There is lack of investigation capturing the complex mechanical interaction of tissue-engineered intervertebral disk (IVD) constructs in physiologically relevant environmental conditions. In this study, mechanical characterization of anisotropic electrospinning (ES) substrates made of polycaprolactone (PCL) was carried out in wet and dry conditions and viability of human bone marrow derived mesenchymal stem cells (hMSCs) seeded within double layers of ES PCL were also studied. Cyclic compression of IVD-like constructs composed of an agarose core confined by ES PCL double layers was implemented using a bioreactor and the cellular response to the mechanical stimulation was evaluated. Tensile tests showed decrease of elastic modulus of ES PCL as the angle of stretching increased, and at 60 deg stretching angle in wet, the maximum ultimate tensile strength (UTS) was observed. Based on the configuration of IVD-like constructs, the calculated circumferential stress experienced by the ES PCL double layers was 40 times of the vertical compressive stress. Confined compression of IVD-like constructs at 5% and 10% displacement dramatically reduced cell viability, particularly at 10%, although cell presence in small and isolated area can still be observed after mechanical conditioning. Hence, material mechanical properties of tissue-engineered scaffolds, composed of fibril structure of polymer with low melting point, are affected by the testing condition. Circumferential stress induced by axial compressive stimulation, conveyed to the ES PCL double layer wrapped around an agarose core, can affect the viability of cells seeded at the interface, depending on the mechanical configuration and magnitude of the load.

Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties. To reconstruct cartilage tissue, articular chondrocytes from bovine humeral heads were embedded in agarose gel and cultured in vitro for up to 4 weeks. The nFCD of the cartilage was determined using the MRI gadolinium exclusion method. The equilibrium modulus was determined using a compressive stress relaxation test, and the dynamic modulus was determined by a dynamic compression test. The equilibrium compressive modulus and dynamic modulus of the tissue-engineered cartilage increased with an increase in culture time. The nFCD value—as determined with the [ Gd-DTPA 2 − ] measurement using the MRI technique—increased with culture time. In the regression analysis, nFCD showed significant correlations with equilibrium compressive modulus and dynamic modulus. From these results, gadolinium-enhanced MRI measurements can serve as a useful predictor of the biomechanical properties of tissue-engineered cartilage.

Mechanical and computational models consisting of flow channels with convergent and oscillating constrictions have been applied to study the dynamics of human vocal fold vibration. To the best of our knowledge, no mechanical model has been studied using a material substitute with similar physical properties to the human vocal fold for surgical experimentation. In this study, we design and develop a mechanical larynx with agarose as a vocal fold substitute, and assess its suitability for surgical experimentation. Agarose is selected as a substitute for the vocal fold as it exhibits similar nonlinear hyperelastic characteristics to biological soft tissue. Through uniaxial compression and extension tests, we determined that agarose of 0.375% concentration most closely resembles the vocal fold mucosa and ligament of a 20-year old male for small tensile strain with an R 2 value of 0.9634 and root mean square error of 344.05 ± 39.84 Pa . Incisions of 10 mm lengthwise and 3 mm in depth were created parallel to the medial edge on the superior surface of agar phantom. These were subjected to vibrations of 80, 130, and 180 Hz, at constant amplitude of 0.9 mm over a period of 10 min each in the mechanical larynx model. Lateral expansion of the incision was observed to be most significant for the lower frequency of 80 Hz. This model serves as a basis for future assessments of wound closure techniques during microsurgery to the vocal fold.

Relaxation indentation experiments using atomic force microscopy (AFM) are used to obtain viscoelastic material properties of soft samples. The quasilinear viscoelastic (QLV) model formulated by Fung ( 1972, “Stress Strain History Relations of Soft Tissues in Simple Elongation,” in Biomechanics, Its Foundation and Objectives, Prentice-Hall, Englewood Cliffs, NJ, pp. 181–207 ) for uniaxial compression data was modified for the indentation test data in this study. Hertz contact mechanics was used for the instantaneous deformation, and a reduced relaxation function based on continuous spectrum is used for the time-dependent part in the model. The modified QLV indentation model presents a novel method to obtain viscoelastic properties from indentation data independent of relaxation times of the test. The major objective of the present study is to develop the QLV indentation model and implement the model on AFM indentation data for 1% agarose gel and a viscoelastic polymer using spherical indenter.

