A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets.

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.

Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network.

Achieving hemostasis following vascular injury requires the rapid accumulation of platelets and fibrin. Here we used a combination of confocal intravital imaging, genetically engineered mice, and antiplatelet agents to determine how variations in the extent of platelet activation following vascular injury arise from the integration of different elements of the platelet-signaling network. Two forms of penetrating injury were used to evoke the hemostatic response. Both produced a hierarchically organized structure in which a core of fully activated platelets was overlaid with an unstable shell of less-activated platelets. This structure emerged as hemostasis was achieved and persisted for at least 60 minutes following injury, its organization at least partly reflecting agonist concentration gradients. Thrombin activity and fibrin formation were found primarily in the innermost core. As proposed previously, greater packing density in the core facilitated contact-dependent signaling and limited entry of plasma-borne molecules visualized with fluorophores coupled to dextran and albumin. Blocking contact-dependent signaling or inhibiting thrombin reduced the size of the core, while the shell was heavily influenced by adenosine 5'-diphosphate and regulators of Gi2-mediated signaling. Thus, the hemostatic response is shown to produce a hierarchical structure arising, in part, from distinct elements of the platelet-signaling network.

Addressable microfluidics technology for non-sacrificial analysis of biomaterial implants in vivo.

Tissue regeneration-promoting and drug-eluting biomaterials are commonly implanted into animals as a part of late-stage testing before committing to human trials required by the government. Because the trials are very expensive (e.g., they can cost over a billion U.S. dollars), it is critical for companies to have the best possible characterization of the materials' safety and efficacy before it goes into a human. However, the conventional approaches to biomaterial evaluation necessitate sacrificial analysis (i.e., euthanizing a different animal for measuring each time point and retrieving the implant for histological analysis), due to the inability to monitor how the host tissues respond to the presence of the material in situ. This is expensive, inaccurate, discontinuous, and unethical. In contrast, our manuscript presents a novel microfluidic platform potentially capable of performing non-disruptive fluid manipulations within the spatial constraints of an 8 mm diameter critical calvarial defect-a "gold standard" model for testing engineered bone tissue scaffolds in living animals. In particular, here, addressable microfluidic plumbing is specifically adapted for the in vivo implantation into a simulated rat's skull, and is integrated with a combinatorial multiplexer for a better scaling of many time points and/or biological signal measurements. The collected samples (modeled as food dyes for proof of concept) are then transported, stored, and analyzed ex vivo, which adds previously-unavailable ease and flexibility. Furthermore, care is taken to maintain a fluid equilibrium in the simulated animal's head during the sampling to avoid damage to the host and to the implant. Ultimately, future implantation protocols and technology improvements are envisioned toward the end of the manuscript. Although the bone tissue engineering application was chosen as a proof of concept, with further work, the technology is potentially versatile enough for other in vivo sampling applications. Hence, the successful outcomes of its advancement should benefit companies developing, testing, and producing vaccines and drugs by accelerating the translation of advanced cell culturing tech to the clinical market. Moreover, the nondestructive monitoring of the in vivo environment can lower animal experiment costs and provide data-gathering continuity superior to the conventional destructive analysis. Lastly, the reduction of sacrifices stemming from the use of this technology would make future animal experiments more ethical.

A Minireview of Microfluidic Scaffold Materials in Tissue Engineering.

