In vitro bone growth responds to local mechanical strain in three-dimensional polymer scaffolds

Mechanical stimulation plays a key role in healing and remodelling of bone tissue in vivo, and is used in bone tissue regeneration strategies in vitro. Although macroscopic compression of three-dimensional (3-D) seeded constructs can increase bone formation, it is not yet reported how this response is related to differences in local mechanical strains inside the scaffolds. In this study, we experimentally test the hypothesis that differences in local average of heterogeneous strains in a polymer scaffold will correlate with induced differences in the local biological response.Twenty-four poly(l-lactic acid) porous scaffolds seeded with rat bone cells were cultured first for 2 and 3 weeks under static conditions, respectively. Then for 1 week, half of the scaffolds were cyclically compressed (1.5%, 1 Hz), 1 h daily, with continuous perfusion (0.1 ml/min). The remaining half was kept under static conditions. The pore-surface strains in the scaffolds at the start of culture were calculated with micro-finite element modelling based on micro-Computed Tomography (μCT) images. The locations of mineralized nodules were determined from μCT images and coupled to the calculated strains.Detectable mineralized nodules (>10³ μm³) were only present in the loaded samples. Averages of absolute principal strains at the start of culture were significantly higher at nodule sites than at sites without a nodule.The results support the hypothesis that regenerating bone tissue in a 3-D porous scaffold responds to local mechanical strain. The methodology presented in this study can contribute design optimisation of tissue regeneration strategies relying on mechanical stimulation.     

Promotion of human mesenchymal stem cell differentiation on bioresorbable polycaprolactone/biphasic calcium phosphate composite scaffolds for bone tissue engineering

An artificial construct mimicking the intrinsic properties of the natural extracellular matrix in bones has been considered an ideal platform for bone tissue engineering, as it can present an appropriate microenvironment and regulate cell behaviours. In this report, we introduce biodegradable composite scaffolds consisting of polycaprolactone (PCL) and biphasic calcium phosphate (BCP). The scaffolds were fabricated by a salt-leaching process, and the ability of the scaffolds to facilitate osteogenic differentiation was investigated using human mesenchymal stem cells (hMSCs). The scaffolds had an inter-connected porous structure with quadrilateral pores of approximately 200 ～ 500 μm in width. The mechanical properties of the scaffolds changed as the BCP content was increased in the starting mixture. In the hMSC experiment, although we found that hMSCs adhered to the surface, as well as the inside, of the scaffolds, the incorporated BCP did not increase the proliferation of the hMSCs over 7 days in culture. Interestingly, the alkaline phosphatase (ALP) activity was 4 times higher on the PCL/BCP composite scaffold (0.12 ± 0.03 nmol/min/μg protein) thanon the PCL scaffold (0.03 ± 0.01 nmol/min/μg protein), suggesting that BCP can aid in generating a local environment that promotes bone regeneration. Therefore, a strategy combining polymers and ceramics can be considered a useful platform for bone tissue engineering.     

Experimental and finite element analysis of the rat ulnar loading model-correlations between strain and bone formation following fatigue loading

The rat forelimb compression model has been used widely to study bone response to mechanical loading. We used strain gages to assess load sharing between the ulna and radius in the forelimb of adult Fisher rats. We used histology and peripheral quantitative computed tomography (pQCT) to quantify ulnar bone formation 12 days after in vivo fatigue loading. Lastly, we developed a finite element model of the ulna to predict the pattern of surface strains during compression. Our findings indicate that at the mid-shaft the ulna carries 65% of the applied compressive force on the forelimb. We observed large variations in fatigue-induced bone formation over the circumference and length of the ulna. Bone formation was greatest 1-2 mm distal to the mid-shaft. At the mid-shaft, we observed woven bone formation that was greatest medially. Finite element analysis indicated a strain pattern consistent with a compression-bending loading mode, with the greatest strains occurring in compression on the medial surface and lesser tensile strains occurring laterally. A peak strain of -5190 microepsilon (for 13.3N forelimb compression) occurred 1-2 mm distal to the mid-shaft. The pattern of bone formation in the longitudinal direction was highly correlated to the predicted peak compressive axial strains at seven cross-sections (r2 = 0.89, p = 0.014). The in-plane pattern of bone formation was poorly correlated to the predicted magnitude of axial strain at 51 periosteal locations (r2 = 0.21, p < 0.001), because the least bone formation was observed where tensile strains were highest. These findings indicate that the magnitude of bone formation after fatigue loading is greatest in regions of high compressive strain.     

