Collagen Fiber Architecture Research Articles

Decreased Mechanical Properties of Heart Valve Tissue Constructs Cultured in Platelet Lysate as Compared to Fetal Bovine Serum

In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered three-dimensional tissue constructs cultured in PL as a substitute for FBS. Our results show that tissue constructs that were cultured in PL and FBS produce similar amounts of collagen, glycosoaminoglycans, and collagen crosslinks, and that the cellular phenotype remains unchanged. Nevertheless, mechanical testing showed that the ultimate tensile strength in PL constructs was on average approximately three times lower as compared to FBS (0.25 vs. 0.74 MPa, respectively, p<0.01), and also the elastic modulus was almost three times lower (1.33 MPa of PL constructs vs. 3.94 MPa of FBS constructs, p<0.01). Additional tests indicated that this difference might be explained by different collagen fiber architecture possibly due to increased production of matrix-degrading proteases by cells cultured in PL. In summary, our results indicate that PL is not preferred for the culture of strong heart valve tissue constructs.

Bone Marrow Aspirate Concentrate (BMAC) as a Biological Adjunct to the Surgical Treatment of Osteochondral Lesions of the Talus

Osteochondral lesions of the talus are common injuries, particularly in athletes. Traditional surgical treatment paradigms for osteochondral lesions include reparative and restorative techniques. The concern with reparative techniques (ie, microfracture and drilling) is that the fibrocartilaginous infill may ultimately fail over time, thereby causing recurrence in symptoms and a progression of arthritis. The current authors have developed a treatment algorithm whereby reparative techniques are reserved for lesions ≤8 mm in diameter, beyond which replacement strategies (ie, autologous osteochondral plug transplantation) comprise the preferred treatment strategy. In an effort to address the concerns of fibrocartilage degradation over time, the authors are currently studying the use of bone marrow aspirate concentrate (BMAC) as a means of improving the tissue quality and collagen fiber architecture of the differentiated repair cartilage. When used as an adjunct to the autologous osteochondral plug transplantation procedure, BMAC may facilitate infill of the graft-host bone and cartilage, thereby reducing the theoretical potential of synovial fluid inflow under normal joint hydrostatics, which could undermine the graft over an extended period. This article will evaluate the use of BMAC in the surgical treatment of osteochondral lesions of the talus.

Arthroscopic Laser Meniscectomy in a Gas Medium

Laboratory investigations demonstrate the theoretical feasibility of utilizing CO2 laser energy for arthroscopic resection of the knee meniscus. Infrared light of 10.6 μm wavelength is sufficiently absorbed by fibrocartilage with byproducts of heat, water vapor, and a small residue of carbon ash. The remaining meniscus rim demonstrates viable chondrocytes in close proximity to the margin of resection, and gross collagen fiber architecture is preserved. The depth of penetration of the laser beam can be controlled by limiting the duration of exposure. Arthroscopic application of CO2 laser energy requires a gas medium. Carbon dioxide and nitrogen have proven to be satisfactory insufflation agents, with no lasting untoward effects noted in a clinical series of diagnostic arthroscopic procedures. The cost of laser generators and the lack of an ideal delivery system are limiting factors in clinical applications of this cutting mode for meniscectomy.

Prolyl 3-Hydroxylase 1 Null Mice Display Abnormalities in Fibrillar Collagen-rich Tissues Such as Tendons, Skin, and Bones

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.

Immunohistochemical and scanning electron microscopic comparison of the collagen network constructions between pig, goat and chicken livers

The distribution and three-dimensional architecture of collagen fibers were compared between pig, goat and chicken livers. Immunohistochemical staining revealed that collagen type I was identified in the interlobular connective tissue region and intralobular areas in pigs and goats. Type III collagen was also identified in the interlobular connective tissue region and intralobular sinusoidal walls. In the chicken liver, only the circumference region of the vessels was immunostained with collagen type I and III antibodies and the interlobular connective tissue wall could not be distinguished clearly. In the intralobular region, collagen type I antibody immunoreacted around the hepatic cells but collagen type III antibody immunoreacted weakly. In the NaOH macerated specimen, well-developed collagen bundles formed the prominent interlobular walls in pigs. In contrast, the wall in the goat liver comprised a thin layer of the bundles. In the chicken liver, there were no notable collagen septa between lobules. The intralobular collagen construction was quite different between the animals, indicating a fragile collagen fibril networks in pigs, a robust framework in goats and dense fabric-like septa in chickens. These results indicate that the distinct collagen frameworks may contribute to the histological strength of the livers in each of the animal species.

