Design Of Experimental Protocols Research Articles

Exploring visual feedback modalities in augmented reality to control the walking speed of children with cerebral palsy. Experimental design protocol

Exploring visual feedback modalities in augmented reality to control the walking speed of children with cerebral palsy. Experimental design protocol

Effectiveness of Using Compression Garments in Winter Racing Sports: A Narrative Review.

Nowadays, compression garments (CGs) are widely used in winter racing sports, such as speed skating, short-track speed skating, alpine skiing, and cross-country skiing. However, the effect of wearing CGs on athletic performance in these specific sports is still not fully examined. Thus, the aim of this narrative review is to summarize the research and application of CGs in winter racing sports and to discuss how the CGs help athletes improve their performance in an integrative manner (i.e., physiology, aerodynamics, and biomechanics). A total of 18 experimental studies dedicated to CGs in winter racing sports were identified from the peer-review scientific literature. The main findings are as follows. (1) Currently, CG studies have mainly focused on drag reduction, metabolism, muscle function, strength performance, and fatigue recovery. (2) The results of most studies conducted in wind tunnels showed that, for cylindrical structures similar to the human body, clothing with rough surfaces can reduce air drag. Notably, the effect of CGs on drag reduction in real competition has not been fully explored in the literature. (3) Compression can reduce muscle vibrations at high impact and help athletes control the center of pressure movement, a function that is important for alpine skiing. Future studies are needed to improve current understanding of the effects of compression clothing microstructure on drag reduction and their stretching in different parts of the body. Furthermore, the design of experimental protocol must be consistent with those during the competition, thus providing a full discussion on energy metabolism, fatigue, and recovery affected by CGs.

Formulation and optimization of quinoa starch nanoparticles: Quality by design approach for solubility enhancement of piroxicam

In the present study piroxicam loaded starch nanoparticles were prepared to enhance the solubility of piroxicam by nanoprecipitation technique. The preparation of nanoparticles was carried out as per central composite experimental design protocol, having concentration of starch and drug as independent variables and particle size and polydispersity index (PdI) as dependent variables. The particle size and PdI of piroxicam loaded starch nanoparticles was found to be between 282–870 nm and 0.339–0.772, respectively. After the characterization by FT-IR, TGA, XRD and SEM studies, the optimized batch was evaluated for in-vitro release study, anti-inflammatory activity and anti-oxidant activity. The in-vitro anti-inflammatory activity of piroxicam loaded starch nanoparticles was found to be more than the pure drug piroxicam whereas the anti-oxidant activity of starch is found greater than starch nanoparticles. In-vitro release study showed 98.8% release in 2 h dissolution study following supercase II transport mechanism of drug release.

Warming effects on methane fluxes differ between two alpine grasslands with contrasting soil water status

Soil moisture plays a vital role in regulating the direction and magnitude of methane (CH4) fluxes. However, it remains unclear whether the responses of CH4 fluxes to climate warming exhibit difference between dry and moist ecosystems. Based on standardized manipulative experiments (i.e., consistent experimental design and measurement protocols), here we explored warming effects on growing season CH4 fluxes in two alpine grasslands with contrasting water status on the Tibetan Plateau. We observed that experimental warming enhanced CH4 uptake in the relatively arid alpine steppe, but had no significant effects on CH4 emission in the moist swamp meadow. The distinct responses of CH4 fluxes were associated with the different warming effects on biotic and abiotic factors related to CH4 oxidation and production processes. Warming decreased soil water-filled pore space (WFPS) and increased the pmoA gene abundance and CH4 oxidation potential in the alpine steppe, which together led to a significant increase in CH4 uptake at this alpine steppe site. However, warming-induced enhancement in CH4 oxidation potential might be counteracted by the simultaneously increased CH4 production potential in the swamp meadow, which could then result in insignificant warming effects on CH4 emission at this swamp meadow site. Based on a meta-analysis of warming effects on CH4 fluxes across the entire Tibetan Plateau, we found that the entire alpine grasslands could absorb an extra 0.042 Tg CH4 (1 Tg = 1012 g) per growing season if soil temperature increased by 1 °C. These findings demonstrate that warming effects on CH4 fluxes differ between two alpine grasslands with contrasting moisture conditions and the entire alpine grasslands may not trigger a positive CH4 feedback to climate system with moderate warming.

