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Objective To assess the actual xylitol content in sugar-free chewing gums available in a market of Saudi Arabia and investigate its effect on acid production and pH change in vitro. Materials and methods: A total of 29 different brands of xylitol-containing sugar-free chewing gums were collected from five major grocery stores in Saudi Arabia. Xylitol was extracted and its concentration was determined using the D-Sorbitol/Xylitol Enzymatic Kit (Megazyme; Bray, Wicklow, Ireland). The pH of the extracts and the amount of acid production for each product was measured after 15-minute and 30-minute incubation with Streptococcus mutans. Descriptive analysis, concentrations, and one-way analysis of variance (ANOVA) with the least significant difference (LSD) as multiple comparisons were performed. Results: The xylitol content in grams was clearly stated on the labels of 16 products, while it was stated in percentages on the labels of ten products. The mean pH of most of the tested products was 5.857 ± 0.096. Significant differences in pH were recorded among 20 products (p ≤ 0.05). Highly significant differences in pH (p = 0.001) were observed in five of the products. Conclusion: The results of this study indicate, using an in vitro model, that xylitol can significantly affect salivary pH. The actual xylitol content in most brands of chewing gums currently available in the markets of Saudi Arabia is less than the concentration recommended for prevention of caries. Accurate information with proper labeling is required to enable dental professionals to take correct and informed decisions about recommending the use of these products.

In order to achieve a high sample throughput, permeation experiments are often carried out using 96-well sandwich plates. Even though agitation is regarded as important, permeation studies in 96-well format are often carried out without agitation since orbital shaking, the most common agitation method for 96-well plates, has been reported to create difficulties (e.g., well-to-well cross-talk), and high cost and low availability limits the use of other agitation techniques (e.g., magnetic stirring). This study investigates how orbital shaking and magnetic stirring affect the apparent permeability of model compounds with different water-solubilities (methylene blue, carbamazepine, and albendazole) using a novel 96-well sandwich plate comprising a cellulose-hydrate membrane (PermeaPlain (R) plate). Orbital shaking was found less efficient than magnetic stirring in terms of homogeneously distributing a small volume of dye within the donor compartment. Furthermore, in terms of achieving maximum trans-barrier flux, magnetic stirring was found a more effective agitation method than orbital shaking. Obviously, with orbital shaking the medium in the bottom compartment of the sandwich plates never was mixed in-phase. The impact of insufficient mixing on permeation was found strongest with the most lipophilic compound, which correlates with literature reports that the contribution of the unstirred water layer towards the overall resistance of the barrier is most expressed in case of lipophilic drugs. Finally, it was tested how different liquid volumes in the bottom compartment of the plates affect the well-to-well cross-talk during permeation experiments under orbital shaking. This study revealed that 250-300 mu L should be used in the bottom compartment of the sandwich plates to reduce well-to-well cross-talk when using orbital shaking for agitation. (C) 2021 Published by Elsevier Inc. on behalf of American Pharmacists Association.

The neural circuits of the central nervous system are the regulatory pathways for feeling, motion control, learning, and memory, and their dysfunction is closely related to various neurodegenerative diseases. Despite the growing demand for the unraveling of the physiology and functional connectivity of the neural circuits, their fundamental investigation is hampered because of the inability to access the components of neural circuits and the complex microenvironment. As an alternative approach, in vitro human neural circuits show principles of in vivo human neuronal circuit function. They allow access to the cellular compartment and permit real-time monitoring of neural circuits. In this review, we summarize recent advances in reconstituted in vitro neural circuits using engineering techniques. To this end, we provide an overview of the fabrication techniques and methods for stimulation and measurement of in vitro neural circuits. Subsequently, representative examples of in vitro neural circuits are reviewed with a particular focus on the recapitulation of structures and functions observed in vivo, and we summarize their application in the study of various brain diseases. We believe that the in vitro neural circuits can help neuroscience and the neuropharmacology. Statement of significance Despite the growing demand to unravel the physiology and functional connectivity of the neural circuits, the studies on the in vivo neural circuits are frequently limited due to the poor accessibility. Furthermore, single neuron-based analysis has an inherent limitation in that it does not reflect the full spectrum of the neural circuit physiology. As an alternative approach, in vitro engineered neural circuit models have arisen because they can recapitulate the structural and functional characteristics of in vivo neural circuits. These in vitro neural circuits allow the mimicking of dysregulation of the neural circuits, including neurodegenerative diseases and traumatic brain injury. Emerging in vitro engineered neural circuits will provide a better understanding of the (patho-)physiology of neural circuits.