In 2020, nearly 107,000 people in the U.S needed a lifesaving organ transplant, but due to a limited number of donors, only ∼35% of them have actually received it. Thus, successful bio-manufacturing of artificial tissues and organs is central to satisfying the ever-growing demand for transplants. However, despite decades of tremendous investments in regenerative medicine research and development conventional scaffold technologies have failed to yield viable tissues and organs. Luckily, microfluidic scaffolds hold the promise of overcoming the major challenges associated with generating complex 3D cultures: 1) cell death due to poor metabolite distribution/clearing of waste in thick cultures; 2) sacrificial analysis due to inability to sample the culture non-invasively; 3) product variability due to lack of control over the cell action post-seeding, and 4) adoption barriers associated with having to learn a different culturing protocol for each new product. Namely, their active pore networks provide the ability to perform automated fluid and cell manipulations (e.g., seeding, feeding, probing, clearing waste, delivering drugs, etc.) at targeted locations in-situ. However, challenges remain in developing a biomaterial that would have the appropriate characteristics for such scaffolds. Specifically, it should ideally be: 1) biocompatible-to support cell attachment and growth, 2) biodegradable-to give way to newly formed tissue, 3) flexible-to create microfluidic valves, 4) photo-crosslinkable-to manufacture using light-based 3D printing and 5) transparent-for optical microscopy validation. To that end, this minireview summarizes the latest progress of the biomaterial design, and of the corresponding fabrication method development, for making the microfluidic scaffolds.

Review of Low-Cost 3D Bioprinters: State of the Market and Observed Future Trends.

Three-dimensional (3D) bioprinting has become mainstream for precise and repeatable high-throughput fabrication of complex cell cultures and tissue constructs in drug testing and regenerative medicine, food products, dental and medical implants, biosensors, and so forth. Due to this tremendous growth in demand, an overwhelming amount of hardware manufacturers have recently flooded the market with different types of low-cost bioprinter models-a price segment that is most affordable to typical-sized laboratories. These machines range in sophistication, type of the underlying printing technology, and possible add-ons/features, which makes the selection process rather daunting (especially for a nonexpert customer). Yet, the review articles available in the literature mostly focus on the technical aspects of the printer technologies under development, as opposed to explaining the differences in what is already on the market. In contrast, this paper provides a snapshot of the fast-evolving low-cost bioprinter niche, as well as reputation profiles (relevant to delivery time, part quality, adherence to specifications, warranty, maintenance, etc.) of the companies selling these machines. Specifically, models spanning three dominant technologies-microextrusion, droplet-based/inkjet, and light-based/crosslinking-are reviewed. Additionally, representative examples of high-end competitors (including up-and-coming microfluidics-based bioprinters) are discussed to highlight their major differences and advantages relative to the low-cost models. Finally, forecasts are made based on the trends observed during this survey, as to the anticipated trickling down of the high-end technologies to the low-cost printers. Overall, this paper provides insight for guiding buyers on a limited budget toward making informed purchasing decisions in this fast-paced market.

An Introductory Overview of Image-Based Computational Modeling in Personalized Cardiovascular Medicine.

Cardiovascular diseases account for the number one cause of deaths in the world. Part of the reason for such grim statistics is our limited understanding of the underlying mechanisms causing these devastating pathologies, which is made difficult by the invasiveness of the procedures associated with their diagnosis (e.g., inserting catheters into the coronal artery to measure blood flow to the heart). Likewise, it is also difficult to design and test assistive devices without implanting them in vivo. However, with the recent advancements made in biomedical scanning technologies and computer simulations, image-based modeling (IBM) has arisen as the next logical step in the evolution of non-invasive patient-specific cardiovascular medicine. Yet, due to its novelty, it is still relatively unknown outside of the niche field. Therefore, the goal of this manuscript is to review the current state-of-the-art and the limitations of the methods used in this area of research, as well as their applications to personalized cardiovascular investigations and treatments. Specifically, the modeling of three different physics - electrophysiology, biomechanics and hemodynamics - used in the cardiovascular IBM is discussed in the context of the physiology that each one of them describes and the mechanisms of the underlying cardiac diseases that they can provide insight into. Only the "bare-bones" of the modeling approaches are discussed in order to make this introductory material more accessible to an outside observer. Additionally, the imaging methods, the aspects of the unique cardiac anatomy derived from them, and their relation to the modeling algorithms are reviewed. Finally, conclusions are drawn about the future evolution of these methods and their potential toward revolutionizing the non-invasive diagnosis, virtual design of treatments/assistive devices, and increasing our understanding of these lethal cardiovascular diseases.

