Optimizing Bioink Composition for Human Chondrocyte Expression of Lubricin.

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage regeneration.

Micronutrient optimization for tissue engineered articular cartilage production of type II collagen.

Tissue Engineering of cartilage has been hampered by the inability of engineered tissue to express native levels of type II collagen in vitro. Inadequate levels of type II collagen are, in part, due to a failure to recapitulate the physiological environment in culture. In this study, we engineered primary rabbit chondrocytes to express a secreted reporter, Gaussia Luciferase, driven by the type II collagen promoter, and applied a Design of Experiments approach to assess chondrogenic differentiation in micronutrient-supplemented medium. Using a Response Surface Model, 240 combinations of micronutrients absent in standard chondrogenic differentiation medium, were screened and assessed for type II collagen promoter-driven Gaussia luciferase expression. While the target of this study was to establish a combination of all micronutrients, alpha-linolenic acid, copper, cobalt, chromium, manganese, molybdenum, vitamins A, E, D and B7 were all found to have a significant effect on type II collagen promoter activity. Five conditions containing all micronutrients predicted to produce the greatest luciferase expression were selected for further study. Validation of these conditions in 3D aggregates identified an optimal condition for type II collagen promoter activity. Engineered cartilage grown in this condition, showed a 170% increase in type II collagen expression (Day 22 Luminescence) and in Young's tensile modulus compared to engineered cartilage in basal media alone.Collagen cross-linking analysis confirmed formation of type II-type II collagen and type II-type IX collagen cross-linked heteropolymeric fibrils, characteristic of mature native cartilage. Combining a Design of Experiments approach and secreted reporter cells in 3D aggregate culture enabled a high-throughput platform that can be used to identify more optimal physiological culture parameters for chondrogenesis.

Water reclamation from anodizing wastewaters by removing reactive silica with adsorption and precipitation methods.

Water stress is a current environmental menace mainly driven by over exploitation of aquifers, which is triggering poor water quality with high concentration of minerals in extracted groundwater. Particularly, silica is widespread in natural water supplies due to weathering processes of silicates occurring in contact with water, light, air, and other factors. However, due to groundwater over extraction the concentration of silica has increased during the last years in aquifer reservoirs from Aguascalientes State (México). In this context, it is very important to note that the removal of silica compounds from water is challenging and different methods can be used to avoid embedding problems in different industries. In the present work, the removal of reactive silica from synthetic solutions as well as from real wastewaters from an industrial anodizing process was studied using adsorption and chemical precipitation methods. Twelve commercial materials of different nature were used for adsorption tests, while seven precipitant agents were applied in the precipitation experiments. Adsorption tests were performed in batch systems with constant stirring at 30 °C and at different pH values (7 and 9). Precipitation experiments were carried out in batch systems and the best conditions for silica removal were found using an L9 orthogonal array of the Taguchi method employing molar ratio, pH of wastewater, stirring time and temperature as experimental factors. Adsorption results showed that Ferrolox (Iron (III) hydroxide-base adsorbent) was the most efficient sorbent for reactive silica removal from synthetic solutions and the anodizing wastewater. Also, the reactive silica adsorption was higher at pH 9 as compared to that measured at pH 7 and the adsorbed quantity at pH 9 was 16.22 and 11.25 mg/g for the synthetic solution and anodizing wastewater, respectively. According to molecular simulation, the main interaction between Ferrolox and silica species was related to the formation of hydroxo-complexes and to the interaction of Fe with oxygen of silica species. Additionally, magnesium chloride was the best precipitating reagent for reactive silica achieving up to 87% removal. According to ANOVA analysis of Taguchi method, pH was the most influential factor during the precipitation of reactive silica with a variance value of 81.42, while values lower than 3 were obtained for the rest of parameters. Overall, the present work is reporting for the first time the removal of reactive silica from anodizing wastewaters with promising results that can be implemented at full scale for water reclamation, which may significantly contribute to manage water reservoir in the region sustainably.

Biomaterial composition and stiffness as decisive properties of 3D bioprinted constructs for type II collagen stimulation.

Gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) are frequently used biomaterials for 3D bioprinting, with individual well-established material characteristics. To identify an ideal combination of GelMA and HAMA for chondrogenesis, a novel, primary human chondrocyte COL2A1-Gaussia luciferase reporter system (HuCol2gLuc) was developed. With this non-destructive, high-throughput temporal assay, Gaussia luciferase is secreted from the cells and used as a proxy for measuring type II collagen production. GelMA:HAMA ratios were screened using the reporter system before proceeding to 3D bioprinting. This method is efficient, saving on time and materials, resulting in a streamlined process of biomaterial optimization. The screen revealed that the addition of HAMA to GelMA improved chondrogenesis over GelMA (15%) alone. Storage moduli were measured using dynamic mechanical analysis of the same GelMA:HAMA ratios and established an initial threshold for chondrogenesis of ∼30kPa. To determine if biomaterial storage moduli impact cell mobility, human primary chondrocytes transduced with green fluorescent protein (GFP) were 3D bioprinted in either 1:1 or 2:1 ratios with storage moduli of 32kPa and 57.9kPa, respectively. We found that reduced cell mobility, in the stiffer biomaterial, had higher type II collagen expression, than the softer material with more cell mobility. Finally, after 3D bioprinting with HuCol2gLuc cells we successfully identified an optimal combination (2:1) of GelMA:HAMA and photo-crosslinking time (38s) for chondrogenesis. STATEMENT OF SIGNIFICANCE: One challenge of 3D bioprinting is identifying ideal biomaterials that stimulate articular cartilage development. To identify an optimal combination of gelatin methacrylate and hyaluronic acid methacrylate for chondrogenesis we developed a primary human chondrocyte type II collagen Gaussia luciferase reporter cell (HuCol2gLuc). This non-destructive, high-throughput assay uses a secreted Gaussia luciferase as a proxy for temporal type II collagen production. This reporter system streamlines the biomaterial optimization process before 3D bioprinting. We also used it to determine the level of stiffness required for chondrogenesis. And for the first time, we quantified chondrocyte mobility in a 3D bioprinted construct. Together these results indicate that a biomaterial with a higher storage modulus and less cell mobility, improves chondrogenesis.

Competitive adsorption of pollutants from anodizing wastewaters to promote water reuse.

Anodizing wastewater contains principally phosphate (PO43-) anions according to previous studies, but with the purpose to promote water reuse in this type of industry, a complete characterization of wastewater was made to remove other anions and cations also present in significant concentration. Particularly, the adsorption of sodium (Na+), potassium (K+), fluoride (F-), sulfate (SO42-) and phosphate (PO43-) was studied using different sorbents such as: coconut shell activated carbon, bone char, bituminous coal activated carbon, natural zeolite, silica, anionic and cationic exchange resins, a coated manganese-calcium zeolite, coconut shell activated carbon containing iron and iron hydroxide. All sorbents were characterized using FT-IR spectroscopy, potentiometric titration, nitrogen adsorption isotherms at 77 K, X-ray diffraction and SEM/EDX analysis to study the adsorption mechanism. The adsorption studies were performed in batch systems under constant agitation using both standard solutions of each ion and real anodizing wastewater. Results showed that, in general, the adsorption of all anions and cations is higher when mono-component standard solutions were used, since in the anodizing wastewater all species are competing for the active sites of the adsorbent. Na+ present in anodizing wastewater was efficiently adsorbed on coated manganese-calcium zeolite (20.55 mg/g) and natural zeolite (18.55 mg/g); while K+ was poorly adsorbed on all sorbents (less than 0.20 mg/g). Anions such as F-, SO42- and PO43-, were better adsorbed on the anionic resin (0.17, 45.38 and 2.92 mg/g, respectively), the iron hydroxide (0.14, 7.96 and 2.87 mg/g, respectively) and the bone char (0.34, 8.71 and 0.27 mg/g, respectively). All these results suggest that adsorption is a promising tertiary treatment method to achieve water reuse in the anodizing industry.

Enhancement of infectivity of insect cell-derived La Crosse Virus by human serum.

Given the dual life cycle of arboviruses in insect and animal hosts and the importance of serum factors as a first line antiviral defense, we have examined the outcome of interactions between the arbovirus La Crosse Virus (LACV) and human serum. To mimic the life cycle between species, we used LACV derived from insect (I-LACV) and human keratinocyte (HaCaT) cells. Incubation of I-LACV with normal human serum did not result in neutralization, but instead stabilized I-LACV virions and enhanced the amount of infectious virus. Enhanced infectivity was also seen with heat-inactivated serum devoid of complement activity and with serum from a range of animals including mouse, ferret, and non-human primates. Depletion of antibodies from serum resulted in loss of enhancement of infectivity and sucrose gradient sedimentation assays showed IgG co-sedimenting with I-LACV particles. In agreement with our results with I-LACV, HaCaT-derived LACV was not neutralized by complement or antibodies in normal human serum. However, in contrast to I-LACV, HaCaT-derived LACV infectivity was stable when incubated alone and treatment with serum did not enhance infectivity. Our results indicate that LACV derived from insect cells differs substantially from virus derived from human cells, with I-LACV being dependent on serum factors to enhance infectivity. These findings suggest that understanding differential composition of insect versus animal cell-derived LACV may form the foundation for potential new antiviral approaches.

La Crosse Virus Infection of Human Keratinocytes Leads to Interferon-Dependent Apoptosis of Bystander Non-Infected Cells In Vitro.

