Abdominal Aortic Aneurysm Pathomechanics: Current Understanding and Future Directions.

Abdominal aortic aneurysms (AAAs) are permanent, local expansions of the abdominal segment of the aorta that can potentially be fatal if progressing to rupture. AAAs are rarely found in patients under age 60, but are more common in older age groups, occurring in 2-3% of the whole population. Their rupture produces up to 14,000 deaths annually in the United States alone. Because aneurysmal rupture is a mechanical phenomenon, in recent years there has been a major effort among researchers to investigate the biologic and mechanical processes surrounding AAA progression and rupture. In addition to the basic science importance of understanding AAA pathophysiology, much of this research has been directed toward the development of accurate clinical criteria for assessing the risk of rupture on a patient-by-patient basis. This review first summarizes degenerative changes of the aorta wall associated with AAA pathogenesis. Current understanding of hemodynamics, transport, and wall mechanics in AAAs is then described, and open questions in aneurysm research are discussed along with potential directions in which further understanding could lead to improved clinical evaluation and management decision processes.

Histology and Biaxial Mechanical Behavior of Abdominal Aortic Aneurysm Tissue Samples.

Abdominal aortic aneurysms (AAAs) represent permanent, localized dilations of the abdominal aorta that can be life-threatening if progressing to rupture. Evaluation of risk of rupture depends on understanding the mechanical behavior of patient AAA walls. In this project, a series of patient AAA wall tissue samples have been evaluated through a combined anamnestic, mechanical, and histopathologic approach. Mechanical properties of the samples have been characterized using a novel, strain-controlled, planar biaxial testing protocol emulating the in vivo deformation of the aorta. Histologically, the tissue ultrastructure was highly disrupted. All samples showed pronounced mechanical stiffening with stretch and were notably anisotropic, with greater stiffness in the circumferential than the axial direction. However, there were significant intrapatient variations in wall stiffness and stress. In biaxial tests in which the longitudinal stretch was held constant at 1.1 as the circumferential stretch was extended to 1.1, the maximum average circumferential stress was 330 ± 70 kPa, while the maximum average axial stress was 190 ± 30 kPa. A constitutive model considering the wall as anisotropic with two preferred directions fit the measured data well. No statistically significant differences in tissue mechanical properties were found based on patient gender, age, maximum bulge diameter, height, weight, body mass index, or smoking history. Although a larger patient cohort is merited to confirm these conclusions, the project provides new insight into the relationships between patient natural history, histopathology, and mechanical behavior that may be useful in the development of accurate methods for rupture risk evaluation.

Seamless, axially aligned, fiber tubes, meshes, microbundles and gradient biomaterial constructs.

A new electrospinning apparatus was developed to generate nanofibrous materials with improved organizational control. The system functions by oscillating the deposition signal (ODS) of multiple collectors, allowing significantly improved nanofiber control by manipulating the electric field which drives the electrospinning process. Other electrospinning techniques designed to impart deposited fiber organizational control, such as rotating mandrels or parallel collector systems, do not generate seamless constructs with high quality alignment in sizes large enough for medical devices. In contrast, the ODS collection system produces deposited fiber networks with highly pure alignment in a variety of forms and sizes, including flat (8 × 8 cm(2)), tubular (1.3 cm diameter), or rope-like microbundle (45 µm diameter) samples. Additionally, the mechanism of our technique allows for scale-up beyond these dimensions. The ODS collection system produced 81.6 % of fibers aligned within 5° of the axial direction, nearly a four-fold improvement over the rotating mandrel technique. The meshes produced from the 9 % (w/v) fibroin/PEO blend demonstrated significant mechanical anisotropy due to the fiber alignment. In 37 °C PBS, aligned samples produced an ultimate tensile strength of 16.47 ± 1.18 MPa, a Young's modulus of 37.33 MPa, and a yield strength of 7.79 ± 1.13 MPa. The material was 300 % stiffer when extended in the direction of fiber alignment and required 20 times the amount of force to be deformed, compared to aligned meshes extended perpendicular to the fiber direction. The ODS technique could be applied to any electrospinnable polymer to overcome the more limited uniformity and induced mechanical strain of rotating mandrel techniques, and greatly surpasses the limited length of standard parallel collector techniques.