Automated Addressable Microfluidic Device for Minimally Disruptive Manipulation of Cells and Fluids within Living Cultures.

Cell culturing experiments are ubiquitous to the study of biology, development of new medical treatments, and the biomanufacturing industry. However, there are still major technological barriers limiting the advancement of knowledge and ballooning the experimental costs associated with these systems. For example, currently, it is difficult to perform nondisruptive monitoring and control of the cells in the cultured samples. This often necessitates the use of sacrificial assays and results in product inconsistency. To resolve these bottlenecks, we present a prototype "addressable" microfluidic technology capable of spatiotemporal fluid and cell manipulations within living cultures. As a proof-of-concept, we demonstrate its ability to perform additive manufacturing by seeding cells in spatial patterns (including co-culturing multiple cell types) and subtractive manufacturing by removing surface adherent cells via the focused flow of trypsin. Additionally, we show that the device can sample fluids and perform cell "biopsies" (which can be subsequently sent for ex situ analysis), from any location within its culture chamber. Finally, the on-chip plumbing is completely automated using external electronics. This opens the possibility of performing long-term computer-driven experiments, where the cell behavior is modulated in response to the minimally disruptive observations (e.g., fluid sampling and cell biopsies) throughout the entire duration of the cultures. A limitation of the presented α prototype is that it is only two-dimensional (2D). However, technology serves as a foundation for ultimately extending the concept to three-dimensional (3D). Another limitation of the device is that it is currently made from poly(dimethylsiloxane) (PDMS), while more work needs to be done to manufacture from a material that degrades away or allow the cells to lay down the tissue matrix. Unfortunately, the existing biodegradable materials are typically not strong enough for the fabrication of microfluidic valves. Hence, new ones need to be developed before this technology can become mainstream. Yet, it is the hope of the authors that this will be achieved soon, and the microfluidic plumbing technology will eventually be scaled up to 3D, to overcome the limitations of the conventional cell culturing platforms.

Is the endothelial cell responsible for the thrombus core and shell architecture?

Ischemia leading to heart attacks and strokes is the major cause of deaths in the world. This report explores the possibility that intracellular material from ruptured endothelial cells is partially responsible for the heterogeneous core-and-shell blood clot architecture, typically observed using intravital microscopy. As evidence, we present a fluid dynamic argument that platelet agonists emanating from the injury cannot activate platelets in the thrombus core, given that they would have to travel against flow of blood escaping into the extravascular. Furthermore, we demonstrate visual evidence that the core material appears to be continuous and originating from the damaged endothelium. Finally, we present a mechanism, illustrating the steps of platelet recruitment into the thrombus and sealing of the injury. If correct, the model presented herein will be beneficial to the understanding and treatment of heart attacks, strokes and hemophilia.

In vivo measurement of blood clot mechanics from computational fluid dynamics based on intravital microscopy images.

Ischemia which leads to heart attacks and strokes is one of the major causes of death in the world. Whether an occlusion occurs or not depends on the ability of a growing thrombus to resist flow forces exerted on its structure. This manuscript provides the first known in vivo measurement of how much stress a clot can withstand, before yielding to the surrounding blood flow. Namely, Lattice-Boltzmann Method flow simulations are performed based on 3D clot geometries, which are estimated from intravital microscopy images of laser-induced injuries in cremaster microvasculature of live mice. In addition to reporting the blood clot yield stresses, we also show that the thrombus "core" does not experience significant deformation, while its "shell" does. This indicates that the shell is more prone to embolization. Therefore, drugs should be designed to target the shell selectively, while leaving the core intact to minimize excessive bleeding. Finally, we laid down a foundation for a nondimensionalization procedure which unraveled a relationship between clot mechanics and biology. Hence, the proposed framework could ultimately lead to a unified theory of thrombogenesis, capable of explaining all clotting events. Thus, the findings presented herein will be beneficial to the understanding and treatment of heart attacks, strokes and hemophilia.

Ranking migration cue contributions to guiding individual fibroblasts faced with a directional decision in simple microfluidic bifurcations.

