Engineering the aortic valve extracellular matrix through stages of development, aging, and disease.

For such a thin tissue, the aortic valve possesses an exquisitely complex, multi-layered extracellular matrix (ECM), and disruptions to this structure constitute one of the earliest hallmarks of fibrocalcific aortic valve disease (CAVD). The native valve structure provides a challenging target for engineers to mimic, but the development of advanced, ECM-based scaffolds may enable mechanistic and therapeutic discoveries that are not feasible in other culture or in vivo platforms. This review first discusses the ECM changes that occur during heart valve development, normal aging, onset of early-stage disease, and progression to late-stage disease. We then provide an overview of the bottom-up tissue engineering strategies that have been used to mimic the valvular ECM, and opportunities for advancement in these areas.

Calcific Aortic Valve Disease Is Associated with Layer-Specific Alterations in Collagen Architecture.

Disorganization of the valve extracellular matrix (ECM) is a hallmark of calcific aortic valve disease (CAVD). However, while microarchitectural features of the ECM can strongly influence the biological and mechanical behavior of tissues, little is known about the ECM microarchitecture in CAVD. In this work, we apply advanced imaging techniques to quantify spatially heterogeneous changes in collagen microarchitecture in CAVD. Human aortic valves were obtained from individuals between 50 and 75 years old with no evidence of valvular disease (healthy) and individuals who underwent valve replacement surgery due to severe stenosis (diseased). Second Harmonic Generation microscopy and subsequent image quantification revealed layer-specific changes in fiber characteristics in healthy and diseased valves. Specifically, the majority of collagen fiber changes in CAVD were found to occur in the spongiosa, where collagen fiber number increased by over 2-fold, and fiber width and density also significantly increased. Relatively few fibrillar changes occurred in the fibrosa in CAVD, where fibers became significantly shorter, but did not otherwise change in terms of number, width, density, or alignment. Immunohistochemical staining for lysyl oxidase showed localized increased expression in the diseased fibrosa. These findings reveal a more complex picture of valvular collagen enrichment and arrangement in CAVD than has previously been described using traditional analysis methods. Changes in fiber architecture may play a role in regulating the pathobiological events and mechanical properties of valves during CAVD. Additionally, characterization of the ECM microarchitecture can inform the design of fibrous scaffolds for heart valve tissue engineering.

Engineering approaches to study fibrosis in 3-D in vitro systems.

Fibrotic diseases occur in virtually every tissue of the body and are a major cause of mortality, yet they remain largely untreatable and poorly understood on a mechanistic level. The development of anti-fibrotic agents has been hampered, in part, by the insufficient fibrosis biomimicry provided by traditional in vitro platforms. This review focuses on recent advancements toward creating 3-D platforms that mimic key features of fibrosis, as well as the application of novel imaging and sensor techniques to analyze dynamic extracellular matrix remodeling. Several opportunities are highlighted to apply new tools from the fields of biomaterials, imaging, and systems biology to yield pathophysiologically relevant in vitro platforms that improve our understanding of fibrosis and may enable identification of potential treatment targets.

pH-responsive hydrogels with dispersed hydrophobic nanoparticles for the oral delivery of chemotherapeutics.

Amphiphilic polymer carriers were formed by polymerizing a hydrophilic, pH-responsive hydrogel composed of poly(methacrylic-grafted-ethylene glycol) (P(MAA-g-EG)) in the presence of hydrophobic PMMA nanoparticles. These polymer carriers were varied in PMMA nanoparticle content to elicit a variety of physiochemical properties which would preferentially load doxorubicin, a hydrophobic chemotherapeutic, and release doxorubicin locally in the colon for the treatment of colon cancers. Loading levels ranged from 49% to 64% and increased with increasing nanoparticle content. Doxorubicin loaded polymers were released in a physiological model where low pH was used to simulate the stomach and then stepped to more neutral conditions to simulate the upper small intestine. P(MAA-g-EG) containing nanoparticles were less mucoadhesive as determined using a tensile tester, polymer samples, and fresh porcine small intestine. The cytocompatibility of the polymer materials were assessed using cell lines representing the GI tract and colon cancer and were noncytotoxic at varying concentrations and exposure times.

pH-Responsive Hydrogels with Dispersed Hydrophobic Nanoparticles for the Delivery of Hydrophobic Therapeutic Agents.

To investigate the delivery of hydrophobic therapeutic agents, a new class of polymer carriers was synthesized. These carriers are composed of two components: (i) a pH-responsive hydrogel composed of methacrylic acid grafted with poly(ethylene glycol) tethers, P(MAA-g-EG), and (ii) hydrophobic poly(methyl methacrylate) (PMMA) nanoparticles. Before the P(MAA-g-EG) hydrogel was crosslinked, PMMA nanoparticles were added to the solution and upon exposure to UV light they were photoencapsulated throughout the P(MAA-g-EG) hydrogel structure. The pH-responsive behavior of P(MAA-g-EG) is capable of triggered release of a loaded therapeutic agent, such as a low molecular weight drug or protein, when it passes from the stomach (low pH) to upper small intestine (neutral pH). The introduction of PMMA nanoparticles into the hydrogel structure affected the swelling behavior, therapeutic agent loading efficiency, and solute release profiles. In equilibrium swelling conditions the swelling ratio of nanoparticle-containing hydrogels decreased with increasing nanoparticle content. Loading efficiencies of the model therapeutic agent fluorescein ranged from 38 - 51 % and increased with increasing hydrophobic content. Release studies from neat P(MAA-g-EG) and the ensuing P(MAA-g-EG) hydrogels containing nanoparticles indicated that the transition from low pH (2.0) to neutral pH (7.0) triggered fluorescein release. Maximum fluorescein release depended on the structure and hydrophobicity of the carriers used in these studies.

Amphiphilic Interpenetrating Networks for the Delivery of Hydrophobic, Low Molecular Weight Therapeutic Agents.

To investigate the delivery of hydrophobic therapeutic agents, a novel class of interpenetrating networks (IPNs) were synthesized and composed of two networks: methacrylic acid grafted with poly(ethylene glycol) tethers, P(MAA-g-EG), and poly(n-butyl acrylate) (PBA). The hydrophilic P(MAA-g-EG) networks are pH-responsive hydrogels capable of triggered release of an encapsulated therapeutic agent, such as a low molecular weight drug or a protein, when it passes from the stomach (low pH) to upper small intestine (neutral pH). PBA is a hydrophobic homopolymer that can affect the IPN swelling behavior, the therapeutic agent loading efficiencies in IPNs, and solute release profiles from IPNs. In dynamic swelling conditions, IPNs had greater swelling ratios than P(MAA-g-EG), but in equilibrium swelling conditions the IPN swelling ratio decreased with increasing PBA content. Loading efficiencies of the model therapeutic agent fluorescein ranged from 21 - 44%. Release studies from neat P(MAA-g-EG) and the ensuing IPNs indicated that the transition from low pH (2.0) to neutral pH (7.0) triggered fluorescein release. Maximum fluorescein release depended on the structure and hydrophilicity of the carriers used in these studies.