraceae Plants on Pancreatic Lipase Followed by Phenolic Content Identification through Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS).

Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective strategies in obesity treatment is the inhibition of PNLIP, which is possible to be achieved by specific phenolic compounds occurring in high abundance in some plants. In this study, a multidisciplinary approach is presented investigating the PNLIP inhibitory effect of 33 plants belonging in the Asteraceae botanical family. In the first stage of the study, a rapid and cost-efficient PNLIP assay in a 96-microwell plate format was developed and important parameters were optimized, e.g., the enzyme substrate. Upon PNLIP assay optimization, aqueous and dichloromethane Asteraceae plant extracts were tested and a cut-off inhibition level was set to further analyze only the samples with a significant inhibitory effect (inhibitory rate > 40%), using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) method. Specifically, a metabolomic suspect screening was performed and 69 phenolic compounds were tentatively identified, including phenolic acids, flavonoids, flavonoid-3-O-glycosides, and flavonoid-7-O-glycosides, amongst others. In the case of aqueous extracts, phytochemicals known for inducing PNLIP inhibitory effect, e.g., compounds containing galloyl molecules or caffeoylquinic acids, were monitored in Chrysanthemum morifolium, Grindella camporum and Hieracium pilosella extracts. All in all, the presented approach combines in vitro bioactivity measurements to high-end metabolomics to identify phenolic compounds with potential medicinal and/or dietary applications.

Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models.

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAirTM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAirTM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

Peptide-based targeted cancer therapeutics: Design, synthesis and biological evaluation.

Cancer is the leading cause for human mortality together with cardiovascular diseases. Abl (Abelson) tyrosine kinases play a fundamental role in transducing various signals that control proliferation, survival, migration and invasion in several cancers such as Chronic Myeloid Leukemia (CML), breast cancer and brain cancer. For these reasons Abl tyrosine kinases are considered important biological targets in drug discovery. In this study a series of lysine-based oligopeptides with expected Abl inhibitory activity were designed resembling the binding of FDA-approved drugs (i.e. of Imatinib and Nilotinib), synthesized, purified by High Performance Liquid Chromatography (HPLC), analyzed by mass spectrometry (MS) and biologically tested in vitro in CML (AR-230 and K-562), breast cancers (MDA-MB 231 and MDA-MB 468) and glioblastoma cell lines (U87 and U118). The solid-phase peptide synthesis (SPPS) by Fmoc (9-fluorenylmethoxycarbonyl) chemistry was used to synthesize target compounds. AutoDock Vina was applied for simulation binding to Abl. The biological activities were measured evaluating cytotoxic effect, induction of apoptosis and inhibition of cancer cells migration. The new peptides exhibited different concentration-dependent antiproliferative effect against the tumor cell lines after 72 h treatment. The most promising results were obtained with the U87 glioblastoma cell line where a significant reduction of the migration ability was detected with one compound (H-Lys1-Lys2-Lys3-NH2).

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the "2 out of 3" Defined Approach.

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the "2 out of 3" defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these dif-ferent testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applica-bility domain of the kDPRA.

Synthesis, In Vitro Testing, and Biodistribution of Surfactant-Free Radioactive Nanoparticles for Cancer Treatment.

New forms of cancer treatment, which are effective, have simple manufacturing processes, and easily transportable, are of the utmost necessity. In this work, a methodology for the synthesis of radioactive Gold-198 nanoparticles without the use of surfactants was described. The nuclear activated Gold-198 foils were transformed into H198AuCl4 by dissolution using aqua regia, following a set of steps in a specially designed leak-tight setup. Gold-198 nanoparticles were synthesized using a citrate reduction stabilized with PEG. In addition, TEM results for the non-radioactive product presented an average size of 11.0 nm. The DLS and results for the radioactive 198AuNPs presented an average size of 8.7 nm. Moreover, the DLS results for the PEG-198AuNPs presented a 32.6 nm average size. Cell line tests showed no cytotoxic effect in any period and the concentrations were evaluated. Furthermore, in vivo testing showed a high biological uptake in the tumor and a cancer growth arrest.

The porcine abattoir blood model-Evaluation of platelet function for in-vitro hemocompatibility investigations.

BACKGROUND: The major obstacle of blood-contacting medical devices is insufficient hemocompatibility, particularly thrombogenicity and platelet activation. Pre-clinical in-vitro testing allows for the evaluation of adverse thrombogenicity-related events, but is limited, among others, by the availability and quantity of human blood donations. The use of animal blood is an accepted alternative for several tests; however, animal and particularly abattoir blood might present species-specific differences to human blood as well as elevated blood values, and pre-activated platelets due to stressed animals and non-standardized blood collection. MATERIAL & METHODS: To this end, we investigated porcine abattoir blood in comparison to human donor blood with the focus on platelet pre-activation and remaining activation potential. By means of light transmission aggregometry, aggregation kinetics of platelet rich plasma after stimulation with three different concentrations of each adenosine diphosphate (ADP) (5 µM, 10 µM, 20 µM) and collagen (2.5 µg/ml, 5 µg/ml, 10 µg/ml) were monitored. RESULTS: The activation with collagen revealed no significant differences in platelet behavior of the two species. In contrast, stimulation with ADP resulted in a lower maximum aggregation and a high disaggregation for porcine abattoir blood. The latter is a species-specific phenomenon of porcine platelets. Variations within each study cohort were comparable for human and abattoir pig. CONCLUSION: The similarities in platelet activation following collagen stimulation and the preservation of the porcine-specific reaction to ADP prove a general functionality of the abattoir blood. This finding provides a first step towards the complete validation of the porcine abattoir blood model.