While mechanical stimulation of cells seeded within scaffolds is widely thought to be beneficial, the amount of benefit observed is highly variable between experimental systems. Although studies have investigated specific experimental loading protocols thought to be advantageous for cartilage growth, less is known about the physical stimuli (e.g., pressures, velocities, and local strains) cells experience during these experiments. This study used results of a literature survey, which looked for patterns in the efficacy of mechanical stimulation of chondrocyte seeded scaffolds, to inform the modeling of spatial patterns of physical stimuli present in mechanically stimulated constructs. The literature survey revealed a large variation in conditions used in mechanical loading studies, with a peak to peak strain of 10% (i.e., the maximum amount of deformation experienced by the scaffold) at 1 Hz on agarose scaffolds being the most frequently studied parameters and scaffold. This loading frequency was then used as the basis for simulation in the finite element analyses. 2D axisymmetric finite element models of 2 × 4 mm 2 scaffolds with 360 modulus/permeability combinations were constructed using COMSOL MULTIPHYSICS software. A time dependent coupled pore pressure/effective stress analysis was used to model fluid/solid interactions in the scaffolds upon loading. Loading was simulated using an impermeable frictionless loader on the top boundary with fluid and solid displacement confined to the radial axis. As expected, all scaffold materials exhibited classic poro-elastic behavior having pressurized cores with low fluid flow and edges with high radial fluid velocities. Under the simulation parameters of this study, PEG scaffolds had the highest pressure and radial fluid velocity but also the lowest shear stress and radial strain. Chitosan and KLD-12 simulated scaffold materials had the lowest radial strains and fluid velocities, with collagen scaffolds having the lowest pressures. Parametric analysis showed maximum peak pressures within the scaffold to be more dependent on scaffold modulus than on permeability and velocities to depend on both scaffold properties similarly. The dependence of radial strain on permeability or modulus was more complex; maximum strains occurred at lower permeabilities and moduli, and the lowest strain occurred at the stiffest most permeable scaffold. Shear stresses within all scaffolds were negligible. These results give insight into the large variations in metabolic response seen in studies involving mechanical stimulation of cell-seeded constructs, where the same loading conditions produce very different results due to the differences in material properties.

A method has been developed to restore cartilage defects by culturing autologous chondrocytes to create a three dimensional tissue and then implanting the cultured tissue. In this kind of approach, it is important to characterize the dynamic mechanical behavior of the regenerated cartilaginous tissue, because these tissues need to bear various dynamic loadings in daily life. The objectives of this study were to evaluate in detail the dynamic viscoelastic responses of chondrocyte-seeded agarose gel cultures in compression and torsion (shear) and to determine the relationships between these mechanical responses and biochemical composition. The results showed that both the dynamic compressive and shear stiffness of the cultured constructs increased during culture. The relative energy dissipation in dynamic compression decreased, whereas that in dynamic shear increased during culture. Furthermore, correlation analyses showed that the sulfated glycosaminoglycan (sGAG) content of the cultured construct showed significant correlations with the dynamic modulus in both compression and shear situations. On the other hand, the loss tangent in dynamic compression, which represents the relative energy dissipation capability of the constructs, showed a low correlation with the sGAG content, whereas this capability in shear exhibited moderate correlation. In conclusion, we explored the dynamic viscoelasticity of the tissue-engineered cartilage in dynamic compression and shear, and determined correlations between viscoelasticity and biochemical composition.

