A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair.

3D bioprinting has begun to show great promise in advancing the development of functional tissue/organ replacements. However, to realize the true potential of 3D bioprinted tissues for clinical use requires the fabrication of an interconnected and effective vascular network. Solving this challenge is critical, as human tissue relies on an adequate network of blood vessels to transport oxygen, nutrients, other chemicals, biological factors and waste, in and out of the tissue. Here, we have successfully designed and printed a series of novel 3D bone scaffolds with both bone formation supporting structures and highly interconnected 3D microvascular mimicking channels, for efficient and enhanced osteogenic bone regeneration as well as vascular cell growth. Using a chemical functionalization process, we have conjugated our samples with nano hydroxyapatite (nHA), for the creation of novel micro and nano featured devices for vascularized bone growth. We evaluated our scaffolds with mechanical testing, hydrodynamic measurements and in vitro human mesenchymal stem cell (hMSC) adhesion (4 h), proliferation (1, 3 and 5 d) and osteogenic differentiation (1, 2 and 3 weeks). These tests confirmed bone-like physical properties and vascular-like flow profiles, as well as demonstrated enhanced hMSC adhesion, proliferation and osteogenic differentiation. Additional in vitro experiments with human umbilical vein endothelial cells also demonstrated improved vascular cell growth, migration and organization on micro-nano featured scaffolds.

Vortical Structures Promote Atheroprotective Wall Shear Stress Distributions in a Carotid Artery Bifurcation Model.

Carotid artery diseases, such as atherosclerosis, are a major cause of death in the United States. Wall shear stresses are known to prompt plaque formation, but there is limited understanding of the complex flow structures underlying these stresses and how they differ in a pre-disposed high-risk patient cohort. A 'healthy' and a novel 'pre-disposed' carotid artery bifurcation model was determined based on patient-averaged clinical data, where the 'pre-disposed' model represents a pathological anatomy. Computational fluid dynamic simulations were performed using a physiological flow based on healthy human subjects. A main hairpin vortical structure in the internal carotid artery sinus was observed, which locally increased instantaneous wall shear stress. In the pre-disposed geometry, this vortical structure starts at an earlier instance in the cardiac flow cycle and persists over a much shorter period, where the second half of the cardiac cycle is dominated by perturbed secondary flow structures and vortices. This coincides with weaker favorable axial pressure gradient peaks over the sinus for the 'pre-disposed' geometry. The findings reveal a strong correlation between vortical structures and wall shear stress and imply that an intact internal carotid artery sinus hairpin vortical structure has a physiologically beneficial role by increasing local wall shear stresses. The deterioration of this beneficial vortical structure is expected to play a significant role in atherosclerotic plaque formation.

Shear stress metrics associated with pro-atherogenic high-risk anatomical features in a carotid artery bifurcation model.

BACKGROUND: Diseases associated with atherosclerotic plaques in the carotid artery are a major cause of deaths in the United States. Blood-flow-induced shear-stresses are known to trigger plaque formation. Prior literature suggests that the internal carotid artery sinus is prone to atherosclerosis, but there is limited understanding of why only certain patients are predisposed towards plaque formation. METHODS: We computationally investigate the effect of vessel geometry on wall-shear-stress distribution by comparing flowfields and wall-shear-stress-metrics between a low-risk and a novel predisposed high-risk carotid artery bifurcation anatomy. Both models were developed based on clinical risk estimations and patient-averaged anatomical features. The high-risk geometry has a larger internal carotid artery branching angle and a lower internal-to-carotid-artery-diameter-ratio. A patient-averaged physiological carotid artery inflow waveform is used. FINDINGS: The high-risk geometry experiences stronger flow separation in the sinus. Furthermore, it experiences a more equal flow split at the bifurcation, thereby reducing internal carotid artery flowrate and increasing atherosclerosis-prone low-velocity areas. Lowest time-averaged-wall-shear-stresses are present at the sinus outer wall, where plaques are often found, for both geometries. The high-risk geometry has significantly high, unfavorable oscillatory-shear-index values not found in the low-risk geometry. High oscillatory-shear-index areas are located at the vessels outside walls distal to the bifurcation and on the sinus wall. INTERPRETATION: These results highlight the effectiveness of oscillatory-shear-index, to augment classical time-averaged-wall-shear-stress, in evaluating pro-atherogenic geometry features. Furthermore, the flow split at the bifurcation is a promising clinical indicator for atherosclerosis risk as it can be directly accessed using clinical imaging, whereas shear-stress-metrics cannot.

An in vitro analysis of the effect of geometry-induced flows on endothelial cell behavior in 3D printed small-diameter blood vessels.

Clinical recovery from vascular diseases has increasingly become reliant upon the successful fabrication of artificial blood vessels (BVs) or vascular prostheses due to the shortage of autologous vessels and the high incidence of vessel graft diseases. Even though many attempts at the clinical implementation of large artificial BVs have been reported to be successful, the development of small-diameter BVs remains one of the significant challenges due to the limitation of micro-manufacturing capacity in complexity and reproducibility, as well as the development of thrombosis. The present study aims to develop 3D printed small-diameter artificial BVs that recapitulate the longitudinal geometric elements in the native BVs using biocompatible polylactic acid (PLA). As their intrinsic physical properties are crystallinity dependent, we used two PLA filaments with different crystallinity to investigate the suitability of their physical properties in the micro-manufacturing of BVs. To explore the mechanism of venous thrombosis, our study provided a preliminarily comparative analysis of the effect of geometry-induced flows on the behavior of human endothelial cells (ECs). Our results showed that the adhered healthy ECs in the 3D printed BV exhibited regulated patterns, such as elongated and aligned parallel to the flow direction, as well as geometry-induced EC response mechanisms that are associated with hemodynamic shear stresses. Furthermore, the computational fluid dynamics simulation results provided insightful information to predict velocity profile and wall shear stress distribution in the geometries of BVs in accordance with their spatiotemporally-dependent cell behaviors. Our study demonstrated that 3D printed small-diameter BVs could serve as suitable candidates for fundamental BV studies and hold great potential for clinical applications.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix.

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size. The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a 'thick and sticky' state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process. The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer.   A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section.

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows (1 - 7). Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis (4 - 6, 8 - 13). Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations. A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies (4 - 6). Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-à-vis those under unperturbed flows (5). These stent failures have been classified as "Types I-to-IV" based on failure considerations and clinical severity (14). This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts ("Type IV") in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements (15 - 18). Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data (5, 6, 19 - 26).