Functional Collagen Fiber Architecture of the Pulmonary Heart Valve Cusp

Defects in the pulmonary valve (PV) occur in a variety of forms of congenital heart diseases. Quantitative information on PV collagen fiber architecture, and particularly its response to diastolic forces, is necessary for the design and functional assessment of approaches for PV repair and replacement. This necessity is especially the case for novel tissue-engineered PV, which rely on extensive in-vivo remodeling for long-term function. Porcine PV and aortic valves (AV) were fixed under a 0 to 90 mm Hg transvalvular pressure. After dissection from the root, small-angle light-scattering measurements were conducted to quantify the collagen fiber architecture and changes with increasing applied transvalvular pressure over the entire cusp. Histomorphologic measurements were also performed to assess changes in cuspal layer thickness with pressure. While the PV and AV displayed anticipated structural similarities, they also presented important functionally related differences. In the unloaded state, the AV cusp demonstrated substantial regional variations in fiber alignment, whereas the PV was surprisingly uniform. Further, the AV demonstrated substantially larger changes in collagen fiber alignment with applied transvalvular pressure compared with the PV. Overall, the AV collagen fiber network demonstrated greater ability to respond to applied transvalvular pressure. A decrease in crimp amplitude was the predominant mechanism for improvement in the degree of orientation of the collagen fibers in both valves. This study clarified the major similarities and differences between the PV and the AV. While underscoring how the PV can serve as an appropriate replacement of the diseased AV, the observed structural differences may also indicate limits to the ability of the PV to fully duplicate the AV. Moreover, quantitative data from this study on PV functional architecture will benefit development of tissue-engineered PV by defining the critical fiber architectural characteristics.

Material Properties of the Axillary Pouch of the Glenohumeral Capsule: Is Isotropic Material Symmetry Appropriate?

Inconclusive findings regarding the collagen fiber architecture and the material properties of the glenohumeral capsule make it unclear whether the material symmetry of the glenohumeral capsule is isotropic or anisotropic. The overall objective of this work was to use a combined experimental and computational protocol to characterize the mechanical properties of the axillary pouch of the glenohumeral capsule and to determine the appropriate material symmetry. Two perpendicular tensile and finite simple shear deformations were applied to a series of tissue samples from the axillary pouch of the glenohumeral capsule. An inverse finite element optimization routine was then used to determine the material coefficients for an isotropic hyperelastic constitutive model by simulating the experimental conditions. There were no significant differences between the material coefficients obtained from the two perpendicular tensile deformations or finite simple shear deformations. Furthermore, stress-stretch relationships predicted by utilizing the material coefficients from one direction were able to predict the responses of the same tissue sample in the perpendicular direction. These similarities between the longitudinal and transverse material behaviors of the tissue imply that the capsule may be considered an isotropic material. However, differences did exist between the material coefficients obtained from the tensile and shear loading conditions. Therefore, a more advanced constitutive model is needed to predict both the tensile and shear responses of the material.

Effects of mechanical loading on collagen propeptides processing in cartilage repair

Injured articular cartilage has poor reparative capabilities and if left untreated may develop into osteoarthritis. Unsatisfactory results with conventional treatment methods have brought as an alternative treatment the development of matrix autologous chondrocyte transplants (MACTs). Recent evidence proposes that the maintenance of the original phenotype by isolated chondrocytes grown in a scaffold transplant is linked to mechanical compression, because macromolecules, particularly collagen, of the extracellular matrix have the ability to 'self-assemble'. In load-bearing tissues, collagen is abundantly present and mechanical properties depend on the collagen fibre architecture. Study of the active changes in collagen architecture is the focus of diverse fields of research, including developmental biology, biomechanics and tissue engineering. In this review, the structural model of collagen assembly is presented in order to understand how scaffold geometry plays a critical role in collagen propeptide processing and chondrocyte development. When physical forces are applied to different cell-based scaffolds, the resulting specific twist of the scaffolds might be accompanied by changes in the fibril pattern synthesis of the new collagen. The alteration in the scaffolds due to mechanical stress is associated with cellular signalling communication and the preservation of N-terminus procollagen moieties, which would regulate both the collagen synthesis and the diameter of the fibre. The structural difference would also affect actin stabilization, cytoskeleton remodelling and proteoglycan assembly. These effects seemed to be dependent on the magnitude and duration of the physical stress. This review will contribute to the understanding of mechanisms for collagen assembly in both a natural and an artificial environment.

