Ziying Yin

Active tissue stiffness modulation controls valve interstitial cell phenotype and osteogenic potential in 3D culture.

UNLABELLED Calcific aortic valve disease (CAVD) progression is a highly dynamic process whereby normally fibroblastic valve interstitial cells (VIC) undergo osteogenic differentiation, maladaptive extracellular matrix (ECM) composition, structural remodeling, and tissue matrix stiffening. However, how VIC with different phenotypes dynamically affect matrix properties and how the altered matrix further affects VIC phenotypes in response to physiological and pathological conditions have not yet been determined. In this study, we develop 3D hydrogels with tunable matrix stiffness to investigate the dynamic interplay between VIC phenotypes and matrix biomechanics. We find that VIC populated within hydrogels with valve leaflet like stiffness differentiate towards myofibroblasts in osteogenic media, but surprisingly undergo osteogenic differentiation when cultured within lower initial stiffness hydrogels. VIC differentiation progressively stiffens the hydrogel microenvironment, which further upregulates both early and late osteogenic markers. These findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive RhoA/ROCK signaling pathway, delays the osteogenic differentiation process. Therefore, direct ECM biomechanical modulation can affect VIC phenotypes towards and against osteogenic differentiation in 3D culture. These findings highlight the importance of the homeostatic maintenance of matrix stiffness to restrict pathological VIC differentiation. STATEMENT OF SIGNIFICANCE We implement 3D hydrogels with tunable matrix stiffness to investigate the dynamic interaction between valve interstitial cells (VIC, major cell population in heart valve) and matrix biomechanics. This work focuses on how human VIC responses to changing 3D culture environments. Our findings identify a dynamic positive feedback loop that governs acceleration of VIC calcification, which is the hallmark of calcific aortic valve disease. Temporal stiffening of pathologically lower stiffness matrix back to normal level, or blocking the mechanosensitive signaling pathway, delays VIC osteogenic differentiation. Our findings provide an improved understanding of VIC-matrix interactions to aid in interpretation of VIC calcification studies in vitro and suggest that ECM disruption resulting in local tissue stiffness decreases may promote calcific aortic valve disease.

Simultaneous MR elastography and diffusion acquisitions: Diffusion-MRE (dMRE)

To present a new technique for concurrent MR elastography (MRE) and diffusion MRI: diffusion‐MRE (dMRE).

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

In this article, based on the invited talk at the “Tissue Science 2012” meeting in Chicago on October 1-3, 2012, we describe some examples of characterization of engineered cartilage and bone tissue using magnetic resonance spectroscopy and imaging. Two different models of engineered cartilage and engineered bone tissue constructs were used for these studies: 1) chondrocyte based cartilage tissue engineering constructs: human and bovine chondrocytes seeded in alginate beads (Hydrogel scaffold model) or bovine chondrocytes grown as pellets (scaffold free model); 2) mesenchymal stem cell (MSC) based cartilage and bone tissue engineering constructs: human mesenchymal stem cell (HMSCs) seeded in cartilage biomimetic scaffolds (collagen/chitosan scaffold integrated with extracellular matrix of cartilage) or HMSCs seeded in collagen/chitosan scaffolds. Magnetic resonance spectroscopy and imaging experiments using 9.4 T (400 MHz proton frequency), 11.7 T (500 MHz proton frequency) or 14.1 T (600 MHz proton frequency) MR spectrometers/Imager were performed on these constructs over two to four weeks of tissue culture time. Specifically, water suppressed proton NMR spectroscopy; proton and sodium multi-quantum coherence spectroscopy and proton T1, T2 and ADC parametric MRI were used to study the chondrogenesis and osteogenesis of these tissues. We found that the change in MR relaxation and diffusion coefficient parameters correlate well with the growth of engineered tissues. We found that the MR parameters and the change in these parameters in growing tissue are strongly influenced by the choice of scaffolds. We also found as expected that the tissue-engineered cartilage lacked order or preference in collagen orientation. Further work is underway to elucidate these findings. We anticipate that in future, MRI will augment histological and immunohistochemical techniques by providing a complimentary and real time quantitative assessment of engineered tissue growth at all growth stages: (i) cell seeding to pre implantation; (ii) preclinical validation studies post implantation in small and large animal models; (iii) clinical studies of performance of engineered tissues.