Recent research on Gut-Brain Axis (GBA) has suggested that the gut luminal environment, including the dietary components and commensal microbiota, could affect behavior, emotion, and cognitive abilities in the brain. Various signaling pathways exist between the gut and the brain, and several studies have implied exosomes could mediate the communication. Current research on GBA has relied heavily on animal models, making the research challenging. Recent advances in organ-on-a-chip technology could be a solution for GBA research. In present work, we have developed a modular microfluidic chip, where gut epithelial and brain endothelial cells were co-cultured to form the gut epithelial barrier and the Blood-Brain Barrier (BBB) interconnected via microfluidic channels. These modules can be easily assembled and disassembled, and cell barriers were well formed when co-cultured under appropriate fluidic flow. Responses to microbial byproducts were consistent with previously known observations of interaction between gut epithelium and BBB. We observed the transport of fluorescently labeled exosomes across the gut barrier towards the BBB. Our results suggest this model can be used as a novel in vitro model of GBA for studying the interaction between the gut and the brain.

Using a novel in vitro model system combining biochemical/histologic with bioengineering approaches has provided significant insights into the physiology of fetal membrane weakening and rupture along with potential mechanistic reasons for lack of efficacy of currently clinically used agents to prevent preterm premature rupture of the membranes (pPROM) and preterm births. Likewise, the model has also facilitated screening of agents with potential for preventing pPROM and preterm birth.

Purpose Additive manufacturing (AM) is an innovation in today's medical field. It helps create a patient-specific 3D model of bones, nerves, organs and blood vessel. Now, doctors and surgeons have successfully applied this technology to plan different types of treatments and also cancers. The purpose of this paper is to study the requirements and applications of AM for cancer treatment. Methods Today AM is used for the application of regenerative medicine. It can help develop and improve in-vitro models in the tumour microenvironment. Thus, to identify its applications, related research papers on AM for cancer are studied. Results 3D printing technology has a unique capability to manufacture tumour models. It helps to understand the status of the whole tumor. Here in this paper, we studied the capabilities of AM technologies for cancer treatment. This study discusses necessary process steps, followed by 3D printing and finally identified significant applications of these technologies for cancer treatment with a brief description. Conclusion 3D printed tumor helps to provide faithful studies on metastasis. These models create a promising platform to construct biomimetic models. By accurate manufacturing of in vitro model, this technology seems to be the best tool to facilitate complex treatment, surgery and therapies. It shows great potential for the printing of organs. This technology is time-saving and avoids time-consuming processes. It has been analysed that AM helps in reducing the duration of treatment of a cancer patient. Researchers are focusing on reducing pain during therapies. They have also tested 3D printed tumor models for different drugs, to eliminate the risks. These models play a significant role in courses of treatments.

The anticoccidial activity of thymol, carvacrol, and saponins was assessed in an in vitro model of coccidiosis. Eimeria spp. sporozoites were collected from field samples, characterized and used for 2 different invasion assays on Madin-Darby Bovine Kidney cells (MDBK). The cells were challenged with 5 x 104 sporozoites without (control) or with various treatments: saponins (10 ppm), thymol and carvacrol (7 ppm each), or a combination of saponins, thymol, and carvacrol at 2 doses; MIX 1 (saponins 5 ppm, thymol 3.5 ppm and carvacrol 3.5 ppm) and MIX 2 (saponins 10 ppm, thymol 7 ppm and carvacrol 7 ppm). The treated cells were incubated at 37°C for 24 hours (invasion assay 1) and for 2, 24, and 48 hours (invasion assay 2). The efficiency of invasion was determined by counting the sporozoites left in the supernatant that were not able to invade the cells, while intracellular Eimeria DNA was detected by qPCR to confirm the data. Data were analyzed with ANOVA and differences were considered significant when p-value was ≤ 0.05. Data from invasion assay 1 showed that the thymol and carvacrol-containing blends significantly reduced invasion, especially in combination with saponins at the highest dose. Saponins alone did not have a strong inhibiting activity but acted synergistically with the other molecules. Interestingly, in invasion assay 2 it was found that the effect of the highest dose of the blend of saponins, thymol and carvacrol was already visible at 2 hours post infection, while the other treatments were significantly successful at 24 hours post infection. The invasion assay protocol was designed to screen molecules in vitro starting from field fecal samples and it can respresent a potential tool in Eimeria research. Moreover this study shows that invasion in MDBK cells by Eimeria sporozoites is inhibited in presence of thymol, carvacrol and saponins, thus highlighting the anticoccidial potential of these compounds.

Implantation represents a critical step for embryonic development and pregnancy. Its success depends on the complex interplay between a receptive endometrium and a competent embryo. Implantation-related events remain hardly accessible, making implantation a true "black box" in developmental biology. Improved in vitro models are becoming useful experimental tools, as they are considerably more accessible than in vivo models, easier to manipulate, and permit the use of human cells or tissues, thus increasing the translational value of the studies. In this Review, we briefly summarize the relevant cell types and structures involved into the process of implantation, in order to outline which compartments are indispensable for creating the perfect in vitro model. We also critically address advantages and limitations of available models and assess their application potential. Moreover, we examine the chances and challenges brought by the latest approaches to recapitulate the endometrial compartment, as well as by peri-implantational embryoids.

