Macro- to Microscale Strain Transfer in Fibrous Tissues is Heterogeneous and Tissue-Specific

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35–70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15–25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.     

Transfer of macroscale tissue strain to microscale cell regions in the deformed meniscus

Cells within fibrocartilaginous tissues, including chondrocytes and fibroblasts of the meniscus, ligament, and tendon, regulate cell biosynthesis in response to local mechanical stimuli. The processes by which an applied mechanical load is transferred through the extracellular matrix to the environment of a cell are not fully understood. To better understand the role of mechanics in controlling cell phenotype and biosynthetic activity, this study was conducted to measure strain at different length scales in tissue of the fibrocartilaginous meniscus of the knee joint, and to define a quantitative parameter that describes the strain transferred from the far-field tissue to a microenvironment surrounding a cell. Experiments were performed to apply a controlled uniaxial tensile deformation to explants of porcine meniscus containing live cells. Using texture correlation analyses of confocal microscopy images, two-dimensional Lagrangian and principal strains were measured at length scales representative of the tissue (macroscale) and microenvironment in the region of a cell (microscale) to yield a strain transfer ratio as a measure of median microscale to macroscale strain. The data demonstrate that principal strains at the microscale are coupled to and amplified from macroscale principal strains for a majority of cell microenvironments located across diverse microstructural regions, with average strain transfer ratios of 1.6 and 2.9 for the maximum and minimum principal strains, respectively. Lagrangian strain components calculated along the experimental axes of applied deformations exhibited considerable spatial heterogeneity and intersample variability, and suggest the existence of both strain amplification and attenuation. This feature is consistent with an in-plane rotation of the principal strain axes relative to the experimental axes at the microscale that may result from fiber sliding, fiber twisting, and fiber-matrix interactions that are believed to be important for regulating deformation in other fibrocartilaginous tissues. The findings for consistent amplification of macroscale to microscale principal strains suggest a coordinated pattern of strain transfer from applied deformation to the microscale environment of a cell that is largely independent of these microstructural features in the fibrocartilaginous meniscus.     

Strain transfer through the aortic valve

The complex structural organization of the aortic valve (AV) extracellular matrix (ECM) enables large and highly nonlinear tissue level deformations. The collagen and elastin (elastic) fibers within the ECM form an interconnected fibrous network (FN) and are known to be the main load-bearing elements of the AV matrix. The role of the FN in enabling deformation has been investigated and documented. However, there is little data on the correlation between tissue level and FN-level strains. Investigating this correlation will help establish the mode of strain transfer (affine or nonaffine) through the AV tissue as a key feature in microstructural modeling and will also help characterize the local FN deformation across the AV sample in response to applied tissue level strains. In this study, the correlation between applied strains at tissue level, macrostrains across the tissue surface, and local FN strains were investigated. Results showed that the FN strain distribution across AV samples was inhomogeneous and nonuniform, as well as anisotropic. There was no direct transfer of the deformation applied at tissue level to the fibrous network. Loading modes induced in the FN are different than those applied at the tissue as a result of different local strains in the valve layers. This nonuniformity of local strains induced internal shearing within the FN of the AV, possibly exposing the aortic valve interstitial cells (AVICs) to shear strains and stresses.     

Finite Element Modeling Predictions of Region-specific Cell-matrix Mechanics in the Meniscus

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of ~0.07 for the rounded inner cells, as compared to levels of 0.02–0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.     

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.     

