Hydrogel Extrusion Speed Measurements for the Optimization of Bioprinting Parameters.

Three-dimensional (3D) bioprinting is the use of computer-controlled transfer processes for assembling bioinks (cell clusters or materials loaded with cells) into structures of prescribed 3D organization. The correct bioprinting parameters ensure a fast and accurate bioink deposition without exposing the cells to harsh conditions. This study seeks to optimize pneumatic extrusion-based bioprinting based on hydrogel flow rate and extrusion speed measurements. We measured the rate of the hydrogel flow through a cylindrical nozzle and used non-Newtonian hydrodynamics to fit the results. From the videos of free-hanging hydrogel strands delivered from a stationary print head, we inferred the extrusion speed, defined as the speed of advancement of newly formed strands. Then, we relied on volume conservation to evaluate the extrudate swell ratio. The theoretical analysis enabled us to compute the extrusion speed for pressures not tested experimentally as well as the printing speed needed to deposit hydrogel filaments of a given diameter. Finally, the proposed methodology was tested experimentally by analyzing the morphology of triple-layered square-grid hydrogel constructs printed at various applied pressures while the printing speeds matched the corresponding extrusion speeds. Taken together, the results of this study suggest that preliminary measurements and theoretical analyses can simplify the search for the optimal bioprinting parameters.

The 3D Printing and Evaluation of Surgical Guides with an Incorporated Irrigation Channel for Dental Implant Placement.

Dental implant insertion requires the preparation of the implant bed via surgical drilling. During this stage, irrigation is essential to avoid thermal damage to the surrounding bone. Surgical guides enhance the accuracy of the implant site preparation, but they mask the drilling site, hampering coolant delivery. A variety of designs are aimed at improving the coolant access to the target site. Using standard dental implant simulation software, this paper presents an in-house design and 3D printing workflow for building surgical guides that incorporate a coolant channel directed toward the entry point of the burr. The proposed design was evaluated in terms of the bone temperature elevations caused by drilling performed at 1500 rpm, under an axial load of 2 kg, and irrigation with 40 mL/min of saline solution at 25 °C. Temperature measurements were performed on porcine femoral pieces, in the middle of the cortical bone layer, at 1 mm from the edge of the osteotomy. The mean temperature rise was 3.2 °C for a cylindrical sleeve guide, 2.7 °C for a C-shaped open-sleeve guide, and 2.1 °C for the guide with an incorporated coolant channel. According to a one-way ANOVA, the differences between these means were marginally insignificant (p = 0.056). The individual values of the peak temperature change remained below the bone damage threshold (10 °C) in all cases. Remarkably, the distribution of the recorded temperatures was the narrowest for the guide with internal irrigation, suggesting that, besides the most effective cooling, it provides the most precise control of the intraosseous temperature. Further studies could test different design variants, experimental models (including live animals), and might involve computer simulations of the bone temperature field.

Learning Effects in Air Displacement Plethysmography.

Air displacement plethysmography (ADP) is a widespread technique for assessing global obesity in both health and disease. The reliability of ADP has been demonstrated by studies focused on duplicate trials. The present study was purported to evaluate learning effects on the reliability of body composition assessment using the BOD POD system, the sole commercially available ADP instrument. To this end, quadruplicate trials were performed on a group of 105 subjects (51 women and 54 men). We estimated measurement error from pairs of consecutive trials-(1,2), (2,3), and (3,4)-to test the hypothesis that early measurements are subject to larger errors. Indeed, statistical analysis revealed that measures of reliability inferred from the first two trials were inferior to those computed for the other pairs of contiguous trials: for percent body fat (%BF), the standard error of measurement (SEM) was 1.04% for pair (1,2), 0.71% for pair (2,3), and 0.66% for pair (3,4); the two-way random effects model intraclass correlation coefficient (ICC) was 0.991 for pair (1,2), and 0.996 for pairs (2,3) and (3,4). Our findings suggest that, at least for novice subjects, the first ADP test should be regarded as a practice trial. When the remaining trials were pooled together, the reliability indices of single ADP tests were the following: ICC = 0.996, SEM = 0.70%, and minimum detectable change (MDC) = 1.93% for %BF, and ICC = 0.999, SEM = 0.49 kg, and MDC = 1.35 kg for fat-free mass (FFM). Thus, the present study pleads for eliminating learning effects to further increase the reliability of ADP.