Purpose Despite proven benefits of in-utero spina bifida (SB) repair, ≥ 30% of children at birth have Chiari II malformation or cerebrospinal fluid (CSF) leakage from the repair site. Our study's purpose was to determine CSF pressures in the myelomeningocele sac during mid-gestation in order to design an in-vitro model for evaluating different surgical methods used for watertight closure during in-utero SB repair. Methods CSF pressures were measured during in-utero SB repair at mid-gestation. An in-vitro chicken thigh model, simulating fetal tissue, tested watertight closure when attached to the base of a water column. Primary closure methods were evaluated using defect sizes of 20 × 3 mm for minimal traction or 20 × 8 mm for moderate traction. Additionally, 3 common in-utero repair patches were compared using 15 × 15 mm defects. Results Using 6–12.5 cm pre-determined CSF pressures, 165 in-vitro experiments were performed. Regardless of methodology we found that in 66 primary-based closures that minimal versus moderate wound edge traction provided better seals. The locking method was superior to the non-locking technique for watertight closure in 99 patch-based closures. Conclusions Minimal wound edge traction was best for primary closures, and locking sutures ideal for patch-based closures, however surgical techniques should be individualized to improve upon clinical outcomes.

Shiga toxin-producing Escherichia coli (STEC) are known to adapt and survive acidic conditions. Acid-adaptation also may result in cross-protection against other stressors present in foods, such as reduced water activity (aw), refrigeration conditions, or the presence of antimicrobials. Three independent experiments were conducted with non-adapted (control) and acid-adapted E. coli O157:H7 and non-O157:H7 serogroups (E. coli O26, O45, O103, O111, O121 and O145) in vitro and in situ. Experiment 1: STEC cocktails in media were left untreated (non-adapted; control) or adapted (pH 5.0), exposed to mild acidic conditions (pH 4.5) or reduced aw (0.88 and 0.75) for 4 days at 24 °C, and evaluated for survival. Experiment 2: Non-adapted and acid-adapted STEC cocktails were subjected to desiccation (28 days at 24 °C) on sterile paper disks, and remaining pathogen populations were determined. Experiment 3: Control and acid-adapted STEC were subjected to mild acidic conditions (pH 4.5) and low aw (0.88 or 0.78) in ground beef slurries (GBS; 4 days at 24 °C) and surviving populations enumerated. Results from experiment 1 demonstrated that populations of non-O157:H7 STEC serogroups O45, O103, O111 and O121 were significantly different from populations of E. coli O157:H7, and non-O157:H7 STEC serogroups O26 and O145. In contrast, when STEC serogroups were acid-adapted, all populations of non-O157:H7 STEC were significantly different from populations of E. coli O157:H7. Results of experiments in media with aw of 0.88 or 0.78 demonstrated that there was no significant difference between E. coli O157:H7 and any of the non-O157:H7 STEC, regardless of acid-adaptation. In experiment 2, exposure to desiccation resulted in no significant difference in survival between E. coli O157:H7 and non-O157:H7 STEC, regardless of acid-adaptation. Experiment 3 in acidified GBS demonstrated that all non-O157:H7 STEC behaved similarly to E. coli O157:H7, except for E. coli O145, which had a higher reduction than O157:H7. There were no significant differences in bacterial populations associated with GBS with modified aw. Collectively, these results suggest than non-O157:H7 STEC behave similarly to E. coli O157:H7 when exposed to low pH (

Background Perfluorocarbon (PFC)-nanoemulsions (NE) are a convenient tool for 19F magnetic resonance imaging in cell and animal experiments. Typical preparation methods, like high-pressure homogenization or microfluidization, produce nanoemulsions in mL-scale. However, experiments usually require only miniscule amounts of PFC-NE, several 100 µL. For site-specific imaging tissue-specific ligands, e.g. peptides or antibodies, are covalently bound to the NE surface. This requires the use of expensive functionalized phospholipids containing reactive groups (e.g. maleimide), which often deteriorate quickly in liquid storage, rendering the manufacturing process highly cost-inefficient. A technique to manufacture storage stable NE that maintain their functionality for coupling of various ligands is desired. Methods and results Different PFC-NE formulations and preparation techniques were compared and the most suitable of these was tested in short-, as well as long-term stability tests. Droplet size stability was investigated by dynamic light scattering and cryogenic transmission electron microscopy over 1.5 a. Surface modifiability was assessed by a fluorescence assay. The utility of these NE was proven in an in vitro model. Conclusion The established PFC-NE platform offers a cost-efficient way to produce larger amounts of long-term storable imaging agents, which can be surface-modified on demand for application in targeted 19F MRI.

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

The Caco-2 permeability assay is a well-accepted in vitro model to evaluate compounds' potential for oral absorption at early discovery. However, for many lipophilic compounds, no meaningful Caco-2 data could be generated due to their low solubility in assay buffer and/or poor recovery from the assay. In our previous study, we reported an organic catch approach to improve compound recovery. To further reduce compound loss and increase solubility in aqueous buffer, we explored the addition of bovine serum albumin (BSA). However, in contrast to the commonly used BSA level at 4%, a lower level of BSA was selected in an effort to minimize the potential risk of missing the identification of efflux substrates, and to avoid the extensive sample cleanup needed for 4% BSA. Through a systematic evaluation, it was found that 0.5% BSA was effective in enhancing compound solubility and reducing nonspecific binding, which allowed reliable assessment of the permeability and efflux potential for lipophilic compounds. Also, with an optimized sample handling process, no extra sample cleanup was required before liquid chromatography-mass spectrometry (LC-MS) analysis. The implementation of this assay has enabled accurate permeability assessment for compounds that had poor solubility and/or poor mass balance under the non-BSA assay conditions.