Directed cell migration in complex micro-environments, such as in vivo pores, is important for predicting locations of artificial tissue growth and optimizing scaffold architectures. Yet, the directional decisions of cells facing multiple physiochemical cues have not been characterized. Hence, we aim to provide a ranking of the relative importance of the following cues to the decision-making of individual fibroblast cells: chemoattractant concentration gradient, channel width, mitosis, and contact-guidance. In this study, bifurcated micro-channels with branches of different widths were created. Fibroblasts were then allowed to travel across these geometries by following a gradient of platelet-derived growth factor-BB (PDGF-BB) established inside the channels. Subsequently, a combination of statistical analysis and image-based diffusion modeling was used to report how the presence of multiple complex migration cues, including cell-cell influences, affect the fibroblast decision-making. It was found that the cells prefer wider channels over a higher chemoattractant gradient when choosing between asymmetric bifurcated branches. Only when the branches were symmetric in width did the gradient become predominant in directing which path the cell will take. Furthermore, when both the gradient and the channels were symmetric, contact guidance became important for guiding the cells in making directional choices. Based on these results we were able to rank these directional cues from most influential to the least as follows: mitosis > channel width asymmetry > chemoattractant gradient difference > and contact-guidance. It is expected that these results will benefit the fields of regenerative medicine, wound healing and developmental biology.

Numerical accuracy comparison of two boundary conditions commonly used to approximate shear stress distributions in tissue engineering scaffolds cultured under flow perfusion.

INTRODUCTION: Flow-induced shear stresses have been found to be a stimulatory factor in pre-osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of the scaffolds, whole scaffold calculations of the local shear forces are computationally intensive. Instead, representative volume elements (RVEs), which are obtained by extracting smaller portions of the scaffold, are commonly used in literature without a numerical accuracy standard. OBJECTIVE: Hence, the goal of this study is to examine how closely the whole scaffold simulations are approximated by the two types of boundary conditions used to enable the RVEs: "wall boundary condition" (WBC) and "periodic boundary condition" (PBC). METHOD: To that end, lattice Boltzmann method fluid dynamics simulations were used to model the surface shear stresses in 3D scaffold reconstructions, obtained from high-resolution microcomputed tomography images. RESULTS: It was found that despite the RVEs being sufficiently larger than 6 times the scaffold pore size (which is the only accuracy guideline found in literature), the stresses were still significantly under-predicted by both types of boundary conditions: between 20% and 80% average error, depending on the scaffold's porosity. Moreover, it was found that the error grew with higher porosity. This is likely due to the small pores dominating the flow field, and thereby negating the effects of the unrealistic boundary conditions, when the scaffold porosity is small. Finally, it was found that the PBC was always more accurate and computationally efficient than the WBC. Therefore, it is the recommended type of RVE.

Cell Sequence and Mitosis Affect Fibroblast Directional Decision-Making During Chemotaxis in Microfluidic Mazes.

INTRODUCTION: Directed fibroblast migration is central to highly proliferative processes in regenerative medicine and developmental biology. However, the mechanisms by which single fibroblasts affect each other's directional decisions, while chemotaxing in microscopic pores, are not well understood. METHODS: We explored effects of cell sequence and mitosis on fibroblast platelet-derived growth factor-BB (PDGF-BB)-induced migration in microfluidic mazes with two possible through paths: short and long. Additionally, image-based modeling of the chemoattractant's diffusion, consumption and decay, was used to explain the experimental observations. RESULTS: It both cases, the cells displayed behavior that is contradictory to expectation based on the global chemoattractant gradient pre-established in the maze. In case of the sequence, the cells tend to alternate when faced with a bifurcation: if a leading cell takes the shorter (steeper gradient) path, the cell following it chooses the longer (weaker gradient) path, and vice versa. Image-based modeling of the process showed that the local PDGF-BB consumption by the individual fibroblasts may be responsible for this phenomenon. Additionally, it was found that when a mother cell divides, its two daughters go in opposite directions (even if it means migrating against the chemoattractant gradient and overcoming on-going cell traffic). CONCLUSIONS: It is apparent that micro-confined fibroblasts modify each other's directional decisions in a manner that is counter-intuitive to what is expected from classical chemotaxis theory. Consequently, accounting for these effects could lead to a better understanding of tissue generation in vivo, and result in more advanced engineered tissue products in vitro.