Using A-Mode Ultrasound to Assess the Body Composition of Soccer Players: A Comparative Study of Prediction Formulas.

For elite athletes, monitoring body composition is important for maximizing performance without health risks. Amplitude (A)-mode ultrasound (AUS) has attracted increasing attention as an alternative to skinfold thickness measurements commonly used for assessing the amount of body fat in athletes. AUS accuracy and precision, however, depend on the formula used to predict body fat percentage (%BF) from subcutaneous fat layer thicknesses. Therefore, this study evaluates the accuracy of the 1-point biceps (B1), 9-sites Parrillo, 3-sites Jackson and Pollock (JP3), and 7-sites Jackson and Pollock (JP7) formulas. Relying on the previous validation of the JP3 formula in college-aged male athletes, we took AUS measurements in 54 professional soccer players (aged 22.9 ± 3.83 y, mean ± SD) and compared the results given by different formulas. The Kruskal-Wallis test indicated significant differences (p < 10-6), and Conover's post hoc test revealed that the JP3 and JP7 data come from the same distribution, whereas the data given by B1 and P9 differ from all the others. Lin's concordance correlation coefficients for B1 vs. JP7, P9 vs. JP7, and JP3 vs. JP7 were 0.464, 0.341, and 0.909, respectively. The Bland-Altman analysis indicated mean differences of -0.5 %BF between JP3 and JP7, 4.7 %BF between P9 and JP7, and 3.1 %BF between B1 and JP7. This study suggests that JP7 and JP3 are equally valid, whereas P9 and B1 overestimate %BF in athletes.

Mathematical and computational models in spheroid-based biofabrication.

Ubiquitous in embryonic development, tissue fusion is of interest to tissue engineers who use tissue spheroids or organoids as building blocks of three-dimensional (3D) multicellular constructs. This review presents mathematical models and computer simulations of the fusion of tissue spheroids. The motivation of this study stems from the need to predict the post-printing evolution of 3D bioprinted constructs. First, we provide a brief overview of differential adhesion, the main morphogenetic mechanism involved in post-printing structure formation. It will be shown that clusters of cohesive cells behave as an incompressible viscous fluid on the time scale of hours. The discussion turns then to mathematical models based on the continuum hydrodynamics of highly viscous liquids and on statistical mechanics. Next, we analyze the validity and practical use of computational models of multicellular self-assembly in live constructs created by tissue spheroid bioprinting. Finally, we discuss the perspectives of the field as machine learning starts to reshape experimental design, and modular robotic workstations tend to alleviate the burden of repetitive tasks in biofabrication. STATEMENT OF SIGNIFICANCE: Bioprinted constructs are living systems, which evolve via morphogenetic mechanisms known from developmental biology. This review presents mathematical and computational tools devised for modeling post-printing structure formation. They help achieving a desirable outcome without expensive optimization experiments. While previous reviews mainly focused on assumptions, technical details, strengths, and limitations of computational models of multicellular self-assembly, this article discusses their validity and practical use in biofabrication. It also presents an overview of mathematical models that proved to be useful in the evaluation of experimental data on tissue spheroid fusion, and in the calibration of computational models. Finally, the perspectives of the field are discussed in the advent of robotic biofabrication platforms and bioprinting process optimization by machine learning.

The impact of subject positioning on body composition assessments by air displacement plethysmography evaluated in a heterogeneous sample.

