Progress in developing microphysiological systems for biological product assessment.

Microphysiological systems (MPS), also known as miniaturized physiological environments, have been engineered to create and study functional tissue units capable of replicating organ-level responses in specific contexts. The MPS has the potential to provide insights about the safety, characterization, and effectiveness of medical products that are different and complementary to insights gained from traditional testing systems, which can help facilitate the transition of potential medical products from preclinical phases to clinical trials, and eventually to market. While many MPS are versatile and can be used in various applications, most of the current applications have primarily focused on drug discovery and testing. Yet, there is a limited amount of research available that demonstrates the use of MPS in assessing biological products such as cellular and gene therapies. This review paper aims to address this gap by discussing recent technical advancements in MPS and their potential for assessing biological products. We further discuss the challenges and considerations involved in successful translation of MPS into mainstream product testing.

Comparison of Engineered Liver 3D Models and the Role of Oxygenation for Patient-Derived Tumor Cells and Immortalized Cell Lines Cocultured with Tumor Stroma in the Detection of Hepatotoxins.

In metabolically active tumors, responses of cells to drugs are heavily influenced by oxygen availability via the surrounding vasculature alongside the extracellular matrix signaling. The objective of this study is to investigate hepatotoxicity by replicating critical features of hepatocellular carcinoma (HCC). This includes replicating 3D structures, metabolic activities, and tumor-specific markers. The internal environment of spheroids comprised of cancerous human patient-derived hepatocytes using microparticles is modulated to enhance the oxygenation state and recreate cell-extracellular matrix interactions. Furthermore, the role of hepatic stellate cells in maintaining hepatocyte survival and function is explored and hepatocytes from two cellular sources (immortalized and patient-derived) to create four formulations with and without microparticles are utilized. To investigate drug-induced changes in metabolism and apoptosis in liver cells, coculture spheroids with and without microparticles are exposed to three hepatotoxic drugs. The use of microparticles increases levels of apoptotic markers in both liver models under drug treatments. This coincides with reduced levels of anti-apoptotic proteins and increased levels of pro-apoptotic proteins. Moreover, cells from different origins undergo apoptosis through distinct apoptotic pathways in response to identical drugs. This 3D microphysiological system offers a viable tool for liver cancer research to investigate mechanisms of apoptosis under different microenvironmental conditions.

Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly, function, and responses.

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.

Generation of Oxygenating Fluorinated Methacrylamide Chitosan Microparticles to Increase Cell Survival and Function in Large Liver Spheroids.

Despite advances in the development of complex culture technologies, the utility, survival, and function of large 3D cell aggregates, or spheroids, are impeded by mass transport limitations. The incorporation of engineered microparticles into these cell aggregates offers a promising approach to increase spheroid integrity through the creation of extracellular spaces to improve mass transport. In this study, we describe the formation of uniform oxygenating fluorinated methacrylamide chitosan (MACF) microparticles via a T-shaped microfluidic device, which when incorporated into spheroids increased extracellular spacing and enhanced oxygen transport via perfluorocarbon substitutions. The addition of MACF microparticles into large liver cell spheroids supported the formation of stable and large spheroids (>500 µm in diameter) made of a heterogeneous population of immortalized human hepatoma (HepG2) and hepatic stellate cells (HSCs) (4 HepG2/1 HSC), especially at a 150:1 ratio of cells to microparticles. Further, as confirmed by the albumin, urea, and CYP3A4 secretion amounts into the culture media, biological functionality was maintained over 10 days due to the incorporation of MACF microparticles as compared to controls without microparticles. Importantly, we demonstrated the utility of fluorinated microparticles in reducing the number of hypoxic cells within the core regions of spheroids, while also promoting the diffusion of other small molecules in and out of these 3D in vitro models.

Advances in removing mass transport limitations for more physiologically relevant in vitro 3D cell constructs.