The study of human cardiomyopathies and the development and testing of new therapies has long been limited by the availability of appropriate in vitro model systems. Cardiomyocytes are highly specialized cells whose internal structure and contractile function are sensitive to the local microenvironment and the combination of mechanical and biochemical cues they receive. The complementary technologies of human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) and microphysiological systems (MPS) allow for precise control of the genetics and microenvironment of human cells in in vitro contexts. These combined systems also enable quantitative measurement of mechanical function and intracellular organization. This review describes relevant factors in the myocardium microenvironment that affect CM structure and mechanical function and demonstrates the application of several engineered microphysiological systems for studying development, disease, and drug discovery.

Traditionally, bone tissue engineering has been used for creating implants for bone regeneration, but recently, it is increasingly applied to create three-dimensional (3D) in vitro bone models to study bone physiology and pathology. For 3D in vitro bone models, the engineered extracellular matrix organization should resemble the in vivo physiological bone structure because this is often the hallmark in skeletal pathologies — an issue that has not been solved yet. In this review article, we define the extracellular matrix requirements for an optimal 3D in vitro model based on the most recent advances on bone structure. To meet these requirements, osteoclasts, osteocytes, and mature osteoblasts should work together under physiological conditions, and the formed extracellular matrix should be analyzed and optimized at multiple length scales.

A static in vitro model was used to assess walnuts and peanuts macronutrient digestion of with two different particle size. Nuts were digested under different intestinal conditions of pH (6 or 7), bile concentration (1–10 mM) and pancreatic concentration (1000 to 4000 LU/g fat) the matrix degradation index (MDI), proteolysis and lipolysis were analyzed. Results showed that nuts particle size affects proteolysis and MDI the most; intestinal pH was more relevant in free fatty acids release. Lipolysis extent was lower under suboptimal intestinal conditions of pH 6 and bile salts 1 mM, and in peanuts it was lower than walnuts (567, 585, 134 and 398 mg FFA/g fat in large and small walnuts, and large and small peanuts, respectively). The higher the pancreatic concentration the higher the proteolysis extent in walnuts; in peanuts, protein digestibility was limited even at high pancreatic concentration at pH 6 and bile concentration 1 mM.

Purpose To develop an in vitro model to determine pre-lens non-invasive break-up time (NIBUT) and to subsequently use this method to compare the NIBUT over contemporary daily disposable (DD) contact lenses (CL). Methods Three silicone hydrogel (SH) and two conventional hydrogel (CH) DD CLs were incubated in an artificial tear solution (ATS). A model blink cell (MBC) was utilised to mimic intermittent air exposure. CLs were repeatedly submerged for 3 seconds (s) and exposed to air for 10 s over periods of 2, 6, 12, and 16 hours (h). NIBUTs (n = 4) were determined out of the blister pack (T0) and at the end of each incubation period. Results Overall, nesofilcon A showed the longest NIBUTs (p Conclusion NIBUT values reduced gradually over time and varying levels of deposition impacted measured pre-lens NIBUTs. While NIBUT of CH materials are longer immediately out of the blister pack, after tear film exposure, the NIBUTs obtained using this methodology became very similar.

Breast cancer is a global health issue and the second leading cause of cancer death in women. Breast cancer tends to migrate to bone and causes bone metastases which is ultimately the cause of death. Here, we report the use of FTIR to identify spectral biomarkers of cancer progression on 3D in vitro model of breast cancer bone metastasis. Our results indicate that the following spectral biomarkers can monitor cancer progression, for example, lipids (CH2 asymmetric/CH2 symmetric stretch), Amide I/Amide II, and RNA/DNA. Principal component analysis also confirmed the involvement of protein, lipids and nucleic acids in cancer progression on sequential culture. The collective observations from this study suggest successful application of FTIR as a non-invasive and accurate method to identify biochemical changes in cancer cells during the progression of breast cancer bone metastasis.

In the past two decades, significant advances have been achieved for the emergence and rapid growth of in vivo-like in vitro cell culture platforms, especially organ-on-a-chip, which are inspired by the human organs with the goal to provide a new insight into fundamental biology and pharmaceutics. The basis for the reconstruction of in vivo-like in vitro model are stemmed from the advance of the microfabrication techniques and microfluidic systems to achieve physiological structural microenvironment and dynamic biochemical/mechanical stimuli. Here, we aim to highlight recent advances of enabling technologies for in vitro culture platform to simulate organ-specific microstructure or dynamic stimuli. These technologies allowed to construct physiological tissue or organ microstructures and tissue-tissue interfaces and to reproduce dynamic biochemical and mechanical microenvironments to guide functional tissue or organ formation.