INTRODUCTION: This study sought to evaluate the impact of subject positioning on body composition assessments by air displacement plethysmography using the BOD POD®. METHODS: Eighty-two adults (42 men and 40 women), aged 26.1 ± 8.4 y (mean ± standard deviation), body mass index = 23.6 ± 4.8 kg/m2, were assessed by repeated measurements in two different positions: relaxed (legs apart, back away from the rear) and compact (legs together, arms near the body, back touching the rear). We relied on Bland-Altman analysis to quantify the agreement between results recorded in the two positions. Using body surface charts, we tested the hypothesis that posture-induced variability stems from differences in exposed skin area. RESULTS: Switching from compact to relaxed position resulted in a bias of -197 mL for body volume, -1.53% for percent body fat, and 1.085 kg for fat-free mass. The body surface area in contact with air was larger in relaxed position by 3632 ± 522 cm2. When body volume was expressed in terms of the actual area of exposed skin in the compact position, the percent body fat bias became 0.08%, with a 95% confidence interval of (-0.14, 0.29)%. CONCLUSIONS: Subject posture is a source of significant variability in air displacement plethysmography. The disagreement between results obtained in different positions can be eliminated by adjusting the surface area artifact, suggesting that subject positioning in the BOD POD® should be controlled to avoid changes in the amount of air maintained under isothermal conditions by the body.

Reliability of Repeated Trials Protocols for Body Composition Assessment by Air Displacement Plethysmography.

Air displacement plethysmography (ADP) is fast, accurate, and reliable. Nevertheless, in about 3% of the cases, standard ADP tests provide rogue results. To spot these outliers and improve precision, repeated trials protocols have been devised, but few works have addressed their reliability. This study was conducted to evaluate the test-retest reliabilities of two known protocols and a new one, proposed here. Ninety-two healthy adults (46 men and 46 women) completed six consecutive ADP tests. To evaluate the reliability of single measurements, we used the results of the first two tests; for multiple measures protocols, we computed the test result from trials 1-3 and the retest result from trials 4-6. Bland-Altman analysis revealed that the bias and the width of the 95% interval of agreement were smaller for multiple trials than for single ones. For percent body fat (%BF)/fat-free mass, the technical error of measurement was 1% BF/0.68 kg for single trials and 0.62% BF/0.46 kg for the new protocol of multiple trials, which proved to be the most reliable. The minimal detectable change (MDC) was 2.77% BF/1.87 kg for single trials and 1.72% BF/1.26 kg for the new protocol.

3D Bioprinting of Model Tissues That Mimic the Tumor Microenvironment.

The tumor microenvironment (TME) influences cancer progression. Therefore, engineered TME models are being developed for fundamental research and anti-cancer drug screening. This paper reports the biofabrication of 3D-printed avascular structures that recapitulate several features of the TME. The tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells (106 cells/mL); it is embedded in the same type of hydrogel containing primary cells-tumor-associated fibroblasts isolated from the peritumoral environment and peripheral blood mononuclear cells. Hoechst staining of cryosectioned tissue constructs demonstrated that cells remodeled the hydrogel and remained viable for weeks. Histological sections revealed heterotypic aggregates of malignant and peritumoral cells; moreover, the constituent cells proliferated in vitro. To investigate the interactions responsible for the experimentally observed cellular rearrangements, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods. Although unable to replicate the complexity of the TME, the approach presented here enables the self-assembly and co-culture of several cell types of the TME. Further studies will evaluate whether the bioprinted constructs can evolve in vivo in animal models. If they become connected to the host vasculature, they may turn into a fully organized TME.

Using Portable Ultrasound to Monitor the Neuromuscular Reactivity to Low-Frequency Electrical Stimulation.

Neuromuscular electrical stimulation (NMES) is useful for muscle strengthening and for motor restoration of stroke patients. Using a portable ultrasound instrument, we developed an M-mode imaging protocol to visualize contractions elicited by NMES in the quadriceps muscle group. To quantify muscle activation, we performed digital image processing based on the Teager-Kaiser energy operator. The proposed method was applied for 35 voluntary patients (18 women and 17 men), of 63.8 ± 14.1 years and body mass index (BMI) 30.2 ± 6.70 kg/m2 (mean ± standard deviation). Biphasic, rectangular electric pulses of 350 µs duration were applied at two frequencies (60 Hz and 120 Hz), and ultrasound was used to assess the sensory threshold (ST) and motor threshold (MT) amplitude of the NMES signal. The MT was 23.4 ± 4.94 mA, whereas the MT to ST ratio was 2.69 ± 0.57. Linear regression analysis revealed that MT correlates poorly with body mass index (R2 = 0.004) or with the thickness of the subcutaneous adipose tissue layer that covers the treated muscle (R2 = 0.013). Our work suggests that ultrasound is suitable to visualize neuromuscular reactivity during electrotherapy. The proposed method can be used in the clinic, enabling the physiotherapist to establish personalized treatment parameters.

