Calcification alters the viscoelastic properties of tendon fascicle bundles depending on matrix content.

Tendon fascicle bundles are often used as biological grafts and thus must meet certain quality requirements, such as excluding calcification, which alters the biomechanical properties of soft tissues. In this work, we investigate the influence of early-stage calcification on the mechanical and structural properties of tendon fascicle bundles with varying matrix content. The calcification process was modeled using sample incubation in concentrated simulated body fluid. Mechanical and structural properties were investigated using uniaxial tests with relaxation periods, dynamic mechanical analysis, as well as magnetic resonance imaging and atomic force microscopy. Mechanical tests showed that the initial phase of calcification causes an increase in the elasticity, storage, and loss modulus, as well as a drop in the normalized value of hysteresis. Further calcification of the samples results in decreased modulus of elasticity and a slight increase in the normalized value of hysteresis. Analysis via MRI and scanning electron microscopy showed that incubation alters fibrillar relationships within the tendon structure and the flow of body fluids. In the initial stage of calcification, calcium phosphate crystals are barely visible; however, extending the incubation time for the next 14 days results in the appearance of calcium phosphate crystals within the tendon structure and leads to damage in its structure. Our results show that the calcification process modifies the collagen-matrix relationships and leads to a change in their mechanical properties. These findings will help to understand the pathogenesis of clinical conditions caused by calcification process, leading to the development of effective treatments for these conditions. STATEMENT OF SIGNIFICANCE: This study investigates how calcium mineral deposition in tendons affects their mechanical response and which processes are responsible for this phenomenon. By analyzing the elastic and viscoelastic properties of animal fascicle bundles affected by calcification induced via incubation in concentrated simulated body fluid, the study sheds light on the relationship between structural and biochemical changes in tendons and their altered mechanical response. This understanding is crucial for optimizing tendinopathy treatment and preventing tendon injury. The findings provide insights into the calcification pathway and its resulting changes in the biomechanical behaviors of affected tendons, which have been previously unclear.

An in-House System for the Precise Measurement of Electrical Potentials and Mechanical Properties of Soft Tissues: Design and Validation Using Adult Mammalian Tendon Fascicle Bundles.

Tissues, such as skin, bones, and tendons, exhibit a piezoelectric effect, which may be an important phenomenon in terms of tissue renewal and regeneration as well as the possibility of modifying their mechanical behavior. In this article, we present the design and development of an in-house system for the precise measurement of electrical potentials and mechanical properties of tendons. The system was validated using tendon fascicle bundles derived from positional as well as energy-storing tendons from various adult mammals (porcine, bovine, and deer samples). The presented system is able to capture changes in elastic and viscoelastic properties of tissue as well as its time-voltage response and, thus, may be used in a broad spectrum of future studies to uncover factors influencing piezoelectric phenomena in tendons. This, in turn, will help to optimize current methods used in physiotherapy and postoperative treatment for effective tendon recovery.

Rehydration of the Tendon Fascicle Bundles Using Simulated Body Fluid Ensures Stable Mechanical Properties of the Samples.

In this work, we investigate the influence of dehydration and subsequent rehydration of tendon fascicle bundles on their structural and mechanical properties by using distilled water, 0.9% NaCl, 10% NaCl, SBF, and double concentrated SBF (SBFx2). The properties of tendon fascicle bundles were investigated by means of uniaxial tests with relaxation periods and hysteresis for samples with various interfascicular matrix content, dissected from the anterior and posterior areas of bovine tendon. Uniaxial tests with relaxation periods and analysis of sample geometry and weight showed that dehydration alters the modulus of elasticity dependent on the interfascicular matrix content and influences the viscoelastic properties of tendon fascicle bundles. Tensile and relaxation tests revealed that changes resulting from excessive sample drying can be reversed by rehydration in an SBF bath solution for elastic strain range above the toe region. Rehydration in SBF solution led to minor differences in mechanical properties when compared to control samples. Moreover, anterior samples with greater interfascicular matrix content, despite their lower stiffness, are less sensitive to sample drying. The obtained results allow us to limit the discrepancies in the measurement of mechanical properties of wet biological samples and can be useful to researchers investigating soft tissue mechanics and the stability of transplant materials.

The dispersion of viscoelastic properties of fascicle bundles within the tendon results from the presence of interfascicular matrix and flow of body fluids.

In this work, we investigate differences in the mechanical and structural properties of tendon fascicle bundles dissected from different areas of bovine tendons. The properties of tendon fascicle bundles were investigated by means of uniaxial tests with relaxation periods and hysteresis, dynamic mechanical analysis (DMA), as well as magnetic resonance imaging (MRI). Uniaxial tests with relaxation periods revealed greater elastic modulus, hysteresis, as well as stress drop during the relaxation of samples dissected from the posterior side of the tendon. However, the normalized stress relaxation curves did not show a statistically significant difference in the stress drop between specimens cut from different zones or between different strain levels. Using dynamic mechanical analysis, we found that fascicle bundles dissected from the anterior side of the tendon had lower storage and loss moduli, which could result from altered fluid flow within the interfascicular matrix (IFM). The lower water content, diffusivity, and higher fractional anisotropy of the posterior part of the tendon, as observed using MRI, indicates a different structure of the IFM, which controls the flow of fluids within the tendon. Our results show that the viscoelastic response to dynamic loading is correlated with fluid flow within the IFM, which was confirmed during analysis of the MRI results. In contrast to this, the long-term relaxation of tendon fascicle bundles is controlled by viscoplasticity of the IFM and depends on the spatial distribution of the matrix within the tendon. Comparison of results from tensile tests, DMA, and MRI gives new insight into tendon mechanics and the role of the IFM. These findings may be useful in improving the diagnosis of tendon injury and effectiveness of medical treatments for tendinopathies.