Bulk stress distributions in the pore space of sphere-packed beds under Darcy flow conditions.

In this paper, bulk stress distributions in the pore space of columns packed with spheres are numerically computed with lattice Boltzmann simulations. Three different ideally packed and one randomly packed configuration of the columns are considered under Darcy flow conditions. The stress distributions change when the packing type changes. In the Darcy regime, the normalized stress distribution for a particular packing type is independent of the pressure difference that drives the flow and presents a common pattern. The three parameter (3P) log-normal distribution is found to describe the stress distributions in the randomly packed beds within statistical accuracy. In addition, the 3P log-normal distribution is still valid when highly porous scaffold geometries rather than sphere beds are examined. It is also shown that the 3P log-normal distribution can describe the bulk stress distribution in consolidated reservoir rocks like Berea sandstone.

3D tissue-engineered construct analysis via conventional high-resolution microcomputed tomography without X-ray contrast.

As the field of tissue engineering develops, researchers are faced with a large number of degrees of freedom regarding the choice of material, architecture, seeding, and culturing. To evaluate the effectiveness of a tissue-engineered strategy, histology is typically done by physically slicing and staining a construct (crude, time-consuming, and unreliable). However, due to recent advances in high-resolution biomedical imaging, microcomputed tomography (µCT) has arisen as a quick and effective way to evaluate samples, while preserving their structure in the original state. However, a major barrier for using µCT to do histology has been its inability to differentiate between materials with similar X-ray attenuation. Various contrasting strategies (hardware and chemical staining agents) have been proposed to address this problem, but at a cost of additional complexity and limited access. Instead, here we suggest a strategy for how virtual 3D histology in silico can be conducted using conventional µCT, and we provide an illustrative example from bone tissue engineering. The key to our methodology is an implementation of scaffold surface architecture that is ordered in relation to cells and tissue, in concert with straightforward image-processing techniques, to minimize the reliance on contrasting for material segmentation. In the case study reported, µCT was used to image and segment porous poly(lactic acid) nonwoven fiber mesh scaffolds that were seeded dynamically with mesenchymal stem cells and cultured to produce soft tissue and mineralized tissue in a flow perfusion bioreactor using an osteogenic medium. The methodology presented herein paves a new way for tissue engineers to identify and distinguish components of cell/tissue/scaffold constructs to easily and effectively evaluate the tissue-engineering strategies that generate them.

Simulation of intrathrombus fluid and solute transport using in vivo clot structures with single platelet resolution.

The mouse laser injury thrombosis model provides up to 0.22 µm-resolved voxel information about the pore architecture of the dense inner core and loose outer shell regions of an in vivo arterial thrombus. Computational studies were conducted on this 3D structure to quantify transport within and around the clot: Lattice Boltzmann method defined vessel hemodynamics, while passive Lagrangian Scalar Tracking with Brownian motion contribution simulated diffusive-convective transport of various inert solutes (released from lumen or the injured wall). For an input average lumen blood velocity of 0.478 cm/s (measured by Doppler velocimetry), a 0.2 mm/s mean flow rate was obtained within the thrombus structure, most of which occurred in the 100-fold more permeable outer shell region (calculated permeability of the inner core was 10(-11) cm(2)). Average wall shear stresses were 80-100 dyne/cm(2) (peak values >200 dyne/cm(2)) on the outer rough surface of the thrombus. Within the thrombus, small molecule tracers (0.1 kDa) experienced ~70,000 collisions/s and penetrated/exited it in about 1 s, whereas proteins (~50 kDa) had ~9000 collisions/s and required about 10 s (tortuosity ~2-2.5). These simulations help define physical processes during thrombosis and constraints for drug delivery to the thrombus.

