Extracellular Fluid Flow Research Articles

From Cancer Immunoediting to New Strategies in Cancer Immunotherapy: The Roles of Immune Cells and Mechanics in Oncology.

For the last three decades, the concept of immunoediting has evolved to characterize our increasing understanding of the interactions between cells from the immune system and cancer development. Elucidating the role of immune cells in the progression of cancer has been very challenging due to their dual role; the immune system can either suppress tumor formation by killing cancer cells, or it can also promote tumor growth. Revealing how immune cells are hampered by the tumor microenvironment and how they aid tumor progression has signaled strategies to reverse these effects and control cancer cell growth; this has been the advent of immunotherapy design. More recently, the role of physical forces in the process of immunoediting has been highlighted by multiple studies focusing on understanding how force changes in the stiffness of the extracellular matrix and fluid flow shear stress contribute to tumor development. Using models in vitro that incorporate biomechanical components, it has been shown that these physical aspects are not only important during the formation and growth of primary tumors, but in the metastatic process as well. In this way, we have also gained insight into the interactions occurring within the vascular system, which are highly affected by the dynamics of physical collisions between cells and by shear forces. Here, we review the concept of cancer immunoediting with an emphasis on biomechanics and conclude with a summary on current immunotherapies and potential new strategies.

Vertebrate Left-Right Asymmetry: What Can Nodal Cascade Gene Expression Patterns Tell Us?

Laterality of inner organs is a wide-spread characteristic of vertebrates and beyond. It is ultimately controlled by the left-asymmetric activation of the Nodal signaling cascade in the lateral plate mesoderm of the neurula stage embryo, which results from a cilia-driven leftward flow of extracellular fluids at the left-right organizer. This scenario is widely accepted for laterality determination in wildtype specimens. Deviations from this norm come in different flavors. At the level of organ morphogenesis, laterality may be inverted (situs inversus) or non-concordant with respect to the main body axis (situs ambiguus or heterotaxia). At the level of Nodal cascade gene activation, expression may be inverted, bilaterally induced, or absent. In a given genetic situation, patterns may be randomized or predominantly lacking laterality (absence or bilateral activation). We propose that the distributions of patterns observed may be indicative of the underlying molecular defects, with randomizations being primarily caused by defects in the flow-generating ciliary set-up, and symmetrical patterns being the result of impaired flow sensing, on the left, the right, or both sides. This prediction, the reasoning of which is detailed in this review, pinpoints functions of genes whose role in laterality determination have remained obscure.

A computational model of amoeboid cell swimming

Amoeboid cells propel by generating pseudopods that are finger-like protrusions of the cell body that continually grow, bifurcate, and retract. Pseudopod-driven motility of amoeboid cells represents a complex and multiscale process that involves bio-molecular reactions, cell deformation, and cytoplasmic and extracellular fluid motion. Here we present a 3D model of pseudopod-driven swimming of an amoeba suspended in a fluid without any adhesion and in the absence of any chemoattractant. Our model is based on front-tracking/immersed-boundary methods, and it combines large deformation of the cell, a coarse-grain model for molecular reactions, and cytoplasmic and extracellular fluid flow. The predicted shapes of the swimming cell from our model show similarity with experimental observations. We predict that the swimming behavior changes from random-like to persistent unidirectional motion, and that the swimming speed increases, with increasing cell deformability and protein diffusivity. The unidirectionality in cell swimming is observed without any external cues and as a direct result of a change in pseudopod dynamics. We find that pseudopods become preferentially focused near the front of the cell and appear in greater numbers with increasing cell deformability and protein diffusivity, thereby increasing the swimming speed and making the cell shape more elongated. We find that the swimming speed is minimum when the cytoplasm viscosity is close to the extracellular fluid viscosity. We further find that the speed increases significantly as the cytoplasm becomes less viscous compared with the extracellular fluid, resembling the viscous fingering phenomenon observed in interfacial flows. While these results support the notion that softer cells migrate more aggressively, they also suggest a strong coupling between membrane elasticity, membrane protein diffusivity, and fluid viscosity.

Modeling mechanical interactions in growing populations of rod-shaped bacteria

Advances in synthetic biology allow us to engineer bacterial collectives with pre-specified characteristics. However, the behavior of these collectives is difficult to understand, as cellular growth and division as well as extra-cellular fluid flow lead to complex, changing arrangements of cells within the population. To rationally engineer and control the behavior of cell collectives we need theoretical and computational tools to understand their emergent spatiotemporal dynamics. Here, we present an agent-based model that allows growing cells to detect and respond to mechanical interactions. Crucially, our model couples the dynamics of cell growth to the cell’s environment: Mechanical constraints can affect cellular growth rate and a cell may alter its behavior in response to these constraints. This coupling links the mechanical forces that influence cell growth and emergent behaviors in cell assemblies. We illustrate our approach by showing how mechanical interactions can impact the dynamics of bacterial collectives growing in microfluidic traps.

