Publications (87)221.12 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Collagen is used extensively for tissue engineering due to its prevalence in connective tissues and its role in defining tissue biophysical and biological signalling properties. However, traditional collagen-based materials fashioned from atelocollagen and telocollagen have lacked collagen densities, multi-scale organization, mechanical integrity, and proteolytic resistance found within tissues in vivo. Here, highly interconnected low-density matrices of D-banded fibrils were created from collagen oligomers, which exhibit fibrillar as well as suprafibrillar assembly. Confined compression then was applied to controllably reduce the interstitial fluid while maintaining fibril integrity. More specifically, low-density (3.5 mg mL(-1)) oligomer matrices were densified to create collagen-fibril constructs with average concentrations of 12.25 mg mL(-1) and 24.5 mg mL(-1). Control and densified constructs exhibited nearly linear increases in ultimate stress, Young's modulus, and compressive modulus over the ranges of 65 to 213 kPa, 400 to 1.26 MPa, and 20 to 150 kPa, respectively. Densification also increased construct resistance to collagenase degradability. Finally, this process was amenable to creating high-density cellularized tissues; all constructs maintained high cell viability (at least 97%) immediately following compression as well as after 1 day and 7 days of culture. This method, which integrates the suprafibrillar assembly capacity of oligomers and controlled fluid reduction by confined compression, supports the rational and scalable design of a broad range of collagen-fibril materials and cell-encapsulated tissue constructs for tissue engineering applications.
- [Show abstract] [Hide abstract] ABSTRACT: Biological tissues and biomaterials are often defined by unique spatial gradients in physical properties that impart specialized function over hierarchical scales. The structure of these materials forms continuous transitional gradients and discrete local microenvironments between adjacent (or within) tissues, and across matrix–cell boundaries, which is difficult to replicate with common scaffold systems. Here, the matrix densification of collagen leading to gradients in density, mechanical properties, and fibril morphology is studied. High-density regions form via a fluid pore pressure and flow-driven mechanism, with increased relative fibril density (10×), mechanical properties (20×, to 94.40 ± 18.74 kPa), and maximum fibril thickness (1.9×, to >1 μm) compared to low-density regions, while maintaining porosity and fluid/mass transport to support viability of encapsulated cells. Similar to the organization of the articular cartilage zonal structure, it is found that high-density collagen regions induce cell and nuclear alignment of primary chondrocytes. Chondrocyte gene expression is maintained in collagen matrices, and no phenotypic changes are observed as a result of densification. Collagen densification provides a tunable platform for the creation of gradient systems to study complex cell–matrix interactions. These methods are easily generalized to compression and boundary condition modalities useful to mimic a broad range of tissues.
- [Show abstract] [Hide abstract] ABSTRACT: The elastic properties of engineered biomaterials and tissues impact their post-implantation repair potential and structural integrity, and are critical to help regulate cell fate and gene expression. The measurement of properties (e.g., stiffness or shear modulus) can be attained using elastography, which exploits noninvasive imaging modalities to provide functional information of a material indicative of the regeneration state. In this review, we outline the current leading elastography methodologies available to characterize the properties of biomaterials and tissues suitable for repair and mechanobiology research. We describe methods utilizing magnetic resonance, ultrasound, and optical coherent elastography, highlighting their potential for longitudinal monitoring of implanted materials in vivo, in addition to spatiotemporal limits of each method for probing changes in cell-laden constructs. Micro-elastography methods now allow acquisitions at length scales approaching 5–100 μm in two and three dimensions. Many of the methods introduced in this review are therefore capable of longitudinal monitoring in biomaterials and tissues approaching the cellular scale. However, critical factors such as anisotropy, heterogeneity and viscoelasity—inherent in many soft tissues—are often not fully described and therefore require further advancements and future developments.
- [Show abstract] [Hide abstract] ABSTRACT: The in vivo measurement of articular cartilage deformation is essential to understand how mechanical forces distribute throughout the healthy tissue and change over time in the pathologic joint. Displacements or strain may serve as a functional imaging biomarker for healthy, diseased, and repaired tissues, but unfortunately intratissue cartilage deformation in vivo is largely unknown. Here, we directly quantified for the first time deformation patterns through the thickness of tibiofemoral articular cartilage in healthy human volunteers. Magnetic resonance imaging acquisitions were synchronized with physiologically relevant compressive loading and used to visualize and measure regional displacement and strain of tibiofemoral articular cartilage in a sagittal plane. We found that compression (of 1/2 body weight) applied at the foot produced a sliding, rigid-body displacement at the tibiofemoral cartilage interface, that loading generated subject- and gender-specific and regionally complex patterns of intratissue strains, and that dominant cartilage strains (approaching 12%) were in shear. Maximum principle and shear strain measures in the tibia were correlated with body mass index. Our MRI-based approach may accelerate the development of regenerative therapies for diseased or damaged cartilage, which is currently limited by the lack of reliable in vivo methods for noninvasive assessment of functional changes following treatment.