Reliability of body composition assessment using A-mode ultrasound in a heterogeneous sample.

BACKGROUND/OBJECTIVES: Several studies have addressed the validity of ultrasound (US) for body composition assessment, but few have evaluated its reliability. This study aimed to determine the reliability of percent body fat (%BF) estimates using A-mode US in a heterogeneous sample. SUBJECTS/METHODS: A group of 144 healthy adults (81 men and 63 women), 30.4 (10.1) years (mean (SD)), BMI 24.6 (4.7) kg/m2, completed 6 consecutive measurements of the subcutaneous fat layer thickness at 8 anatomical sites. The measurements were done, alternatively, by two testers, using a BodyMetrix™ instrument. To compute %BF, 4 formulas from the BodyView™ software were applied: 7-sites Jackson and Pollock, 3-sites Jackson and Pollock, 3-sites Pollock, and 1-point biceps. RESULTS: The formula with the most anatomic sites provided the best reliability quantified by the following measures: intraclass correlation coefficient (ICC) = 0.979 for Tester 1 (T1) and 0.985 for T2, technical error of measurement (TEM) = 1.07% BF for T1 and 0.89% BF for T2, and minimal detectable change (MDC) = 2.95% BF for T1, and 2.47% BF for T2. The intertester bias was -0.5% BF, whereas the intertester ICC was 0.972. The intertester MDC was 3.43% BF for the entire sample, 3.24% BF for men, and 3.65% BF for women. CONCLUSIONS: A-mode US is highly reliable for %BF assessments, but it is more precise for men than for women. Examiner performance is a source of variability that needs to be mitigated to further improve the precision of this technique.

Using Sacrificial Cell Spheroids for the Bioprinting of Perfusable 3D Tissue and Organ Constructs: A Computational Study.

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.

Evaluation of the influence of patient positioning on the reliability of lateral cephalometry.

Two-dimensional cephalometry is widely used for monitoring orthodontic treatments and for quantifying the outcome of maxillofacial surgery. Despite careful use of a cephalostat, successive radiographs might differ due to slight differences in patient posture. This study evaluates the reliability of lateral cephalometric measurements and estimates the impact of patient positioning on this reliability. We studied cephalograms of 104 patients; 31 of them had two radiographs because the first was deemed unsuitable for cephalometric analysis. Using AudaxCeph 3.0 (Audax, Ljubljana, Slovenia), two observers traced each cephalogram twice, one month apart. We evaluated intra- and interobserver agreement via Bland-Altman analysis, intraclass correlation coefficient (ICC), standard error of measurement, and smallest detectable difference (SDD). First, we studied the reliability of the hard tissue part of the Tweed-Merrifield analysis for 73 single cephalograms and for the better ones of patients with two exposures. Then, we studied 31 unsatisfactory cephalograms, and the ones recorded at improved patient posture. Although intraobserver bias was less than 0.5° or 0.3 mm, interobserver bias was significant for most measurements. Intraobserver reliability was high (ICC > 0.9), whereas interobserver reliability was good (ICC > 0.83) except for FMPA, FMIA and OP. Head rotations and inclinations had little impact on reliability (e.g., interobserver SDD decreased for 3 of 11 measurements). We conclude that averaging the positions of bilateral structures enables a reliable cephalometric analysis in spite of imprecise patient posture. Retaking cephalograms is ethically questionable in such cases.

The secondary structure and the thermal unfolding parameters of the S-layer protein from Lactobacillus salivarius.