Time-frequency analyses of fluid–solid interaction under sinusoidal translational shear deformation of the viscoelastic rat cerebrum

During normal extracellular fluid (ECF) flow in the brain glymphatic system or during pathological flow induced by trauma resulting from impacts and blast waves, ECF–solid matter interactions result from sinusoidal shear waves in the brain and cranial arterial tissue, both heterogeneous biological tissues with high fluid content. The flow in the glymphatic system is known to be forced by pulsations of the cranial arteries at about 1 Hz. The experimental shear stress response to sinusoidal translational shear deformation at 1 Hz and 25% strain amplitude and either 0% or 33% compression is compared for rat cerebrum and bovine aortic tissue. Time-frequency analyses aim to correlate the shear stress signal frequency components over time with the behavior of brain tissue constituents to identify the physical source of the shear nonlinear viscoelastic response. Discrete fast Fourier transformation analysis and the novel application to the shear stress signal of harmonic wavelet decomposition both show significant 1 Hz and 3 Hz components. The 3 Hz component in brain tissue, whose magnitude is much larger than in aortic tissue, may result from interstitial fluid induced drag forces. The harmonic wavelet decomposition locates 3 Hz harmonics whose magnitudes decrease on subsequent cycles perhaps because of bond breaking that results in easier fluid movement. Both tissues exhibit transient shear stress softening similar to the Mullins effect in rubber. The form of a new mathematical model for the drag force produced by ECF–solid matter interactions captures the third harmonic seen experimentally.

Role for Mechanotransduction in Macrophage and Dendritic Cell Immunobiology.

Tissue homeostasis is not only controlled by biochemical signals but also through mechanical forces that act on cells. Yet, while it has long been known that biochemical signals have profound effects on cell biology, the importance of mechanical forces has only been recognized much more recently. The types of mechanical stress that cells experience include stretch, compression, and shear stress, which are mainly induced by the extracellular matrix, cell-cell contacts, and fluid flow. Importantly, macroscale tissue deformation through stretch or compression also affects cellular function.Immune cells such as macrophages and dendritic cells are present in almost all peripheral tissues, and monocytes populate the vasculature throughout the body. These cells are unique in the sense that they are subject to a large variety of different mechanical environments, and it is therefore not surprising that key immune effector functions are altered by mechanical stimuli. In this chapter, we describe the different types of mechanical signals that cells encounter within the body and review the current knowledge on the role of mechanical signals in regulating macrophage, monocyte, and dendritic cell function.

Intracellular Ca2+ increase in endothelial cells induced by shock wave irradiation

Shock wave irradiations induce bioeffects including angiogenesis and bone healing. However, physical mechanisms underlying those bioeffects remain elusive. Extracorporeal shock wave treatment (ESWT) is one of well-known shock wave therapies. In ESWT, shock waves destroy kidney stones with cavitation and negative pressures. Energy flux density of shock waves in ESWT is around 0.1—1 mJ/mm2. In this study, shock waves with energy flux density of 0.001 mJ/mm2, around 1/100 of ESWT, were irradiated on endothelial cells. In those cells, intracellular Ca2+ increase, i.e. a typical physiological response, was observed. Along the intracellular Ca2+ increase, extracellular fluid flow was not present. As well, both plasma membrane permeabilization and cell detachment were not detected. On the other hand, actin cytoskeletons, which link extracellular substrates and cellular components, were suggested to involve in the intracellular Ca2+ increase. Thus, it was supposed that shock waves initiated intracellular Ca2+ increase by loading forces on actin cytoskeletons. Although some previous studies have suggested that extracellular fluid flow could involve in shock wave induced bioeffects, intracellular Ca2+ increase seems to rely on extracellular fluid flow.

Cilia play a role in breaking left-right symmetry of the sea urchin embryo.

Left-right asymmetry of bilaterian animals is established during early development. In mice, frogs and fishes, the ciliated left-right organizer plays an essential role in establishing bilateral asymmetry, and leftward flow of extracellular fluid generated by ciliary motion results in Nodal activity on the left side. However, H(+) /K(+) -ATPase activity is also involved in the determination of left-right asymmetry in a variety of animals, and it has been thought to be an ancestral mechanism in deuterostomes. In sea urchin, the determination of the left-right asymmetry based on H(+) /K(+) -ATPase activity was already clarified, but it remains to be uncovered whether ciliary motion is involved in the left-right asymmetry of the embryo. Here, we show evidence that ciliary motion is involved in the establishment of left-right asymmetry of sea urchin embryo. Furthermore, we show that the initial cilia generated on small micromeres during the early stage of embryogenesis may be involved in this process. These results suggest that the cilia-mediated mechanism for the determination of left-right asymmetry may be acquired at the base of the deuterostomes.