- [Show abstract] [Hide abstract] ABSTRACT: The lubricating proteoglycan, lubricin, facilitates the remarkable low friction and wear properties of articular cartilage in the synovial joints of the body. Lubricin lines the joint surfaces and plays a protective role as a boundary lubricant in sliding contact; decreased expression of lubricin is associated with cartilage degradation and the pathogenesis of osteoarthritis. An unmet need for early osteoarthritis treatment is the development of therapeutic molecules that mimic lubricin function and yet are also resistant to enzymatic degradation common in the damaged joint. Here, we engineered a lubricin mimic (mLub) that is less susceptible to enzymatic degradation and binds to the articular surface to reduce friction. mLub was synthesized using a chondroitin sulfate backbone with type II collagen and hyaluronic acid (HA) binding peptides to promote interaction with the articular surface and synovial fluid constituents. In vitro and in vivo characterization confirmed the binding ability of mLub to isolated type II collagen and HA, and to the cartilage surface. Following trypsin treatment to the cartilage surface, application of mLub, in combination with purified or commercially available hyaluronan, reduced the coefficient of friction, and adhesion, to control levels as assessed over macro-to micro-scales by rheometry and atomic force microscopy. In vivo studies demonstrate an mLub residency time of less than 1 week. Enhanced lubrication by mLub reduces surface friction and adhesion, which may suppress the progression of degradation and cartilage loss in the joint. mLub therefore shows potential for treatment in early osteoarthritis following injury.
- [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study is to evaluate the ability of machine learning to discriminate between magnetic resonance images (MRI) of normal and pathological human articular cartilage obtained under standard clinical conditions. An approach to MRI classification of cartilage degradation is proposed using pattern recognition and multivariable regression in which image features from MRIs of histologically scored human articular cartilage plugs were computed using weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHRM). The WND-CHRM method was first applied to several clinically available MRI scan types to perform binary classification of normal and osteoarthritic osteochondral plugs based on the OARSI histological system. In addition, the image features computed from WND-CHRM were used to develop a multiple linear least-squares regression model for classification and prediction of an OARSI score for each cartilage plug. The binary classification of normal and osteoarthritic plugs yielded results of limited quality with accuracies between 36% and 70%. However, multiple linear least-squares regression successfully predicted OARSI scores and classified plugs with accuracies as high as 86%. The present results improve upon the previously-reported accuracy of classification using average MRI signal intensities and parameter values. MRI features detected by WND-CHRM reflect cartilage degradation status as assessed by OARSI histologic grading. WND-CHRM is therefore of potential use in the clinical detection and grading of osteoarthritis. Published by Elsevier Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: PurposeThis work evaluates the ability of MRIcontrasts to discriminate between normal and pathological human osteochondral plugscharacterized by the OARSI histological system.Methods Normal and osteoarthritic human osteochondralplugswere scored using the OARSI histological system and imaged at 3T using MRI sequences measuring T1, T2, and T2* relaxation times, magnetization transfer, anddiffusion. The classification accuracies of quantitative MRI parameters and corresponding weighted image contrasts were evaluated.Classification models based on the Mahalanobis distance metric for each MRI measurement were trained and validated using leave-one-out cross-validation with plugsgrouped according to OARSI histological grade and score. MRI measurements used for classification were performed using a region-of-interest analysis which included superficial, deep, and full-thickness cartilage.ResultsThe best classifiers based on OARSI grade and score were T1- and T2-weighted image contrast, which yielded accuracies of 0.68 and 0.75, respectively. Classification accuracies using OARSI score-based group membership were generally higher compared with grade-based group membership.ConclusionsMRI-based classification—either using quantitative MRI parameters or weighted image contrast—is able to detect early osteoarthritic tissue changes as classified by the OARSI histological system.These findings suggest the benefit of incorporating quantitative MRI acquisitions in a comprehensive clinical evaluation of OA. This article is protected by copyright. All rights reserved
- [Show abstract] [Hide abstract] ABSTRACT: Visualizing the three-dimensional morphology and spatial patterning of cells embedded deep within dense connective tissues of the musculoskeletal system has been possible only by utilizing destructive techniques. Here we utilize fructose-based clearing solutions to image cell connectivity and deep tissue-scale patterning in situ by standard confocal microscopy. Optical clearing takes advantage of refractive index matching of tissue and the embedding medium to visualize light transmission through a broad range of bovine and whole mount murine tissues, including cartilage, bone, and ligament, of the head and hindlimb. Using non-destructive methods, we show for the first time intercellular chondrocyte connections throughout the bulk of cartilage, and we reveal in situ patterns of osteocyte processes and the lacunar-canalicular system deep within mineralized cortical bone. Optical clearing of connective tissues is expected to find broad application for the study of cell responses in normal physiology and disease pathology.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Functional imaging refers broadly to the visualization of organ or tissue physiology using medical image modalities. In load-bearing tissues of the body, including articular cartilage lining the bony ends of joints, changes in strain, stress, and material properties occur in osteoarthritis (OA), providing an opportunity to probe tissue function through the progression of the disease. Here, biomechanical measures in cartilage and related joint tissues are discussed as key imaging biomarkers in the evaluation of OA. Emphasis will be placed on the (1) potential of radiography, ultrasound, and magnetic resonance imaging to assess early tissue pathomechanics in OA, (2) relative utility of kinematic, structural, morphological, and biomechanical measures as functional imaging biomarkers, and (3) improved diagnostic specificity through the combination of multiple imaging biomarkers with unique contrasts, including elastography and quantitative assessments of tissue biochemistry. In comparison to other modalities, magnetic resonance imaging provides an extensive range of functional measures at the tissue level, with conventional and emerging techniques available to potentially to assess the spectrum of preclinical to advance OA.