Cyclic mechanical compression increases mineralization of cell-seeded polymer scaffolds in vivo

Despite considerable documentation of the ability of normal bone to adapt to its mechanical environment, very little is known about the response of bone grafts or their substitutes to mechanical loading even though many bone defects are located in load-bearing sites. The goal of this research was to quantify the effects of controlled in vivo mechanical stimulation on the mineralization of a tissue-engineered bone replacement and identify the tissue level stresses and strains associated with the applied loading. A novel subcutaneous implant system was designed capable of intermittent cyclic compression of tissue-engineered constructs in vivo. Mesenchymal stem cell-seeded polymeric scaffolds with 8 weeks of in vitro preculture were placed within the loading system and implanted subcutaneously in male Fisher rats. Constructs were subjected to 2 weeks of loading (3 treatments per week for 30 min each, 13.3 N at 1 Hz) and harvested after 6 weeks of in vivo growth for histological examination and quantification of mineral content. Mineralization significantly increased by approximately threefold in the loaded constructs. The finite element method was used to predict tissue level stresses and strains within the construct resulting from the applied in vivo load. The largest principal strains in the polymer were distributed about a modal value of -0.24% with strains in the interstitial space being about five times greater. Von Mises stresses in the polymer were distributed about a modal value of 1.6 MPa, while stresses in the interstitial tissue were about three orders of magnitude smaller. This research demonstrates the ability of controlled in vivo mechanical stimulation to enhance mineralized matrix production on a polymeric scaffold seeded with osteogenic cells and suggests that interactions with the local mechanical environment should be considered in the design of constructs for functional bone repair.     

Predicting the stress distribution within scaffolds with ordered architecture

Current tissue engineering technologies involve the seeding of cells on porous scaffolds, within which the cells can proliferate and differentiate, when cultured in bioreactors. The flow of culture media through the scaffolds generates stresses that are important for both cell differentiation and cell growth. A recent study [Appl. Phys. Lett. 97 (2010), 024101] showed that flow-induced stresses inside highly porous and randomly structured scaffolds follow a three-point gamma probability density function (p.d.f.). The goal of the present study is to further investigate whether the same p.d.f. can also describe the distribution of stresses in structured porous scaffolds, what is the range of scaffold porosity for which the distribution is valid, and what is the physical reason for such behavior. To do that, the p.d.f. of flow-induced stresses in different scaffold geometries were calculated via flow dynamics simulations. It was found that the direction of flow relative to the internal architecture of the scaffolds is important for stress distributions. The stress distributions follow a common distribution within statistically acceptable accuracy, when the flow direction does not coincide with the direction of internal structural elements of the scaffold.     

Porous bioactive glass scaffolds for local drug delivery in osteomyelitis: development and in vitro characterization

A new bioactive glass-based scaffold was developed for local delivery of drugs in case of osteomyelitis. Bioactive glass having a new composition was prepared and converted into porous scaffold. The bioactivity of the resulting scaffold was examined by in vitro acellular method. The scaffolds were loaded with two different drugs, an antibacterial or antifungal drug. The effects of the size of the scaffold, drug concentration, and dissolution medium on drug release were studied. The scaffolds were further coated with a degradable natural polymer, chitosan, to further control the drug release. Both the glass and scaffold were bioactive. The scaffolds released both the drugs for 6 weeks, in vitro. The results indicated that the bigger the size and the higher the drug concentration, the better was the release profile. The scaffolds appeared to be suitable for local delivery of the drugs in cases of osteomyelitis.     

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.     