Transcatheter Aortic Valves rely on the tissue-stent interaction to ensure that the valve is secured within the aortic root. Aortic stenosis presents with heavily calcified leaflets and it has been proposed that this calcification also acts to secure the valve, but this has never been quantified. In this study, we developed an in vitro calcified aortic root model to quantify the role of calcification on the tissue-stent interaction. The in vitro model incorporated artificial calcifications affixed to the leaflets of porcine aortic heart valves. A self-expanding nitinol braided stent was deployed into non-calcified and artificially calcified porcine aortic roots and imaged by micro computed tomography. Mechanical tests were then conducted to dislodge the stent from the aortic root and it was found that, in the presence of calcification, there was a significant increase in pullout force (8.59 ± 3.68 N vs. 2.84 ± 1.55 N p = 0.045), stent eccentricity (0.05 ± 0.01 vs. 0.02 ± 0.01, p = 0.049), and coefficient of friction between the stent and aortic root (0.36 ± 0.12 vs. 0.09 ± 0.05, p = 0.018), when compared to non-calcified roots. This study quantifies for the first time the impact of calcification on the friction between the aortic tissue and transcatheter aortic valve stent, showing the role of calcification in anchoring the valve stent in the aortic root.

The field of stomach-directed therapeutics and diagnosis is still hampered by the lack of reliable in vitro models that closely mimic the gastric mucosa where gastric cancer cells are generally confined. Here we propose a rapid, complex, and innovative 3D in vitro model of the gastric mucosa, by extending a conventional gastric monolayer model to an inner stratum of the mucosa – the lamina propria. The developed model comprises normal stomach fibroblasts embedded in a 3D RGD-modified alginate hydrogel prepared on the basolateral side of a Transwell® insert, mimicking the extracellular matrix and cellular component of the lamina propria, onto which a moderately differentiated adenocarcinoma stomach cell line (MKN74) was seeded, reproducing the physiological conditions of the gastric barrier. The integrity and functionality of the in vitro model was evaluated through permeability studies of FITC-dextran and 200 nm fluorescent polystyrene nanoparticles at gastric conditions. Nanoparticle transport was pH-dependent and strongly impacted by the biomimetic lamina propria, highlighting that a gastric extracellular matrix (ECM)-like microenvironment should be integrated in an in vitro permeability model to be adopted as a reliable evaluation tool of innovative therapeutics and diagnosis of gastric diseases. Statement of Significance Current in vitro models of the gastric mucosa are limited to simplistic 2D cell culture systems, which ignore the dimensionality of the stomach wall and make it difficult to reliably test new therapeutic approaches to gastric pathologies. By combining stomach fibroblasts embedded within a 3D RGD-modified alginate hydrogel and epithelial gastric cancer cells in a Transwell® system, we established a new biomimetic model of the stomach mucosa. Epithelial cells recreate the gastric epithelium, while the cell-laden 3D hydrogel recapitulates both the cellular composition and dimensionality of the extracellular matrix of gastric lamina propria. This cellularized 3D model stands as a promising evaluation platform to assist the development of new strategies for the treatment and diagnosis of gastric diseases.

Musculoskeletal injuries in equine athletes represent a leading cause for sport-induced and career-ending injuries because of slow or incomplete tendon repair creating clinical challenges in functional repair. Three-dimensional (3D) tendon constructs provide an in vitro model highly representative of in vivo tendon and provide an ideal strategy for testing therapies to improve tendon repair outcomes. Exploring different 3D construct protocols found in literature led us to consider the need for protocol optimization for horse superficial digital flexor tendon (SDFT) cells. The objective of this study was to assess both cell seeding density and seeding technique for equine SDFT 3D constructs by analyzing functionality and ultrastructure via biochemical, morphological, and mechanical characterization of engineered tendons under varying conditions of cell seeding density and seeding technique. Engineered tendons produced with varying cell seeding densities were assessed for their biochemical, collagen content, and fibril ultrastructure organization analysis. Although not statistically significant, using 300,000 cells per construct produced a more functionally and structurally reproducible construct as compared with 100,000 and 500,000 cells. A localized seeding method produced significantly stronger engineered tendons than those produced using a spread method as assessed by their biomechanical properties and collagen content. Collectively, our findings show evidence that although cell seeding number did not ultimately affect the construct, localized seeding method improved collagen content and mechanical properties for the model tendon. The tendon construct strategy described in this study is now being used as an initial “in vitro” step in the analysis of therapeutic strategies for equine tendon repair.

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.

