Richard G. Spencer

Resveratrol improves health and survival of mice on a high-calorie diet.

Resveratrol (3,5,4′-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing.

Imaging of collagen and proteoglycan in cartilage sections using Fourier transform infrared spectral imaging

OBJECTIVE To test the hypothesis that Fourier transform infrared (FTIR) spectral imaging, coupled with multivariate data processing techniques, can image the spatial distribution of matrix constituents in native and engineered cartilage samples. METHODS Tissue sections from native and trypsin-digested bovine nasal cartilage (BNC) and from engineered cartilage, generated by chick sternal chondrocytes grown in a hollow fiber bioreactor, were placed either on calcium fluoride windows for FTIR analysis or gelatinized microscope slides for histologic analysis. Based on the assumption that cartilage is predominantly chondroitin sulfate (CS) and type II collagen, chemical images were extracted from FTIR spectral imaging data sets using 2 multivariate methods: the Euclidean distance algorithm and a least-squares approach. RESULTS Least-squares analysis of the FTIR data of native BNC yielded a collagen content of 54 +/- 13% and a CS content of 37 +/- 16% (mean +/- SD). Euclidean distance analysis of measurements made on trypsin-digested BNC demonstrated only trace amounts of CS. For engineered cartilage, the CS content was significantly lower (15 +/- 5%), while the collagen content (73 +/- 6%) was significantly higher than biochemically determined values (CS 34%, collagen 5%, protein 61%). These differences are due to the fact that the dimethylmethylene blue assay overestimated the CS content of the tissue because it is not specific for CS, while the FTIR spectral imaging technique overestimated the collagen content because it lacks specificity for different proteins. CONCLUSION FTIR spectral imaging combines histology-like spatial localization with the quantitative capability of bulk chemical analysis. For molecules with a unique spectral signature, such as CS, the FTIR technique coupled with multivariate analysis can define a unique spatial distribution. However, for some applications, the lack of specificity of this technique for different types of proteins may be a limitation.

Adenovirus-Mediated VEGF121 Gene Transfer Stimulates Angiogenesis in Normoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Ischemia

BACKGROUND Administration of angiogenic factors stimulates neovascularization in ischemic tissues. However, there is no evidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis that adenovirus-mediated intramuscular (IM) gene transfer of the 121-amino-acid form of vascular endothelial growth factor (AdCMV.VEGF(121)) could stimulate neovascularization in nonischemic skeletal muscle and consequently attenuate the hemodynamic deficit secondary to surgically induced ischemia. METHODS AND RESULTS Rabbits and rats received IM injections of AdCMV.VEGF(121), AdCMV.Null, or saline in the thigh, 4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF(121), we found 96% and 29% increases in length density of arterioles and capillaries, respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF(121) group was documented, as early as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactive microspheres (AdCMV.VEGF(121)=5.69+/-0.40, AdCMV.Null=2.97+/-0.50, and saline=2.78+/-0.43 mL x min(-1) x 100 g(-1), P<0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV. VEGF(121)=50.58+/-1.48, AdCMV.Null=29.08+/-4.22, saline=11.83+/-1.90, P<0.0001), and improvement of the bioenergetic reserve of the gastrocnemius muscle as assessed by (31)P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemic limb in the AdCMV.VEGF(121) group persisted until it was equalized by spontaneous collateral vessel development in untreated animals. CONCLUSIONS IM administration of AdCMV.VEGF(121) stimulates angiogenesis in normoperfused skeletal muscles, and the newly formed vessels preserve TP after induction of ischemia.

Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants.

Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 × 106 cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure.

Multicomponent T2 relaxation analysis in cartilage.

MR techniques are sensitive to the early stages of osteoarthritis, characterized by disruption of collagen and loss of proteoglycan (PG), but are of limited specificity. Here, water compartments in normal and trypsin‐degraded bovine nasal cartilage were identified using a nonnegative least squares multiexponential analysis of T2 relaxation. Three components were detected: T2,1 = 2.3 ms, T2,2 = 25.2 ms, and T2,3 = 96.3 ms, with fractions w1 = 6.2%, w2 = 14.5%, and w3 = 79.3%, respectively. Trypsinization resulted in increased (P < 0.01) values of T2,2 = 64.2 ms and T2,3 = 149.4 ms, supporting their assignment to water compartments that are bound and loosely associated with PG, respectively. The T2 of the rapidly relaxing component was not altered by digestion, supporting assignment to relatively immobile collagen‐bound water. Relaxation data were simulated for a range of TE, number of echoes, and SNR to guide selection of acquisition parameters and assess the accuracy and precision of experimental results. Based on this, the expected experimental accuracy of measured T2s and associated weights was within 2% and 4% respectively, with precision within 1% and 3%. These results demonstrate the potential of multiexponential T2 analysis to increase the specificity of MR characterization of cartilage. Magn Reson Med, 2009.

Response of engineered cartilage tissue to biochemical agents as studied by proton magnetic resonance microscopy.

