Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.

To design more effective tissue-engineered heart valve replacements, the replacement tissue may need to mimic the biaxial stress-strain behavior of native heart valve tissue. This study characterized the planar biaxial properties of tissue-engineered valve leaflets and native aortic valve leaflets. Fibrin-based valve equivalent (VE) and porcine aortic valve (PAV) leaflets were subjected to incremental biaxial stress relaxation testing, during which fiber alignments were measured, over a range of strain ratios. Results showed that VE leaflets exhibited a modulus and fiber reorientation behavior that correlated with strain ratio. In contrast, PAV leaflets maintained their relaxed modulus and fiber alignment when exposed to nonequibiaxial strain, but exhibited changes in stress relaxation. In uniaxial and equi-biaxial tension, there were few observed differences in relaxation behavior between VE and PAV leaflets, despite differences in the modulus and fiber reorientation. Likewise, in both tissues there was similar relaxation response in the circumferential and radial directions in biaxial tension, despite different moduli in these two directions. This study presents some fundamental differences in the mechanical response to biaxial tension of fibrin-based tissue-engineered constructs and native valve tissue. It also highlights the importance of using a range of strain ratios when generating mechanical property data for valvular and engineered tissues. The data presented on the stress-strain, relaxation, and fiber reorientation of VE tissue will be useful in future efforts to mathematically model and improve fibrin-based tissue-engineered constructs.

Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.

Heart valve replacements composed of living tissue that can adapt, repair, and grow with a patient would provide a more clinically beneficial option than current inert replacements. Bioartificial valves were produced by entrapping human dermal fibroblasts within a fibrin gel. Using a mold design that presents appropriate mechanical constraints to the cell-induced fibrin gel compaction, gross fiber alignment (commissure-to-commissure alignment in the leaflets and circumferential alignment in the root) and the basic geometry of a native aortic valve were obtained. After static incubation on the mold in complete medium supplemented with transforming growth factor beta 1, insulin, and ascorbate, collagen fibers produced by the entrapped cells were found to coalign with the fibrin based on histological analyses. The resultant tensile mechanical properties were anisotropic. Ultimate tensile strength and tensile modulus of the leaflets in the commissural direction were 0.53 and 2.34 MPa, respectively. The constructs were capable of withstanding backpressure commensurate with porcine aortic valves in regurgitation tests (330 mmHg) and opened and closed under physiological pressure swings of 10 and 20 mmHg, respectively. These data support proof of principle of using cell-remodeled fibrin gel to produce tissue-engineered valve replacements.

Cell sourcing and culture conditions for fibrin-based valve constructs.

Cell sourcing for tissue-engineered heart valves remains a critical issue. In this work, human dermal fibroblasts (HDF) or porcine valve interstitial cells (PVIC) were entrapped in adherent fibrin disk constructs and harvested at 3 and 5 weeks. We compared the fibrin remodeling abilities of each cell type in Dulbecco's Modified Eagle's Medium (DMEM) and DMEM/F12 supplemented with transforming growth factor beta (TGF), and the response of PVIC to DMEM/F12 supplemented with fibroblast growth factor (FGF), a combination of FGF and TGF, and TGF with varying ascorbic acid (AA) concentrations. Culture media were supplemented with serum, insulin, AA, a fibrinolysis inhibitor, and antibiotics. DMEM maximized collagen and elastin deposition by HDF, while DMEM/F12 with FGF yielded the highest fibrin remodeling response by PVIC. HDF degraded fibrin slower than PVIC, and PVIC constructs had higher cellularity than HDF constructs in DMEM and DMEM/F12 at 3 weeks. FGF addition increased collagen content, collagen deposited per cell, and collagen as percentage of total protein compared to medium supplemented with TGF or TGF and FGF. AA addition increased collagen deposition by PVIC, but there was no dose dependence between 50 and 150 microg/mL AA. These results collectively show that PVIC are able to remodel fibrin faster and exhibit greater mechanical stiffening compared to HDF. Conditions for increased collagen deposition are also identified toward the engineering of valve constructs.

Decorin regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development.

Tendon function involves the development of an organized hierarchy of collagen fibrils. Small leucine-rich proteoglycans have been implicated in the regulation of fibrillogenesis and decorin is the prototypic member of this family. Decorin-deficient mice demonstrate altered fibril structure and mechanical function in mature skin and tail tendons. However, the developmental role(s) of decorin needs to be elucidated. To define these role(s) during tendon development, tendons (flexor digitorum longus) were analyzed ultrastructurally from postnatal day 10 to 90. Decorin-deficient tendons developed abnormal, irregularly contoured fibrils. Finite mixture modeling estimated that the mature tendon was a three-subpopulation mixture of fibrils with characteristic diameter ranges. During development, in each subpopulation the mean diameter was consistently larger in mutant mice. Also, diameter distributions and the percentage of fibrils in each subpopulation were altered. Biomechanical analyses demonstrated that mature decorin-deficient tendons had significantly reduced strength and stiffness; however, there was no reduction in immature tendons. Expression of decorin and biglycan, a closely related family member, was analyzed during development. Decorin increased with development while biglycan decreased. Spatially, both had a comparable localization throughout the tendon. Biglycan expression increased substantially in decorin-deficient tendons suggesting a potential functional compensation. The accumulation of structural defects during fibril growth, a period associated with decorin expression and low biglycan expression, may be the cause of compromised mechanical function in the absence of decorin. Our findings indicate that decorin is a key regulatory molecule and that the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development.

Influence of decorin and biglycan on mechanical properties of multiple tendons in knockout mice.