Concurrent 3D acquisition of diffusion tensor imaging and magnetic resonance elastography displacement data (DTI-MRE): Theory and in vivo application

To introduce a newly developed technique (DTI‐MRE) for the simultaneous acquisition of diffusion tensor imaging (DTI) and 3D‐vector field magnetic resonance elastography (MRE) data, and to demonstrate its feasibility when applied in vivo to the mouse brain.

Concurrent 3D acquisition of diffusion tensor imaging and magnetic resonance elastography displacement data (DTI-MRE)

To introduce a newly developed technique (DTI‐MRE) for the simultaneous acquisition of diffusion tensor imaging (DTI) and 3D‐vector field magnetic resonance elastography (MRE) data, and to demonstrate its feasibility when applied in vivo to the mouse brain.

Assessment of glenoid chondral healing: comparison of microfracture to autologous matrix-induced chondrogenesis in a novel rabbit shoulder model.

BACKGROUND Management of glenohumeral arthrosis in young patients is a considerable challenge, with a growing need for non-arthroplasty alternatives. The objectives of this study were to develop an animal model to study glenoid cartilage repair and to compare surgical repair strategies to promote glenoid chondral healing. METHODS Forty-five rabbits underwent unilateral removal of the entire glenoid articular surface and were divided into 3 groups--untreated defect (UD), microfracture (MFx), and MFx plus type I/III collagen scaffold (autologous matrix-induced chondrogenesis [AMIC])--for the evaluation of healing at 8 weeks (12 rabbits) and 32 weeks (33 rabbits) after injury. Contralateral shoulders served as unoperated controls. Tissue assessments included 11.7-T magnetic resonance imaging (long-term healing group only), equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-μCT), and histologic investigation (grades on International Cartilage Repair Society II scoring system). RESULTS At 8 weeks, x-ray attenuation, thickness, and volume did not differ by treatment group. At 32 weeks, the T2 index (ratio of T2 values of healing to intact glenoids) was significantly lower for the MFx group relative to the AMIC group (P = .01) whereas the T1ρ index was significantly lower for AMIC relative to MFx (P = .01). The micro-computed tomography-derived repair tissue volume was significantly higher for MFx than for UD. Histologic investigation generally suggested inferior healing in the AMIC and UD groups relative to the MFx group, which exhibited improvements in both integration of repair tissue with subchondral bone and tidemark formation over time. DISCUSSION Improvements conferred by AMIC were limited to magnetic resonance imaging outcomes, whereas MFx appeared to promote increased fibrous tissue deposition via micro-computed tomography and more hyaline-like repair histologically. The findings from this novel model suggest that MFx promotes biologic resurfacing of full-thickness glenoid articular injury.

Diffusion tensor MRI phantom exhibits anomalous diffusion

This paper reports diffusion weighted MRI measurements of cyclohexane in a novel diffusion tensor MRI phantom composed of hollow coaxial electrospun fibers (average diameter 10.2 μm). Recent studies of the phantom demonstrated its potential as a calibration standard at low b values (less than 1000 s/mm<;sup>2<;/sup>) for mean diffusivity and fractional anisotropy. In this paper, we extend the characterization of cyclohexane diffusion in this heterogeneous, anisotropic material to high b values (up to 5000 s/mm<;sup>2<;/sup>), where the apparent diffusive motion of the cyclohexane exhibits anomalous behavior (i.e., the molecular mean squared displacement increases with time raised to the fractional power 2α/β). Diffusion tensor MRI was performed at 9.4 T using an Agilent imaging scanner and the data fit to a fractional order Mittag-Leffler (generalized exponential) decay model. Diffusion along the fibers was found to be Gaussian (2α/β=l), while diffusion across the fibers was sub-diffusive (2α/β<;l). Fiber tract reconstruction of the data was consistent with scanning electron micrograph images of the material. These studies suggest that this phantom material may be used to calibrate MR systems in both the normal (Gaussian) and anomalous diffusion regimes.