Spheroids and organoids are promising models for biomedical applications ranging from human disease modeling to drug discovery. A main goal of these 3D cell-based platforms is to recapitulate important physiological parameters of their in vivo organ counterparts. One way to achieve improved biomimetic architectures and functions is to culture cells at higher density and larger total numbers. However, poor nutrient and waste transport lead to low stability, survival, and functionality over extended periods of time, presenting outstanding challenges in this field. Fortunately, important improvements in culture strategies have enhanced the survival and function of cells within engineered microtissues/organs. Here, we first discuss the challenges of growing large spheroids/organoids with a focus on mass transport limitations, then highlight recent tools and methodologies that are available for producing and sustaining functional 3D in vitro models. This information points toward the fact that there is a critical need for the continued development of novel cell culture strategies that address mass transport in a physiologically relevant human setting to generate long-lasting and large-sized spheroids/organoids.

Fluorinated Chitosan Microgels to Overcome Internal Oxygen Transport Deficiencies in Microtissue Culture Systems.

Poor oxygen transport is a major obstacle currently for 3D microtissue culture platforms, which at this time cannot be grown large enough to be truly physiologically relevant and replicate adult human organ functions. To overcome internal oxygen transport deficiencies, oxygenating microgels are formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water-in-oil miniemulsion method. Microgels that are on the order of a cell diameter (≈10 µm) are formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it is demonstrated that incorporating oxygenating microgels at ratios ranging from 50:1 to 400:1 (# of cells:# of microgels) into dense human fibroblast-based spheroids facilitated the growth of larger human cell-based spheroids, especially at the highest incorporation percentages (50:1), which lacked defined hypoxic cores. Quantification of total double-stranded (ds)-DNA, a measure of number of live cells, demonstrated similar results to hypoxia quantification, showing more ds-DNA due incorporation of oxygenating microgels. Finally, oxygen concentrations are measured at different depths within spheroids directly and confirmed higher oxygen partial pressures due to chitosan-PFC microspheres.

The Effect of Lucite Glass Reinforcement on the Properties of Conventional Glass-Ionomer Filling Materials.

STATEMENT OF THE PROBLEM: The usage of glass ionomer cements (GICs) restorative materials are very limited due to lack of flexural strength and toughness. PURPOSE: The aim of this study was to investigate the effect of using a leucite glass on a range of mechanical and optical properties of commercially available conventional glass ionomer cement. MATERIALS AND METHOD: Ball milled 45µm leucite glass particles were incorporated into commercial conventional GIC, Ketac-Molar Easymix (KMEm). The characteristics of the powder particles were observed under scanning electron microscopy. The samples were made for each experimental group; KMEm and lucite- modified Ketac-Molar easy Mix (LMKMEm) according to manufacturer's instruction then were collected in damp tissue and stored in incubator for 1 hour. The samples were divided into two groups, one stored in distilled water for 24 hours and the others for 1 week.10 samples were made for testing biaxial flexural strength after 1 day and 1 week, with a crosshead speed of 1mm/min, calculated in MPa. The hardness (Vickers hardness tester) of each experimental group was also tested. To evaluate optical properties, 3 samples were made for each experimental group and evaluated with a spectrophotometer. The setting time of modified GIC was measured with Gillmore machine. RESULT: The setting time in LMKMEm was 8 minutes. The mean biaxial flexural strength was LMKMEm/ 1day: 24.13±4.14 MPa, LMKMEm/ 1 week: 24.22±4.87 MPa KMEm/1day:28.87±6.31 MPa and KMEm/1 week: 26.65±5.82 MPa which were not statistically different from each other. The mean Vickers hardness was LMKMEm: 403±66 Mpa and KMEm: 358±22 MPa; though not statistically different from each other. The mean total transmittance (Tt) was LMKMEm: 15.9±0.7, KMEm: 22.3±1.2, the mean diffuse transmittance (Td) was LMKMEm: 12.2±0.5, KMEm: 18.0±0.5 which were statistically different from each other. CONCLUSION: Leucite glass can be incorporated with a conventional GIC without interfering with setting time. Yet, it did not improve the mechanical and optical properties of the GIC.