Surface layer (S-layer) proteins have been identified in the cell envelope of many organisms, such as bacteria and archaea. They self-assemble, forming monomolecular crystalline arrays. Isolated S-layer proteins are able to recrystallize into regular lattices, which proved useful in biotechnology. Here we investigate the structure and thermal unfolding of the S-layer protein isolated from Lactobacillus salivarius 16 strain of human origin. Using circular dichroism (CD) spectroscopy, and the software CDSSTR from DICHROWEB, CONTINLL from CDPro, as well as CDNN, we assess the fractions of the protein's secondary structural elements at temperatures ranging between 10 and 90 °C, and predict the tertiary class of the protein. To study the thermal unfolding of the protein, we analyze the temperature dependence of the CD signal in the far- and near-UV domains. Fitting the experimental data by two- and three-state models of thermal unfolding, we infer the midpoint temperatures, the temperature dependence of the changes in Gibbs free energy, enthalpy, and entropy of the unfolding transitions in standard conditions, and the temperature dependence of the equilibrium constant. We also estimate the changes in heat capacity at constant pressure in standard conditions. The results indicate that the thermal unfolding of the S-layer protein from L. salivarius is highly cooperative, since changes in the secondary and tertiary structures occur simultaneously. The thermodynamic analysis predicts a "cold" transition, at about -3 °C, of both the secondary and tertiary structures. Our findings may be important for the use of S-layer proteins in biotechnology and in biomedical applications.

Shape changes of bioprinted tissue constructs simulated by the Lattice Boltzmann method.

Tissue engineers seek to build living tissue constructs for replacing or repairing damaged tissues. Computational methods foster tissue engineering by pointing out dominant mechanisms involved in shaping multicellular systems. Here we apply the Lattice Boltzmann (LB) method to study the fusion of multicellular constructs. This process is of interest in bioprinting, in which multicellular spheroids or cylinders are embedded in a supportive hydrogel by a computer-controlled device. We simulated post-printing rearrangements of cells, aiming to predict the shape and stability of certain printed structures. To this end, we developed a two-dimensional LB model of a multicellular system in a hydrogel. Our parallel computing code was implemented using the Portable Extensible Toolkit for Scientific Computation (PETSc). To validate the LB model, we simulated the fusion of multicellular cylinders in a contiguous, hexagonal arrangement. Our two-dimensional LB simulation describes the evolution of the transversal cross section of the construct built from three-dimensional multicellular cylinders whose length is much larger than their diameter. Fusion eventually gave rise to a tubular construct, in qualitative agreement with bioprinting experiments. Then we simulated the time course of a defect in a bioprinted tube. To address practical problems encountered in tissue engineering, we also simulated the evolution of a planar construct, as well as of a bulky, perfusable construct made of multicellular cylinders. The agreement with experiments indicates that our LB model captures certain essential features of morphogenesis, and, therefore, it may be used to test new working hypotheses faster and cheaper than in the laboratory.

Computational study of the self-assembly of two different cell populations in contact with a biomaterial.

The organisation of a heterotypic multicellular system is intensely studied in developmental biology, tissue engineering and regenerative medicine.To address this problem, we have created a computational model of a biological system made of two cell populations of various cohesivities, and simulated its evolution on the surface of biomaterials of different adhesivities. To this end, it was necessary to extend our SIMMMC application with algorithms that treat two cell types. We have observed, in accordance with experiments that, depending on the strength of cell-substrate adhesion, different structures emerge by the self-assembly of the two cell populations. The agreement with experimental results validates the extended version of the SIMMMC application, suggesting that this tool might offer useful insights for tissue engineers.

Computational study of the potential role of chemotaxis in enhancing the cell seeding of tissue engineering scaffolds.