In situ cell–matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (～2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Modeling interlamellar interactions in angle-ply biologic laminates for annulus fibrosus tissue engineering

Mechanical function of the annulus fibrosus of the intervertebral disc is dictated by the composition and microstructure of its highly ordered extracellular matrix. Recent work on engineered angle-ply laminates formed from mesenchymal stem cell (MSC)-seeded nanofibrous scaffolds indicates that the organization of collagen fibers into planes of alternating alignment may play an important role in annulus fibrosus tissue function. Specifically, these engineered tissues can resist tensile deformation through shearing of the interlamellar matrix as layers of collagen differentially reorient under load. In the present work, a hyperelastic constitutive model was developed to describe the role of interlamellar shearing in reinforcing the tensile response of biologic laminates, and was applied to experimental results from engineered annulus constructs formed from MSC-seeded nanofibrous scaffolds. By applying the constitutive model to uniaxial tensile stress–strain data for bilayers with three different fiber orientations, material parameters were generated that characterize the contributions of extrafibrillar matrix, fibers, and interlamellar shearing interactions. By 10 weeks of in vitro culture, interlamellar shearing accounted for nearly 50% of the total stress associated with uniaxial extension in the anatomic range of ply angle. The model successfully captured changes in function with extracellular matrix deposition through variations in the magnitude of model parameters with culture duration. This work illustrates the value of engineered tissues as tools to further our understanding of structure–function relations in native tissues and as a test-bed for the development of constitutive models to describe them.     

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

Electrospun fibrous mats with high porosity as potential scaffolds for skin tissue engineering

Diffusional limitations of mass transport have adverse effects on engineering tissues that normally have high vascularity and cellularity. The current electrospinning method is not always successful to create micropores to encourage cell infiltration within the scaffold, especially when relatively large-sized pores are required. In this study, a slow rotating frame cylinder was developed as the collector to extend the pore size and increase the porosity of electrospun fibrous scaffolds. Fibrous mats with porosity as high as 92.4% and average pore size of 132.7 microm were obtained. Human dermal fibroblasts (HDFs) were seeded onto these mats, which were fixed on a cell-culture ring to prevent the shrinkage and contraction during the incubation. The viability test indicated that significantly more HDFs were generated on highly porous fibrous mats. Toluidine blue staining showed that the highly porous scaffold provided mechanical support for cells to maintain uniform distribution. The cross-section observations indicated that cells migrated and infiltrated more than 100 microm inside highly porous fibrous mats after 5 d incubation. The immunohistochemistry analysis demonstrated that cells began secreting collagen, which is the main constituent of extracellular matrix. It is supposed that highly porous electrospun fibrous scaffolds could be constructed by this elaboration and may be used for skin tissue engineering.     

Structural properties of scaffolds: Crucial parameters towards stem cells differentiation

Tissue engineering is a multidisciplinary field that applies the principles of engineering and life-sciences for regeneration of damaged tissues. Stem cells have attracted much interest in tissue engineering as a cell source due to their ability to proliferate in an undifferentiated state for prolonged time and capability of differentiating to different cell types after induction. Scaffolds play an important role in tissue engineering as a substrate that can mimic the native extracellular matrix and the properties of scaffolds have been shown to affect the cell behavior such as the cell attachment, proliferation and differentiation. Here, we focus on the recent reports that investigated the various aspects of scaffolds including the materials used for scaffold fabrication, surface modification of scaffolds, topography and mechanical properties of scaffolds towards stem cells differentiation effect. We will present a more detailed overview on the effect of mechanical properties of scaffolds on stem cells fate.     

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.     

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.     

An overview of the different approaches used in the development of meniscal tissue engineering

The menisci are important fibrocartilaginous structures which give lubrication, shock absorption, nutrition and stabilisation to the knee joint, and also help transfer load. The meniscus' extracellular matrix possesses a complex architecture which is not uniform throughout the tissue. The inner third of the meniscus is composed of hyaline cartilage and the outer meniscus is composed of fibrocartilage. In a mature meniscus only the outer 10-25% is vascularised. There are various types of pathology associated with the meniscus. Previously, surgical techniques used to be considered as conventional treatment for meniscal lesions. However lesions in the avascular regions of the meniscus would rarely heal appropriately. It has been found that total menisectomies in patients may increase their chance of suffering from osteoarthritis in the future. Meniscal tissue engineering has been developed in an attempt to help improve the healing potential of avascular meniscal regions. Many different concepts and approaches have been tried and tested, such as the application of natural and synthetic scaffolds, mesenchymal stem cells, growth factors, fibrin glue and more. The objective of this review is to summarise the different approaches that have been used in the development of meniscal tissue engineering. The focus of this review is to evaluate the strengths and weaknesses of the studies that have been carried out, and from there determine what we have learnt from them in order to further the development in meniscal tissue engineering.     