OBJECTIVE To test the hypothesis that magnetic resonance imaging (MRI) results correlate with the biochemical composition of cartilage matrix and can therefore be used to evaluate natural tissue development and the effects of biologic interventions. METHODS Chondrocytes harvested from day-16 chick embryo sterna were inoculated into an MRI-compatible hollow-fiber bioreactor. The tissue that formed over a period of 2-4 weeks was studied biochemically, histologically, and with MRI. Besides natural development, the response of the tissue to administration of retinoic acid, interleukin-1beta (IL-1beta), and daily dosing with ascorbic acid was studied. RESULTS Tissue wet and dry weight, glycosaminoglycan (GAG) content, and collagen content all increased with development time, while tissue hydration decreased. The administration of retinoic acid resulted in a significant reduction in tissue wet weight, proteoglycan content, and cell number and an increase in hydration as compared with controls. Daily dosing with ascorbic acid increased tissue collagen content significantly compared with controls, while the administration of IL-1beta resulted in increased proteoglycan content. The water proton longitudinal and transverse relaxation rates correlated well with GAG and collagen concentrations of the matrix as well as with tissue hydration. In contrast, the magnetization transfer value for the tissue correlated only with total collagen. Finally, the self-diffusion coefficient of water correlated with tissue hydration. CONCLUSION Parameters derived from MR images obtained noninvasively can be used to quantitatively assess the composition of cartilage tissue generated in a bioreactor. We conclude that MRI is a promising modality for the assessment of certain biochemical properties of cartilage in a wide variety of settings.

Electron paramagnetic resonance oxygen mapping (EPROM): direct visualization of oxygen concentration in tissue.

Tissue oxygen content is a central parameter in physiology but is difficult to measure. We report a novel procedure for spatial mapping of oxygen by electron paramagnetic resonance (EPR) utilizing a spectral‐spatial imaging data set, in which an EPR spectrum is obtained from each image volume element. From this data set, spatial maps corresponding to local spin density and maximum EPR spectral line amplitude are generated. A map of local EPR spectral linewidth is then computed. Because linewidth directly correlates with oxygen concentration, the linewidth image provides a map of oxygenation. This method avoids a difficulty inherent in other oxygen content mapping techniques using EPR, that is, the unwanted influence of local spin probe density on the image. We provide simulation results and data from phantom studies demonstrating the validity of this method. We then apply the method to map oxygen content in rat tail tissue and vasculature. This method provides a new, widely applicable, approach to direct visualization of oxygen concentration in living tissue. Magn Reson Med 43:804–809, 2000.

The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study

The proximal and distal parts of sterna of chick embryos represent cartilage undergoing endochondral ossification and hyaline cartilage, respectively. Cartilage explants from both regions were exposed for 20 min to pulsed low-intensity ultrasound (PLIUS) with an intensity of 30 mW. cm(-2) (spatial average-temporal average) at a frequency of 1.5 MHz, with a pulse burst frequency of 1 kHz and burst duration of 200 micros. Histological and immunohistochemical analysis was performed on days 1, 3, 5 and 7 after treatment. An anabolic effect of PLIUS on matrix production was shown by an increase of up to 10% to 20% in quantitative immunohistochemical staining for type II collagen and aggrecan in the two parts of the sternum. PLIUS also increased type X collagen staining by up to 10% in certain regions of the proximal part of the sternum. Staining for type X collagen was negative in the distal part of the sternum in both PLIUS and control groups. These results suggest that PLIUS may stimulate bone formation by increasing hypertrophy of chondrocytes directed to terminal differentiation. However, PLIUS did not induce hypertrophy in hyaline cartilage; moreover, increased matrix synthesis indicates a potential role in cartilage repair.

Magnetic Resonance Imaging of Chondrocytes Labeled with Superparamagnetic Iron Oxide Nanoparticles in Tissue-Engineered Cartilage

The distribution of cells within tissue-engineered constructs is difficult to study through nondestructive means, such as would be required after implantation. However, cell labeling with iron-containing particles may prove to be a useful approach to this problem, because regions containing such labeled cells have been shown to be readily detectable using magnetic resonance imaging (MRI). In this study, we used the Food and Drug Administration-approved superparamagnetic iron oxide (SPIO) contrast agent Feridex in combination with transfection agents to label chondrocytes and visualize them with MRI in two different tissue-engineered cartilage constructs. Correspondence between labeled cell spatial location as determined using MRI and histology was established. The SPIO-labeling process was found not to affect the phenotype or viability of the chondrocytes or the production of major cartilage matrix constituents. We believe that this method of visualizing and tracking chondrocytes may be useful in the further development of tissue engineered cartilage therapeutics.

Fourier transform infrared imaging spectroscopic analysis of tissue engineered cartilage: histologic and biochemical correlations

The composition of cartilage is predictive of its in vivo performance. Therefore, the ability to assess its primary macromolecular components, proteoglycan (PG) and collagen, is of great importance. In the current study, we hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier-transform infrared imaging spectroscopy (FT-IRIS). The cartilage was grown from chondrocytes within a hollow fiber bioreactor (HFBR) system previously used extensively to study cartilage development. FT-IRIS analysis showed a gradient of PG content, with the highest content in the center near the nutritive fibers and the lowest near the interior surface of the HFBR. Further, we found significantly greater PG content in the region near culture medium inflow (45.0%) as compared to the outflow region (24.7%) (p<0.001). This difference paralleled the biochemically determined glycosaminoglycan difference of 42.6% versus 27.8%. In addition, FT-IRIS-determined PG content at specific positions within the tissue sections correlated with histologically determined PG content (R=0.73, p=0.007). In summary, FT-IRIS determination of PG correlates with histological determination of PG and yields quantitatively similar results to biochemical determination of glycosaminoglycan in developing cartilage.