Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells

The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-β3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5 ± 0.21%w/w vs. 0.94 ± 0.03%w/w) and annulus (1.09 ± 0.09%w/w vs. 0.59 ± 0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.     

Dynamic deformational loading results in selective application of mechanical stimulation in a layered, tissue-engineered cartilage construct

The application of dynamic physiologic loading to a bilayered chondrocyte-seeded agarose construct with a 2% (wt/vol) top layer and 3% (wt/vol) bottom layer was hypothesized to (1) improve overall construct properties and (2) result in a tissue that mimics the mechanical inhomogeneity of native cartilage. Dynamic loading over the 28 day culture period was found to significantly increase bulk mechanical and biochemical properties versus free-swelling culture. The initial depth-distribution of the compressive Young's modulus (EY) reflected the intrinsic properties of the gel in each layer and a similar trend to the native tissue, with a softer 2% gel layer and a much stiffer 3% gel layer. After 28 days in culture, free-swelling conditions maintained this general trend while loaded constructs possessed a reverse profile, with significant increases in EY observed only in the 2% gel. Histological analysis revealed preferential matrix formation in the 2% agarose layer, with matrix localized more pericellularly in the 3% agarose layer. Finite element modeling revealed that, prior to significant matrix elaboration, the 2% layer experiences increased mechanical stimuli (fluid flow and compressive strain) during loading that may enhance chondrocyte stimulation and nutrient transport in that layer, consistent with experimental observations. From these results, we conclude that due to the limitations in 3% agarose, the use of this type of bilayered construct to construct depth-dependent inhomogeneity similar to the native tissue is not likely to be successful under long-term culture conditions. Our study underscores the importance of other physical properties of the scaffold that may have a greater influence on interconnected tissue formation than intrinsic scaffold stiffness.     

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Dynamic compressive loading of image-guided tissue engineered meniscal constructs

This study investigated the hypothesis that dynamic compression loading enhances tissue formation and increases mechanical properties of anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were seeded in 2%w/v alginate, crosslinked with CaSO(4), injected into μCT based molds, and post crosslinked with CaCl(2). Samples were loaded via a custom bioreactor with loading platens specifically designed to load anatomically shaped constructs in unconfined compression. Based on the results of finite element simulations, constructs were loaded under sinusoidal displacement to yield physiological strain levels. Constructs were loaded 3 times a week for 1 h followed by 1 h of rest and loaded again for 1 h. Constructs were dynamically loaded for up to 6 weeks. After 2 weeks of culture, loaded samples had 2-3.2 fold increases in the extracellular matrix (ECM) content and 1.8-2.5 fold increases in the compressive modulus compared with static controls. After 6 weeks of loading, glycosaminoglycan (GAG) content and compressive modulus both decreased compared with 2 week cultures by 2.3-2.7 and 1.5-1.7 fold, respectively, whereas collagen content increased by 1.8-2.2 fold. Prolonged loading of engineered constructs could have altered alginate scaffold degradation rate and/or initiated a catabolic cellular response, indicated by significantly decreased ECM retention at 6 weeks compared with 2 weeks. However, the data indicates that dynamic loading had a strikingly positive effect on ECM accumulation and mechanical properties in short term culture.     

A mathematical model of tissue-engineered cartilage development under cyclic compressive loading

In this work a coupled model of solute transport and uptake, cell proliferation, extracellular matrix synthesis and remodeling of mechanical properties accounting for the impact of mechanical loading is presented as an advancement of a previously validated coupled model for free-swelling tissue-engineered cartilage cultures. Tissue-engineering constructs were modeled as biphasic with a linear elastic solid, and relevant intrinsic mechanical stimuli in the constructs were determined by numerical simulation for use as inputs of the coupled model. The mechanical dependent formulations were derived from a calibration and parametrization dataset and validated by comparison of normalized ratios of cell counts, total glycosaminoglycans and collagen after 24-h continuous cyclic unconfined compression from another dataset. The model successfully fit the calibration dataset and predicted the results from the validation dataset with good agreement, with average relative errors up to 3.1 and 4.3 %, respectively. Temporal and spatial patterns determined for other model outputs were consistent with reported studies. The results suggest that the model describes the interaction between the simultaneous factors involved in in vitro tissue-engineered cartilage culture under dynamic loading. This approach could also be attractive for optimization of culture protocols, namely through the application to longer culture times and other types of mechanical stimuli.     

