In vivo biological activity of exendin (1-30).

Activation of the glucagon-like peptide-1 (GLP-1) receptor on pancreatic beta cells by GLP-1 and exendin-4 increases insulin secretion. Exendin-4 is 39 amino acids long, unlike GLP-1 which has 30 amino acids. Because of its non-mammalian (lizard) origin and unique C-terminal sequence, exendin-4 may be immunogenic in humans. We showed previously that the C terminally truncated exendin peptide exendin (1-30) has a reduced affinity for the GLP-1 receptor and a diminished ability to increase intracellular cAMP in insulinoma cells. Here we show that daily intraperitoneal injection of exendin (1-30) (1 nmol/kg) for 20 d followed by 31 d twice daily to Lepr(db)/Lepr(db) (db/db) mice significantly reduced the amount of visceral fat relative to saline-treated controls and improved HbA1C (control 9.5 +/- 0.2% vs treated 7.9 +/- 0.2%, p = 0.001) but was not as effective as exendin-4. To examine the ability of exendin (1-30) to stimulate beta-cell growth, we injected one group of 3-mo-old Fisher rats with exendin (1-30) (1 nmol/kg) and another group with saline for 8 d. We observed no change in beta-cell area, but did see a change in the number of islets with nuclei positive for BrdU [10.7 +/- 1.8% exendin (1-30) vs 6.5 +/- 0.5% control].

Growth and integration of neocartilage with native cartilage in vitro.

Poor integration of neocartilage with recipient has been a major obstacle to articular cartilage restoration. An in vitro study was designed to provide insights regarding the integration process. Cartilage explants and chondrocytes were harvested from the distal sternum of 16-day-old chick embryos. Four million chondrocytes and one 1mm(3) explant were centrifuged together in a 0.75ml tube. In the constructs, consisting of cartilage explant and chondrocyte pellet, isolated chondrocytes attached to the surface of the explant at the beginning of the culture, followed by significant chondrocyte death at the interface between chondrocyte pellet and explant. Chondrocyte apoptosis was seen almost exclusively at this interface. Meanwhile, the interface was a zone with active extracellular matrix deposition as demonstrated by immunohistochemistry. By two weeks, the junction of neocartilage and native cartilage explant had formed an acellular zone with collagen fibrils orientated parallel with the surface of the cartilage explant. In conclusion, chondrocyte death leads to acellularity and fibril network reorganization at the neocartilage/explant interface, and impacts the quality of cartilage repair as abnormal matrix remodeling implies.

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 x 10(6) 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.

The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production.

This study was designed to examine the effects of pulsed low-intensity ultrasound (PLIUS) on chondrocyte viability, proliferation, matrix production and gene expression. Chondrocytes were isolated from the distal part of the sternum of 16-day-old chick embryos and cultured in alginate beads. PLIUS at 2 mW/cm(2) (group PLIUS(2)) and 30 mW/cm(2) (group PLIUS(30)) was applied to chondrocytes for a single 20-min treatment. A control group was treated without PLIUS. The viability of chondrocytes was not affected by exposure to PLIUS. PLIUS influenced chondrocyte proliferation in an intensity-dependent manner. By day 7 after application of PLIUS, the gene expression and synthesis of aggrecan was the same as in the controls. At this same time point, the expression and synthesis of type II collagen was not different between the controls and PLIUS(30), but was increased in PLIUS(2). PLIUS was shown to inhibit the expression of type X collagen. This inhibition of chondrocyte hypertrophy may prove to be significant in the management of cartilage degeneration.

Matrix fixed-charge density as determined by magnetic resonance microscopy of bioreactor-derived hyaline cartilage correlates with biochemical and biomechanical properties.

OBJECTIVE: To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow- fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage. METHODS: Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests. RESULTS: S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties. CONCLUSION: HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.

Experimental demonstration of quantitation errors in MR spectroscopy resulting from saturation corrections under changing conditions.