Simulation of planar soft tissues using a structural constitutive model: Finite element implementation and validation

Computational implementation of physical and physiologically realistic constitutive models is critical for numerical simulation of soft biological tissues in a variety of biomedical applications. It is well established that the highly nonlinear and anisotropic mechanical behaviors of soft tissues are an emergent behavior of the underlying tissue microstructure. In the present study, we have implemented a structural constitutive model into a finite element framework specialized for membrane tissues. We noted that starting with a single element subjected to uniaxial tension, the non-fibrous tissue matrix must be present to prevent unrealistic tissue deformations. Flexural simulations were used to set the non-fibrous matrix modulus because fibers have little effects on tissue deformation under three-point bending. Multiple deformation modes were simulated, including strip biaxial, planar biaxial with two attachment methods, and membrane inflation. Detailed comparisons with experimental data were undertaken to insure faithful simulations of both the macro-level stress–strain insights into adaptations of the fiber architecture under stress, such as fiber reorientation and fiber recruitment. Results indicated a high degree of fidelity and demonstrated interesting microstructural adaptions to stress and the important role of the underlying tissue matrix. Moreover, we apparently resolve a discrepancy in our 1997 study (Billiar and Sacks, 1997. J. Biomech. 30 (7), 753–756) where we observed that under strip biaxial stretch the simulated fiber splay responses were not in good agreement with the experimental results, suggesting non-affine deformations may have occurred. However, by correctly accounting for the isotropic phase of the measured fiber splay, good agreement was obtained. While not the final word, these simulations suggest that affine fiber kinematics for planar collagenous tissues is a reasonable assumption at the macro level. Simulation tools such as these are imperative in the design and simulation of native and engineered tissues.     

Engineering porous scaffolds using gas-based techniques

Scaffolds are used in tissue engineering as a matrix for the seeding and attachment of human cells. The creation of porosity in three-dimensional (3D) structures of scaffolds plays a critical role in cell proliferation, migration, and differentiation into the specific tissue while secreting extracellular matrix components. These pores are used to transfer nutrients and oxygen and remove wastes produced from the cells. The lack of oxygen and nutrient supply impedes the cell migration more than 500μm from the surface. The physical properties of scaffolds such as porosity and pore interconnectivity can improve mass transfer and have a great impact on the cell adhesion and penetration into the scaffolds to form a new tissue. Various techniques such as electrospinning, freeze-drying, and solvent casting/salt leaching have been used to create porosity in scaffolds. The major issues in these methods include lack of 3D structure, control on pore size, and pore interconnectivity. In this review, we provide a brief overview of gas-based techniques that have been developed for creating porosity in scaffolds.     

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.     

In vivo tissue engineering of musculoskeletal tissues

Tissue engineering of musculoskeletal tissues often involves the in vitro manipulation and culture of progenitor cells, growth factors and biomaterial scaffolds. Though in vitro tissue engineering has greatly increased our understanding of cellular behavior and cell-material interactions, this methodology is often unable to recreate tissue with the hierarchical organization and vascularization found within native tissues. Accordingly, investigators have focused on alternative in vivo tissue engineering strategies, whereby the traditional triad (cells, growth factors, scaffolds) or a combination thereof are directly implanted at the damaged tissue site or within ectopic sites capable of supporting neo-tissue formation. In vivo tissue engineering may offer a preferential route for regeneration of musculoskeletal and other tissues with distinct advantages over in vitro methods based on the specific location of endogenous cultivation, recruitment of autologous cells, and patient-specific regenerated tissues.