It is estimated that 10% of the worldwide population suffers from chronic kidney disease (CKD) with a rising tendency. Patients with CKD have limited treatment options and novel therapies that could halt or even reverse the progression of CKD are urgently needed.Bioprinting is considered one of the most promising approaches to generate novel 3D in vitro models and organ-like constructs to investigate underlying pathomechanism of kidney diseases. This study aimed at establishing a method to isolate primary renal cells in an easy and reproducible way. These cells were used in a new bioprinting platform laying the foundation for the development of a 3D renal tubulointerstitium model for in vitro studies. Primary murine tubular (pmTECs), endothelial and fibroblast cells were successfully isolated, but further optimization is required for the culture and expansion of primary endothelial cells. Therefore, an endothelial cell line (HUVECs) and pmTECs were combined with polysaccharide biomaterial ink solutions and processed with a microfluidic 3D bioprinter, leading to high cell viability and metabolic activity. Core-shell bioprinted constructs with HUVECs and pmTECs were manufactured mimicking tubules.In conclusion, microfluidic bioprinting strategy could be used to build a novel 3D kidney in vitro model.

Three-dimensional cellular aggregates can mimic the natural microenvironment of tissues and organs and obtaining them through controlled and reproducible processes is mandatory for scaling up and implementing drug cytotoxicity and efficacy tests, as well as tissue engineering protocols. The purpose of this work was to develop and evaluate the performance of a device with two different geometries fabricated by additive manufacturing. The methodology was based on casting a microwell array insert using a non-adhesive hydrogel to obtain highly regular microcavities to standardize spheroid formation and morphology. Spheroids of dental pulp stem cells, bone marrow stromal cells and embryonic stem cells showing high cell viability and average diameters of around 253, 220, and 500 μm, respectively, were produced using the device with the geometry considered most adequate. The cell aggregates showed sphericity indexes above 0.9 and regular surfaces (solidity index higher than 0.96). Around 1000 spheroids could be produced in a standard six-well plate. Overall, these results show that this method facilitates obtaining a large number of uniform, viable spheroids with pre-specified average diameters and through a low-cost and reproducible process for a myriad of applications.

During diabetes, the characteristic hyperglycemia can induce red blood cell glycation. Several researchers have proposed different protocols to perform an in vitro model to study this phenomenon. In this article, some of the most important in vitro glycation protocols available in the bibliography were compared to each other. The incubation parameters as the suspension medium, glucose concentration, red blood cell concentration, time, and temperature were analyzed. Also, several assays were carried out in our laboratory, and glycated hemoglobin, erythrocyte aggregation and viscoelasticity were determined for the protocol validation. Based on the bibliographic analysis and our experimental results, an optimal protocol for in vitro glycation of red blood cells is presented.

Precise detection of brain regions harboring heightened electrical activity plays a central role in the understanding and treatment of diseases such as epilepsy. Superparamagnetic iron oxide nanoparticles (SPIONs) react to magnetic fields by aggregating and represent interesting candidates as new sensors for neuronal magnetic activity. We hypothesized that SPIONs in aqueous solution close to active brain tissue would aggregate proportionally to neuronal activity. We tested this hypothesis using an in vitro model of rat brain slice with different levels of activity. Aggregation was assessed with dynamic light scattering (DLS) and magnetic resonance imaging (MRI). We found that increasing brain slice activity was associated with higher levels of aggregation as measured by DLS and MRI, suggesting that the magnetic fields from neuronal tissue could induce aggregation in nearby SPIONs in solution. MRI signal change induced by SPIONs aggregation could serve as a powerful new tool for detection of brain electrical activity.

Human milk oligosaccharides (HMOs) are a unique class of non-digestible carbohydrates present in the mother milk, which play a key role in the development of infant gut microbiota, epithelial barrier and immune function. The deficiency of HMOs in the bovine milk-based infant formula has been widely recognized as a major culprit for the much higher incidence of immune disorders of formula-fed infants. This report was to give an up-to-date review on the structure characteristics of HMOs and the possible mechanisms, and strategies for their cellular uptake, and metabolism by the gut bacteria and the associated effects on the infant gut microbiome, and immune function. Most previous studies have been carried out in animals or in vitro model systems on the utilization strategies for HMOs in infant bacteria and their roles in infant microbiome, and gut immune function. A few HMO molecules have been synthesized artificially and applied in infant formulas.

Calcification is among the principal causes of biological heart valve substitute failure. Glycosaminoglcans (GAGs) are negatively charged molecules, possessing anticoagulation and anti-inflammatory activity that make them a potential solution against calcification. In the present work, the anticalcification potential of heparin was investigated under constant supersaturation conditions with respect to hydroxyapatite (Ca 5 (PO 4 ) 3 OH; HAP). Heparin concentration in the supersaturated solutions was in the range between 0.25 and 3 ppm, at pH 7.40 and 37 °C. Heparin showed inhibitory activity, which was attributed to adsorption at the active crystal growth centres. Heparin concentration as low as ca. 50 μM, reduced the rate of HAP crystal growth by more than 80%, while further increase (up to 200 μM) failed to completely inhibit the process beyond 90%. Heparin uptake studies at equilibrium conditions and analysis of the kinetics data in the presence of heparin, strongly suggest that the inhibition is due to the adsorption of heparin onto the HAP crystals, which resulted in the poisoning of the active growth sites.