Evaluations of tendon mechanical behavior based on biochemical and structural arrangement have implications for designing tendon specific treatment modalities or replacement strategies. In addition to the well studied type I collagen, other important constituents of tendon are the small proteoglycans (PGs). PGs have been shown to vary in concentration within differently loaded areas of tendon, implicating them in specific tendon function. This study measured the mechanical properties of multiple tendon tissues from normal mice and from mice with knock-outs of the PGs decorin or biglycan. Tail tendon fascicles, patellar tendons (PT), and flexor digitorum longus tendons (FDL), three tissues representing different in vivo loading environments, were characterized from the three groups of mice. It was hypothesized that the absence of decorin or biglycan would have individual effects on each type of tendon tissue. Surprisingly, no change in mechanical properties was observed for the tail tendon fascicles due to the PG knockouts. The loss of decorin affected the PT causing an increase in modulus and stress relaxation, but had little effect on the FDL. Conversely, the loss of biglycan did not significantly affect the PT, but caused a reduction in both the maximum stress and modulus of the FDL. These results give mechanical support to previous biochemical data that tendons likely are uniquely tailored to their specific location and function. Variances such as those presented here need to be further characterized and taken into account when designing therapies or replacements for any one particular tendon.

Investigating tendon fascicle structure-function relationships in a transgenic-age mouse model using multiple regression models.

Proper replacement or repair of damaged tendons or ligaments requires functionally engineered tissue that mimics their native mechanical properties. While tendon structure-function relationships are generally assumed, there exists little quantitative evidence of the roles of distinct tendon components in tendon function. Previous work has used linear correlations to assess the independent, univariate effects of one structural or one biochemical variable on mechanics. The current study's objective was to simultaneously and rigorously evaluate the relative contributions of seven different structural and compositional variables in predicting tissue mechanical properties through the use of multiple regression statistical models. Structural, biochemical, and mechanical analysis were all performed on tail tendon fascicles from different groups of transgenic mice, which provide a reproducible, noninvasive, in vivo model of changes in tendon structure and composition. Interestingly, glycosaminoglycan (GAG) content was observed to be the strongest predictor of mechanical properties. GAG content was also well correlated with collagen content and mean collagen fibril diameter. Collagen fibril area fraction was a significant predictor only of material properties. Therefore, in a large multivariate model, GAG content was the largest predictor of mechanical properties, perhaps both through direct influence and indirectly through its correlation with collagen content and fibril structure.

Strain-rate sensitive mechanical properties of tendon fascicles from mice with genetically engineered alterations in collagen and decorin.

Tendons have complex mechanical behaviors that are nonlinear and time dependent. It is widely held that these behaviors are provided by the tissue composition and structure. It is generally thought that type I collagen provides the primary elastic strength to tendon while proteoglycans, such as decorin, play a role in failure and viscoelastic properties. This study sought to quantify such structure-function relationships by comparing tendon mechanical properties between normal mice and mice genetically engineered for altered type I collagen content and absence of decorin. Uniaxial tensile ramp to failure experiments were performed on tail tendon fascicles at two strain rates, 0.5%/s and 50%/s. Mutations in type I collagen led to reduced failure load and stiffness with no changes in failure stress, modulus or strain rate sensitivity. Fascicles without decorin had similar elastic properties to normal fascicles, but reduced strain rate sensitivity. Fascicles from immature mice, with increased decorin content compared to adult fascicles, had inferior elastic properties but higher strain rate sensitivity. These results showed that tendon viscoelasticity is affected by decorin content but not by collagen alterations. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity.

Mechanical strength of repairs of the hip piriformis tendon.

Mechanical properties of repairs of the piriformis tendon to the proximal femur were examined in a cadaveric model. Four constructs were separately tested: a suture anchor in the proximal femur, the anchor to suture interface, the suture in tendon interface, and a bone bridge style repair. The weakest interface was the anchor in the bone in cases in which the bone quality was poor. In specimens with high bone quality, the weakest interface was the suture in the anchor. A positive correlation was seen between Singh score for each femur and failure load of the anchor-bone constructs. Therefore, repairing the short external rotators after procedures involving a posterior approach to the hip may be warranted in patients with good bone quality.

Methods for quasi-linear viscoelastic modeling of soft tissue: application to incremental stress-relaxation experiments.

The quasi-linear viscoelastic (QLV) model was applied to incremental stress-relaxation tests and an expression for the stress was derived for each step. This expression was used to compare two methods for normalizing stress data prior to estimating QLV parameters. The first and commonly used normalization method was shown to be strain-dependent. Thus, a second normalization method was proposed and shown to be strain-independent and more sensitive to QLV time constants. These analytical results agreed with representative tendon data. Therefore, this method for normalizing stress data was proposed for future studies of incremental stress-relaxation, or whenever comparing stress-relaxation at different strains.

Effect of altered matrix proteins on quasilinear viscoelastic properties in transgenic mouse tail tendons.

Tendons have complex mechanical behaviors that are viscoelastic, nonlinear, and anisotropic. It is widely held that these behaviors are provided for by the tissue's composition and structure. However, little data are available to quantify such structure-function relationships. This study quantified tendon mechanical behaviors, including viscoelasticity and nonlinearity, for groups of mice that were genetically engineered for altered extracellular matrix proteins. Uniaxial tensile stress-relaxation experiments were performed on tail tendon fascicles from the following groups: eight week old decorin knockout, eight week old reduced type I collagen, three week old control, and eight week old control. Data were fit using Fung's quasilinear viscoelastic model, where the model parameters represent the linear viscoelastic and nonlinear elastic response. The viscoelastic properties demonstrated a larger and faster stress relaxation for the decorin knockout and a smaller and slower stress relaxation for the three week control. The elastic parameter, A, in the eight week control group was significantly greater than in the collagen reduction and three week control groups. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity. Future studies should directly correlate composition and structure with tendon mechanics for the design and evaluation of tissue-engineered constructs or tendon repairs.