Woojin M. Han

Homologous structure–function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials.

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.

Nanoparticle coatings for enhanced capture of flowing cells in microtubes.

Recently, a flow-based selectin-dependent method for the capture and enrichment of specific types of cells (CD34+ hematopoetic stem and progenitor cells and human leukemia HL60) from peripheral blood was demonstrated. However, these devices depend on a monolayer of selectin protein, which has been shown to have a maximum binding efficiency as a function of surface area. A novel surface coating of colloidal silica nanoparticles was designed that alters the surface roughness resulting in increased surface area. The nanoparticles were adhered using either an inorganic titanate resinous coating or an organic polymer of poly-L-lysine. Using Alexa Fluor 647 conjugated P-selectin, an increase in protein adsorption of up to 35% when compared to control was observed. During perfusion experiments using P-selectin-coated microtubes, we observed increased cell capture and greatly decreased rolling velocity at equivalent protein concentration compared to nonparticle control. Atomic force microscopy showed increased surface roughness consistent with the nanoparticle mean diameter, suggesting a monolayer of particles. These results support the coatings potential to improve existing cell capture implantable devices for a variety of therapeutic and scientific uses.

An Injectable Nucleus Pulposus Implant Restores Compressive Range of Motion in the Ovine Disc

Study Design. Investigation of injectable nucleus pulposus (NP) implant. Objective. To assess the ability of a recently developed injectable hydrogel implant to restore nondegenerative disc mechanics through support of NP functional mechanics. Summary of Background Data. Although surgical intervention for low back pain is effective for some patients, treated discs undergo altered biomechanics and adjacent levels are at increased risk for accelerated degeneration. One potential treatment as an alternative to surgery for degenerated disc includes the percutaneous delivery of agents to support NP functional mechanics. The implants are delivered in a minimally invasive fashion, potentially on an outpatient basis, and do not preclude later surgical options. One of the challenges in designing such implants includes the need to match key NP mechanical behavior and mimic the role of native nondegenerate NP in spinal motion. Methods. The oxidized hyaluronic acid gelatin implant material was prepared. In vitro mechanical testing was performed in mature ovine bone-disc-bone units in 3 stages: intact, discectomy, and implantation versus sham. Tested samples were cut axially for qualitative structural observations. Results. Discectomy increased axial range of motion (ROM) significantly compared with intact. Hydrogel implantation reduced ROM 17% (P < 0.05) compared with discectomy and returned ROM to intact levels (ROM intact 0.71 mm, discectomy 0.87 mm, postimplantation 0.72 mm). Although ROM for the hydrogel implant group was statistically unchanged compared with the intact disc, ROM for sham discs, which received a discectomy and no implant, was significantly increased compared with intact. The compression and tension stiffness were decreased with discectomy and remained unchanged for both implant and sham groups as expected because the annulus fibrosus was not repaired. Gross morphology images confirmed no ejection of NP implant. Conclusion. An injectable implant that mimics nondegenerate NP has the potential to return motion segment ROM to normal subsequent to injury.

Effect of orientation and targeted extracellular matrix degradation on the shear mechanical properties of the annulus fibrosus.

The intervertebral disc experiences combinations of compression, torsion, and bending that subject the disc substructures, particularly the annulus fibrosus (AF), to multidirectional loads and deformations. Combined tensile and shear loading is a particularly important loading paradigm, as compressive loads place the AF in circumferential hoop tension, and spine torsion or bending induces AF shear. Yet the anisotropy of AF mechanical properties in shear, as well as important structure-function mechanisms governing this response, are not well-understood. The objective of this study, therefore, was to investigate the effects of tissue orientation and enzymatic degradation of glycosaminoglycan (GAG) and elastin on AF shear mechanical properties. Significant anisotropy was found: the circumferential shear modulus, Gθz, was an order of magnitude greater than the radial shear modulus, Grθ. In the circumferential direction, prestrain significantly increased the shear modulus, suggesting an important role for collagen fiber stretch in shear properties for this orientation. While not significant and highly variable, ChABC treatment to remove GAG increased the circumferential shear modulus compared to PBS control (p=0.15). Together with the established literature for tensile loading of fiber-reinforced GAG-rich tissues, the trends for changes in shear modulus with ChABC treatment reflect complex, structure-function relationships between GAG and collagen that potentially occur over several hierarchical scales. Elastase digestion did not significantly affect shear modulus with respect to PBS control; further contributing to the notion that circumferential shear modulus is dominated by collagen fiber stretch. The results of this study highlight the complexity of the structure-function relationships that govern the mechanical response of the AF in radial and circumferential shear, and provide new and more accurate data for the validation of material models and tissue-engineered disc replacements.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop microengineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, aging, and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue engineered constructs (hetTECs) with microscale non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical, and mechanobiological benchmarks of native tissue. Our tissue engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating, and regenerating fibrous tissues.Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Mechanical properties of the extra-fibrillar matrix of human annulus fibrosus are location and age dependent

