Tyler Novak

Cell encapsulation in a magnetically aligned collagen–GAG copolymer microenvironment

Engineered tissue microenvironments impart specialized cues that drive distinct cellular phenotypes and function. Microenvironments with defined properties, such as mechanical properties and fibril alignment, can elicit specific cellular responses that emulate those observed in vivo. Collagen- and glycosaminoglycan (GAG)-based tissue matrices have been popularized due to their biological ubiquity in a broad range of tissues and the ability to tune structure and mechanical properties through a variety of processes. Here, we investigate the combined effects of static magnetic fields, and GAG and cell encapsulation, on the structure (e.g. collagen fibril orientation) and material properties of collagen matrices. We found that magnetic fields align the collagen-GAG matrix, alter equilibrium mechanical properties and provide a method for encapsulating cells within a three-dimensional aligned matrix. Cells are encapsulated prior to polymerization, allowing for controlled cell density and eliminating the need for cell seeding. Increased relative GAG concentrations reduced the ability to magnetically align collagen fibrils, in part through a mechanism involving increased viscosity and polymerization time of the collagen-GAG solution. This work provides a functional design space for the development of pure collagen and hybrid collagen-GAG matrices in the presence of magnetic fields. Additionally, this work shows that magnetic fields are effective for the fabrication of collagen constructs with controlled fibril orientation, and can be coupled with GAG incorporation to modulate mechanical properties and the response of embedded cells.

Optical clearing in collagen- and proteoglycan-rich osteochondral tissues.

OBJECTIVE Recent developments in optical clearing and microscopy technology have enabled the imaging of intact tissues at the millimeter scale to characterize cells via fluorescence labeling. While these techniques have facilitated the three-dimensional (3D) cellular characterization within brain and heart, study of dense connective tissues of the musculoskeletal system have been largely unexplored. Here, we quantify how optical clearing impacted the cell and tissue morphology of collagen-, proteoglycan-, and mineral-rich cartilage and bone from the articulating knee joint. METHODS Water-based fructose solutions were used for optical clearing of bovine osteochondral tissues, followed by imaging with transmission and confocal microscopy. To confirm preservation of tissue structure during the clearing process, samples were mechanically tested in unconfined compression and visualized by cryo-SEM. RESULTS Optical clearing enhanced light transmission through cartilage, but not subchondral bone regions. Fluorescent staining and immunolabeling was preserved through sample preparations, enabling imaging to cartilage depths five times deeper than previously reported, limited only by the working distance of the microscope objective. Chondrocyte volume remained unchanged in response to, and upon the reversal, of clearing. Equilibrium modulus increased in cleared samples, and was attributed to exchange of interstitial fluid with the more viscous fructose solution, but returned to control levels upon unclearing. In addition, cryo-SEM-based analysis of cartilage showed no ultrastructural changes. CONCLUSION We anticipate large-scale microscopy of diverse connective tissues will enable the study of intact, 3D interfaces (e.g., osteochondral) and cellular connectivity as a function of development, disease, and regeneration, which have been previously hindered by specimen opacity.

Prestress as an optimal biomechanical parameter for needle penetration

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.

Direct Noninvasive Measurement and Numerical Modeling of Depth-Dependent Strains in Layered Agarose Constructs

Biomechanical factors play an important role in the growth, regulation, and maintenance of engineered biomaterials and tissues. While physical factors (e.g. applied mechanical strain) can accelerate regeneration, and knowledge of tissue properties often guide the design of custom materials with tailored functionality, the distribution of mechanical quantities (e.g. strain) throughout native and repair tissues is largely unknown. Here, we directly quantify distributions of strain using noninvasive magnetic resonance imaging (MRI) throughout layered agarose constructs, a model system for articular cartilage regeneration. Bulk mechanical testing, giving both instantaneous and equilibrium moduli, was incapable of differentiating between the layered constructs with defined amounts of 2% and 4% agarose. In contrast, MRI revealed complex distributions of strain, with strain transfer to softer (2%) agarose regions, resulting in amplified magnitudes. Comparative studies using finite element simulations and mixture (biphasic) theory confirmed strain distributions in the layered agarose. The results indicate that strain transfer to soft regions is possible in vivo as the biomaterial and tissue changes during regeneration and maturity. It is also possible to modulate locally the strain field that is applied to construct-embedded cells (e.g. chondrocytes) using stratified agarose constructs.