Predicting the stress distribution within scaffolds with ordered architecture.

Current tissue engineering technologies involve the seeding of cells on porous scaffolds, within which the cells can proliferate and differentiate, when cultured in bioreactors. The flow of culture media through the scaffolds generates stresses that are important for both cell differentiation and cell growth. A recent study [Appl. Phys. Lett. 97 (2010), 024101] showed that flow-induced stresses inside highly porous and randomly structured scaffolds follow a three-point gamma probability density function (p.d.f.). The goal of the present study is to further investigate whether the same p.d.f. can also describe the distribution of stresses in structured porous scaffolds, what is the range of scaffold porosity for which the distribution is valid, and what is the physical reason for such behavior. To do that, the p.d.f. of flow-induced stresses in different scaffold geometries were calculated via flow dynamics simulations. It was found that the direction of flow relative to the internal architecture of the scaffolds is important for stress distributions. The stress distributions follow a common distribution within statistically acceptable accuracy, when the flow direction does not coincide with the direction of internal structural elements of the scaffold.

Computational modeling of flow-induced shear stresses within 3D salt-leached porous scaffolds imaged via micro-CT.

Flow-induced shear stresses have been found to be a stimulatory factor in pre-osteoblastic cells seeded in 3D porous scaffolds and cultured under continuous flow perfusion. However, due to the complex internal structure of porous scaffolds, analytical estimation of the local shear forces is impractical. The primary goal of this work is to investigate the shear stress distributions within Poly(l-lactic acid) scaffolds via computation. Scaffolds used in this study are prepared via salt leeching with various geometric characteristics (80-95% porosity and 215-402.5microm average pore size). High resolution micro-computed tomography is used to obtain their 3D structure. Flow of osteogenic media through the scaffolds is modeled via lattice Boltzmann method. It is found that the surface stress distributions within the scaffolds are characterized by long tails to the right (a positive skewness). Their shape is not strongly dependent on the scaffold manufacturing parameters, but the magnitudes of the stresses are. Correlations are prepared for the estimation of the average surface shear stress experienced by the cells within the scaffolds and of the probability density function of the surface stresses. Though the manufacturing technique does not appear to affect the shape of the shear stress distributions, presence of manufacturing defects is found to be significant: defects create areas of high flow and high stress along their periphery. The results of this study are applicable to other polymer systems provided that they are manufactured by a similar salt leeching technique, while the imaging/modeling approach is applicable to all scaffolds relevant to tissue engineering.

Boundary slip and wetting properties of interfaces: correlation of the contact angle with the slip length.

Correlations between contact angle, a measure of the wetting of surfaces, and slip length are developed using nonequilibrium molecular dynamics for a Lennard-Jones fluid in Couette flow between graphitelike hexagonal-lattice walls. The fluid-wall interaction is varied by modulating the interfacial energy parameter epsilonr=epsilonsfepsilonff and the size parameter sigmar=sigmasfsigmaff, (s=solid, f=fluid) to achieve hydrophobicity (solvophobicity) or hydrophilicity (solvophilicity). The effects of surface chemistry, as well as the effects of temperature and shear rate on the slip length are determined. The contact angle increases from 25 degrees to 147 degrees on highly hydrophobic surfaces (as epsilonr decreases from 0.5 to 0.1), as expected. The slip length is functionally dependent on the affinity strength parameters epsilonr and sigmar: increasing logarithmically with decreasing surface energy epsilonr (i.e., more hydrophobic), while decreasing with power law with decreasing size sigmar. The mechanism for the latter is different from the energetic case. While weak wall forces (small epsilonr) produce hydrophobicity, larger sigmar smoothes out the surface roughness. Both tend to increase the slip. The slip length grows rapidly with a high shear rate, as wall velocity increases three decades from 100 to 10(5) ms. We demonstrate that fluid-solid interfaces with low epsilonr and high sigmar should be chosen to increase slip and are prime candidates for drag reduction.