Role of cell-associated matrix in the development of free-swelling and dynamically loaded chondrocyte-seeded agarose gels

Chondrocytes embedded in agarose and subjected to dynamic deformational loading produce a functional matrix with time in culture, but there is usually a delay in the development of significant differences compared to free swelling. In this study, we hypothesized that the initial presence of a cell-associated matrix would expedite construct development in response to dynamic deformational loading. Seeded samples with enzymatically isolated chondrocytes and chondrons (the chondrocyte and its pericellular matrix) and examined the effects of seeding density and dynamic loading on the development of tissue properties. At 60 million/ml, dynamic loading significantly augmented the development of material properties in chondrocyte- and chondron-seeded constructs. Biochemical content and histological analysis indicated that the deposition of GAG, link protein and collagens are affected by the pre-existing cell-associated matrix of the chondron-seeded samples. The pericellular matrix associated with the chondrons did not expedite the development of material properties in response to deformational loading, disproving our hypothesis. The relative concentration and distribution of matrix proteins may play a major role in the disparate responses observed for the chondrocyte- and chondron-seeded cultures. In further support of these findings, culturing chondrocytes in agarose for two weeks prior to the application of deformational loading also did not exhibit expedited construct development.     

Anisotropic Poisson's ratio and compression modulus of cortical bone determined by speckle interferometry

Young's modulus and Poisson's ratios of 6mm-sized cubes of equine cortical bone were measured in compression using a micro-mechanical loading device. Surface displacements were determined by electronic speckle pattern-correlation interferometry. This method allows for non-destructive testing of very small samples in water. Analyses of standard materials showed that the method is accurate and precise for determining both Young's modulus and Poisson's ratio. Material properties were determined concurrently in three orthogonal anatomic directions (axial, radial and transverse). Young's modulus values were found to be anisotropic and consistent with values of equine cortical bone reported in the literature. Poisson's ratios were also found to be anisotropic, but lower than those previously reported. Poisson's ratios for the radial-transverse and transverse-radial directions were 0.15+/-0.02, for the axial-transverse and axial-radial directions 0.19+/-0.04, and for the transverse-axial and radial-axial direction 0.09+/-0.02 (mean+/-SD). Cubes located only millimetres apart had significantly different elastic properties, showing that significant spatial variation occurs in equine cortical bone.     

Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation

One approach to functional tissue engineering of cartilage is to utilize bioreactors to provide environmental conditions that stimulate chondrogenesis in cells cultured on biomaterial scaffolds. We report the combined use of a three-dimensional in vitro model and a novel bioreactor with perfusion of culture medium and mechanical stimulation in long-term studies of cartilage development and function. To engineer cartilage, scaffolds made of a non-woven mesh of polyglycolic acid (PGA) were seeded with bovine calf articular chondrocytes, cultured for an initial 30-day period under free swelling conditions, and cultured for an additional 37 day period in one of the three groups: (1) free-swelling, (2) static compression (on 24 h/day, strain control, static offset 10%), and (3) dynamic compression (on 1 h/day; off 23 h/day; strain control, static offset 2%, dynamic strain amplitude 5%; frequency 0.3 Hz). Constructs were sampled at timed intervals and assessed with respect to structure, biochemical composition, and mechanical function. Mechanical simulation had little effect on the compositions, morphologies and on mechanical properties of construct interiors discs, but it resulted in distincly different properties of the peripheral rings and face sides. Contructs cultured with mechanical loading maintained their cylindrical shape with flat and parallel top and bottom surfaces, and retained larger amounts of GAG. The modular bioreactor system with medium perfusion and mechanical loading can be utilized to define the conditions of cultivation for functional tissue engineering of cartilage.     

Effect of fiber orientation and strain rate on the nonlinear uniaxial tensile material properties of tendon