Platelet receptor recognition and cross-talk in collagen-induced activation of platelets.

Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.

Scanning electron microscopic observation

In the process of hepatic fibrosis, the accumulation of collagen fibers is strongly related to the hepatic function. The aim of this study was to investigate the three-dimensional architecture of the collagen network in the liver of rats with hepatic fibrosis. Healthy adult male Wistar rats (n = 32) were randomly divided into a control group (n = 16) and a hepatic fibrosis group (n = 16). In the control group, the rats were treated with peanut oil while the rats in hepatic fibrosis group were treated for 10 weeks with 60% CCl(4) diluted in peanut oil. The quantity of collagen fibers was detected by Western blotting; distribution of the collagen was detected by sirius red staining and polarized microscope; the three-dimensional architecture of collagen in the liver was observed under the scanning electron microscope after fixed tissues were treated with cell-maceration using NaOH. Statistical analysis was performed using the u test. The quantity of collagen fibers increased significantly in the hepatic fibrosis group. With the aggravation of hepatic fibrosis, collagen fibers gradually accumulated. They interlaced the reticulation compartment and formed a round or ellipse liver tissue conglomeration like a grape framework that was disparate and wrapped up the normal liver lobule. The deposition of collagen fibers was obvious in adjacent hepatic parenchyma, especially around the portal tracts. Our experiment showed the collagen proliferation and displays clearly the three-dimensional architecture of collagen fibers in rat liver with hepatic fibrosis by scanning electron microscope. It can provide a morphological foundation for the mechanisms of changed haemodynamics and portal hypertension in hepatic fibrosis.

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional (2)H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo.

Collagen Fiber Architecture of the Periodontal Ligament in Equine Cheek Teeth

The objective of this study was to examine the spatial arrangement of the fiber apparatus of the equine periodontium which is supposed to meet two contrary requirements: (1) to attach the tooth firmly and elastically to the alveolar bone; and, to be appropriately remodeled and reconstructed in order to facilitate the prolonged eruption of the tooth. Specimens of periodontal ligament were obtained from the buccal and lingual/palatal aspects of the first molars from the maxilla and mandible of 12 horses. The animals were assigned to three age groups. Histological sections were prepared from three specific horizontal levels of the periodontal ligament and examined with conventional and polarized-light microscopy. At the gingival level, collagen fascicles (diameter > 200 microm) were densely packed. Their spatial alignment was the same in all age groups. The architecture of the collagen fiber apparatus differed at the middle and apical levels in the three age groups. There was a clear distinction between fibers, bundles, and fascicles. Bundling of collagen fibers, density of the fiber arrangement, and collagen fascicles with an alveolo-cemental orientation increased with age. The collagen fiber apparatus of the equine periodontal ligament is highly adaptive, responding continuously to the dynamic changes in the periodontal environment. Site-specific arrangements and age-dependent structural variations are assumed to maintain tooth support as the reserve crown gradually decreases in length with progressive dental attrition. Most of the age-dependent changes to the periodontal ligament in teeth examined in this study occurred at the apical level. The apical region of young teeth had no periodontal attachment, while the roots of older teeth were firmly attached to the alveolus. When evaluating periodontal ligament development, the individual tooth's 'dental age' should be considered rather than the animal's age to account for individual tooth eruption times.

Etiology of inguinal hernia: ultrastructure of rectus sheath revisited

In the last decade, in the search for abdominal-wall hernia etiology, attention has been brought to alterations in the connective tissue ultrastructure as the probable etiological factor. These may cause weakening of connective tissue, which in turn may form ground for hernia formation. To investigate this hypothesis in depth, we compared the ultrastructure of the connective tissue in hernia patients and the control group. The study group consisted of five patients with primary inguinal hernia (Nyhus II = 4, Nyhus IIIa = 1). Another five patients posted for emergency appendectomy created the control group. Tissue specimens, harvested intraoperatively from the rectus muscle sheath (RAMS) and fixed in 4% glutaraldehyde, underwent staining by the Masson, H-E and methylene blue techniques and were assessed by microscopy (light and scanning electron). The examinations showed significant differences in the rectus sheath ultrastructure. They included altered architecture, placement and quantity of collagen and elastic fibers, differences in the caliber of individual fibers and disrupted ground matter-to-fiber ratio. In patients with hernias, chaotic arrangement of collagen fibers was seen, as well as their thinning and a decrease in the general amount of elastic fibers, replaced by ground matter. Our research has shown significant differences in the structure of the RAMS between patients with hernias and healthy individuals. This supports the theory linking connective tissue alterations with the etiology of hernia, and stating that these alterations include connective tissue at locations distant from the hernia site as well, as the rectus sheath itself does not form a hernial defect.