Resident cells in the skin serve as the first innate line of defense against insect-borne pathogens, but the role of these cell types in promoting or limiting arbovirus replication is not completely understood. Here, we have examined the outcome of infection of cultured human keratinocyte cells with La Crosse virus (LACV), using a spontaneously transformed cell line, HaCaT. In single cycle infections, keratinocyte HaCaT cells supported rapid and high level LACV replication, resulting in high virus yields and extensive caspase-dependent cell death. By contrast, multi-cycle LACV replication in HaCaT cells was restricted by an antiviral response elicited by the production of both IFN-ß and IFN-λ. During low multiplicity LACV infections, HaCaT cell death was seen in non-infected bystander cells. Media from LACV-infected cells induced caspase-dependent killing of naïve non-infected HaCaT cells, and this bystander cell death was relieved by IFN-ß neutralizing antibodies or by an inhibitor of JAK-STAT signaling. Naïve HaCaT cells showed dose-dependent killing by treatment with exogenous IFN-ß but not IFN-λ. Our data suggest a model whereby keratinocytes produce IFNs which limit virus spread through both antiviral signaling and by induction of bystander cell death of potential new target cells for infection.

Validation of the Clock Drawing Test Scoring Method in older adults with neurocognitive disorder / Validación del método de calificación del Test del Dibujo del Reloj en adultos mayores con trastorno neurocognitivo

Abstract: Introduction: The Clock Drawing Test (CDT) is a widely used instrument for identifying neurocognitive disorders (NCDs) in older adults. However, there is insufficient evidence to determine the best scoring method, since current quantitative methods involve the assignment of numerical values, while qualitative ones do not allow for objectivity in the diagnosis. Parsey & Schmitter-Edgecombe (2011) proposed a scoring scheme which, in addition to providing a score of the patient's performance, permits error analysis, thereby making it possible to identify potential underlying cognitive difficulties. Objective: The purpose of this study was to validate the CDT scoring scheme proposed by Parsey & Schmitter-Edgecombe (2011) for screening for NCDs in Mexican older adults. Method: There were 167 participants: 58 cognitively healthy subjects (CH), 52 with mild neurocognitive disorder (mild-NCD), and 57 with major neurocognitive disorder (major-NCD).The CDT scoring method was compared with the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment in Spanish (MoCA-S). Inter- and intra-observer reliability and construct validity were determined and the sensitivity and specificity of this method were calculated. Results: The X - age was 75 years (SD ± 8 years) and the X - educational attainment was 10.7 years (SD ± 5.2 years). Internal reliability was .750, with an intraclass correlation coefficient of .774. The cut-off point for the CDT in mild-NCD was 14 points (sensitivity: 40%, specificity: 70%) and 12 points for major-NCD (sensitivity: 90%, specificity: 95%).The most frequent errors in the CDT were: graphic, conceptual, spatial, and/or planning difficulties. Discussion and conclusion: This method makes it possibly to quickly and easily explore the cognitive status of the patient. It contains ideal psychometric properties for the detection of patients with major-NCD, in addition to offering the possibility of analyzing performance errors and underlying cognitive difficulties.

Resumen: Introducción: El Test del Dibujo del Reloj (TDR) es un instrumento ampliamente utilizado para identificar trastornos neurocognitivos (TNC) en adultos mayores. Sin embargo, no existe suficiente evidencia para determinar el mejor método para calificarlo, ya que los métodos cuantitativos actuales se abocan a la asignación de valores numéricos, mientras que los cualitativos no permiten objetividad en el diagnóstico. Parsey y Schmitter-Edgecombe (2011) propusieron un método de calificación que, además de proporcionar un puntaje de la ejecución del paciente, permite el análisis de los errores y, con ello, la identificación de las potenciales dificultades cognitivas subyacentes. Objetivo: El objetivo de este estudio fue validar el método de calificación del TDR propuesto por Parsey y Schmitter-Edgecombe (2011) para el tamizaje del TNC en adultos mayores mexicanos. Método: Se contó con 167 participantes: 58 cognitivamente sanos (CS), 52 con trastorno neurocognitivo leve (TNC-leve) y 57 con trastorno neurocognitivo mayor (TNC-mayor). El método de calificación del TDR se comparó con el Examen Mínimo del Estado Mental (MMSE) y la Evaluación Cognitiva de Montreal en español (MoCA-E). Se determinó la confiabilidad inter e intra-observador y la validez de constructo, y se calcularon la sensibilidad y la especificidad de este método. Resultados: La X - de edad fue de 75 años (DE ± 8 años) y la X - de escolaridad fue de 10.7 años (DE ± 5.2 años). La confiabilidad interna fue de .750, con un coeficiente de correlación intraclase de .774. El punto de corte para el TDR en TNC-leve fue de 14 puntos (sensibilidad: 40%, especificidad: 70%) y 12 puntos para TNC-mayor (sensibilidad: 90%, especificidad: 95%). Los errores más frecuentes en el TDR fueron: dificultades gráficas, conceptuales y espaciales, y/o de planeación. Discusión y conclusión: Este método permite explorar breve y ágilmente el estado cognitivo del paciente y posee propiedades psicométricas ideales para la detección de pacientes con TNC-mayor, además de ofrecer la posibilidad de analizar los errores que presentan en el desempeño y las dificultades cognitivas subyacentes.