A combined experimental-numerical approach was adopted to characterize glucose and oxygen uptake and lactate production by bovine articular chondrocytes in a model system. For a wide range of cell concentrations, cells in agarose were supplemented with either low or high glucose medium. During an initial culture phase of 48 h , oxygen was monitored noninvasively using a biosensor system. Glucose and lactate were determined by medium sampling. In order to quantify glucose and oxygen uptake, a finite element approach was adopted to describe diffusion and uptake in the experimental model. Numerical predictions of lactate, based on simple relations for cell metabolism, were found to agree well for low glucose, but not for high glucose medium. Oxygen did not play a role in either case. Given the close association between chondrocyte energy metabolism and matrix synthesis, a quantifiable prediction of utilization can present a valuable contribution in the optimization of tissue engineering conditions.

The complex modulus E * and elastic modulus E ′ of agarose gels (2% to 4%) are measured with a dynamic mechanical analyzer in frequency sweep shear sandwich mode between 0.1 and 20 Hz. The data showed that E * and E ′ increase with frequency according to a power law which can be described by a fractional derivative model to characterize the dynamic viscoelasticity of the gel. The functions between the model parameters including storage modulus coefficient H and the power law exponent (β) and the agarose concentration are established. A molecular basis for the application of the fractional derivative model to gel polymers is also discussed. Such an approach can be useful in tissue culture studies employing dynamic pressurization or for validation of magnetic resonance elastography.

A primary mechanism of solute transport in articular cartilage is believed to occur through passive diffusion across the articular surface, but cyclical loading has been shown experimentally to enhance the transport of large solutes. The objective of this study is to examine the effect of dynamic loading within a theoretical context, and to investigate the circumstances under which convective transport induced by dynamic loading might supplement diffusive transport. The theory of incompressible mixtures was used to model the tissue (gel) as a mixture of a gel solid matrix (extracellular matrix/scaffold), and two fluid phases (interstitial fluid solvent and neutral solute), to solve the problem of solute transport through the lateral surface of a cylindrical sample loaded dynamically in unconfined compression with frictionless impermeable platens in a bathing solution containing an excess of solute. The resulting equations are governed by nondimensional parameters, the most significant of which are the ratio of the diffusive velocity of the interstitial fluid in the gel to the solute diffusivity in the gel R g , the ratio of actual to ideal solute diffusive velocities inside the gel R d , the ratio of loading frequency to the characteristic frequency of the gel f ^ , and the compressive strain amplitude ε 0 . Results show that when R g > 1 , R d < 1 , and f ^ > 1 , dynamic loading can significantly enhance solute transport into the gel, and that this effect is enhanced as ε 0 increases. Based on representative material properties of cartilage and agarose gels, and diffusivities of various solutes in these gels, it is found that the ranges R g > 1 , R d < 1 correspond to large solutes, whereas f ^ > 1 is in the range of physiological loading frequencies. These theoretical predictions are thus in agreement with the limited experimental data available in the literature. The results of this study apply to any porous hydrated tissue or material, and it is therefore plausible to hypothesize that dynamic loading may serve to enhance solute transport in a variety of physiological processes.

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 × (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading ( 100 ± 16 kPa versus 15 ± 8 kPa , p < 0.0001 ) . This represented a 21-fold increase over the equilibrium modulus of day 0 4.8 ± 2.3 kPa . Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 ( p < 0.0001 and p = 0.002 , respectively). [S0148-0731(00)00703-2]

Low-concentration biogels, which provide an extracellular matrix for cells in vitro, are involved in a number of important cell biological phenomena, such as cell motility and cell differentiation. In order to characterize soft tissues, which collapse under their own weight, we developed and standardized a new experimental device that enabled us to analyze the mechanical properties of floating biogels with low concentrations, i.e., with values ranging from 2 g/L to 5 g/L. In order to validate this approach, the mechanical responses of free floating agarose gel samples submitted to compression as well as stretching tests were quantified. The values of the Young’s moduli, measured in the range of 1000 to 10,000 Pa, are compared to the values obtained from other experimental techniques. Our results showed indeed that the values we obtained with our device closely match those obtained independently by performing compression tests on an Instron device. Thus, the floating gel technique is a useful tool first to characterize and then to model soft tissues that are used in biological science to study the interaction between cell and extracellular matrix.