Sunflower Seed and Acne Vulgaris.

BACKGROUND: Regardless of the overall association between diet and acne which cannot be easily ignored, there might be an association between specific nutrients and acne development or improvement. OBJECTIVES: The aim of this study was to assess the effect of dietary intake of sunflower seeds on acne severity and the pattern of acne lesions. PATIENTS AND METHODS: In a randomized controlled trial, 50 patients aged 15 - 30 years old with acne vulgaris were enrolled through consecutive convenient sampling, in a dermatology clinic in Ardabil, Iran. They were randomly allocated into two trial arms. Those in the control group were asked to stop eating sunflower seeds if they did before. In the intervention group, they consumed 25 g sunflower-containing food daily for seven days. The primary outcome of interest was 10% increase/decrease in the baseline acne severity index (ASI), sustained to the end of the follow-up period on day 14. RESULTS: The mean ASI did not change significantly through the study period in the control group, but it increased in the sunflower group from 62 at the baseline to 86.8 after two weeks (P < 0.001). The ASI mean change was 24.8 in the sunflower group compared to 4.9 in the control group (P < 0.001). The global acne grading score (GAGS) did not significantly change in any of the groups and the difference in the change of GAGS was not significant between the groups (2.4 in the sunflower group versus 1.6 in the control group). Twenty two subjects (88%) in the sunflower group versus 9 (36%) in the control group had at least 10% increment in ASI throughout the follow-up period (P < 0.001). The relative risk of developing the primary outcome in taking the sunflower seed intervention was 2.4 (95% CI: 1.4 - 4.2). The observed risk difference was 0.52 (95% CI: 0.29 - 0.75). CONCLUSIONS: Sunflower seed intake appears to aggravate acne vulgaris; however, further evidence is needed to ban sunflower seed intake in patients with acne. Considering the observed potential negative effect in this trial, future randomized clinical trials may base their design on randomly assigning the exposed patients to give up use of sunflower seed intake.

Effects of three surface conditioning techniques on repair bond strength of nanohybrid and nanofilled composites.

BACKGROUND: Repair bond strength of different composite resins has been assessed in few studies. In addition, reports on the efficacy of surface treatments are debated. Therefore, this in vitro study was conducted to evaluate the effect of three surface treatments on two nanocomposites versus a microhybrid composite. MATERIALS AND METHODS: In this experimental study, 135 composite blocks (45 specimens per composite) of microhybrid (Filtek Supreme Z250, 3M ESPE, USA), nanohybrid (Filtek Supreme XT, 3M ESPE), and nanofilled (Filtek Supreme Z350, 3M ESPE) were thermocycled (5000 rounds) and then surface roughened (except in a control group of 9 specimens of three composite types). Each composite type was divided into three subgroups of surface treatments: (1) Bur abrading and phosphoric acid (PA) etching, (2) sandblasting and PA etching, and (3) hydrofluoric etching and silane application (n = 15 × 9, complying with ISO TR11405). Composite blocks were repaired with the same composite type but of a different color. Microtensile bond strength and modes of failure were analyzed statistically using two-way analyses of variance, Tukey and Chi-square tests (α = 0.05). RESULTS: There were significant differences between three composite resins (P < 0.0001) and treatment techniques (P < 0.0001). Their interaction was nonsignificant (P = 0.228). The difference between nanofilled and nanohybrid was not significant. However, the microhybrid composite showed a significantly higher bond strength (Tukey P < 0.05). Sandblasting was significantly superior to the other two methods, which were not different from each other. CONCLUSION: Within the limitations of this in vitro study, it seems that microhybrid composite might have higher repair strengths than two evaluated nanocomposites. Among the assessed preparation techniques, sandblasting followed by PA etching might produce the highest bond strength.