Histologic evaluation of therapeutic responses in ischemic myocardium elicited by dual growth factor delivery from composite glycosaminoglycan hydrogels.

In this project, the ability of dual growth factor-preloaded, silk-reinforced, composite hyaluronic acid-based hydrogels to elicit advantageous histologic responses when secured to ischemic myocardium was evaluated in vivo. Reinforced hydrogels containing both Vascular Endothelial Growth Factor (VEGF) and Platelet-derived Growth Factor (PDGF) were prepared by crosslinking chemically modified hyaluronic acid and heparin with poly(ethylene glycol)-diacrylate around a reinforcing silk mesh. Composite patches were sutured to the ventricular surface of ischemic myocardium in Sprague-Dawley rats, and the resulting angiogenic response was followed for 28 days. The gross appearance of treated hearts showed significantly reduced ischemic area and fibrous deposition compared to untreated control hearts. Histologic evaluation showed growth factor delivery to restore myofiber orientation to pre-surgical levels and to significantly increase elicited microvessel density and maturity by day 28 in infarcted myocardial tissue (p < 0.05). In addition, growth factor delivery reduced cell apoptosis and decreased the density of elicited mast cells and both CD68+ and anti-inflammatory CD163+ macrophages. These findings suggest that HA-based, dual growth factor-loaded hydrogels can successfully induce a series of beneficial responses in ischemic myocardium, and offer the potential for therapeutic improvement of ischemic myocardial remodeling.

Microvascular maturity elicited in tissue treated with cytokine-loaded hyaluronan-based hydrogels.

Hydrogels composed of crosslinked, chemically modified hyaluronic acid (HA), gelatin (Gtn) and heparin (Hp) were preloaded with vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) or platelet derived growth factor (PDGF) either individually or in combination with VEGF and implanted into the Balb/c mouse ear pinna. At 7 and 14 days post-surgery, elicited vascular maturity levels were quantified using immunohistochemical (IHC) staining techniques and reported as a vascular maturity index (VMI). At both time points, it was discovered that the dual cytokine combinations elicited greater maturity levels than that of cytokine administered individually. For example, VEGF and KGF-containing HA:Hp implants at day 7 yielded VMI values of -0.1375 and -0.092, respectively, whereas their combination resulted in a VMI of 0.176 (p<0.007). At day 7, only one of the seven HA:Hp experimental cases yielded a positive VMI (VEGF+KGF), whereas four of the seven HA:Hp cases yielded positive VMI values at day 14, indicating a sustained maturity response. The same general trends were found to exist in tissue treated with HA:Hp:Gtn experimental implants. Differences in elicited maturity also existed between tissue treated with HA:Hp and HA-containing hydrogels (VMI=0.176 for HA:Hp-VEGF+KGF vs. -0.064 for HA-VEGF+KGF, p<0.012), and these differences are thought to result from differences in characteristic cytokine release rates. This result also suggests that the presentation of multiple growth factors (GFs) on immobilized Hp may actively contribute to cytokine related signal transduction, a characteristic that may be exploited in the future to improve the efficacy of cytokine-loaded implants towards tissue regeneration therapeutic strategies.

Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants.

Crosslinked hyaluronan (HA) hydrogels preloaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), were employed to elicit new microvessel growth in vivo. As a major glycosaminoglycan (GAG) component of extracellular matrix (ECM), HA is an excellent biopolymeric building block for new biomimetic, biocompatible therapeutic materials. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Histologic analysis showed that each of the groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.001). Treatment groups receiving either co-delivery of both KGF and VEGF, an HA hydrogel lacking a growth factor or HA hydrogels containing a single cytokine were statistically unchanged with time, whereas treatment with KGF alone produced continuing increases in vascularization from day 7 to day 14. Strikingly, presentation of both VEGF and KGF in crosslinked HA generated intact microvessel beds with well-defined borders. In addition, an additive response to co-delivery of both cytokines in the HA hydrogel was observed. The HA hydrogels containing KGF+VEGF produced the greatest angiogenic response of any treatment group tested (NI=5.4 at day 14, where NI is a neovascularization index). This was 33% greater vessel density than in the next largest treatment group, that received HA+KGF (NI=4.0, p<0.002). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and KGF from crosslinked HA films.

Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF.

Controlled release of human vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate growth factor (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (15 kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to 21%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel growth at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.

Stimulation of in vivo angiogenesis using dual growth factor-loaded crosslinked glycosaminoglycan hydrogels.

Crosslinked, chemically modified hyaluronan (HA) hydrogels pre-loaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1), were employed to elicit new microvessel growth in vivo, in both the presence and absence of heparin (Hp) in the gels. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Analysis of neovascularization showed that each of the treatment groups receiving an implant, except for HA/Hp at day 14, demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.015). Treatment groups receiving either Ang-1 alone, or aqueous co-delivery of both Ang-1 and VEGF, were statistically unchanged with time. In contrast, film delivery of both growth factors produced continuing increases in vascularization from day 7 to day 14 in the absence of Hp, but decreases in its presence. However, presentation of both VEGF and Ang-1 in crosslinked HA gels containing Hp generated intact microvessel beds with well-defined borders. The HA hydrogels containing Ang-1+VEGF produced the greatest angiogenic response of any treatment group tested at day 14 (NI=7.44 in the absence of Hp and 4.67 in its presence, where NI is a neovascularization index). Even in the presence of Hp, this had 29% greater vessel density than the next largest treatment group receiving HA/Hp+VEGF (NI=3.61, p=0.04). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and Ang-1 from crosslinked HA films.

Silk-hyaluronan-based composite hydrogels: a novel, securable vehicle for drug delivery.

A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs.

Release of growth factors, cytokines and therapeutic molecules by hyaluronan-based hydrogels.

Hyaluronan (HA) is a highly biocompatible biopolymer that is widely used for a variety of therapeutic purposes including surgical preparations, adhesion prevention, viscosupplementation and drug and cytokine delivery. Delivery can be accomplished effectively when HA-based carriers are synthesized in the form of hydrogels, though doing so normally requires chemical modification of the native HA structure. Solute delivery from HA-based gels can be either "simple", that is from a gel not including separate components intended to control release, or "regulated" when specific components are included for that purpose. A variety of modified forms of HA have been developed and used for delivery of desired molecules in therapeutic, clinical, veterinary and laboratory research environments, and the number of such applications is likely to grow in future years.

Stimulation of in vivo angiogenesis by in situ crosslinked, dual growth factor-loaded, glycosaminoglycan hydrogels.

As part of a study of elicited angiogenesis, hyaluronan (HA)-based hydrogels crosslinked by polyethylene glycol diacrylate (PEGDA) were loaded with combinations of the cytokine growth factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) and platelet-derived growth factor (PDGF). GF release in vivo was controlled by covalent incorporation of thiol-modified heparin into thiolated HA hydrogels, which were injected into the ear pinnae of mice and allowed to crosslink in situ. GF release in vivo was controlled by covalent incorporation of thiol-modified heparin in the gels. The ears were harvested at 7 or 14 days post-implantation, and vascularization evaluated via a Neovascularization Index (NI). The study demonstrates that in situ gelling implants produced no gross inflammation, redness or swelling, and an improved tolerance compared to HA-based dry film implants. All treatments showed significantly more vascularization than either contralateral ears or ears receiving a sham surgery. The maximum response was observed after 14 days in the ears receiving 0.3% Hp, gelatin-containing gels loaded with VEGF + KGF (NI = 3.91). The study revealed injected growth factor-loaded HA-based hydrogels can successfully produce localized controllable vascularization, while minimizing tissue necrosis, polymorphonuclear leukocytes and inflammation. The ability to target and controllably release growth factors can prove a useful tool in specific diseased tissue/organ angiogenesis.

Effect of gelatin on heparin regulation of cytokine release from hyaluronan-based hydrogels.