The mechanical behavior of the annulus fibrosus (AF) of the intervertebral disc can be modeled as a mixture of fibers, extra‐fibrillar matrix (EFM), ions, and fluid. However, the properties of the EFM have not been measured directly. We measured mechanical properties of the human EFM at several locations, determined the effect of age and degeneration, and evaluated whether changes in EFM properties correspond to AF compositional changes. EFM mechanical properties were measured using a method that combines osmotic loading and confined compression. AF samples were dissected from several locations, and mechanical properties were correlated with age, degeneration, and composition. EFM modulus was found to range between 10 and 50 kPa, increasing nonlinearly with compression magnitude and being highest in the AF outer‐anterior region. EFM properties were not correlated with composition or degeneration. However, the EFM modulus, its relative contribution to tissue modulus, and model parameters were correlated with age. These measurements will result in more accurate predictions of deformations in the intervertebral disc. Additionally, parameters such as permeability and diffusivity used for biotransport analysis of glucose and other solutes depend on EFM deformation. Consequently, the accuracy of biotransport simulations will be greatly improved.

Micro-scale strain transfer in fiber-reinforced native tissues and cell-seeded aligned nanofibrous scaffolds

Mechanical signals are essential in regulating cell functions such as viability, differentiation, proliferation, and extracellular matrix (ECM) production in load-bearing tissues. However, the current understanding of how macroscopic tissue level strain is transferred to cells is confounded by the highly variable strain fields that arise within the ECM of both native and cell-seeded nanofibrous scaffolds. Moreover, it is unclear how these transmission mechanisms relate to native load bearing tissues. The current study investigates how applied macroscopic tensile strain is transferred to the intercellular ECM and cell nuclei in meniscus, tendon, single lamellar AF, and MSC-seeded scaffolds. The mean microscopic Lagrangian and principal strains in the loading direction (E22 and e2) of all native tissues and scaffolds were attenuated from the applied strains by 50% and 30-40% respectively. In aligned scaffold, a significant correlation was observed between the mean nuclear strain and E22 (r2=0.98), where 100% of E22 transferred to nuclei. Less pronounced strain attenuation in scaffolds compared to native tissues is likely due to more homogeneous microstructure and lack of ECM. In addition, the presence of pericellular matrix in native tissues, along with dense ECM, may shield and regulate strain transfer from the ECM to the subcellular space.

Multi-Scale Structural and Tensile Mechanical Response of Annulus Fibrosus to Osmotic Loading

The annulus fibrosus (AF) is a multi-lamellar fibrocartilagenous ring in the intervertebral disc. The variation of biochemical composition from the outer to the inner AF is largely responsible for the heterogeneous mechanical properties. In vitro tissue-level studies require mechanical testing in aqueous buffers to avoid tissue dehydration. The varying glycosaminoglycan (GAG) contents from outer to inner AF suggest that the response to high and low PBS osmolarity may also be different with radial position. Previous studies in tendon and ligament have been conflicting: soaking tendon fascicles in PBS decreased tensile modulus1 and treating ligament in buffer had no effect on modulus.2Copyright

Micro-Scale Strain Transfer in Fiber-Reinforced Native Tissue Is Distinct From Cell-Seeded Aligned Nanofibrous Scaffolds

Mechanical signals influence cell viability, differentiation, proliferation, and extracellular matrix (ECM) production in load-bearing tissues. However, the current understanding of how macroscopic tissue level strain is transferred to cells is confounded by the highly variable strain fields that arise within the ECM of these tissues. In tendon and outer annulus fibrosus (AF), microscale strains in the ECM can be significantly lower than the applied strains.1,2,3 In meniscus, both strain amplification and attenuation were observed at the microscale level.4© 2012 ASME