Ethical Considerations in Animal Research: The Principle of 3R's.

In the last century, progress in the knowledge of human diseases, their diagnosis and treatment have grown exponentially, due in large part to the introduction and use of laboratory animals. Along with this important progress, the need to provide training and guidance to the scientific community in all aspects related to the proper use of experimental animals has been indispensable. Animal research committees play a primary role in evaluating experimental research protocols, from their feasibility to the rational use of animals, but above all in seeking animal welfare. The Institutional Committee for the Care and Use of Animals (IACUC) has endeavored to share several relevant aspects in conducting research with laboratory animals. Here, we present and discuss the topics that we consider of utmost importance to take in the account during the design of any experimental research protocol, so we invite researchers, technicians, and undergraduate and graduate students to dive into the fascinating subject of proper animal care and use for experimentation. The main intention of these contributions is to sensitize users of laboratory animals for the proper and rational use of them in experimental research, as well as to disseminate the permitted and unpermitted procedures in laboratory animals. In the first part, the significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are addressed.

Sex complicates the effect and treatment of heat stress

Global temperature is continuing to rise, and resultant heat stress (HS) poses a major threat to human health. Using large animal models, we have shown that 12 and 24 h, and 7 d of heat exposure induced oxidative stress and impaired autophagy in oxidative skeletal muscle but not glycolytic muscle. We and others have implicated mitochondrial dysfunction in this process. We hypothesized that treatment with a mitochondrially targeted antioxidant, MitoQ, would decrease oxidative stress and alleviate cellular dysfunction during HS. To address this hypothesis, 3 mo old castrated male pigs (59 ± 1.0m kg; n=8/group) were administered either placebo or MitoQ (40 mg/day) divided into two daily doses every 12 h beginning 48 h prior to environmental treatment and continuing through environmental exposure. Half of the placebo‐ and half of the MitoQ‐treated pigs were assigned to thermoneutral (TN; 20°C) or HS (35°C) conditions for 24 h. The red portion of the semitendinosus (STR) was collected for subsequent analysis. Unexpectedly, malondialdehyde (MDA) concentration, a marker of oxidative stress, was unaffected by treatment or environment. Additionally, LC3A/B‐I was decreased 0.5‐fold (p<0.05) during HS, LC3A/B‐II was similar, and the ratio of LC3A/B‐II/I was increased 1.5‐fold (p<0.05) in HS; p62 was similar between treatment groups. These data suggest increased degradation of autophagosomes in HS males, which may explain maintenance of redox balance but conflicts with previous findings in female pigs; a scenario which raises the possibility of a sex effect on the muscular response to HS. To probe this idea, we compared STR from these HS males to archived HS STR from female pigs treated with a similar experimental design and environmental protocol described above. We confirmed our previous findings of oxidative stress and dysfunctional autophagy in females, and maintenance of redox balance with increased degradation of autophagosomes in males. In total, these data suggest that muscle from female pigs is more susceptible to detrimental effects of environment‐induced hyperthermia than muscle from male pigs.Support or Funding InformationSupported by USDA grant 2017‐05931

Multi-target optimization of solid phase microextraction to analyse key flavour compounds in wort and beer

Despite the literature comprises numerous studies dealing with the analysis of wort and beer flavour-related compounds by HS-SPME followed by GC–MS quantification, no generalized consensus exists regarding the optimal conditions for the extraction procedure. The complex chemistry nature of these matrices, the number of analytes, as well as the number and interactions among parameters affecting the extraction performance, requires the adoption of optimal experimental design protocols. This aspect is often overlooked and often not properly addressed in practice. Therefore, in the present work, the optimal conditions under which a range of wort and beer analytes can be extracted and quantified were analysed. The optimal extraction conditions were presented at two levels of aggregation: global (untargeted) and key-flavour analysis. Experimental data was generated by Definitive-Screening-Design, followed by model development and optimization. Both approaches were compared and critically analysed. For vicinal-diketones group, a complete validation study for the optimal conditions is presented.