Tendons are exposed to complex loading scenarios that can only be quantified by mathematical models, requiring a full knowledge of tendon mechanical properties. This study measured the anisotropic, nonlinear, elastic material properties of tendon. Previous studies have primarily used constant strain-rate tensile tests to determine elastic modulus in the fiber direction. Data for Poisson's ratio aligned with the fiber direction and all material properties transverse to the fiber direction are sparse. Additionally, it is not known whether quasi-static constant strain-rate tests represent equilibrium elastic tissue behavior. Incremental stress-relaxation and constant strain-rate tensile tests were performed on sheep flexor tendon samples aligned with the tendon fiber direction or transverse to the fiber direction to determine the anisotropic properties of toe-region modulus (E0), linear-region modulus (E), and Poisson's ratio (v). Among the modulus values calculated, only fiber-aligned linear-region modulus (E1) was found to be strain-rate dependent. The E1 calculated from the constant strain-rate tests were significantly greater than the value calculated from incremental stress-relaxation testing. Fiber-aligned toe-region modulus (E(1)0 = 10.5 +/- 4.7 MPa) and linear-region modulus (E1 = 34.0 +/- 15.5 MPa) were consistently 2 orders of magnitude greater than transverse moduli (E(2)0 = 0.055 +/- 0.044 MPa, E2 = 0.157 +/- 0.154 MPa). Poisson's ratio values were not found to be rate-dependent in either the fiber-aligned (v12 = 2.98 +/- 2.59, n = 24) or transverse (v21 = 0.488 +/- 0.653, n = 22) directions, and average Poisson's ratio values in the fiber-aligned direction were six times greater than in the transverse direction. The lack of strain-rate dependence of transverse properties demonstrates that slow constant strain-rate tests represent elastic properties in the transverse direction. However, the strain-rate dependence demonstrated by the fiber-aligned linear-region modulus suggests that incremental stress-relaxation tests are necessary to determine the equilibrium elastic properties of tendon, and may be more appropriate for determining the properties to be used in elastic mathematical models.     

Analysis of the passive mechanical properties of rat carotid arteries

The passive mechanical properties of rat carotid arteries were studied in vitro. Using a tensile testing machine and a piston pump, intact segments of carotid arteries were subjected to large deformations both in the longitudinal and circumferential directions. Internal pressure, external diameter, length and longitudinal force were measured during the experiment and compared with the in vivo dimensions of the segments prior to excision. The anisotropic mechanical properties of the vessel wall material were analyzed using incremental elastic moduli and incremental Poisson's ratios. The results suggest that there is a characteristic deformation pattern common to all vessels investigated which is highly correlated with the conditions of loading that occur in vivo. That is, under average physiological deformation of the vessel, the longitudinal force is nearly independent of internal pressure. In this range of loading the circumferential incremental elastic modulus is nearly independent of longitudinal strain. However, the longitudinal and radial incremental elastic moduli vary significantly with deformation in this direction. The values of the moduli in all three directions increase with raising internal pressure. The weak coupling between circumferential and longitudinal direction in the wall material of carotid arteries is shown by the small value of the corresponding incremental Poisson's ratios.     

Influence of retinoic acid on the ultrastructure and hyaluronic acid synthesis of adult human epidermis in whole skin organ culture

Normal human skin was maintained in organ culture under chemically defined conditions. All-trans retinoic acid was added to the culture medium at the final concentration of 5 mumol/l. After 5 days in culture samples were either harvested for electron microscopy or labeled with 3H-glucosamine for 24 h. After labeling, epidermis was separated from dermis and both tissue compartments were analyzed for the content of 3H-labeled glycosaminoglycans (GAGs) using CPC-precipitation and thin layer chromatography after enzymatic degradation into specific disaccharides. Retinoic acid caused a marked change in the epidermal tissue architecture. The epidermal cells were flattened and contained fewer desmosomes and tonofilaments than control explants. Retinoic acid induced accumulation of fine granular material in the intercellular spaces in the upper, and less dense, flocculent material in the lower epidermis. The analysis of 3H-glycosaminoglycans showed that in the epidermis retinoic acid elevated the amount of labeled hyaluronate by 70%, whereas sulfated GAGs were not significantly increased. In dermis the incorporation of 3H-glucosamine into neither hyaluronate nor sulfated GAGs was stimulated by the retinoic acid. It is concluded that retinoic acid significantly modifies the differentiation of normal adult human epidermis by decreasing cytoskeleton components and by inducing the synthesis of new intercellular material, at least a part of which is hyaluronic acid. As a consequence, the cohesion between the epidermal cells was apparently weakened.     