Background Mushrooms have been valuable as edible and medical provisions for humankind. It is attributed not only to the quality of their taste but also to their biological activities. The extracts, compounds, and metabolites have a lot of biological activities such as antidiabetic, antioxidative, antitumor, and anti-inflammatory effects. The potent anti-inflammatory activity is associated with a multitude of signaling pathways. However, there are a few review papers on the anti-inflammatory activity of mushrooms. Scope and approach Herein, the anti-inflammatory potential of mushroom is critically discussed using in vitro and animal models. Moreover, the mechanisms of anti-inflammatory activity are summarized. The current status of clinical studies evaluating anti-inflammatory activity of mushroom is also presented. Key findings and conclusion It is believed that insights about these advances may encourage researchers to discover new phytochemical components and further study specific bioactivities of mushroom.

Biomaterials for applications in the urinary tract are challenged with both biofilm formation and encrustation, two highly interconnected processes. While great effort has been achieved developing promising materials there is only a limited choice of sophisticated in vitro models that are available to analyse the performance of biomaterials prior to performing delicate and expensive in vivo studies. In this study we present a complex BioEncrustation model that imitates both the processes of multi-species biofilm formation and encrustation in vitro. The resulting crystalline biofilms are compared to the deposits found on explanted ureteral stent surfaces (in vivo situation) and to deposits formed in an experimental set up that does not contain bacteria (Encrustator®). Further focus of this study is dedicated to employing the developed BioEncrustation model to evaluate the effect multifunctional coatings impose on the processes of biofilm formation and encrustation under in vitro conditions. The investigated TANP coating combines unspecific and broad band specific antibacterial properties with a degrading polymer matrix that is intended to inhibit crystal formation. The coating was prepared on both polyurethane and silicone tubes and the subsequent results of the in vitro BioEncrustation analyses reveal a promising potential for employing the coating to render ureteral stent surfaces more biocompatible.

NMDA receptor (NMDAr) hypofunction has been widely used as a schizophrenia model. Decreased activation of NMDAr is associated with a disrupted excitation/inhibition balance in the prefrontal cortex and with alterations in gamma synchronization. Our aim was to investigate whether this phenomenon could be reproduced in the spontaneous oscillatory activity generated by the local prefrontal network in vitro and, if so, to explore the effects of antipsychotics on the resulting activity. Extracellular recordings were obtained from prefrontal cortex slices bathed in in vivo-like ACSF solution. Slow (1 Hz) oscillations consisting of interspersed Up (active) and Down (silent) states spontaneously emerged. Fast-frequency oscillations (15-90 Hz) occurred during Up states. We explored the effects of the NMDAr antagonist MK-801 on the spontaneously generated activity. Bath-applied MK-801 induced a dose-dependent decrease in Up-state duration and in the frequency of Up states. However, the beta/gamma power during Up states significantly increased; this increase was in turn prevented by the antipsychotic drug clozapine. The increased beta/gamma power with NMDAr blockade implies that NMDAr activation in physiological conditions prevents hypersynchronization in this frequency range. High-frequency hypersynchronization following NMDAr blockade occurring in cortical slices suggests that-at least part of-the underlying mechanisms of this schizophrenia feature persist in the local cortical circuit, even in the absence of long-range cortical or subcortical inputs. The observed action of clozapine decreasing hypersynchronization in the local circuit may be one of the mechanisms of action of clozapine in preventing schizophrenia symptoms derived from NMDA hypofunction.

Chagas disease, caused by Trypanosoma cruzi, is an important global public health problem which, despite partial efficacy of benznidazole (Bz) in acute phase, urgently needs an effective treatment. Cardiotoxicity is a major safety concern for conduction of more accurate preclinical drug screening platforms. Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are a reliable model to study genetic and infectious cardiac alterations and may improve drug development. Herein, we introduce hiPSC-CM as a suitable model to study T. cruzi heart infection and to predict the safety and efficacy of anti-T. cruzi drugs.

Purpose\ud To develop a mechanical model in which a contact lens is swept over ocular surface cells under conditions that mimic the force and speed of the blink, and to investigate the resulting biological changes.\ud \ud Methods\ud A computer controlled mechanical instrument was developed to hold a dish containing 3D cultured stratified human ocular surface epithelial cells, across which an arm bearing a contact lens was swept back and forth repeatedly at a speed and force mimicking the human blink. Cells were subjected to repeated sweep cycles for up to 1 h at a speed of 120 mm/s with or without an applied force of 19.6 mN (to mimic pressure exerted by upper eyelid), after which the cell layer thickness was measured, the cell layer integrity was investigated using fluorescent quantum dots (6 and 13 nm) and the phosphorylation levels of various protein kinases were analyzed by human phospho-kinase arrays. Data for selected kinases were further quantitated by enzyme immunoassays.\ud \ud Results\ud The thickness of the cell layers did not change after exposure to sweep cycles with or without applied force. Quantum dots (6 and 13 nm) were able to penetrate the layers of cells exposed to sweep cycles but not layers of untreated control cells. The phosphorylation levels of HSP27 and JNK1/2/3 increased for cells exposed to sweep cycles with applied force compared to untreated control cells.\ud \ud Conclusions\ud The in vitro mechanical instrument is a useful tool to investigate the effects of blinking on the ocular surface.