Small scale design of experiment investigation of equilibrium solubility in simulated fasted and fed intestinal fluid

It is widely recognised that drug solubility within the gastrointestinal tract (GIT) differs from values determined in a simple aqueous buffer and to circumvent this problem measurement in biorelevant fluids is determined. Biorelevant fluids are complex mixtures of components (sodium taurocholate, lecithin, sodium phosphate, sodium chloride, pancreatin and sodium oleate) at various concentrations and pH levels to provide systems simulating fasted (FaSSIF) or fed (FeSSIF) intestinal media. Design of Experiment (DoE) studies have been applied to investigate FaSSIF and FeSSIF and indicate that a drug's equilibrium solubility varies over orders of magnitude, is influenced by the drug type and individual or combinations of media components, with some of these interactions being drug specific. Although providing great detail on the drug media interactions these studies are resource intensive requiring up to ninety individual experiments for FeSSIF. In this paper a low sample number or reduced DoE system has been investigated by restricting components with minimal solubility impact to a single value and only investigating variations in the concentrations of sodium taurocholate, lecithin, sodium oleate, pH and additionally in the case of fed media, monoglyceride. This reduces the experiments required to ten (FaSSIF) and nine (FeSSIF). Twelve poorly soluble drugs (Ibuprofen, Valsartan, Zafirlukast, Indomethacin, Fenofibrate, Felodipine, Probucol, Tadalafil, Carvedilol, Aprepitant, Bromocriptine and Itraconazole) were investigated and the results compared to published DoE studies and literature solubility values in human intestinal fluid (HIF), FaSSIF or FeSSIF. The solubility range determined by the reduced DoE is statistically equivalent to the larger scale published DoE results in over eighty five percent of the cases. The reduced DoE range also covers HIF, FaSSIF or FeSSIF literature solubility values. In addition the reduced DoE provides lowest measured solubility values that agree with the published DoE values in ninety percent of the cases. However, the reduced DoE only identified single and in some cases none of the major components influencing solubility in contrast to the larger published DoE studies which identified multiple individual components and component interactions. The identification of significant components within the reduced DoE was also dependent upon the drug and system under investigation. The study demonstrates that the lower experimental number reduces statistical power of the DoE to resolve the impact of media components on solubility. However, in a situation where only the solubility range is required the reduced DoE can provide the desired information, which will be of benefit during in vitro development studies. Further refinements are possible to extend the reduced DoE protocol to improve biorelevance and application into areas such as PBPK modelling.

Does antibiotic use accelerate or retard cutaneous repair? A systematic review in animal models.

BackgroundThe presence of infections is one of the main factors that leads to delays in healing or non-closure of cutaneous wounds. Although the goal of antibiotic use is to treat or prevent infection, there is currently no agreement on the effectiveness of these products.AimThe aim of this study was to evaluate the efficacy of antibiotic use during the healing process of skin wounds in animal models not intentionally infected, as well as to analyze the advances and limitations of the studies carried out in this field.Main methodsThis systematic review was performed according to the PRISMA guidelines, using a structured search on the MedLine (PubMed) and Scopus platforms to retrieve studies published until August 29, 2018, 13:35p.m. The studies included were limited to those that used excision or incision wound models and that were not intentionally infected. The data for the animal models, antibiotic used, and the main results of the studies were extracted, and compared where possible. Bias analysis and methodological quality assessments were examined through the SYRCLE’s Risk of Bias tool.Key findingsTwenty-seven studies were selected. Overall, the effects of the antibiotic on the wound decreased inflammatory cell infiltration and promoted an increased number of fibroblasts, extracellular matrix constituents, re-epithelialization and tissue strength. A great deal of important information about the methodology was not presented, such as: the statistical analysis used, the animal model (sex and age), antibiotic dosage, blinding and randomization of the animals chosen.SignificanceBased on the results found, we believe that antibiotic therapy can be considered a viable alternative for the treatment of cutaneous wounds. However, current evidence obtained from the methodological quality analysis points towards a high risk of bias. This is due to the incomplete characterization of the experimental design and treatment protocol, which compromises the reproducibility of the studies.