Nonuniform swelling-induced residual strains in articular cartilage

Swelling effects in cartilage originate from an interstitial osmotic pressure generated by the presence of negatively charged proteoglycans in the tissue. This swelling pressure gives rise to a non-zero residual strain in the cartilage solid matrix in the absence of externally applied loads. Previous studies have quantified swelling effects in cartilage as volumetric or dimensional change of excised samples in varying osmotically active solutions. This study presents a new optical technique for measuring two-dimensional swelling-induced residual strain fields in planar samples of articular cartilage attached to the bone (i.e., in situ). Osmotic loading was applied to canine cartilage bone samples by equilibration in external baths of varying NaCl concentration. Non-zero swelling-induced strains were measured in physiological saline, giving evidence of the existence of residual strains in articular cartilage. Only one component of planar strain (i.e., in thickness direction) was found to be non-zero. This strain was found to be highly non-uniform in the thickness direction, with evidence of compressive strain in the deep zone of cartilage and tensile strain in the middle and surface zones. The obtained results can be used to characterize the material properties of the articular cartilage solid matrix, with estimated values of 26 M Pa for the tensile modulus for middle zone cartilage. The method provides the basis to obtain material properties of the cartilage solid matrix from a simple, free-swelling test and may be useful for quantifying changes in cartilage properties with injury, degeneration and repair.     

Computational modeling of chemical reactions and interstitial growth and remodeling involving charged solutes and solid-bound molecules

Mechanobiological processes are rooted in mechanics and chemistry, and such processes may be modeled in a framework that couples their governing equations starting from fundamental principles. In many biological applications, the reactants and products of chemical reactions may be electrically charged, and these charge effects may produce driving forces and constraints that significantly influence outcomes. In this study, a novel formulation and computational implementation are presented for modeling chemical reactions in biological tissues that involve charged solutes and solid-bound molecules within a deformable porous hydrated solid matrix, coupling mechanics with chemistry while accounting for electric charges. The deposition or removal of solid-bound molecules contributes to the growth and remodeling of the solid matrix; in particular, volumetric growth may be driven by Donnan osmotic swelling, resulting from charged molecular species fixed to the solid matrix. This formulation incorporates the state of strain as a state variable in the production rate of chemical reactions, explicitly tying chemistry with mechanics for the purpose of modeling mechanobiology. To achieve these objectives, this treatment identifies the specific theoretical and computational challenges faced in modeling complex systems of interacting neutral and charged constituents while accommodating any number of simultaneous reactions where reactants and products may be modeled explicitly or implicitly. Several finite element verification problems are shown to agree with closed-form analytical solutions. An illustrative tissue engineering analysis demonstrates tissue growth and swelling resulting from the deposition of chondroitin sulfate, a charged solid-bound molecular species. This implementation is released in the open-source program FEBio (www.febio.org). The availability of this framework may be particularly beneficial to optimizing tissue engineering culture systems by examining the influence of nutrient availability on the evolution of inhomogeneous tissue composition and mechanical properties, the evolution of construct dimensions with growth, the influence of solute and solid matrix electric charge on the transport of cytokines, the influence of binding kinetics on transport, the influence of loading on binding kinetics, and the differential growth response to dynamically loaded versus free-swelling culture conditions.     

Static and dynamic compression regulate cartilage metabolism of PRoteoGlycan 4 (PRG4)

The boundary lubrication function of articular cartilage is mediated in part by molecules at the articular surface and in synovial fluid, encoded by Prg4. The objective of this study was to determine whether static and dynamic compression regulate PRG4 biosynthesis by cartilage explants. Articular cartilage disks were harvested to include the articular surface from immature bovines. Some disks were subjected to 24 h (day 1) of loading, followed by 72 h (days 2-4) of free-swelling culture to assess chondrocyte responses following unloading. Loading consisted of 6 or 100 kPa of static compression, with or without superimposed dynamic compression (10 or 300 kPa peak amplitude, 0.01 Hz). Other disks were cultured free-swelling as controls. PRG4 secretion into culture medium was inhibited by all compression protocols during day 1. Following unloading, cartilage previously subjected to dynamic compression to 300 kPa exhibited a rebound effect, secreting more PRG4 than did controls, while cartilage previously subjected to 100 kPa static loading secreted less PRG4. Immunohistochemistry revealed that all compression protocols also affected the number of cells expressing PRG4. The paradigm that mechanical stimuli regulate biosynthesis in cartilage appears operative not only for load bearing matrix constituents, but also for PRG4 molecules mediating lubrication.     