3D Microtissues Mimic the Architecture, Estradiol Synthesis, and Gap Junction Intercellular Communication of the Avascular Granulosa.

Humans are consistently exposed to thousands of untested chemicals that have been detected in the follicular fluid of the ovaries, and can disrupt reproductive health. Human granulosa cells (GCs) are the functional unit of the ovarian follicle with steroidogenic and signaling activities, and play a pivotal role in oocyte development. During follicle progression, GCs multiply to form a 3D avascular structure, and establish gap junction intercellular communication (GJIC) that is critical to maintaining optimal viability and function. We developed a high-throughput in vitro platform of human GCs for the screening of chemicals that can impact GJIC and estradiol (E2) production of human granulosa. Our granulosa 3D microtissues fabricated with human ovarian granulosa-like tumor KGN cells are multicell-layered structures that mimic the avascular granulosa layers surrounding the oocyte. These microtissues robustly expressed the steroidogenic CYP19 aromatase enzyme and GJIC intercellular membrane channel, connexin 43. Granulosa microtissues produced E2 at rates comparable to primary human GCs as previously reported. E2 production was suppressed by the CYP19 inhibitor, letrozole, and induced by CYP19 activators, bisphenol A at 100 µM, and genistein at 100 µM. Granulosa microtissues displayed active GJIC function, as demonstrated by the connexin 43-dependent diffusion of calcein fluorescent dye from microtissue surface to the core using high-throughput confocal microscopy in conjunction with our open-sourced automated image analysis tool. Overall, our 3D human granulosa screening platform is highly promising for predictive and efficient in vitro toxicity testing to screen for chemicals that contaminate follicular fluid and may affect fertility.

Experimental analysis of early periprosthetic femoral fractures with uncemented straight hip stems.

BACKGROUND: The periprosthetic femoral fracture is one of the most severe complications after total hip arthroplasty and is associated with an increased mortality. The underlying causes and the patient- and implant-specific risk factors of periprosthetic femoral fractures remain insufficiently understood. The aim of this study was to gain a more profound understanding of the underlying fracture mechanisms and to provide experimental datasets for validation of computational models. METHODS: Six cadaveric femurs were implanted with straight hip stems (Zweymueller design) and loaded until fracture reproducing the clinically relevant load cases stumbling and sideways fall. Displacements and the strain distribution on the surface of the femurs and implants, as well as the fracture load and implant subsidence were measured. FINDINGS: For the load case stumbling the mean fracture load was 6743 N and two different mechanisms leading to fracture could be identified: high subsidence with low femoral bending and small subsidence with high femoral bending. For the load case sideways fall the mean fracture load was 1757 N and both tested femurs fractured due to a rotation of the hip stem around its own axis. The detailed datasets provided by this study can be used in future computational models. INTERPRETATION: We demonstrated that the underlying fracture mechanisms of periprosthetic femoral fractures can be fundamentally different in the load case stumbling. The seating and exact position of the hip stem in the femur may correlate with implant subsidence and therefore lead to different types of fracture mechanisms resulting in different patient-specific fracture risks.

3D model-assisted instrumentation of the pediatric spine: a technical note.

BACKGROUND: Instrumentation of the pediatric spine is challenging due to anatomical constraints and the absence of specific instrumentation, which may result in iatrogenic injury and implant failure, especially in occipito-cervical constructs. Therefore, preoperative planning and in vitro testing of instrumentation may be necessary. METHODS: In this paper, we present a technical note on the use of 1:1 scale patient-specific 3D printed spinal models for preoperative assessment of feasibility of spinal instrumentation with conventional spinal implants in pediatric spinal pathologies. RESULTS: The printed 3D models fully matched the intraoperative anatomy and allowed a preoperative confirmation of the feasibility of the planned instrumentation with conventional screws for adult patients. In addition, the possibility of intraoperative model assessment resulted in better intraoperative sense of spinal anatomy and easier freehand screw insertion, thereby reducing the potential for iatrogenic injury. All 3D models were printed at the surgical department at a very low cost, and the direct communication between the surgeon and the dedicated specialist allowed for multiple models or special spinal segments to be printed for more detailed consideration. CONCLUSIONS: Our technical note highlights the critical steps for preoperative virtual planning and in vitro testing of spinal instrumentation on patient-specific 3D printed models at 1:1 scale. The simple and affordable method helps to better visualize pediatric spinal anatomy and confirm the suitability of preplanned conventional spinal instrumentation, thereby reducing X-ray exposure and intraoperative complications in freehand screw insertion without navigation.