Fourth cycle breeding and testing strategy for Pinus taeda in the NC State University Cooperative Tree Improvement Program

The Cooperative Tree Improvement Program at North Carolina State University used the differential evolution algorithm developed for animal breeding (Kinghorn, Genet Sel Evol 43(1):4, Kinghorn 2011) to design the mating in the 4th cycle of loblolly pine (Pinus taeda L.) breeding. The mate selection algorithm optimizes genetic gain while putting constraints on progeny inbreeding and coancestry level. The final mating list (4th cycle population) is expected to produce an index breeding value 63% higher than the candidate population mated at random. The average inbreeding coefficient in the candidate population was 0.000146. It increased to 0.000285 in the 4th cycle progeny. Only eight crosses out of 748 with progeny inbreeding value greater than zero were included in the mating list. One parent with a high index breeding value was used in 13 matings. A high majority of 421 individuals (75%) were mated two or more times; the remaining were mated once. The Cooperative also made significant changes in experimental design protocols to increase the efficiency of progeny testing by implementing an alpha-cyclic incomplete row-column design. Progeny test series across years were connected with a rolling front testing strategy by overlapping genetic entries across years. The Cooperative is expected to complete the 4th cycle in about 14 years, a significant reduction compared to previous cycles. With availability of high-throughput genotyping platforms, the Cooperative is moving toward implementation of genomic selection in the 5th cycle and beyond.

Defining the viability of tardigrades with a molecular sensor related to death.

The design of experimental protocols that use animal models to assess the impact of a stress on a population or to determine the life span expectancy impact can be time-consuming due to the need for direct observations of dead and living animals. These experiments are usually based on the detectable activity of animals such as food intake or mobility and can sometimes produce either under- or overestimated results. The tardigrade Hypsibius exemplaris is an emerging model for the evolutionary biology of the tardigrade phylum because of its convenient laboratory breeding and the recent introduction of new molecular tools. In this report, we describe the use of a new fluorescent dye that can specifically stain dead tardigrades. Furthermore, we also monitored the absence of a toxic side effect of the death-linked fluorescent dye on tardigrade populations. Finally, we conclude that tardigrade experiments that require survival counting of the Hypsibius exemplaris species can be greatly improved by using this technique in order to limit underestimation of alive animals.

Experimental models in Chagas disease: a review of the methodologies applied for screening compounds against Trypanosoma cruzi.

One of the main problems of Chagas disease (CD), the parasitic infection caused by Trypanosoma cruzi, is the lack of a completely satisfactory treatment, which is currently based on two old nitroheterocyclic drugs (i.e., nifurtimox and benznidazole) that show important limitations for treating patients. In this context, many laboratories look for alternative therapies potentially applicable to the treatment, and therefore, research in CD chemotherapy works in the design of experimental protocols for detecting molecules with activity against T. cruzi. Phenotypic assays are considered the most valuable strategy for screening these antiparasitic compounds. Among them, in vitro experiments are the first step to test potential anti-T. cruzi drugs directly on the different parasite forms (i.e., epimastigotes, trypomastigotes, and amastigotes) and to detect cytotoxicity. Once the putative trypanocidal drug has been identified in vitro, it must be moved to in vivo models of T. cruzi infection, to explore (i) acute toxicity, (ii) efficacy during the acute infection, and (iii) efficacy in the chronic disease. Moreover, in silico approaches for predicting activity have emerged as a supporting tool for drug screening procedures. Accordingly, this work reviews those in vitro, in vivo, and in silico methods that have been routinely applied during the last decades, aiming to discover trypanocidal compounds that contribute to developing more effective CD treatments.

Biofluid Metabolomics in Preterm Birth Research.

This article presents an account of the research carried out so far in the use of metabolomics to find biomarkers of preterm birth (PTB) in fetal, maternal, and newborn biofluids. Metabolomic studies have employed mainly nuclear magnetic resonance spectroscopy or mass spectrometry-based methodologies to analyze, on one hand, prenatal biofluids (amniotic fluid, maternal urine/maternal blood, cervicovaginal fluid) to identify predictive biomarkers of PTB, and on the other hand, biofluids collected at or after birth (amniotic fluid, umbilical cord blood, newborn urine, and newborn blood, maternal blood, or breast milk) to assess and follow up the health status of PTB babies. Besides advancing on the biochemical knowledge of PTB metabolism mainly during the in utero period and at birth, the work carried out has also helped to identify important requirements related to experimental design and analytical protocol that need to be addressed, if translation of these biomarkers to the clinic is to be envisaged. An outlook of possible future developments for the translation of laboratory results to the clinic is presented.

