Mrignayani Kotecha

Nanomole-scale protein solid-state NMR by breaking intrinsic 1H T1 boundaries

We present an approach that accelerates protein solid-state NMR 5–20-fold using paramagnetic doping to condense data-collection time (to ∼0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic 1H T1 longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional 13C-13C and 13C-15N solid-state NMR spectra for several to tens of nanomoles of β-amyloid fibrils and ubiquitin in 1–2 d.

Efficient low-power heteronuclear decoupling in 13C high-resolution solid-state NMR under fast magic angle spinning.

The use of a low‐power two‐pulse phase modulation (TPPM) sequence is proposed for efficient 1H radio frequency (rf) decoupling in high‐resolution 13C solid‐state NMR (SSNMR) under fast MAS conditions. Decoupling efficiency for different low‐power decoupling sequences such as continuous‐wave (cw), TPPM, XiX, and π‐pulse (PIPS) train decoupling has been investigated at a spinning speed of 40 kHz for 13C CPMAS spectra of uniformly 13C‐ and 15N‐labeled L‐alanine. It was found that the TPPM decoupling sequence, which was originally designed for high‐power decoupling, provides the best decoupling efficiency at low power among all the low‐power decoupling sequences examined here. Optimum performance of the low‐power TPPM sequence was found to be obtained at a decoupling field intensity (ω1) of ∼ωR/4 with a pulse flip angle of ∼π and a phase alternation between ± ϕ(ϕ = ∼20° ), where ωR/2π is the spinning speed. The sensitivity obtained for 13CO2−, 13CH, and 13CH3 in L‐alanine under low‐power TPPM at ω1/2π of 10 kHz was only 5–15% less than that under high‐power TPPM at ω1/2π of 200 kHz, despite the fact that only 0.25% of the rf power was required in low‐power TPPM. Analysis of the 13CH2 signals for uniformly 13C‐ and 15N‐labeled L‐isoleucine under various low‐power decoupling sequences also confirmed superior performance of the low‐power TPPM sequence, although the intensity obtained by low‐power TPPM was 61% of that obtained by high‐power TPPM. 13C CPMAS spectra of 13C‐labeled ubiquitin micro crystals obtained by low‐power TPPM demonstrates that the low‐power TPPM sequence is a practical option that provides excellent resolution and sensitivity in 13C SSNMR for hydrated proteins. Copyright

Application of sodium triple‐quantum coherence NMR spectroscopy for the study of growth dynamics in cartilage tissue engineering

We studied the tissue growth dynamics of tissue‐engineered cartilage at an early growth stage after cell seeding for four weeks using sodium triple‐quantum coherence NMR spectroscopy. The following tissue‐engineering constructs were studied: 1) bovine chondrocytes cultured in alginate beads; 2) bovine chondrocytes cultured as pellets (scaffold‐free chondrocyte pellets); and 3) human marrow stromal cells (HMSCs) seeded in collagen/chitosan based biomimetic scaffolds. We found that the sodium triple‐quantum coherence spectroscopy could differentiate between different tissue‐engineered constructs and native tissues based on the fast and slow components of relaxation rate as well as on the average quadrupolar coupling. Both fast (Tf) and slow (Ts) relaxation times were found to be longer in chondrocyte pellets and biomimetic scaffolds compared to chondrocytes suspended in alginate beads and human articular cartilage tissues. In all cases, it was found that relaxation rates and motion of sodium ions measured from correlation times were dependent on the amount of macromolecules, high cell density and anisotropy of the cartilage tissue‐engineered constructs. Average quadrupolar couplings were found to be lower in the engineered tissue compared to native tissue, presumably due to the lack of order in collagen accumulated in the engineered tissue. These results support the use of sodium triple‐quantum coherence spectroscopy as a tool to investigate anisotropy and growth dynamics of cartilage tissue‐engineered constructs in a simple and reliable way. Copyright

Reduction of water diffusion coefficient with increased engineered cartilage matrix growth observed using MRI

Non-destructive monitoring of tissue-engineered cartilage growth is needed to optimize growth conditions, but extracting quantitative biomarkers of extracellular matrix development remains a technical challenge. MRI provides a non-invasive way to obtain a three dimensional map of growing tissue where the image contrast is based on tissue water relaxation times and the apparent diffusion coefficient (ADC). In this study, bovine chondrocytes were seeded in alginate beads (0, 1, 2, and 4 million cells/ml) and the ADC was measured weekly using diffusion-weighted MRI at 14.1 T over a one-month incubation period. Two groups of tissue-engineering constructs were created: one with ascorbic acid (vitamin C) added as a vitamin cofactor to increase collagen synthesis, and another with no added ascorbic acid. When normalized to the control beads without chondrocytes, the ADC was found to monotonically fall with incubation time (decreasing by up to 40% at 4 weeks), and with the administration of vitamin C. These results reflect the expected development of the extracellular matrix in the tissue-engineered constructs. We conclude that the normalized ADC is a potential biomarker for characterizing engineered cartilage tissue growth.

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.