Hydrodynamics and convection enhanced macromolecular fluid transport in soft biological tissues: Application to solid tumor

This work addresses a theoretical framework for transvascular exchange and extravascular transport of solute macromolecules through soft interstitial space inside a solid tumor. Most of the soft biological tissues show materialistic properties similar to deformable porous material. They exhibit mechanical behavior towards the fluid motion since the solid phase of the tumor tissue gets compressed by the drag force that is associated with the extracellular fluid flow. This paper presents a general view about the transvascular and interstitial transport of solute nutrients inside a tumor in the macroscopic level. Modified Starling׳s equation is used to describe transvascular nutrient transport. On the macroscopic level, motion of extracellular fluid within the tumor interstitium is modeled with the help of biphasic mixture theory and a spherical symmetry solution is given as a simpler case. This present model describes the average interstitial fluid pressure (IFP), extracellular fluid velocity (EFV) and flow rate of extracellular fluid, as well as the deformation of the solid phase of the tumor tissue as an immediate cause of extracellular fluid flow. When the interstitial transport is diffusion dominated, an analytical treatment of advection–diffusion–reaction equation finds the overall nutrient distribution. We propose suitable criteria for the formation of necrosis within the tumor interstitium. This study introduces some parameters that represent the nutrient supply from tumor blood vessels into the tumor extracellular space. These transport parameters compete with the reversible nutrient metabolism of the tumor cells present in the interstitium. The present study also shows that the effectiveness factor corresponding to a first order nutrient metabolism may reach beyond unity if the strength of the distributive solute source assumes positive non-zero values.

Extracellular fluid flow and chloride content modulate H(+) transport by osteoclasts.

BackgroundBone resorption takes place within the basic multicellular units (BMU), and the surface to be resorbed is isolated from adjacent bone surfaces by a sealing zone between osteoclast membrane and bone matrix, which defines the limits of the resorption lacuna. Considering that the extracellular fluid (ECF) in both BMU and the resorption lacuna can be isolated from its surroundings, I hypothesize that flow and ion composition of the bone ECF in these sites might contribute to the regulation of osteoclast H+ secretion. To investigate this hypothesis, I evaluated the H+ secretion properties of individual osteoclasts and osteoclast-like cells (OCL-cells) and investigated whether changes in flow or chloride content of the extracellular solution modify the H+ secretion properties in vitro.ResultsThe results show that 1) osteoclasts are unable to secrete H+ and regulate intracellular pH (pHi) under continuous flow conditions and exhibit progressive intracellular acidification; 2) the cessation of flow coincides with the onset of H+ secretion and subsequent progressive intracellular alkalinization of osteoclasts and OCL-cells; 3) osteoclasts exhibit spontaneous rhythmic oscillations of pHi in non-flowing ECF, 4) pHi oscillations are not abolished by concanamycin, NPPB, or removal of extracellular Na+ or Cl−; 5) extracellular Cl− removal modifies the pattern of oscillations, by diminishing H+ secretion; 6) pHi oscillations are abolished by continuous flowing of ECF over osteoclasts and OCL-cells.ConclusionsThe data suggest, for the first time, that ECF flow and Cl− content have direct effects on osteoclast H+ secretion and could be part of a mechanism determining the onset of osteoclast H+ secretion required for bone resorption.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0066-4) contains supplementary material, which is available to authorized users.

ATP4a is required for development and function of the Xenopus mucociliary epidermis – a potential model to study proton pump inhibitor-associated pneumonia

Proton pump inhibitors (PPIs), which target gastric H+/K+ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/β-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways.

Intraciliary Calcium Oscillations Initiate Vertebrate Left-Right Asymmetry

SummaryBackgroundBilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades is unknown.ResultsHere, we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically-encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink demonstrates that they are essential for LR development.ConclusionsThese findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling.

Symmetry breakage in the vertebrate embryo: When does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.