Fluid-structure interaction computational modelling of realistically reconstructed atherosclerotic plaques to assess their vulnerability to rupture based on collagen fibre architecture

Fluid-structure interaction computational modelling of realistically reconstructed atherosclerotic plaques to assess their vulnerability to rupture based on collagen fibre architecture

Remodeling of the collagen fiber architecture in cardiovascular tissues

The stimulus functions are assumed to depend on the mechanical loading condition within the tissue, i.e., gi = gi(λi, σi). The stimulus functions are specified such that: (1) for a uniaxial loading condition all fiber directions align with the major loading direction, (2) in case of a biaxial loading condition, the fiber families are situated in between the major directions with the main orientation and dispersity depending on the ratio of g1 and g2, and (3) for an equibiaxial loading condition, the fibers are distributed uniformly in the plane of the major directions v1 and v2. The artery is modeled as a thick-walled cylinder, which is inflated by an internal pressure and stretched longitudinally. The aortic valve is modeled as a leaflet with uniform thickness and is loaded by a transvalvular pressure in the diastolic (i.e., closed) configuration.

Micrometer scale Ex Vivo multiphoton imaging of unstained arterial wall structure

We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. This study illustrates the potential of 2PEF/SHG microscopy for pharmacological studies in unlabeled arteries.

The Effect of Matrix Tension-Compression Nonlinearity and Fixed Negative Charges on Chondrocyte Responses in Cartilage

Thorough analyses of the mechano-electrochemical interaction between articular cartilage matrix and the chondrocytes are crucial to understanding of the signal transduction mechanisms that modulate the cell metabolic activities and biosynthesis. Attempts have been made to model the chondrocytes embedded in the collagen-proteoglycan extracellular matrix to determine the distribution of local stress-strain field, fluid pressure and the time-dependent deformation of the cell. To date, these models still have not taken into account a remarkable characteristic of the cartilage extracellular matrix given rise from organization of the collagen fiber architecture, now known as the tension-compression nonlinearity (TCN) of the tissue, as well as the effect of negative charges attached to the proteoglycan molecules, and the cell cytoskeleton that interacts with mobile ions in the interstitial fluid to create osmotic and electro-kinetic events in and around the cells. In this study, we proposed a triphasic, multi-scale, finite element model incorporating the Conewise Linear Elasticity that can describe the various known coupled mechanical, electrical and chemical events, while at the same time representing the TCN of the extracellular matrix. The model was employed to perform a detailed analysis of the chondrocytes' deformational and volume responses, and to quantitatively describe the mechano-electrochemical environment of these cells. Such a model describes contributions of the known detailed micro-structural and composition of articular cartilage. Expectedly, results from model simulations showed substantial effects of the matrix TCN on the cell deformational and volume change response. A low compressive Poisson's ratio of the cartilage matrix exhibiting TCN resulted in dramatic recoiling behavior of the tissue under unconfined compression and induced significant volume change in the cell. The fixed charge density of the chondrocyte and the pericellular matrix were also found to play an important role in both the time-dependent and equilibrium deformation of the cell. The pericellular matrix tended to create a uniform osmolarity around the cell and overall amplified the cell volume change. It is concluded that the proposed model can be a useful tool that allows detailed analysis of the mechano-electrochemical interactions between the chondrocytes and its surrounding extracellular matrix, which leads to more quantitative insights in the cell mechano-transduction.