True MRI assessment of stem cell chondrogenesis in a tissue engineered matrix

Developing a non-invasive method to monitor the growth of tissue-engineered cartilage is of utmost importance for tracking the progress and predicting the success or failure of tissue-engineering approaches. Magnetic Resonance Imaging (MRI) is a leading non-invasive technique suitable for follow-through in preclinical and clinical stages. As complex tissue-engineering approaches are being developed for cartilage tissue engineering, it is important to develop strategies for true non-invasive MRI monitoring that can take into account contributions of the scaffold, cells and extracellular matrix (ECM) using MR parameters. In the current study, we present the preliminary MRI assessment of chondrogenic differentiation of human bone marrow derived stem cells seeded onto a specially designed osteochondral matrix system. We performed water relaxation times (T1 and T2) MRI measurements at 7, 14 and 28 days after cell seeding. The MRI experiments were performed for the tissue-engineered cartilage as well as for acellular scaffolds. We identified that the contribution of the scaffold is the dominant contribution in MR parameters of engineered cartilage and that it hinders observation of the tissue growth. An attempt is made to filter out this contribution, for the first time, in order to make a true observation of tissue growth using MRI.

Characterization of pore structure in biologically functional poly(2-hydroxyethyl methacrylate)-poly(ethylene glycol) diacrylate (PHEMA-PEGDA).

A copolymer composed of poly(2-hydroxyethyl methacrylate) (PHEMA) and poly(ethylene glycol) diacrylate (PEGDA) (PHEMA-PEGDA) is structurally versatile. Its structure can be adjusted using the following porogens: water, sucrose, and benzyl alcohol. Using phase separation technique, a variety of surface architectures and pore morphologies were developed by adjusting porogen volume and type. The water and sucrose porogens were effective in creating porous and cytocompatible PHEMA-PEGDA scaffolds. When coated with collagen, the PHEMA-PEGDA scaffolds accommodated cell migration. The PHEMA-PEGDA scaffolds are easy to produce, non-toxic, and mechanically stable enough to resist fracture during routine handling. The PHEMA-PEGDA structures presented in this study may expedite the current research effort to engineer tissue scaffolds that provide both structural stability and biological activity.

Magnetic resonance spectroscopy and imaging can differentiate between engineered bone and engineered cartilage.

In the situation when both cartilage and its underlying bone are damaged, osteochondral tissue engineering is being developed to provide a solution. In such cases, the ability to non-invasively monitor and differentiate the development of both cartilage and bone tissues is important. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) have been widely used to non-invasively assess tissue-engineered cartilage and tissue-engineered bone. The purpose of this work is to assess differences in MR properties of tissue-engineered bone and tissue-engineered cartilage generated from the same cell-plus-scaffold combination at the early stage of tissue growth. We developed cartilage and bone tissue constructs by seeding human marrow stromal cells (HMSCs, 2 million/ml) in 1:1 collagen/chitosan gel for four weeks. The chondrogenic or osteogenic differentiation of cells was directed with the aid of a culture medium containing chondrogenic or osteogenic growth factors, respectively. The proton and sodium NMR and waterproton T1, T2 and diffusion MRI experiments were performed on these constructs and the control collagen/chitosan gel using a 9.4 T (1H freq. = 400 MHz) and a 11.7 T (1H freq. = 500 MHz) NMR spectrometers. In all cases, the development of bone and cartilage was found to be clearly distinguishable using NMR and MRI. We conclude that MRS and MRI are powerful tools to assess growing osteochondral tissue regeneration.

Calculation of pulsed NMR signal in I = 3/2 quadrupolar spin system.

The rf pulse response of I = 3/2 spin system experiencing first order quadrupolar splitting is studied using density matrix approach. A general expression is derived in terms of spin populations, quadrupole splitting and duration and amplitude of the rf pulse for calculating the NMR signal arising due to the centre line and satellite resonances for the situation where the impressed rf pulse excites the resonances selectively as well as non-selectively. The necessary 4 X 4 transformation matrix obtained analytically by diagonalyzing the Hamiltonian are used to get the expression for the centre line response. The satellite signals are obtained in the same way but by using the numerical values of the roots of the related quartics. The widths of the corresponding pi/2-pulses are calculated for different initial spin populations. The variations of this pulse-width and the corresponding signal amplitude as a function of satellite splitting are studied.

In vivo preclinical cancer and tissue engineering applications of absolute oxygen imaging using pulse EPR

The value of any measurement and a fortiori any measurement technology is defined by the reproducibility and the accuracy of the measurements. This implies a relative freedom of the measurement from factors confounding its accuracy. In the past, one of the reasons for the loss of focus on the importance of imaging oxygen in vivo was the difficulty in obtaining reproducible oxygen or pO2 images free from confounding variation. This review will briefly consider principles of electron paramagnetic oxygen imaging and describe how it achieves absolute oxygen measurements. We will provide a summary review of the progress in biomedical EPR imaging, predominantly in cancer biology research, discuss EPR oxygen imaging for cancer treatment and tissue graft assessment for regenerative medicine applications.