The evolution and conservation of left-right patterning mechanisms

Morphological asymmetry is a common feature of animal body plans, from shell coiling in snails to organ placement in humans. The signaling protein Nodal is key for determining this laterality. Many vertebrates, including humans, use cilia for breaking symmetry during embryonic development: rotating cilia produce a leftward flow of extracellular fluids that induces the asymmetric expression of Nodal. By contrast, Nodal asymmetry can be induced flow-independently in invertebrates. Here, we ask when and why flow evolved. We propose that flow was present at the base of the deuterostomes and that it is required to maintain organ asymmetry in otherwise perfectly bilaterally symmetrical vertebrates.

The Zic2 gene directs the formation and function of node cilia to control cardiac situs

The first molecular herald of organ asymmetry during murine embryogenesis is found at the periphery of the node in early-somite stage embryos. Asymmetric gene expression and calcium accumulation at the node occurs in response to a left-ward flow of extracellular fluid across the node, generated by motile cilia within the pit of the node and likely sensed by immotile cilia in the periphery of the node. The ciliation of node cells is controlled by a cascade of node-restricted transcription factor activity during mid-late gastrulation. Mutation of the murine Zic2 transcription factor is associated with random cardiac situs and a loss of asymmetric gene expression at the early-somite node and in the lateral plate. Zic2 is not expressed in these regions but is transiently expressed in the mid-late gastrula node at the time of ciliogenesis. The cilia of the node are overtly abnormal in Zic2 mutant embryos being dysmorphic and short relative to wild-type littermates. The expression of the Noto, Rfx3, and Foxj1 transcription factors known to regulate ciliogenesis is greatly depleted in the mid-gastrula node of mutants, as is the expression of the Pkd1l1 gene required for cilia function. Zic2 appears to be a component of the gene regulatory network that drives ciliation of node cells during gastrulation.

Symmetry breakage in the frog Xenopus: Role of Rab11 and the ventral‐right blastomere

Vertebrates display asymmetric arrangements of inner organs such as heart and stomach. The Nodal signaling cascade in the left lateral plate mesoderm in all cases directs asymmetric morphogenesis and placement during organogenesis. Mechanisms that lead up to left-asymmetric Nodal induction seem to differ between the vertebrates. Cilia produce a leftward extracellular fluid flow in zebrafish, medaka, mouse, rabbit, and Xenopus embryos during neurulation. In Xenopus, earlier asymmetric cues were described. Some, such as Rab11, apparently act in the zygote. Others were efficiently manipulated in ventral-right cells at the four-cell stage, a lineage presumably independent of the ciliated gastrocoel roof plate (GRP) during neurulation. Here, we show that one- and four-cell manipulations of Rab11 showed equal low efficiencies of left-right disturbances. We also reevaluated the lineage of the GRP. By tracing back future ciliated cells from the gastrula to the four-cell stage, we show that ventral cells contribute to ciliated sensory cells at the border of the GRP. Knockdown of the Nodal inhibitor Coco in the ventral right lineage resulted in embryos with ectopic right-sided Nodal and Pitx2c expression. Together, these experiments support a cilia-based mechanism of symmetry breakage in the frog Xenopus.

Wnt11b Is Involved in Cilia-Mediated Symmetry Breakage during Xenopus Left-Right Development

Breakage of bilateral symmetry in amphibian embryos depends on the development of a ciliated epithelium at the gastrocoel roof during early neurulation. Motile cilia at the gastrocoel roof plate (GRP) give rise to leftward flow of extracellular fluids. Flow is required for asymmetric gene expression and organ morphogenesis. Wnt signaling has previously been involved in two steps, Wnt/ß-catenin mediated induction of Foxj1, a regulator of motile cilia, and Wnt/planar cell polarity (PCP) dependent cilia polarization to the posterior pole of cells. We have studied Wnt11b in the context of laterality determination, as this ligand was reported to activate canonical and non-canonical Wnt signaling. Wnt11b was found to be expressed in the so-called superficial mesoderm (SM), from which the GRP derives. Surprisingly, Foxj1 was only marginally affected in loss-of-function experiments, indicating that another ligand acts in this early step of laterality specification. Wnt11b was required, however, for polarization of GRP cilia and GRP morphogenesis, in line with the known function of Wnt/PCP in cilia-driven leftward flow. In addition Xnr1 and Coco expression in the lateral-most GRP cells, which sense flow and generate the first asymmetric signal, was attenuated in morphants, involving Wnt signaling in yet another process related to symmetry breakage in Xenopus.