Corticoids modulate liposome membrane fluidity and permeability depending on membrane composition and experimental protocol design

Given that literature data may give inconsistent results on the effect of a drug on lipid membrane properties, this work aims to investigate the impact of the liposome composition and experimental protocol design on glucocorticoids (GRs: cortisol, cortisone, fludrocortisone acetate, methylprednisolone, prednisolone and prednisone)-modulating membrane fluidity and permeability. GRs-loaded liposomes consisting of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CHOL) were prepared by reverse phase evaporation technique (REV) at DPPC:CHOL:GR molar ratios of 100:100:2.5, and 100:100:10. The formulations were characterized for their size and homogeneity, encapsulation efficiency and loading rates of GRs, incorporation rates and loading rates of DPPC and CHOL. Changes in DPPC membrane fluidity (CHOL% 0, 10, 20, 30 and 100) after exposure to methylprednisolone were monitored by using 5- and 16-doxyl stearic acids (DSA) as spin probes. For permeability studies, the above-mentioned GRs-loaded liposomes and the preformed liposomes exposed to GRs (2.5 mol%) were compared for the leakage of an encapsulated fluorescent dye, sulforhodamine B (SRB), at 37 °C in buffer (pH 7.5) containing NaCl. The SRB release kinetics were analyzed by the Higuchi model for two release phases (from 0 to 10 h, and from 10 to 48 h). All formulations exhibited a monodispersed size distribution of liposomes with a mean particle value close to 0.4 μm, also the DPPC and CHOL were highly incorporated (>95%). High loading rate values of DPPC and CHOL were also obtained. Except for fludrocortisone acetate (51%) and prednisolone (77%), high loading rate values of GRs were obtained (>81%). Fluidity and permeability studies showed that the GR concentration, CHOL content, experimental protocol design including the period of incubation represent critical parameters to be considered in analyzing the effect of drugs on the membrane properties.

Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus.

The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5–40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25−1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Could diet composition modulate pathological outcomes in schistosomiasis mansoni? A systematic review of in vivo preclinical evidence.

Schistosomiasis and malnutrition are often overlapped in poor communities, resulting in disproportionately high mortality rates. Currently, fragmented data make it difficult to define the relationship between diet and schistosomiasis. Thus, we systematically review the preclinical evidence on the impact of diet in Schistosoma mansoni infection. From a structured search, we recovered 27 original articles. All studies used mice and most of them investigated hypoproteic (70.37%), hyperlipidic (22.22%) or vitamin-deficient (7.41%) diets. Diets based on carbohydrate, zinc or milk supplementation were investigated at a reduced frequency (3.70% each). Hypoproteic diets attenuated parasitic load and granulomatous inflammation, but also reduced host resistance to S. mansoni infection, determining higher mortality rates. By stimulating steatohepatitis, parasitic load and granulomatous inflammation, hyperlipidic diets increase organ damage and mortality in infected animals. Although a high-sugar diet and vitamin restriction potentiate and zinc supplementation attenuates S. mansoni infection, the current evidence for these diets remains inconclusive. Analysis of methodological quality indicated that the current evidence is at high risk of bias due to incomplete characterization of the experimental design, diet composition and treatment protocols. From the bias analysis, we report methodological limitations that should be considered to avoid systematic reproduction of inconsistent and poorly reproducible experimental designs.

Water Adsorption and Dissociation on Polycrystalline Copper Oxides: Effects of Environmental Contamination and Experimental Protocol.

We use ambient-pressure X-ray photoelectron spectroscopy (APXPS) to study chemical changes, including hydroxylation and water adsorption, at copper oxide surfaces from ultrahigh vacuum to ambient relative humidities of ∼5%. Polycrystalline CuO and Cu2O surfaces were prepared by selective oxidation of metallic copper foils. For both oxides, hydroxylation occurs readily, even at high-vacuum conditions. Hydroxylation on both oxides plateaus near ∼0.01% relative humidity (RH) at a coverage of ∼1 monolayer. In contrast to previous studies, neither oxide shows significant accumulation of molecular water; rather, both surfaces show a high affinity for adventitious carbon contaminants. Results of isobaric and isothermic experiments are compared, and the strengths and potential drawbacks of each method are discussed. We also provide critical evaluations of the effects of the hot filament of the ion pressure gauge on the reactivity of gas-phase species, the peak fitting procedure on the quantitative analysis of spectra, and rigorous accounting of carbon contamination on data analysis and interpretation. This work underscores the importance of considering experimental design and data analysis protocols during APXPS experiments with water vapor in order to minimize misinterpretations arising from these factors.