The effect of multiple freeze-thaw cycles on the viscoelastic properties and microstructure of bovine superficial digital flexor tendon.

The most common injuries of the human musculoskeletal system are related to soft tissue structures such as tendons or ligaments. To repair torn structures, surgical intervention and application of a biological or synthetic graft may be required. A typical procedure for the processing, storage, and distribution of soft tissue grafts involves at least two freezing/thawing (F/T) cycles. Even though repeated F/T cycles decrease the mechanical performance and change the structure of tendons, it is unclear whether there exists a maximum number of F/T cycles above which tendons should not be approved for use as a tissue allograft. To fill this research gap, we present an ex vivo study on the effects of repetitive F/T cycles on the biomechanical stability of bovine superficial digital flexor tendon tissue. Using mechanical testing supported with scanning electron microscopy imaging, we show that multiple F/T cycles affect the viscoelastic and structural properties of tissue by significantly reducing its tensile modulus after the 3rd or 4th F/T cycle (depending on the strain range), stress drop during relaxation after the 8th F/T cycle (regardless the strain values), mechanical hysteresis after the 10th F/T cycle, and by causing a significant decrease in collagen fibril diameter. Our results provide a deeper insight into understanding the mechanisms responsible for tissue damage during multiple F/T cycles, and thus, may be useful for the future optimization of tissue storage protocols.

The differences in viscoelastic properties of subtendons result from the anatomical tripartite structure of human Achilles tendon - ex vivo experimental study and modeling.

The human Achilles tendon (AT) is a hierarchical structure macroscopically composed of three subtendons originating from the soleus (SOL) and gastrocnemius (GL, GM) muscles. According to recent reports, the divisible structure of the AT together with diverse material properties of its subtendons are suspected as a probable cause of non-homogeneous stress and strain distribution occurring in loaded AT. Despite numerous investigations on human AT, there is still relatively little knowledge regarding mechanical properties of subtendon-level hierarchy, which is crucial in fully understanding the multiscale relationship which governs tendon mechanics. In this paper we present the first ex vivo study conducted on SOL, GL, and GM subtendons of human AT. We investigate differences in viscoelastic properties of SOL, GM, and GL subtendons in terms of tensile modulus, mechanical hysteresis as well as stress relaxation observed at two different values of strain. Our results show that the most significant differences in mechanical properties exist between subtendon attached to the soleus muscle (SOL) and subtendons originating from the two heads of the gastrocnemius muscle (GM and GL). We used our experimental results to calibrate three different constitutive models: the hyperelastic Yeoh model with power-law flow, the microstructurally motivated Holzapfel-Gasser-Ogden model enhanced with strain-dependent Berström-Boyce flow and the phenomenological elasto-viscoplastic Arruda-Boyce-based model with strain-dependent Berström-Boyce flow supplemented with component representing matrix response. All calibrated models may be applied to commercial FEA software as a sufficient solution for rapid mechanical response modeling of human AT subtendons or for the purpose of future development of comprehensive patient-specific models of human lower limbs. STATEMENT OF SIGNIFICANCE: The divisible structure of the Achilles tendon together with diverse material properties of its subtendons are suspected as a probable cause of non-homogeneous stress and strain distribution occurring in loaded Achilles tendon. Despite numerous investigations on mechanical properties of Achilles tendon, there is still relatively little knowledge regarding mechanical properties of subtendon-level hierarchy, which is crucial in fully understanding the multiscale relationship which governs tendon mechanics. This study is the first reported ex vivo investigation conducted on SOL, GL, and GM human Achilles subtendons. We investigate differences in the viscoelastic properties of individual subtendons and demonstrate that the observed differences should be considered as muscle-dependent. Our experimental research is supported with a modeling study in which we calibrate three different constitutive models.

A 3D Scanning System for Inverse Analysis of Moist Biological Samples: Design and Validation Using Tendon Fascicle Bundles.

In this article, we present the design and validation of a non-contact scanning system for the development of a three-dimensional (3D) model of moist biological samples. Due to the irregular shapes and low stiffness of soft tissue samples, the use of a non-contact, reliable geometry scanning system with good accuracy and repeatability is required. We propose a reliable 3D scanning system consisting of a blue light profile sensor, stationary and rotating frames with stepper motors, gears and a five-phase stepping motor unit, single-axis robot, control system, and replaceable sample grips, which once mounted onto the sample, are used for both scanning and mechanical tests. The proposed system was validated by comparison of the cross-sectional areas calculated based on 3D models, digital caliper, and vision-based methods. Validation was done on regularly-shaped samples, a wooden twig, as well as tendon fascicle bundles. The 3D profiles were used for the development of the 3D computational model of the sample, including surface concavities. Our system allowed for 3D model development of samples with a relative error of less than 1.2% and high repeatability in approximately three minutes. This was crucial for the extraction of the mechanical properties and subsequent inverse analysis, enabling the calibration of complex material models.