Cellular control of connective tissue matrix tension

The biomechanical behavior of connective tissue in response to stretching is generally attributed to the molecular composition and organization of its extracellular matrix. It also is becoming apparent that fibroblasts play an active role in regulating connective tissue tension. In response to static stretching of the tissue, fibroblasts expand within minutes by actively remodeling their cytoskeleton. This dynamic change in fibroblast shape contributes to the drop in tissue tension that occurs during viscoelastic relaxation. We propose that this response of fibroblasts plays a role in regulating extracellular fluid flow into the tissue, and protects against swelling when the matrix is stretched. This article reviews the evidence supporting possible mechanisms underlying this response including autocrine purinergic signaling. We also discuss fibroblast regulation of connective tissue tension with respect to lymphatic flow, immune function, and cancer.

Abstract 3931: Determining extracellular fluid velocity along white matter tracts with injections of increasing concentrations of intracranial albumin using both histological and radiological techniques.

Abstract INTRODUCTION: Tumor cells are passively carried by extracellular fluid flow to regional lymph nodes in most systemic cancers including breast cancer, melanoma and colorectal cancer. Although the brain does not contain lymphatics, cancer spread within the brain may be a function of extracellular fluid velocity. As malignant brain tumors are characterized by a disrupted blood-brain-barrier that leaks albumin from blood vessels into brain parenchyma, we hypothesize that this albumin influx will pull water in osmotically and increase flow rates and glioma cell dissemination down adjacent white matter tracts. High flow rates may increase tumor dissemination via the WMT. This study sought to determine if increasing intracranial albumin concentrations affect WMT flow rates. If true, restricting albumin extravasation (through steroids or VEGF inhibitors) has the potential to reduce glioma dissemination and may have implications for future radiation planning and steroid/VEGF inhibitor dosing. METHODS: Our study examines flow rates in white matter tracts after intracranial administration of albumin. 0.12-6 mg bovine albumin were mixed with 20 μL of 2% Evans Blue and injected stereotactically into the right frontal lobe at the gray-white matter junction over 80 minutes in 15 anesthetized Sprague-Dawley female rats. Seven hours later, the rats were sacrificed and the brains sectioned into 0.5mm coronal slices, and imaged to determine interstitial flow rates. RESULTS: Average anterior-posterior (AP) velocity for rats receiving 0.12 mg and 6.0 mg of albumin were 0.43 and 1.02 mm/hour respectively (p = 0.029). Correlation was observed between administered albumin dose and interstitial fluid velocity (R = 0.57). In vivo MRI results were consistent with ex vivo Evans blue results and demonstrate markedly increased edema (T2) in the hemisphere where albumin was injected. CONCLUSIONS: This study demonstrates higher interstitial fluid flow rates in adjacent white matter tracts in response to albumin-induced-edema. These observations provide insight into expected pathways for glioma dissemination which could be used to tailor radiation plans for individual patients. Furthermore, these findings provide a rationale for interventions to reduce glioma dissemination by limiting the extravasation of albumin through the blood-brain-barrier while local radiation is being administered. Citation Format: Carmen Kut, Vadappuram Chacko, Betty Tyler, Arvind P. Pathak, Zaver Bhujwalla, Elliot R. McVeigh, Xingde Li, Daniel A. Herzka, Stuart A. Grossman. Determining extracellular fluid velocity along white matter tracts with injections of increasing concentrations of intracranial albumin using both histological and radiological techniques. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2013-3931

Biomimetic Collagen–Hydroxyapatite Composite Fabricated via a Novel Perfusion-Flow Mineralization Technique

Prevalent three-dimensional scaffolds for bone tissue engineering are mineralized collagen-hydroxyapatite (Col/HA) composites. Conventional mineralization techniques are either to coat collagen scaffold surfaces with minerals or to simply mix collagen and mineral nanoparticles together. These conventional in vitro collagen mineralization methods are different from the in vivo bone formation process and often result in scaffolds that are not suitable for bone tissue engineering. In this study, a unique perfusion-flow (i.e., dynamic) in conjunction with a previously described polymer-induced liquid-precursor (PILP) method was used to fabricate a porous Col/HA composite. The dynamic flow emulated the physiological extracellular fluid flow containing the mineralization ions, while the PILP method facilitated the deposition of the HA crystals within the collagen fibrils (i.e., intrafibrillar mineralization). By utilizing a dynamic PILP technique to mimic the in vivo bone formation process, the resultant Col/HA composite has a similar structure and compositions like human trabecular bone. A comparison of the dynamic and static mineralization methods revealed that the novel dynamic technique facilitates more efficient and homogenous mineral deposition throughout the Col/HA composite. The dynamic intrafibrillar mineralization method generated stiff Col/HA composites with excellent surface property for cell attachment and growth. The human mesenchymal stem cells cultured on the Col/HA composites quickly remodeled the scaffolds and resulted in constructs with an extensive cell-derived extracellular matrix network.