The influence of fibrous elastomer structure and porosity on matrix organization

Fibrous scaffolds are finding wide use in the field of tissue engineering, as they can be designed to mimic many native tissue properties and structures (e.g., cardiac tissue, meniscus). The influence of fiber alignment and scaffold architecture on cellular interactions and matrix organization was the focus of this study. Three scaffolds were fabricated from the photocrosslinkable elastomer poly(glycerol sebacate) (PGS), with changes in fiber alignment (non-aligned (NA) versus aligned (AL)) and the introduction of a PEO sacrificial polymer population to the AL scaffold (composite (CO)). PEO removal led to an increase in scaffold porosity and maintenance of scaffold anisotropy, as evident through visualization, mechanical testing, and mass loss studies. Hydrated scaffolds possessed moduli that ranged between ～3-240 kPa, failing within the range of properties (<300 kPa) appropriate for soft tissue engineering. CO scaffolds were completely degraded as early as 16 days, whereas NA and AL scaffolds had ～90% mass loss after 21 days when monitored in vitro. Neonatal cardiomyocytes, used as a representative cell type, that were seeded onto the scaffolds maintained their viability and aligned along the surface of the AL and CO fibers. When implanted subcutaneously in rats, a model that is commonly used to investigate in vivo tissue responses to biomaterials, CO scaffolds were completely integrated at 2 weeks, whereas ～13% and ～16% of the NA and AL scaffolds, respectively remained acellular. However, all scaffolds were completely populated with cells at 4 weeks post-implantation. Polarized light microscopy was used to evaluate the collagen elaboration and orientation within the scaffold. An increase in the amount of collagen was observed for CO scaffolds and enhanced alignment of the nascent collagen was observed for AL and CO scaffolds compared to NA scaffolds. Thus, these results indicate that the scaffold architecture and porosity are important considerations in controlling tissue formation.     

Micromechanical study of the load transfer in a polycaprolactone–collagen hybrid scaffold when subjected to unconfined and confined compression

Scaffolds are used in diverse tissue engineering applications as hosts for cell proliferation and extracellular matrix formation. One of the most used tissue engineering materials is collagen, which is well known to be a natural biomaterial, also frequently used as cell substrate, given its natural abundance and intrinsic biocompatibility. This study aims to evaluate how the macroscopic biomechanical stimuli applied on a construct made of polycaprolactone scaffold embedded in a collagen substrate translate into microscopic stimuli at the cell level. Eight poro-hyperelastic finite element models of 3D printed hybrid scaffolds from the same batch were created, along with an equivalent model of the idealized geometry of that scaffold. When applying an 8% confined compression at the macroscopic level, local fluid flow of up to 20 \(\upmu \)m/s and octahedral strain levels mostly under 20% were calculated in the collagen substrate. Conversely unconfined compression induced fluid flow of up to 10 \(\upmu \)m/s and octahedral strain from 10 to 35%. No relevant differences were found amongst the scaffold-specific models. Following the mechanoregulation theory based on Prendergast et al. (J Biomech 30:539–548, 1997.  https://doi.org/10.1016/S0021-9290(96)00140-6), those results suggest that mainly cartilage or fibrous tissue formation would be expected to occur under unconfined or confined compression, respectively. This in silico study helps to quantify the microscopic stimuli that are present within the collagen substrate and that will affect cell response under in vitro bioreactor mechanical stimulation or even after implantation.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

The effect of axial compression and distraction on cervical facet mechanics during anterior shear,flexion, axial rotation,and lateral bending motions

The subaxial cervical facets are important load-bearing structures, yet little is known about their mechanical response during physiological or traumatic intervertebral motion. Facet loading likely increases when intervertebral motions are superimposed with axial compression forces, increasing the risk of facet fracture. The aim of this study was to measure the mechanical response of the facets when intervertebral axial compression or distraction is superimposed on constrained, non-destructive shear, bending and rotation motions. Twelve C6/C7 motion segments (70 ± 13 yr, nine male) were subjected to constrained quasi-static anterior shear (1 mm), axial rotation (4°), flexion (10°), and lateral bending (5°) motions. Each motion was superimposed with three axial conditions: (1) 50 N compression; (2) 300 N compression (simulating neck muscle contraction); and, (3) 2.5 mm distraction. Angular deflections, and principal and shear surface strains, of the bilateral C6 inferior facets were calculated from motion-capture data and rosette strain gauges, respectively. Linear mixed-effects models (α = 0.05) assessed the effect of axial condition. Minimum principal and maximum shear strains were largest in the compressed condition for all motions except for maximum principal strains during axial rotation. For right axial rotation, maximum principal strains were larger for the contralateral facets, and minimum principal strains were larger for the left facets, regardless of axial condition. Sagittal deflections were largest in the compressed conditions during anterior shear and lateral bending motions, when adjusted for facet side.     

Distribution of bone-marrow stromal cells in a 3D scaffold depending on the seeding method and the scaffold inside a surface modification

The distribution of bone-marrow stromal cells (BMSC) was studied in 3D polylactide scaffolds. Seeding of cells into the scaffold by the dynamic method (with the aid of a peristaltic pump) has been shown to provide distribution of cells throughout the entire scaffold volume, unlike the static method of seeding, in which the cell suspension is applied onto the scaffold surface. Unlike the cells seeded into the scaffold by the dynamic method, the cells seeded by the static method practically completely migrate from the scaffold on the dish for the first several days. It is revealed that BMSCs cultivated in 3D polylactide scaffolds modified by fibrin form colonies, whereas BMSCs cultivated inside scaffolds modified by collagen type 1 distribute all over the scaffold volume in the form of individual cells.     

Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.     

Micropatterned biopolymer 3D scaffold for static and dynamic culture of human fibroblasts

During in vivo tissue regeneration, cell behavior is highly influenced by the surrounding environment. Thus, the choice of scaffold material and its microstructure is one of the fundamental steps for a successful in vitro culture. An efficacious method for scaffold fabrication should prove its versatility and the possibility of controlling micro- and nanostructure. In this paper, hyaluronic acid 3D scaffolds were developed through lamination of micropatterned membranes, fabricated after optimization of a soft-lithography method. The scaffold presented here is characterized by a homogeneous hexagonal lattice with porosity of 69%, specific surface area of 287 cm-1, and permeability of 18.9 microm2. The control over the geometry was achieved with an accuracy of 20 mum. This technique allowed not only fabrication of planar 3D scaffolds but also production of thin wall tubular constructs. Mechanical tests, performed on dry tubular scaffolds, show high rupture tensile strength. This construct could be promising not only as engineered vascular grafts but also for regeneration of skin, urethra, and intestinal walls. The biocompatibility of a 3D planar scaffold was tested by seeding human fibroblasts. The cells were cultured in both static and dynamic conditions, in a perfusion bioreactor at different flow rates. Microscope analysis and MTT test showed cell proliferation and viability and a uniform cell distribution likely due to an appropriate lattice structure.     

Determination of mechanical properties of soft tissue scaffolds by atomic force microscopy nanoindentation

While the determination of mechanical properties of a hard scaffold is relatively straightforward, the mechanical testing of a soft tissue scaffold poses significant challenges due in part to its fragility. Here, we report a new approach for characterizing the stiffness and elastic modulus of a soft scaffold through atomic force microscopy (AFM) nanoindentation. Using collagen-chitosan hydrogel scaffolds as model soft tissue scaffolds, we demonstrated the feasibility of using AFM nanoindentation to determine a force curve of a soft tissue scaffold. A mathematical model was developed to ascertain the stiffness and elastic modulus of a scaffold from its force curve obtained under different conditions. The elastic modulus of a collagen-chitosan (80%/20%, v/v) scaffold is found to be 3.69 kPa. The scaffold becomes stiffer if it contains more chitosan. The elastic modulus of a scaffold composed of 70% collagen and 30% chitosan is about 11.6 kPa. Furthermore, the stiffness of the scaffold is found to be altered significantly by extracellular matrix deposited from cells that are grown inside the scaffold. The elastic modulus of collagen-chitosan scaffolds increased from 10.5 kPa on day 3 to 63.4 kPa on day 10 when human foreskin fibroblast cells grew inside the scaffolds. Data acquired from these measurements will offer new insights into understanding cell fate regulation induced by physiochemical cues of tissue scaffolds.     

Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design

The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee. The insufficient vascularization of this tissue prevents it from healing completely after extreme tearing or rupture, creating a need for ACL grafts for reconstruction. The limitations of existing grafts have motivated the investigation of tissue-engineered ACL grafts. A successful tissue-engineered graft must possess mechanical properties similar to the ACL; to date no commercially available synthetic graft has achieved this. To accomplish this goal we have combined the techniques of polymer fiber braiding and twisting to design a novel poly L-lactic acid (PLLA) braid-twist scaffold for ACL tissue engineering. The scaffold is designed to accurately mimic the biomechanical profile and mechanical properties of the ACL. In this study, braid-twist scaffolds were constructed and compared to braided scaffolds and twisted fiber scaffolds. The addition of fiber twisting to the braided scaffold resulted in a significant increase in the ultimate tensile strength, an increase in ultimate strain, and an increase in the length of the toe region in these constructs over scaffolds that were braided. Based on the findings of this study, the braid-twist scaffold studied was found to be a promising construct for tissue engineering of the ACL.     