Chemotaxis is a morphogenetic mechanism that consists of cell movement along a concentration gradient. Since cell adhesion is essential for cell migration and tissue integrity, and morphogenesis also involves chemotactic cell movement, we conducted a study of the influence of adhesion and chemotaxis on cell seeding of scaffolds. We built three-dimensional models of a cell aggregate, respectively of a cell suspension, in the vicinity of a porous scaffold that contained a chemoattractant substance. We assumed that the chemoattractant is released at a steady rate, creating a constant concentration gradient. To study the interplay of adhesion and chemotaxis, we simulated cell seeding using an algorithm based on the Metropolis Monte Carlo method. We varied the chemotactic strength, which describes the extent to which cells tend to move along the chemoattractant gradient. We found that, for the same energetic and geometric conditions, the distribution of cells within the scaffold hinges on the chemotactic strength. Our study suggests that cell seeding of tissue engineering scaffolds may be enhanced by incorporating chemoattractants of controlled release rate into the scaffold.

Circular dichroism and the secondary structure of the ROF2 protein from Arabidopsis thaliana.

The protein ROF2 from the plant Arabidopsis thaliana acts as a heat stress modulator, being involved in the long-term acquired thermotolerance of the plant. Here we investigate the relationship between the biological function and the structure of ROF2, inferred by circular dichroism (CD) spectroscopy. The far-UV CD spectra, analyzed with the CDPro and DICHROWEB program packages, yield the percentages of α-helices, ß-sheets, unordered regions, turns and poly(Pro)II-helices in the secondary structure of ROF2. According to the analysis, the percentages of the structural elements of ROF2 are about 40% for ß-sheets, 30% for unordered regions, 17% for turns, 10% for poly(Pro)II-helices and 3% for α-helices. The near-UV CD spectra suggest that ROF2 proteins can associate, forming super-secondary structures. Our CD experiments performed at temperatures between 5 °C and 97 °C indicate that the thermal denaturation of ROF2 caused by a raise in temperature up to 55 °C is followed by a thermal refolding of the protein as the temperature is raised further. The new secondary structure, acquired around 65 °C, remains stable up to 97 °C. The structural stability of ROF2 at high temperatures might play an important role in the experimentally observed thermotolerance of Arabidopsis thaliana.

Computational study of the interplay of adhesion and chemotaxis in the cell seeding of tissue engineering scaffolds with incorporated chemoattractants.

Cell migration is important in embryogenesis, metastasis and wound healing. Often, morphogenesis involves chemotactic cell movement up or down chemical gradients. We have developed a computational model of a cell suspension in the vicinity of a porous scaffold that incorporates a chemoattractant substance. In order to study the interplay of adhesion and chemotaxis on cell seeding , we developed a computational model of the system and simulated its evolution using a Metropolis Monte Carlo algorithm. We varied the chemotactic strengths of the cells in order to identify the optimal conditions for a rapid and uniform seeding.

Tempolenses with variable magnification for sonic representation of medical data. Application for cardiac signals.

The purpose of this study was to develop the tools and the methodology for a systematic analysis of usefulness of adding sonic representation of data, supplementary to visualization. This paper is mainly dedicated various temporal lenses, including the newly developed lenses with variable magnification, proposed as a tool for a better perception of short events combined with a compression of irrelevant intervals. Sonification procedures are also briefly presented. The programs were tested using various cardiac signals: ECG and heart rate HR both in humans and in rats (experimental data). The results, represented by the sound files, were uploaded in an accessible library, which contains both sonic and visual representation of the signals.

Computer simulations of in vitro morphogenesis.

One of the most important challenges of contemporary biology is understanding how cells assemble into tissues. The complexity of morphogenesis calls for computational tools able to identify the dominant mechanisms involved in shaping tissues. This narrative review presents individual-based computational models that proved useful in simulating phenomena of interest in tissue engineering (TE), a research field that aims to create tissue replacements in the laboratory. First, we briefly describe morphogenetic mechanisms. Then, we present several computational models of cellular and subcellular resolution, along with applications that illustrate their potential to address problems of TE. Finally, we analyze experiments that may be used to validate computational models of tissue constructs made of cohesive cells. Our analysis shows that the models available in the literature are not exploited to their full potential. We argue that, upon validation, a computational model can be used to optimize cell culture conditions and to design new experiments.