The hypothesis that incorporation of small amounts (0.3% w/w) of modified heparin in thiol-modified hyaluronan or HA and gelatin hydrogels would regulate release of cytokine growth factors (GFs) from those gels has been investigated in vitro. In addition, the physiologic response to gel implantation has been evaluated in vivo. Tests were performed with 6 GFs: basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor, platelet-derived growth factor-AA (PDGF), and transforming growth factor-beta 1. Release profiles for all 6 over several weeks were well fit by first order exponential kinetics (R(2) > 0.9 for all cases). The most remarkable result of the experiment was a dramatic variation in the total mass ultimately released, which varied from as much as 90.2% of the initial load for bFGF to as little as 1.8% for PDGF, a 45-fold difference. Furthermore, gels containing either VEGF of Ang-1 produced twice the vascularization response in vivo as gels not containing a growth factor. Thus, those GFs maintained strong physiologic effectiveness.

Perfusion effects and hydrodynamics.

Biological processes within living systems are significantly influenced by the motion of the liquids and gases to which those tissues are exposed. Accordingly, tissue engineers must not only understand hydrodynamic phenomena, but also appreciate the vital role of those phenomena in cellular and physiologic processes both in vitro and in vivo. In particular, understanding the fundamental principles of fluid flow underlying perfusion effects in the organ-level internal environment and their relation to the cellular microenvironment is essential to successfully mimicking tissue behavior. In this work, the major principles of hemodynamic flow and transport are summarized, to provide readers with a physical understanding of these important issues. In particular, since quantifying hemodynamic events through experiments can require expensive and invasive techniques, the benefits that can be derived from the use of computational fluid dynamics (CFD) packages and neural networking (NN) models are stressed. A capstone illustration based on analysis of the hemodynamics of aortic aneurysms is presented as a representative example of this approach, to stress the importance of tissue responses to flow-induced events.

Controlling tissue microenvironments: biomimetics, transport phenomena, and reacting systems.

The reconstruction of tissues ex vivo and production of cells capable of maintaining a stable performance for extended time periods in sufficient quantity for synthetic or therapeutic purposes are primary objectives of tissue engineering. The ability to characterize and manipulate the cellular microenvironment is critical for successful implementation of such cell-based bioengineered systems. As a result, knowledge of fundamental biomimetics, transport phenomena, and reaction engineering concepts is essential to system design and development. Once the requirements of a specific tissue microenvironment are understood, the biomimetic system specifications can be identified and a design implemented. Utilization of novel membrane systems that are engineered to possess unique transport and reactive features is one successful approach presented here. The limited availability of tissue or cells for these systems dictates the need for microscale reactors. A capstone illustration based on cellular therapy for type 1 diabetes mellitus via encapsulation techniques is presented as a representative example of this approach, to stress the importance of integrated systems.

Pulsatile flow in fusiform models of abdoiminal aortic aneurysms: flow fields, velocity patterns and flow-induced wall stresses.

As one important step in the investigation of the mechanical factors that lead to rupture of abdominal aortic aneurysms, flow fields and flow-induced wall stress distributions have been investigated in model aneurysms under pulsatile flow conditions simulating the in vivo aorta at rest. Vortex pattern emergence and evolution were evaluated, and conditions for flow stability were delineated. Systolic flow was found to be forward-directed throughout the bulge in all the models, regardless of size. Vortices appeared in the bulge initially during deceleration from systole, then expanded during the retrograde flow phase. The complexity of the vortex field depended strongly on bulge diameter In every model, the maximum shear stress occurred at peak systole at the distal bulge end, with the greatest shear stress developing in a model corresponding to a 4.3 cm AAA in vivo. Although the smallest models exhibited stable flow throughout the cycle, flow in the larger models became increasingly unstable as bulge size increased, with strong amplification of instability in the distal half of the bulge. These data suggest that larger aneurysms in vivo may be subject to more frequent and intense turbulence than smaller aneurysms. Concomitantly, increased turbulence may contribute significantly to wall stress magnitude and thereby to risk of rupture.