The purpose of this study was to estimate the in vivo pharmacokinetics of meropenem during intermittent-infusion hemodiafiltration (I-HDF) and clarify its optimal dosage and dosing interval in patients receiving I-HDF. The clearance of meropenem by online hemodiafiltration (OL-HDF) and I-HDF was predicted using an in vitro system and assessed to establish whether the results obtained are applicable to clinical cases. In the in vivo study, the mean volume of distribution (Vd), non-I-HDF clearance (CLnon-I-HDF), and I-HDF clearance (CLI-HDF) were 15.80 ± 3.59 l, 1.05 ± 0.27 l/h, and 5.78 ± 1.03 l/h. Dosing regimens of 0.25 g once daily for a MIC of 8 μg/ml and of 0.5 g once daily for a MIC of 16 μg/ml achieved 40% T > MIC. In the in vitro and in vivo studies, observed CLHDF was similar to predictive CLHDF (= Cf/Cp × (QD + QSUB)). In conclusion, adjustments to the dose and interval of meropenem were developed based on the presumed susceptibility of pathogens to meropenem in patients receiving I-HDF. We suggest 0.5 g once daily as an appropriate regimen for empirical treatment.

Two new compounds namely acetylcaloncobaside (1) and friedocaloncobic acid (2) were isolated from the leaves of Caloncoba welwitschii together with six known compounds. Their structures were determined by standard spectroscopic methods including one- and two-dimensional NMR, EI-MS, and HRESIMS. The new compounds were established as kaempferol 4′,7-dimethoxy-3-O-(3″,4″,6″-O-triacetyl)-β- null -glucopyranoside (1) and 15β-hydroxy-3-oxo-28-friedelanoic acid (2). The isolated compounds were evaluated for their ability to inhibit the 3D7 strain of Plasmodium falciparum, using the semi-automated in vitro model with parasite lactate dehydrogenase assay.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. Published version available at https://doi.org/10.1016/j.ijpharm.2017.12.038. The mucus layer covering all mucosal surfaces in our body is the first barrier encountered by drugs before their potential absorption through epithelial tissues, and could thus affect the drugs’ permeability and their effectiveness. Therefore, it is of key importance to have in vitro permeability models that can mimic this specific environment. For this purpose, the novel mucus phospholipid vesicle-based permeation assay (mucus-PVPA) has been developed and used for permeability screening of drugs and formulations. The model proved to be stable under the chosen conditions and demonstrated the ability to discriminate between compounds with different chemical structures and properties. Overall, a decrease in drug permeability was found in the presence of mucus on top of the PVPA barriers, as expected. Moreover, mucoadhesive (chitosan-coated) and mucopenetrating (PEGylated) liposomes were investigated in the newly developed model. The mucus-PVPA was able to distinguish between the different liposomal formulations, confirming the penetration potential of the tested formulations and the related drug permeability. The mucus-PVPA model appears to be a promising in vitro tool able to mimic the environment of mucosal tissues, and could therefore be used for further drug permeability screening and formulation development.

This study sought to determine if proteins associated with pain in humans could be measured using a swine in vitro model of inflammation. This would constitute the first step towards using them as surrogate endpoints to help support effectiveness indications for investigational new animal drugs to control pain in swine. Swine whole blood samples were cultured in vitro with E. coli derived-lipopolysaccharide (LPS) or without LPS for 24 h. Supernatants from these cultures were collected to determine the concentration of proteins associated with pain and whether the levels were altered in response to LPS-induced inflammation. Bradykinin protein levels steadily increased over time due to LPS stimulation and returned to 0 h levels after 6 h of culture. Corticotrophin-releasing factor protein levels were not affected by LPS. Substance-P protein trended towards increasing concentrations after LPS stimulation, following a time-concentration profile similar to that observed with bradykinin. These results suggest that 2 biomarkers may be useful as surrogate endpoints for evaluation of pain.

Despite decades of research and advancements in diagnostic and treatment modalities, cancer remains a major global healthcare challenge. This is due in part to a lack of model systems that allow investigating the mechanisms underlying tumor development, progression, and therapy resistance under relevant conditions in vitro. Tumor cell interactions with their surroundings influence all stages of tumorigenesis and are shaped by both biological and biophysical cues including cell-cell and cell-extracellular matrix (ECM) interactions, tissue architecture and mechanics, and mass transport. Engineered tumor models provide promising platforms to elucidate the individual and combined contributions of these cues to tumor malignancy under controlled and physiologically relevant conditions. This review will summarize current knowledge of the biological and biophysical microenvironmental cues that influence tumor development and progression, present examples of in vitro model systems that are presently used to study these interactions and highlight advancements in tumor engineering approaches to further improve these technologies.