1. AUGMENTATION OF TISSUE GROWTH[unreadable] ANABOLIC EFFECTS OF ULTRASOUND ON CHONDROCYTES AND CARTILAGE[unreadable] Chondrocyte and cartilage explant studies: Pulsed low intensity ultrasound (PLIUS) was applied to chondrocytes. The effect on cell proliferation and on expression of mRNA for collagen II, collagen X, Sox9, and aggrecan was followed for a period of one week in addition to immunostaining. Anabolic effects of PLIUS were documented; similar results were obtained upon application of PLIUS to chick sternal explants.[unreadable] Animal model study: The Hartley guinea pig model of spontaneous age-associated OA was used to test the efficacy of PLIUS. Groups representing early intervention and treatment of established disease were studied. Histologic evaluation was performed using a modified Mankin scale. PLIUS applied to the knee joint of both young and older animals was found to attenuate the development of OA.[unreadable] [unreadable] 2. ASSESSMENT OF CARTILAGE[unreadable] FT-IRIS CARTILAGE EVALUATION, with Dr. Nancy Pleshko[unreadable] Study of Avian-derived HFBR Tissue [unreadable] We hypothesized that PG content and distribution in tissue engineered cartilage could be determined using Fourier transform infrared imaging spectroscopy (FTIRIS). After 4 weeks of cultur, bioreactor cartilage was sectioned. FTIRIS, histology, and biochemical analyses were performed. We found greater PG absorbance for regions near the tissue culture medium inflow and near the center of the HFBR. Correlations with biochemical assays were also established. We found that we could evaluate developing cartilage at the molecular level, demonstrating that FTIRIS determination of PG correlates with histological determination and biochemical analysis. [unreadable] [unreadable] FT-IRIS and MR Microscopy in a Rabbit Model of OA[unreadable] FT-IRIS and selected comparisons with corresponding MR microscopy were undertaken to evaluate articular cartilage in surgically induced osteoarthritic cartilage in the rabbit. FT-IRIS studies of femoral condyle cartilage revealed that the OA cartilage had significantly reduced PG content 2 and 4 weeks post-surgery, collagen fibril orientation changes 2 and 4 weeks post-surgery, and changes in collagen integrity 2 and 10 weeks post-surgery, but no significant changes in collagen content at any time. MR microscopy studies revealed reduced PG content in the OA cartilage 4 weeks post-surgery. A rend toward higher apparent MT exchange rate, km, was also found in the OA cartilage, suggesting changes in collagen structural features. The MR findings for PG and km paralleled the FT-IRIS findings of reduced PG content and altered collagen integrity, respectively. MR studies 12-weeks after surgery also found a trend toward longer T2 values and reduced anisotropy in the deep zone of the OA cartilage, consistent with increased hydration and less ordered collagen. These FT-IRIS and MR microscopy findings highlight the complementary nature of the two techniques.[unreadable] [unreadable] Statistical Comparison of FTIR Spectra[unreadable] FTIR spectra are sensitive to subtle changes in matrix composition of cartilage. However, statistical comparison of two seemingly different spectra from a given sample is required to determine whether apparent spectral differences arise from true changes in the sample, such as degradation, or can be attributed to noise. We addressed this problem with respect to NMR spectra some time ago, and have now performed comparable analyses for the double-beam transmittance and absorbance spectra typically used for characterization of biological tissue. The approach is based upon the fact that typical FTIR spectra are acquired using signal averaging. For the standard deviation of the ratio of spectral band amplitudes in single-scan absorbance spectra in terms of quantities that are observable from the signal-averaged spectrum, we find, after a rather lengthy calculation, a remarkably simple result which permits statistical comparison of absorbance band ratios. [unreadable] [unreadable] IMPROVEMENT IN SENSITIVITY AND SPECIFICITY OF MRI CHARACTERIZATION OF CARTILAGE[unreadable] We tested the hypothesis that MT and T2 values in cartilage may depend upon collagen crosslinking status at constant collagen concentration. We used formalin as an agent which is well-known to cross-link tissue proteins. Samples of bovine nasal cartilage were studied prior to fixation, after 9 weeks immersion in formalin solutions at three formaldehyde concentrations and after NaBH3CN reduction and washing. Apparent MT rate increased by 25.93.7% and 52.87.1% over baseline under these conditions, while T2 decreased by 59.41.1% upon fixation in 10% formalin and was 32.25.2% shorter than initial values after washing.[unreadable] Biochemical assays showed no significant differences in water content, proteoglycan, natural crosslink or collagen content between 0% and 10% formalin-treated samples. We conclude that formalin fixation of cartilage results in significant decreases in T2 and increases in MT parameters which persist after removal of unreacted formaldehyde. The new collagen crosslinks thus created are associated with large changes to MT and T2, indicating that interpretation of MT and T2 values in cartilage must be made with caution; these parameters depend upon collagen crosslinking status in addition to collagen content.[unreadable] [unreadable] 3. PERIARTICULAR STUDIES: MUSCLE[unreadable] The effects on periarticular musculature of injection into the knee joint of the rat[unreadable] Bioenergetic reserve in the quadriceps muscle of the rat in response to intraarticular injection of saline (SAL group), papain (papain-induced arthritis; PIA), and sham injection (SHAM) was compared to that of control animals. The degree of cartilage damage was assessed using histology. The three groups that underwent joint invasion exhibited a decreased rate of NMR-determined metabolic bioenergetic recovery as compared to control animals, but did not differ significantly among themselves.[unreadable] Functionally, the bioenergetic deficit seen in the intervention groups corresponded to decreased ability of the quadriceps muscle to maintain force during 8 minutes of muscle stimulation. Finally, measurements of intramuscular IL-1 and IL-6 indicated that invasion of the joint resulted in inflammation of the quadriceps muscles, including those of the sham&#61485;operated animals, but especially in the SAL and PIA groups. This indicates that invasion of the knee joint results in pathologic changes in periarticular muscle. [unreadable] [unreadable] Development of an improved method to determine chemical exchange rates in muscle[unreadable] We have developed a new method for measuring spin-lattice relaxation times and chemical exchange rate constants in multiple-site exchanging systems. CUPS, for chemical exchange and T1 measurement using progressive saturation, was applied to determine T1's and to analyze exchange among phosphocreatine (PCr), ATP, and inorganic phosphate (Pi), mediated by creatine kinase and ATP synthase, using 31P-MRS. 2-site exchange was analyzed in vitro and in the rat leg, and 3-site exchange was analyzed in the rat heart. The method avoids the use of selective irradiation and complex pulse timings or shapes, resulting in lack of RF-related artifacts and experimental simplicity. The analysis is based on a model that describes the observed magnetization of a chemically exchanging resonance subjected to the one-pulse sequence. It is these curves which are simultaneously fit to the mode.[unreadable] For the in vitro system at 8.45 T, we found T1(PCr)=2.86 s and T1(&#61543;-ATP)=1.72 s. For the rat gastrocnemius at 1.9 T, we found T1(PCr)=6.60 s and T1(&#61543;-ATP)=2.06 s. For the rat heart at 9.4 T, we found T1(PCr)=3.35 s, T1(&#61543;-ATP)=0.69 s, and T1(Pi)=1.83 s. All derived T1 values were within 20% of literature values. Similarly, exchange rates were in the same range as published values.
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