In situ intercellular mechanics of the bovine outer annulus fibrosus subjected to biaxial strains

In situ intercellular strains in the outer annulus fibrosus of bovine caudal discs were determined under two states of biaxial strain. Confocal microscopy was used to track and capture images of fluorescently labelled nuclei at applied Lagrangian strains in the axial direction (E(A)(S)) of 0%, 7.5% and 15% while the circumferential direction (E(C)(S)) was constrained to either 0% or -2.5%. The position of the nuclear centroids were calculated in each image and used to investigate the in situ intercellular mechanics of both lamellar and interlamellar cells. The intercellular Lagrangian strains measured in situ were non-uniform and did not correspond with the biaxial Lagrangian strains applied to the tissue. A row-oriented analysis of intercellular unit displacements within the lamellar layers found that the magnitudes of unit displacements between cells along a row (delta;(II)) were small (|delta;(IIavg)|=1.6% at E(C)(S)=0%, E(A)(S)=15%; |delta;(IIavg)|=3.0% at E(C)(S)=-2.5%, E(A)(S)=15%) with negative unit displacements occurring greater than one-third of the time. Evidence of interlamellar shear and increased intercellular Lagrangian strains among the cells within the interlamellar septa suggested that their in situ mechanical environment may be more complex. The in situ intercellular strains of annular cells were strongly dependent upon the local structure and behaviour of the extracellular matrix and did not correspond with applied tissue strains. This knowledge has immediate relevance for in vitro investigations of disc mechanobiology, and will also provide a base to investigate the mechanical implications of disc degeneration at the cellular level.     

Effects of three-dimensional scaffolds on cell organization and tissue development

Tissue engineering scaffolds play a critical role in regulating the reconstructed human tissue development. Various types of scaffolds have been developed in recent years, including fibrous matrix and foam-like scaffolds. The design of scaffold materials has been investigated extensively. However, the design of physical structure of the scaffold, especially fibrous matrices, has not received much attention. This paper compares the different characteristics of fibrous and foam-like scaffolds, and reviews regulatory roles of important scaffold properties, including surface geometry, scaffold configuration, pore structure, mechanical property and bioactivity. Tissue regeneration, cell organization, proliferation and differentiation under different microstructures were evaluated. The importance of proper scaffold selection and design is further discussed with the examples of bone tissue engineering and stem cell tissue engineering. This review addresses the importance of scaffold microstructure and provides insights in designing appropriate scaffold structure for different applications of tissue engineering.     

Detection of forces and displacements along the axial direction in an optical trap

We present measurements of the forces on, and displacements of, an optically trapped bead along the propagation direction of the trapping laser beam (the axial direction). In a typical experimental configuration, the bead is trapped in an aqueous solution using an oil-immersion, high-numerical-aperture objective. This refractive index mismatch complicates axial calibrations due to both a shift of the trap center along the axial direction and spherical aberrations. In this work, a known DNA template was unzipped along the axial direction and its characteristic unzipping force-extension data were used to determine 1), the location of the trap center along the axial direction; 2), the axial displacement of the bead from the trap center; and 3), the axial force exerted on the bead. These axial calibrations were obtained for trap center locations up to approximately 4 microm into the aqueous solution and with axial bead displacements up to approximately 600 nm from the trap center. In particular, the axial trap stiffness decreased substantially when the trap was located further into the aqueous solution. This approach, together with conventional lateral calibrations, results in a more versatile optical trapping instrument that is accurately calibrated in all three dimensions.     

Ultrasonic measurement of depth-dependent transient behaviors of articular cartilage under compression

We previously reported an ultrasound method for measuring the depth-dependent equilibrium mechanical properties of articular cartilage using quasi-static compression. The objective of this paper was to introduce our recent development for nondestructively measuring the transient depth-dependent strains of full-thickness articular cartilage specimens prepared from bovine patellae. A 50 MHz focused ultrasound transducer was used to collect ultrasound echoes from articular cartilage specimens (n=8) and sponge phantoms with open pores (n=10) during tests of compression and subsequent stress-relaxation. The transient displacements of the tissues at different depths along the compression direction were calculated from the ultrasound echoes using a cross-correlation tracking technique. An LVDT sensor and a load cell were used to measure the overall deformation of the tissue and the applied force, respectively. Results showed that the tissues inside the cartilage layer continued to move during the stress-relaxation phase after the compression was completed. In the equilibrium state, the displacements of the cartilage tissues at the depths of 1/4, 1/2, and 3/4 of the full-thickness reduced by 51%+/-22%, 54%+/-17%, and 50+/-17%, respectively, in comparison with its peak value. However, no similar phenomenon was observed in the sponge phantoms. Our preliminary results demonstrated that this ultrasound method may provide a potential tool for the nondestructive measurement of the transient depth-dependent processes involved in biological and bioengineered soft tissues as well as soft biomaterials under dynamic loading.