Elsevier

Acta Biomaterialia

Volume 7, Issue 1, January 2011, Pages 75-82
Acta Biomaterialia

Mitral valvular interstitial cell responses to substrate stiffness depend on age and anatomic region

https://doi.org/10.1016/j.actbio.2010.07.001Get rights and content

Abstract

The material properties of heart valves depend on the subject’s age, the state of the disease and the complex valvular microarchitecture. Furthermore, valvular interstitial cells (VICs) are mechanosensitive, and their synthesis of extracellular matrix not only determines the valve’s material properties but also provides an adhesive substrate for VICs. However, the interrelationship between substrate stiffness and VIC phenotype and synthetic properties is poorly understood. Given that the local mechanical environment (substrate stiffness) surrounding VICs differs among different age groups and different anatomic regions of the valve, it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness. Therefore, 6-week-, 6-month- and 6-year-old porcine VICs from the center of the mitral valve anterior leaflet (MVAC) and posterior leaflet (PML) were seeded onto poly(ethylene) glycol hydrogels of different stiffnesses and stained for markers of VIC activation (smooth muscle alpha-actin (SMaA)) and collagen synthesis (heat shock protein-47 (HSP47), prolyl 4-hydroxylase (P4H)). Six-week-old MVAC demonstrated decreased SMaA, P4H and HSP47 on stiffer gels, while 6-week-old PML only demonstrated decreased HSP47. Six-month-old MVAC demonstrated no difference between substrates, while 6-month-old PML demonstrated decreased SMaA, P4H and HSP47. Six-year-old MVAC demonstrated decreased P4H and HSP47, while 6-year-old PML demonstrated decreased P4H and increased HSP47. In conclusion, the age-specific and valve-region-specific responses of VICs to substrate stiffness link VIC phenotype to the leaflet regional matrix in which the VICs reside. These data provide further rationale for investigating the role of substrate stiffness in VIC remodeling within diseased and tissue engineered valves.

Introduction

Valve disease afflicts a substantial portion of the population: 1–2% of 26- to 84-year-olds are afflicted by mitral valve disorders [1]. Valve disease incurs significant morbidity and mortality, requiring over 100,000 surgeries in the USA each year [2]. In many of these disease states the mechanical properties of these valves are altered, often contributing to the poor valve function requiring surgical intervention. Valvular interstitial cells (VICs) are the dynamic, living component of heart valves responsible for synthesizing and maintaining the valve matrix composition, which in turn determines the valve’s material behavior. VICs and valves are known to be responsive to changes in their mechanical environment [3], [4], [5]. However, the interplay between matrix-driven material properties such as stiffness and the phenotype and synthetic behavior of VICs, particularly in the mitral valve (MV), has largely been overlooked. Recent work has demonstrated age-related significant changes in valve composition [6], [7], [8] and material properties [9]; other studies have shown substantial heterogeneity in material behavior among the different anatomic regions of the MV [10]. Given that different aged VICs and VICs from different regions of the MV reside in valve tissues with distinct stiffnesses [9], it was hypothesized that there may be an age- and valve-region-specific response of VICs to substrate stiffness.

In order to test this hypothesis, separate groups of VICs from three distinct age groups and from two different regions of the MV were cultured on poly(ethylene) glycol (PEG) hydrogels of two different stiffnesses. After 48 h, the resulting VIC expression of cell phenotype and collagen synthesis markers was assessed using immunocytochemistry (ICC).

PEG hydrogels were chosen for this experiment based on their promise as a potential platform for the design of scaffolds for tissue-engineered heart valves. PEG hydrogels are extremely hydrophilic, providing prevention against protein adsorption, a critical step in the immunogenicity and degradation of bioprosthetics [11]. They are also highly permeable, allowing the exchange of nutrients and waste materials [12]. Their stiffness can be regulated by changing the molecular weight and concentration of PEG [13]. However, one of the factors that makes these gels particularly attractive is the ability to customize them by conjugating to the PEG backbone various peptides, including cell ligands and growth factors, as well as incorporating enzyme-degradable sequences allowing tunability of the degradation rate of the hydrogel. This designer biofunctionality makes PEG hydrogels advantageous for the tissue engineering of heart valves. In the present study PEG hydrogels were conjugated with an Arg–Gly–Asp–Ser (RGDS) peptide, enabling VIC attachment to the hydrogel, and methacrylated heparin, which is necessary for normal VIC morphology [14], [15]. These functionalized PEG hydrogels of two different stiffnesses were formulated to keep the concentration of biological cues constant, thus isolating the effect of stiffness on VIC phenotype.

Section snippets

Synthesis of PEG hydrogel components

PEG-diacrylate (PEG-DA) of 3400 Da MW was synthesized from PEG (Sigma–Aldrich, St. Louis, MO) as previously described [13]. 1H nuclear magnetic resonance (NMR) analysis revealed >95% acrylation. Methacrylated heparin was synthesized as described previously [14]. Briefly, a 10 mg ml–1 solution of heparin (Sigma–Aldrich) dissolved in ultrapure water was reacted with 40 molar excess methacrylic anhydride (Sigma–Aldrich). The pH of the solution was adjusted to 7.5 using 4 M NaOH and stirred for 24 h.

Stiffness of different weight–volume fraction functionalized PEG hydrogels

Uniaxial testing revealed that functionalized 5% weight–volume PEG gels had a mean modulus of 34.5 kPa, while functionalized 15% weight–volume PEG gels had a mean modulus of 323.3 kPa (Fig. 3).

SMaA expression of VICs on gels of different stiffnesses

Not all VICs stained positively for SMaA, as expected. Analysis of the fraction of VICs expressing SMaA (SMaA+ VICs) on the different gels revealed a trend towards decreased SMaA+ VICs on the 15% gels compared to the 5% in the 6-week-old MVAC VICs (Fig. 4, p = 0.1), but there was no difference between gels

Discussion

In this study, mitral VICs grown on functionalized PEG hydrogels demonstrated both age- and valve-region-specific (MVAC vs. PML) responses to substrate stiffness. These results underscore the range of unique phenotypes found in valvular cells and provide compelling motivation for further studies of heart valve mechanobiology.

Conclusions

In this study, the response of MV VICs to substrate stiffness has been investigated for the first time. VIC populations taken from different regions of the same MV and VICs of different ages cultured on PEG hydrogels of different stiffnesses demonstrated age- and valve-region-specific responses to substrate stiffness. These findings should be taken into consideration in the design of an age-specific tissue-engineered heart valve and in future investigations of heart valve mechanobiology.

Acknowledgements

The authors appreciate the assistance of the members of the Grande-Allen laboratory, as well as members of the West laboratory: Michael Cuchiara, Stephanie Nemir, Ph.D., Melissa McHale, Ph.D., Maude Rowland, Jean Altus, Ph.D. and Jerome Saltarelli, Ph.D. The authors also appreciate the counsel of Scott Baggett, Ph.D. regarding statistics and the assistance of Sean Moran, Ph.D. in NMR analysis. This research was supported in part by a Hertz Foundation Graduate Fellowship (E.H.S.), a NIH Ruth

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