- [Show abstract] [Hide abstract] ABSTRACT: 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 matrix 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 3D 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 collagan-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.
- [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study was to compare displacement behavior of cyclically loaded cadaveric human intervertebral discs as measured noninvasively on a clinical 3.0 T and a research 9.4 T MRI system. Intervertebral discs were cyclically compressed at physiologically relevant levels with the same MRI-compatible loading device in the clinical and research systems. Displacement-encoded imaging was synchronized to cyclic loading to measure displacements under applied loading with MRI (dualMRI). Displacements from the two systems were compared individually using linear regression and, across all specimens, using Bland–Altman analysis. In-plane displacement patterns measured at 3.0 T and 9.4 T were qualitatively comparable and well correlated. Bland–Altman analyses showed that over 90% of displacement values within the intervertebral disc regions of interest lay within the limits of agreement. Measurement of displacement using dualMRI using a 3.0 T clinical system is comparable to that of a 9.4 T research system. Additional refinements of software, technique implementation, and image processing have potential to improve agreement between different MRI systems. Despite differences in MRI systems in this initial implementation, this work demonstrates that dualMRI can be reliably implemented at multiple magnetic field strengths, permitting translation of dualMRI for a variety of applications in the study of tissue and biomaterial biomechanics.
- [Show abstract] [Hide abstract] ABSTRACT: 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 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. 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 cryoSEM. 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 5 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, cryoSEM-based analysis of cartilage showed no ultrastructural changes. We anticipate large-scale microscopy of diverse connective tissues will enable the study of intact, three-dimensional interfaces (e.g. osteochondral) and cellular connectivity as a function of development, disease, and regeneration, which have been previously hindered by specimen opacity. Copyright © 2014. Published by Elsevier Ltd.
Conference Paper: Gradient Densification of Collagen Scaffolds
Conference Paper: Controlled Fibrillogenesis for Improved Magnetic Alignment of Collagen
- [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study was to compare displacement behavior of cyclically loaded cadaveric human intervertebral discs as measured noninvasively on a clinical 3.0T and a research 9.4T MRI system. Intervertebral discs were cyclically compressed at physiologically relevant levels with the same MRI-compatible loading device in the clinical and research systems. Displacement-encoded imaging was synchronized to cyclic loading to measure displacements under applied loading with MRI (dualMRI). Displacements from the two systems were compared individually using linear regression and, across all specimens, using Bland-Altman analysis. In-plane displacement patterns measured at 3.0T and 9.4T were qualitatively comparable and well correlated. Bland-Altman analyses showed that over 90% of displacement values within the intervertebral disc regions of interest lay within the limits of agreement. Measurement of displacement using dualMRI using a 3.0T clinical system is comparable to that of a 9.4T research system. Additional refinements of software, technique implementation, and image processing have potential to improve agreement between different MRI systems. Despite differences in MRI systems in this initial implementation, this work demonstrates that dualMRI can be reliably implemented at multiple magnetic field strengths, permitting translation of dualMRI for a variety of applications in the study of tissue and biomaterial biomechanics.
- [Show abstract] [Hide abstract] ABSTRACT: Purpose: Noninvasive assessment of tissue mechanical behavior could enable insights into tissue function in healthy and diseased conditions and permit the development of effective tissue repair treatments. Measurement of displacements under applied loading with MRI (dualMRI) has the potential for such biomechanical characterization on a clinical MRI system. Methods: dualMRI was translated from high-field research systems to a 3T clinical system. Precision was calculated using repeated tests of a silicone phantom. dualMRI was demonstrated by visualizing displacements and strains in an intervertebral disc and compared to T2 measured during cyclic loading. Results: The displacement and strain precisions were 24 µm and 0.3% strain, respectively, under the imaging parameters used in this study. Displacements and strains were measured within the intervertebral disc, but no correlations were found with the T2 values. Conclusion: The translation of dualMRI to a 3T system unveils the potential for in vivo studies in a myriad of tissue and organ systems. Because of the importance of mechanical behavior to the function of a variety of tissues, it's expected that dualMRI implemented on a clinical system will be a powerful tool in assessing the interlinked roles of structure, mechanics, and function in both healthy and diseased tissues.
ウェストラファイエット, Indiana, United States
- Weldon School of Biomedical Engineering
University of California, Davis
Davis, California, United States
- • Department of Orthopaedic Surgery
- • Center for Tissue Regeneration and Repair