Cell transplantation preserves matrix homeostasis: A novel paracrine mechanism

Cell transplantation prevents chamber dilatation, but the underlying molecular mechanisms remain undefined. Structural cardiac remodeling involves matrix degradation from an imbalance of matrix metalloproteinases (MMP) relative to endogenous tissue inhibitors of metalloproteinases (TIMP). We aimed to determine the capacity of cell transplantation to alter extracellular matrix in the failing heart and, in so doing, identify novel paracrine molecular mediators underlying the beneficial effects of cell transplantation on chamber dilatation. Smooth muscle cells were transplanted to the dilating left ventricle of cardiomyopathic hamsters (CTX, n = 15) compared with age-matched media-injected cardiomyopathic (CON, n = 15) and normal hamsters (n = 7). After 5 weeks, left ventricular volume was measured by computerized planimetry. Fibrillar collagen was examined by confocal microscopy. Matrix homeostasis was quantified by measuring MMP/TIMP expression/activity relative to myocardial collagen synthesis (14C-proline uptake). Left ventricular dilatation was attenuated in CTX hearts (P = .02). CTX restored perimysial collagen fiber content and architecture to normal levels. TIMP-2 and TIMP-3 expression were enhanced in CTX (TIMP-2, 195% +/- 42% of CON, P = .02; TIMP-3, 118% +/- 3% of CON, P = .002), and correspondingly, gelatinase MMP-2 activity was reduced (P < .05). The TIMP:MMP ratio was increased in CTX hearts (TIMP-2 to MMP-2, 410% +/- 134% of CON, P = .04, and TIMP-3 to MMP-9, 205% +/- 47% of CON, P = .03), reflecting a reduced capacity for matrix degradation. Collagen synthesis was equivalent (CTX vs CON), suggesting that restored matrix architecture was a function of attenuated matrix degradation. These data provide the first evidence that cell transplantation limits ventricular dilatation in the failing heart through a paracrine-mediated mechanism that preserves extracellular matrix homeostasis.

Improved Prediction of the Collagen Fiber Architecture in the Aortic Heart Valve

Living tissues show an adaptive response to mechanical loading by changing their internal structure and morphology. Understanding this response is essential for successful tissue engineering of load-bearing structures, such as the aortic valve. In this study, mechanically induced remodeling of the collagen architecture in the aortic valve was investigated. It was hypothesized that, in uniaxially loaded regions, the fibers aligned with the tensile principal stretch direction. For biaxial loading conditions, on the other hand, it was assumed that the collagen fibers aligned with directions situated between the principal stretch directions. This hypothesis has already been applied successfully to study collagen remodeling in arteries. The predicted fiber architecture represented a branching network and resembled the macroscopically visible collagen bundles in the native leaflet. In addition, the complex biaxial mechanical behavior of the native valve could be simulated qualitatively with the predicted fiber directions. The results of the present model might be used to gain further insight into the response of tissue engineered constructs during mechanical conditioning.

Cyclic loading response of bioprosthetic heart valves: effects of fixation stress state on the collagen fiber architecture

Biologically derived, chemically modified collagenous tissues are being increasingly used to fabricate cardiac valve prostheses and as biomaterials in cardiovascular repair. A stress-free state during chemical modification has been shown to preserve the collagen fiber architecture of the native tissue, potentially preserving native mechanical properties and improving prostheses durability. However, it is not known if the native collagen fiber architecture is stable during long-term in vivo operation. To address this question, we obtained porcine aortic valves chemically treated at (i) 0mmHg transvalvular pressure (with 40mmHg aortic pressure) and (ii) 4mmHg transvalvular pressure, then subjected the valves to 0,1×106, 50×106, and 200×106 in vitro accelerated wear testing (AWT) cycles. The resulting changes in collagen fiber architecture were quantified using small angle light scattering analysis (SALS). SALS measurements indicated that collagen fibers in the 0mmHg pressure-fixed leaflets became more aligned between 1×106 and 50×106 AWT cycles. In contrast, only minor changes (not statistically significant) in collagen fiber orientation occurred in the 4mmHg pressure-fixed valvular tissue with cycling. It was also noted that although the 0mmHg group was fixed without transvalvular pressure, distention of the root induced significant changes in collagen structure of the leaflets. Overall, our observations suggest that the native collagen fiber crimp of the 0mmHg pressure-fixed leaflets were rapidly lost after only 50×106 AWT cycles (equivalent to ∼1.6 patient years) and thus may not be maintained over a sufficient period of time to be clinically beneficial. Further, the collagen structure of the native aortic valve is exquisitely sensitive to dimensional change in the aortic root—independent of the presence of transvalvular pressure. Our findings also suggest that without in vivo remodeling, any collagenous tissue used to fabricate BHV may undergo similar degenerative, irreversible changes in vivo.