The effects of incubation temperature and experimental design on heart rates of lizard embryos

Many studies of phenotypic plasticity alter environmental conditions during embryonic development, yet only measure phenotypes at the neonatal stage (after embryonic development). However, measuring aspects of embryo physiology enhances our understanding of how environmental factors immediately affect embryos, which aids our understanding of developmental plasticity. While current research on reptile developmental plasticity has demonstrated that fluctuating incubation temperatures affect development differently than constant temperatures, most research on embryo physiology is still performed with constant temperature experiments. In this study, we noninvasively measured embryonic heart rates of the brown anole (Anolis sagrei), across ecologically relevant fluctuating temperatures. We incubated eggs under temperatures measured from potential nests in the field and examined how heart rates change through a diel cycle and throughout embryonic development. We also evaluated how experimental design (e.g., repeated vs. single measures designs, constant vs. fluctuating temperatures) and different protocols (e.g., removing eggs from incubators) might influence heart rate. We found that heart rates were correlated with daily temperature and increased through development. Our findings suggest that experimenters have reasonable flexibility in choosing an experimental design to address their questions; however, some aspects of design and protocol can potentially influence estimations of heart rates. Overall, we present the first ecologically relevant measures of anole embryonic heart rates and provide recommendations for experimental designs for future experiments.

Novel imaging techniques to study the functional organization of the human brain

Despite more than a century of investigation into the cortical organization of motor function, the existence of motor somatotopy is still debated. We review functional magnetic resonance imaging (fMRI) studies examining motor somatotopy in the cerebral cortex. In spite of a substantial overlap of representations corresponding to different body parts, especially in non-primary motor cortices, geographic approaches are capable of revealing somatotopic ordering. From the iconic homunculus in the contralateral primary cortex to the subtleties of ipsilateral somatotopy and its relations with lateralization, we outline potential reasons for the lack of segregation between motor representations. Among these are the difficulties in distinguishing activity that arises from multiple muscular effectors, the need for flexible motor control and coordination of complex movements through functional integration and artefacts in fMRI. Methodological advances with regard to the optimization of experimental design and fMRI acquisition protocols as well as improvements in spatial registration of images and indices aiming at the quantification of the degree of segregation between different functional representations are inspected. Additionally, we give some hints as to how the functional organization of motor function might be related to various anatomical landmarks in brain morphometry.

Application of multiphysics models to efficient design of experiments of solute transport across articular cartilage

Transport of solutes helps to regulate normal physiology and proper function of cartilage in diarthrodial joints. Multiple studies have shown the effects of characteristic parameters such as concentration of proteoglycans and collagens and the orientation of collagen fibrils on the diffusion process. However, not much quantitative information and accurate models are available to help understand how the characteristics of the fluid surrounding articular cartilage influence the diffusion process. In this study, we used a combination of micro-computed tomography experiments and biphasic-solute finite element models to study the effects of three parameters of the overlying bath on the diffusion of neutral solutes across cartilage zones. Those parameters include bath size, degree of stirring of the bath, and the size and concentration of the stagnant layer that forms at the interface of cartilage and bath. Parametric studies determined the minimum of the finite bath size for which the diffusion behavior reduces to that of an infinite bath. Stirring of the bath proved to remarkably influence neutral solute transport across cartilage zones. The well-stirred condition was achieved only when the ratio of the diffusivity of bath to that of cartilage was greater than ≈1000. While the thickness of the stagnant layer at the cartilage-bath interface did not significantly influence the diffusion behavior, increase in its concentration substantially elevated solute concentration in cartilage. Sufficient stirring attenuated the effects of the stagnant layer. Our findings could be used for efficient design of experimental protocols aimed at understanding the transport of molecules across articular cartilage.