Metabolite concentration measurements in in vivo NMR are generally performed under partially saturated conditions, with correction for partial saturation performed after data collection using a measured saturation factor. Here, we present an experimental test of the hypothesis that quantitation errors can occur due to application of such saturation factor corrections in changing systems. Thus, this extends our previous theoretical work on quantitation errors due to varying saturation factors. We obtained results for two systems frequently studied by 31P NMR, the ischemic rat heart and the electrically stimulated rat gastrocnemius muscle. The results are interpreted in light of previous theoretical work which defined the degree of saturation occurring in a one-pulse experiment for a system with given spin-lattice relaxation times, T(1)s, equilibrium magnetizations, M(0)s, and reaction rates. We found that (i) the assumption of constancy of saturation factors leads to quantitation errors on the order of 40% in inorganic phosphate; (ii) the dominant contributor to the quantitation errors in inorganic phosphate is most likely changes in T(1); (iii) T(1) and M(0) changes between control and intervention periods, and chemical exchange contribute to different extents to quantitation errors in phosphocreatine and gamma-ATP; (iv) relatively small increases in interpulse delay substantially decreased quantitation errors for metabolites in ischemic rat hearts; (v) random error due to finite SNR led to approximately 4% error in quantitation, and hence was a substantially smaller contributor than were changes in saturation factors.

Ex vivo magnetic resonance microscopy of an osteochondral transfer.

A 49-year-old woman with right knee pain and a chondral defect on the medial femoral condyle underwent an osteochondral transfer. The patient initially had pain relief, but then sustained a twisting injury and had progressive chondromalacia and pain on the affected side. She subsequently underwent a total knee replacement, and the tissue from the osteochondral transfer (OATS) site was harvested for analysis. In vitro MR microimaging of the excised joint segment revealed undamaged, full-thickness cartilage on the OATS plug, intact cartilage on the posterior condyle, and severely thinned and damaged cartilage on the anterior condyle. Alcian blue-stained sections revealed that proteoglycans were present throughout the OATS core but were nearly absent in the native cartilage. Quantitative T(1) data acquired after equilibration with Gd-DTPA indicated a distribution of matrix fixed charge in the OATS plug and anterior tissue that agreed well with histology and literature observations, while the posterior native cartilage appeared to have fixed charge similar to that of the OATS tissue. Histology revealed poor graft integration between OATS and native cartilage, with a distinct layer of fibrous tissue at the posterior interface. MRI images, by comparison, showed a hypointense feature at the posterior interface but uniform intensity across the anterior interface. Quantitative T(2), magnetization transfer and T(1) data acquired with and without gadolinium contrast showed dependences on depth, location, and pathology that were consistent with measurements reported in the literature for articular cartilage.

Differences in the bioenergetic response of the isolated perfused rat heart to selective beta1- and beta2-adrenergic receptor stimulation.

BACKGROUND: In the heart, striking functional differences exist after stimulation of the beta1- and beta2-adrenergic receptor (AR) subtypes. These may be linked to differences in metabolic response during beta1- and beta2-AR stimulation. METHODS AND RESULTS: The relation between work and metabolism was examined during selective beta1- and beta2-AR stimulation (beta1 and beta2 groups, respectively) in the isolated perfused rat heart. Measurements were made of rate-pressure product (RPP=LV developed pressure x heart rate), phosphorus-containing metabolites, and pH by 31P nuclear magnetic resonance spectroscopy and of O2 consumption by fiber-optic oximetry. Experiments were performed under high constant flow (HCF) and under flow-limiting conditions (constant pressure, CP). Despite substantially greater RPP increases relative to baseline during beta1-AR (HCF, 475%; CP, 150%) than beta2-AR (HCF, 90%; CP, 72%) stimulation, the relative decrease in the intracellular energy charge relative to baseline was similar for the beta1 (HCF, 49%; CP, 64%) and beta2 (HCF, 59%; CP, 55%) groups. For each group, an increase in oxygen consumption (MVO2) occurred commensurate with workload during HCF (beta1, 141%; beta2, 30%). During CP, however, the MVO2 increase was similar (beta1, 39%; beta2, 34%), despite the large RPP difference between the groups. During both protocols, there was greater acidosis during beta1-AR than during beta2-AR stimulation. Thus, at a given workload, intracellular energy charge decreased, and MVO2 (CP) increased to a greater extent during beta2 than beta1-AR stimulation. CONCLUSIONS: The bioenergetic differences are consistent with access to an additional substrate pool during beta1-AR stimulation. This may occur via increased glycogenolysis during beta1-AR stimulation, facilitating increased energy production by oxidative phosphorylation, and under flow-limiting conditions, anaerobic glycolysis.