Dissociated and Reconstituted Cartilage Microparticles in Densified Collagen Induce Local hMSC Differentiation

Decellularized cartilage microparticles, and all associated native signals, are delivered to hMSC populations in a dense, type I collagen matrix. Hybrid usage of native tissue signals and the engineering control of collagen matrices show the ability to induce local infiltration and differentiation of hMSCs. Additionally, the solid cartilage microparticles inhibit bulk cell-mediated contraction of the composite.

Temperature and concentration dependent fibrillogenesis for improved magnetic alignment of collagen gels

Collagen fibrils form the structural basis for a broad range of complex biological tissues and materials. Collagen serves as an ideal natural polymer, formed as gels or matrices, for engineering solutions aimed at the regeneration of tissues following damage or disease. Recapitulation of native tissue hierarchical structure involves the careful consideration of the fibril-microstructure of the target tissue extracellular matrix and the choice of fibrillogenesis conditions that favor spatially-dependent fibril alignment. While magnetic fields non-destructively influence collagen fibrillogenesis and alignment, previous methods have demonstrated only limited control, especially when preparing large volume tissue constructs suitable for implantation. In this study, we investigate the use of temperature-controlled fibrillogenesis over a range of applicable collagen concentrations for improved magnetic alignment of polymerizable collagen-fibril gels. Magnetically aligned collagen gels show that bulk and microscale fibril alignment depend on both polymerization temperature and collagen concentration. The degree of fibril alignment at the microscale increased with decreasing polymerization temperature and collagen concentration. Further, computational simulations suggest that lower polymerization temperatures affect the internal gel temperature distribution and convective fluid velocity, potentially facilitating greater fibril alignment. This work demonstrates improvements in observed fibril anisotropy compared to previous work using similar magnetic field strengths, suggesting that temperature and collagen concentration may be utilized to achieve desired fibril alignment and structural properties. Improved control of collagen-based gel structure may better emulate native tissue structural (alignment) and physical properties, with enhanced potential for repair success in vivo.

Densification of Type I Collagen Matrices as a Model for Cardiac Fibrosis

Cardiac fibrosis is a disease state characterized by excessive collagenous matrix accumulation within the myocardium that can lead to ventricular dilation and systolic failure. Current treatment options are severely lacking due in part to the poor understanding of the complexity of molecular pathways involved in cardiac fibrosis. To close this gap, in vitro model systems that recapitulate the defining features of the fibrotic cellular environment are in need. Type I collagen, a major cardiac extracellular matrix protein and the defining component of fibrotic depositions, is an attractive choice for a fibrosis model, but demonstrates poor mechanical strength due to solubility limits. However, plastic compression of collagen matrices is shown to significantly increase its mechanical properties. Here, confined compression of oligomeric, type I collagen matrices is utilized to resemble defining hallmarks seen in fibrotic tissue such as increased collagen content, fibril thickness, and bulk compressive modulus. Cardiomyocytes seeded on compressed matrices show a strong beating abrogation as observed in cardiac fibrosis. Gene expression analysis of selected fibrosis markers indicates fibrotic activation and cardiomyocyte maturation with regard to the existing literature. With these results, a promising first step toward a facile heart-on-chip model is presented to study cardiac fibrosis.

Temperature Control During Fibrillogenesis Allows for Improved Magnetic Alignment of Collagen

Osteoarthritis (OA), commonly known as ‘wear and tear’ in human joints, affects over 27 million people in the United States [1]. There is currently no encompassing solution for the regeneration of damaged articular cartilage. One potential solution involves the close emulation of the native structure of articular cartilage, with special consideration given to maintaining the distinct organized zonal ultrastructure, characterized by both random and highly aligned zones of collagen fibrils, in order to preserve mechanical and cell signaling properties of the extracellular matrix [2]. Techniques such as electrospinning achieve high degrees of alignment, but do so at the cost of denaturing the collagen molecule [3] that may lead to inferior cell recognition and mechanical strength.Copyright

Augmented and Magnetically-Aligned Type I Collagen-GAG Scaffolds for Cartilage Tissue Engineering

Osteoarthritis (OA) affects over 27 million Americans, causing an annual economic burden of over

Magnetic Alignment of Type I Collagen as a Method for Altering Tensile Mechanical Properties

300 million [1]. Left untreated, local cartilage defects promote cartilage degeneration and serve as a target for clinical and research based interventions [2]. While current treatments have limited success and result in recurring symptoms [3], tissue engineering solutions are promising for cartilage repair.© 2013 ASME