Biao Han

Mechanically and chemically tunable cell culture system for studying the myofibroblast phenotype.

Cell culture systems for studying the combined effects of matrix proteins and mechanical forces on the behavior of soft tissue cells have not been well developed. Here, we describe a new biomimetic cell culture system that allows for the study of mixtures of matrix proteins while controlling mechanical stiffness in a range that is physiological for soft tissues. This system consists of layer-by-layer (LbL)-assembled films of native matrix proteins atop mechanically tunable soft supports. We used hepatic stellate cells, which differentiate to myofibroblasts in liver fibrosis, for proof-of-concept studies. By culturing cells on collagen and lumican LbL-modified hydrogels, we demonstrate that this system is noncytotoxic and offers a valid control substrate, that the hydrogel determines the overall system mechanics, and that the addition of lumican to collagen influences the stellate cell phenotype. LbL-modified hydrogels offer the potential to study the influence of complex environmental factors on soft-tissue cells in culture.

Micromechanical anisotropy and heterogeneity of the meniscus extracellular matrix

To understand how the complex biomechanical functions of the meniscus are endowed by the nanostructure of its extracellular matrix (ECM), we studied the anisotropy and heterogeneity in the micromechanical properties of the meniscus ECM. We used atomic force microscopy (AFM) to quantify the time-dependent mechanical properties of juvenile bovine meniscus at deformation length scales corresponding to the diameters of collagen fibrils. At this scale, anisotropy in the elastic modulus of the circumferential fibers, the major ECM structural unit, can be attributed to differences in fibril deformation modes: uncrimping when normal to the fiber axis, and laterally constrained compression when parallel to the fiber axis. Heterogeneity among different structural units is mainly associated with their variations in microscale fiber orientation, while heterogeneity across anatomical zones is due to alterations in collagen fibril diameter and alignment at the nanoscale. Unlike the elastic modulus, the time-dependent properties are more homogeneous and isotropic throughout the ECM. These results enable a detailed understanding of the meniscus structure-mechanics at the nanoscale, and can serve as a benchmark for understanding meniscus biomechanical functions, documenting disease progression and designing tissue repair strategies. STATEMENT OF SIGNIFICANCE Meniscal damage is a common cause of joint injury, which can lead to the development of post-traumatic osteoarthritis among young adults. Restoration of meniscus function requires repairing its highly heterogeneous and complex extracellular matrix. Employing AFM, this study quantifies the anisotropic and heterogeneous features of the meniscus ECM structure and mechanics. The micromechanical properties are interpreted within the context of the collagen fibril nanostructure and its variation with tissue anatomical locations. These results provide a fundamental structure-mechanics knowledge benchmark, against which, repair and regeneration strategies can be developed and evaluated with respect to the specialized structural and functional complexity of the native tissue.

Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306±84kPa, mean±95% CI) than those of the superior (85±23kPa) and inferior (45±12kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest. Using SEM, this study also found that the surfaces of disc are composed of anteroposteriorly oriented collagen fibers, which are sporadically covered by thinner random fibrils. Such fibrous nature results in both an F-D3/2 indentation response, which is a typical Hertzian response for soft continuum tissue under a spherical tip, and a linear F-D response, which is typical for fibrous tissues, further signifying the high degree of tissue heterogeneity. In comparison, the surface of condyle cartilage is dominated by thinner, randomly oriented collagen fibrils, leading to Hertzian-dominated indentation responses. As the first biomechanical study of murine TMJ, this work will provide a basis for future investigations of TMJ tissue development and osteoarthritis in various murine TMJ models.

Tendon healing affects the multiscale mechanical, structural and compositional response of tendon to quasi-static tensile loading

Tendon experiences a variety of multiscale changes to its extracellular matrix during mechanical loading at the fascicle, fibre and fibril levels. For example, tensile loading of tendon increases its stiffness, with organization of collagen fibres, and increases cell strain in the direction of loading. Although applied macroscale strains correlate to cell and nuclear strains in uninjured tendon, the multiscale response during tendon healing remains unknown and may affect cell mechanosensing and response. Therefore, this study evaluated multiscale structure–function mechanisms in response to quasi-static tensile loading in uninjured and healing tendons. We found that tendon healing affected the macroscale mechanical and structural response to mechanical loading, evidenced by decreases in strain stiffening and collagen fibre realignment. At the micro- and nanoscales, healing resulted in increased collagen fibre disorganization, nuclear disorganization, decreased change in nuclear aspect ratio with loading, and decreased indentation modulus compared to uninjured tendons. Taken together, this work supports a new concept of nuclear strain transfer attenuation during tendon healing and identifies several multiscale properties that may contribute. Our work also provides benchmarks for the biomechanical microenvironments that tendon cells may experience following cell delivery therapies.

Impacts of maturation on the micromechanics of the meniscus extracellular matrix

To elucidate how maturation impacts the structure and mechanics of meniscus extracellular matrix (ECM) at the length scale of collagen fibrils and fibers, we tested the micromechanical properties of fetal and adult bovine menisci via atomic force microscopy (AFM)-nanoindentation. For circumferential fibers, we detected significant increase in the effective indentation modulus, Eind, with age. Such impact is in agreement with the increase in collagen fibril diameter and alignment during maturation, and is more pronounced in the outer zone, where collagen fibrils are more aligned and packed. Meanwhile, maturation also markedly increases the Eind of radial tie fibers, but not those of intact surface or superficial layer. These results provide new insights into the effect of maturation on the assembly of meniscus ECM, and enable the design of new meniscus repair strategies by modulating local ECM structure and mechanical behaviors.

Non-additive impacts of covalent cross-linking on the viscoelastic nanomechanics of ionic polyelectrolyte complexes

This study elucidates the influences of adding covalent cross-linking on the nanomechanical viscoelasticity of ionically cross-linked polyelectrolyte networks. Using layer-by-layer (LbL) assembled PAH/PAA networks, we report how adding covalent amide cross-links changes the degree of swelling, indentation modulus, and force relaxation behaviors with varying solution conditions such as pH and ionic strength, which modulate the ionic cross-link density and fixed charge density. The addition of covalent cross-linking increases both the modulus and degree of elasticity through providing permanent anchorage to the ionically linked networks. Such addition also strongly increases the relaxation times at net neutral, less swollen states. These results together underscore the synergistic interplay between the covalent and ionic cross-links in the viscoelastic nanomechanics of polyelectrolyte networks, potentially enabling the use of these stimulus-responsive materials in mechano-sensitive biomedical and engineering applications.