Anisotropic and inhomogeneous tensile behavior of the human anulus fibrosus: experimental measurement and material model predictions

The anulus fibrosus (AF) of the intervertebral disc exhibits spatial variations in structure and composition that give rise to both anisotropy and inhomogeneity in its material behaviors in tension. In this study, the tensile moduli and Poisson's ratios were measured in samples of human AF along circumferential, axial, and radial directions at inner and outer sites. There was evidence of significant inhomogeneity in the linear-region circumferential tensile modulus (17.4+/-14.3 MPa versus 5.6+/-4.7 MPa, outer versus inner sites) and the Poisson's ratio v21 (0.67+/-0.22 versus 1.6+/-0.7, outer versus inner), but not in the axial modulus (0.8+/-0.9 MPa) or the Poisson's ratios V12 (1.8+/-1.4) or v13 (0.6+/-0.7). These properties were implemented in a linear an isotropic material model of the AF to determine a complete set of model properties and to predict material behaviors for the AF under idealized kinematic states. These predictions demonstrate that interactions between fiber populations in the multilamellae AF significantly contribute to the material behavior, suggesting that a model for th     

Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

The success of stem cell-based cartilage repair requires that the regenerate tissue reach a stable state. To investigate the long-term stability of tissue engineered cartilage constructs, we assessed the development of compressive mechanical properties of chondrocyte and mesenchymal stem cell (MSC)-laden three dimensional agarose constructs cultured in a well defined chondrogenic in vitro environment through 112 days. Consistent with previous reports, in the presence of TGF-β, chondrocytes outperformed MSCs through day 56, under both free swelling and dynamic culture conditions, with MSC-laden constructs reaching a plateau in mechanical properties between days 28 and 56. Extending cultures through day 112 revealed that MSCs did not simply experience a lag in chondrogenesis, but rather that construct mechanical properties never matched those of chondrocyte-laden constructs. After 56 days, MSC-laden constructs underwent a marked reversal in their growth trajectory, with significant declines in glycosaminoglycan content and mechanical properties. Quantification of viability showed marked differences in cell health between chondrocytes and MSCs throughout the culture period, with MSC-laden construct cell viability falling to very low levels at these extended time points. These results were not dependent on the material environment, as similar findings were observed in a photocrosslinkable hyaluronic acid (HA) hydrogel system that is highly supportive of MSC chondrogenesis. These data suggest that, even within a controlled in vitro environment that is conducive to chondrogenesis, there may be an innate instability in the MSC phenotype that is independent of scaffold composition, and may ultimately limit their application in functional cartilage repair.     

Spatial distribution and orientation of dermatan sulfate in human medial collateral ligament

The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.     

Serum‐free,chemically defined medium with TGF‐β3 enhances functional properties of nucleus pulposus cell‐laden carboxymethylcellulose hydrogel constructs

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum‐containing medium formulation was compared to a chemically defined, serum‐free medium to determine the effect on matrix elaboration and functional properties of NP cell‐laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor‐beta 3 (TGF‐β₃). Glycosaminoglycan (GAG) content increased in both TGF‐β₃‐treated groups and was highest for treated, serum‐free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum‐containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum‐free constructs. Type II collagen content was greater in both serum‐free groups and highest in treated, serum‐free constructs. The equilibrium Young's modulus was highest in serum‐free samples supplemented with TGF‐β₃ (18.54 ± 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 ± 0.46 vs. 19.94 ± 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum‐free, TGF‐β₃ supplemented medium, indicating the importance of medium formulation in NP construct development. Biotechnol. Bioeng. 2010; 105: 384–395. © 2009 Wiley Periodicals, Inc.     

Mechanical properties of the gastrocnemius aponeurosis in wild turkeys

In many muscles, the tendinous structures include both an extramuscular free tendon as well as a sheet-like aponeurosis. In both free tendons and aponeuroses the collagen fascicles are oriented primarily longitudinally, along the muscle's line of action. It is generally assumed that this axis represents the direction of loading for these structures. This assumption is well founded for free tendons, but aponeuroses undergo a more complex loading regime. Unlike free tendons, aponeuroses surround a substantial portion of the muscle belly and are therefore loaded both parallel (longitudinal) and perpendicular (transverse) to a muscle's line of action when contracting muscles bulge to maintain a constant volume. Given this biaxial loading pattern, it is critical to understand the mechanical properties of aponeuroses in both the longitudinal and transverse directions. In this study, we use uniaxial testing of isolated tissue samples from the aponeurosis of the lateral gastrocnemius of wild turkeys to determine mechanical properties of samples loaded longitudinally (along the muscle's line of action) and transversely (orthogonal to the line of action). We find that the aponeurosis has a significantly higher Young's modulus in the longitudinal than in the transverse direction. Our results also show that aponeuroses can behave as efficient springs in both the longitudinal and transverse directions, losing little energy to hysteresis. We also test the failure properties of aponeuroses to quantify the likely safety factor with which these structures operate during muscular force production. These results provide an essential foundation for understanding the mechanical function of aponeuroses as biaxially loaded biological springs.