Mechanical analysis of an axially symmetric cylindrical phantom with a spherical heterogeneity for MR elastography

Cylindrical homogenous phantoms for magnetic resonance (MR) elastography in biomedical research provide one way to validate an imaging systems performance, but the simplified geometry and boundary conditions can cloak complexity arising at tissue interfaces. In an effort to develop a more realistic gel tissue phantom for MRE, we have constructed a heterogenous gel phantom (a sphere centrally embedded in a cylinder). The actuation comes from the phantom container, with the mechanical waves propagating toward the center, focusing the energy and thus allowing for the visualization of high-frequency waves that would otherwise be damped. The phantom was imaged and its stiffness determined using a 9.4 T horizontal MRI with a custom build piezo-elastic MRE actuator. The phantom was vibrated at three frequencies, 250, 500, and 750 Hz. The resulting shear wave images were first used to reconstruct material stiffness maps for thin (1 mm) axial slices at each frequency, from which the complex shear moduli μ were estimated, and then compared with forward modeling using a recently developed theoretical model which took μ as inputs. The overall accuracy of the measurement process was assessed by comparing theory with experiment for selected values of the shear modulus (real and imaginary parts). Close agreement is shown between the experimentally obtained and theoretically predicted wave fields.

CHAPTER 20:Magnetic Resonance in the Assessment of Tissue Engineered Cartilage

Magnetic resonance spectroscopy (MRS) and imaging (MRI) are routinely used for non-invasive monitoring and assessment of cartilage regeneration in vitro and in vivo. Cartilage tissue engineering utilizes a combination of three-dimensional porous scaffold, chondrocytes or stem cells, growth factors such as transforming growth factor-β, and growth stimulating conditions to obtain a neocartilage tissue that contains a high level of chondrogenic extracellular matrix proteins, proteoglycans and collagen, type II. Water proton (1H) parametric MRI is commonly applied for monitoring and assessing tissue-engineered cartilage growth at the bench and for in vivo settings. The change in relaxation times (T1, T2 and T1ρ) and apparent diffusion coefficient are correlated with the change in the amount of proteoglycan and collagen in tissueengineered cartilage. In stem cells and scaffold-based engineered cartilage, it has been shown that once the scaffold’s contribution is removed, both T1 and T2 correlate with the amount of matrix regeneration. The cartilage tissue’s functional properties depend on its special composition of extracellular matrix proteins. This arrangement of extracellular matrix is highly anisotropic and one that is the source of cartilage health. In engineered cartilage, tissue anisotropy can be measured using the sodium triple quantum coherence nuclear magnetic resonance-based average quadrupolar coupling (ωQ) or the diffusion tensor imaging based fractional anisotropy parameters. Using these techniques, it has been shown that the engineered cartilage tissues are less anisotropic than the natural cartilage. Glycosaminoglycan (GAG) of proteoglycan is negatively charged and sodium MRI can be used for assessing the GAG amount. The sodium MRI-based fixed charge density (FCD) is found to strongly correlate with the FCD derived from the GAG assay in a tissue-engineered matrix created from stem cell chondrogenesis in polymer–hydrogel hybrid scaffolds. In summary, magnetic resonance technologies offer tools to non-invasively assess the engineered cartilage tissue growth at all stages, in vitro and in vivo, from cell seeding to post-implantation.

Scattering and Diffraction of Elastodynamic Waves in a Concentric Cylindrical Phantom for MR Elastography

Aim: The focus of this paper is to report on the design and construction of a multiply connected phantom for use in magnetic resonance elastography (MRE)-an imaging technique that allows for the noninvasive visualization of the displacement field throughout an object from externally driven harmonic motion-as well as its inverse modeling with a closed-form analytic solution which is derived herein from first principles. Methods: Mathematically, the phantom is described as two infinite concentric circular cylinders with unequal complex shear moduli, harmonically vibrated at the exterior surface in a direction along their common axis. Each concentric cylinder is made of a hydrocolloid with its own specific solute concentration. They are assembled in a multistep process for which custom scaffolding was designed and built. A customized spin-echo-based MR elastography sequence with a sinusoidal motion-sensitizing gradient was used for data acquisition on a 9.4 T Agilent small-animal MR scanner. Complex moduli obtained from the inverse model are used to solve the forward problem with a finite-element method.Results: Both complex shear moduli show a significant frequency dependence (p <; 0.001) in keeping with previous work. Conclusion: The novel multiply connected phantom and mathematical model are validated as a viable tool for MRE studies. Significance: On a small enough scale much of physiology can be mathematically modeled with basic geometric shapes, e.g., a cylinder representing a blood vessel. This study demonstrates the possibility of elegant mathematical analysis of phantoms specifically designed and carefully constructed for biomedical MRE studies.