In this study, we report a novel approach for determining the mechanical properties of mouse aortic valve tissue using a modified micropipette aspiration technique. Importantly, our findings demonstrate the feasibility of this approach in mouse valve tissue. Significant differences were observed in biomechanical properties between annulus and cusp regions of mouse aortic valves. The tensile stiffness was found to be significantly greater in the annulus region (~550–600 kPa) compared to the cusp region (~270–320 kPa) at all stages of postnatal valve development, consistent with previously reported values of canine and porcine valve and aortic sinus stiffness (Thubrikar et al., 1980
; Merryman et al., 2006a
; Liao et al., 2008
). The higher stiffness values observed in the valve annulus are likely due to the regional abundance of collagen, which is associated with high material stiffness, valve durability and strength (Sacks et al., 1997
; Driessen et al., 2003
; Balguid et al., 2008
), and relative paucity of elastic fibers and proteoglycans (Vesely, 1998
). Importantly, the regional differences in valve tissue stiffness observed in our study are in agreement with previously reported modeling-based stress-strain studies that showed differences in stress sharing between valve and aortic root tissue (Grande et al., 1998
). Overall, the findings of the current study demonstrate regional variation in valve tensile stiffness, consistent with regional differences in ECM composition and organization.
Age-related changes in ECM organization, and alterations in matrix remodeling in human valve tissue have been reported previously (Otto et al., 1994
; Rabkin et al., 2001
; Fedak et al., 2003
; Hinton et al., 2006
). Recent studies examining porcine valves demonstrate specific changes in collagen and proteoglycan composition with advanced age, suggesting that age-associated ECM alterations can play a role in valve degeneration (Stephens et al., 2007
; Stephens et al., 2009
). Interestingly, proteoglycan content in valves is known to change with age or disease states (Barber et al., 2001
; Grande-Allen et al., 2003
; Stephens et al., 2008
). Our results indicate that collagen decreases while proteoglycans increase with aging in both cusp and annulus regions of mouse valves. Importantly, regional valve tensile stiffness also decreases at this stage suggesting that latent changes observed in valve biomechanical properties are due in part to matrix compositional changes.
Fibrogenesis abnormalities in valve tissue can manifest as fibrosis (too much collagen) or myxomatous change (too much proteoglycan). Based on our findings, we conclude that mice undergo age-associated degeneration that primarily affects the annulus, similar to degeneration in human valves, but involves proteoglycan accumulation instead of collagen accumulation (Roberts, 1970
; Otto et al., 1994
). We reconcile this important difference by considering the dominant nature of the collagen-rich fibrosa layer in humans and the relatively proteoglycan-rich and collagen-poor mouse aortic valve. While fibrotic disease states may manifest as different changes in ECM composition (Wynn, 2007
), this distinguishing feature between aged human and mouse valves has significant biomechanical implications and warrants further study. Taken together, these findings suggest that impaired ECM maintenance and remodeling with aging, i.e., structural degeneration, results in compromised mechanical valve function. Importantly, studies in targeted mutagenesis mouse models of valve disease will help elucidate the mechanisms underlying maladaptive matrix remodeling and fibrogenesis abnormalities, and improve our understanding of the different manifestations of age-associated changes and valve disease.
One limitation of the current analysis technique is that tensile stiffness determination was based on the assumption that small deformations in the tissue are treated as a half-space layer with infinite lateral dimensions (Theret et al., 1988
). Valve tissue in humans and large animals has a complex layered structure and exhibits non-linear stress-strain relationship and regional anisotropy (Lo et al., 1995
; Stella et al., 2007
; Sacks et al., 2009a
). Micropipette aspiration can be extended to include the multi-layered valve structure (Alexopoulos et al., 2003
), and will be the subject of future studies. The model used in the present study also requires that all tissue samples be at least 4 times the radius of micropipette, for the effects of geometry to be negligible (Aoki et al, 1997
; Ohashi et al, 2005
). In these experiments, the validity of this assumption was confirmed by visual microscopic observation.
In summary, these findings demonstrate that micropipette aspiration in combination with quantitative histology can be used to study region-specific age-related degeneration in mouse valve tissue. Importantly, this method provides opportunities for valve biomechanical testing in targeted mutagenesis mouse models of valve disease, which in turn enables a multidisciplinary approach to the study of AVD pathogenesis and natural history. Ultimately, combining engineering and molecular methodologies will elucidate valve structure and function and potentially inform new therapeutic strategies and bioprosthesis development.