Long repetition time experiments for measurement of concentrations in systems with chemical exchange and undergoing temporal variation-comparison of methods with and without correction for saturation.

The purpose of this paper is to compare two methods for quantifying metabolite concentrations using the one-pulse experiment for a sample undergoing chemical exchange and subject to an intervention or other temporal variation. The methods, LATR-C (Long Acquisition TR (interpulse delay); Corrected for partial saturation) and LATR-NC (Long Acquisition TR; Not Corrected), are compared in terms of signal-to-noise ratio, SNR, per unit time and quantitation errors. Parameters relevant to the isolated perfused rat heart are used as a specific application, although the results are general. We assume throughout that spin-lattice relaxation times, T(1), do not change. For a given flip angle, theta, TR's are calculated which result in maximal SNR per unit time under 10%, 5%, and 1% constraints on quantitation errors. Additional simulations were performed to demonstrate explicitly the dependence of the quantitation errors on TR for a fixed theta. We find (i) if the allowed error is large, and when both metabolite concentrations and rate constants vary, LATR-C permits use of shorter TR, and hence yields greater SNR per unit time, than LATR-NC; (ii) for small allowed error, the two methods give similar TR's and SNR per unit time, so that the simpler LATR-NC experiment may be preferred; (iii) large values of theta result in similar constrained TR's and hence SNR per unit time for the two methods; (iv) the ratio of concentrations of metabolites with similar T(1) exhibit similar errors for the two methods.

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.

Optimized pulse parameters for reducing quantitation errors due to saturation factor changes in magnetic resonance spectroscopy.

We present an analysis of the effects of chemical exchange and changes in T(1) on metabolite quantitation for heart, skeletal muscle, and brain using the one-pulse experiment for a sample which is subject to temporal variation. We use an optimization algorithm to calculate interpulse delay times, TRs, and flip angles, theta, resulting in maximal root-mean-squared signal-to-noise per unit time (S/N) for all exchanging species under 5 and 10% constraints on quantitation errors. The optimization yields TR and theta pairs giving signal-to-noise per unit time close or superior to typical literature values. Additional simulations were performed to demonstrate explicitly the dependence of the quantitation errors on pulse parameters and variations in the properties of the sample, such as may occur after an intervention. We find that (i) correction for partial saturation in accordance with the usual analysis neglecting variations in metabolite concentrations and rate constants may readily result in quantitation errors of 15% or more; the exact degree of error depends upon the details of the system under consideration; (ii) if T(1)'s vary as well, significantly larger quantitation errors may occur; and (iii) optimal values of pulse parameters may minimize errors in quantitation with minimal S/N loss.

Microengineering neocartilage scaffolds.

Advances in micropatterning methodologies have made it possible to create structures with precise architecture on the surface of cell culture substrata. We applied these techniques to fabricate microfeatures (15-65 microm wide; 40 microm deep) on the surface of a flexible, biocompatible polysaccharide gel. The micropatterned polymer gels were subsequently applied as scaffolds for chondrocyte culture and proved effective in maintaining key aspects of the chondrogenic phenotype. These were rounded cell morphology and a positive and statistically significant (p < 0.0001) immunofluorescence assay for the production of type II collagen throughout the maximum culture time of 10 days after cell seeding. Further, cells housed within individual surface features were observed to proliferate, while serial application of chondrocytes resulted in the formation of cellular aggregates. These methods represent a novel approach to the problem of engineering reparative cartilage in vitro.