Francisco J. Muller

Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization

The mechanical behavior and biochemical composition of articular cartilage were studied in an experimental model of joint disuse, in which the canine knee was immobilized in a sling at 90 degrees of flexion. Articular cartilage from the surface zone of the femur was tested in an isometric tensile test and full-thickness cartilage on the tibial plateau was tested in a compressive indentation test. Water, proteoglycan and collagen contents were measured in site-matched samples. Site-specific increases in the tensile moduli (approximately 88% above control values in distal femoral groove) were observed in cartilage after 8 weeks of joint disuse, and after 3 weeks of remobilization following either 4 (approximately 140%, distal and proximal femoral groove) or 8 weeks (approximately 140%, distal femoral groove) of joint disuse. In contrast, the compressive properties of cartilage determined in the indentation test exhibited no change from control values with joint disuse or disuse followed by remobilization. Water contents increased at some sites on the tibia after 8 weeks of joint disuse (approximately 6% of tissue wet weight, posterior site), but not in the surface zone tissue of the femur. Proteoglycan/collagen and cartilage thickness were not found to change with disuse or disuse followed by remobilization. Reduced values for the ratio of proteoglycan:water were observed in the surface zone tissue of the femur (approximately 23%, distal femoral groove) and in the full-thickness tissue of the tibia (approximately 21%, anterior and posterior sites) after periods of joint disuse. In this study, the measured material properties suggest that the articular surface remains intact following periods of disuse or disuse with remobilization. This finding suggests one important difference between this model of joint disuse and other experimental models in which cartilage changes are both progressive and degenerative, such as surgically-induced joint instability.

Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage

Our previous studies have indicated the presence of a macromolecular inhibitor of in vitro mineral growth, as well as a mineral nucleational agent in extracellular matrix fluid aspirated by micropuncture methods from epiphyseal hypertrophic cell cartilage. In this report, new miniaturized methods were used to extract proteinpolysaccharide complexes (PPC) from cartilage, to isolate a light fraction (PPL-C), and further, to separate it into R1, R2, and SR2 subfractions. These methods were applied to PPL-C complexes separated from microdissected epiphyseal cartilages and to cetylpyridinium chloride (CPC) precipitates of extracellular matrix fluid aspirated from similar cartilages. Most of all of the inhibitory action on an in vitro system of mineral growth shown by whole cartilage PPL-C and by cartilage fluid PPC obtained from noncalcifying sites was contained in the R2 fraction which represented (1/4)-[unk] of the total hexuronate. The R2 fraction was diminished or absent from calcified cartilage fluids and from whole calcified epiphyseal septa. The ratio R1 + R2: SR2 ranged from 0.37 to 0.71 in the fluids and whole tissue samples of noncalcified cartilages. The R2 fraction was distinguished from SR2 by a 2- to 3-fold higher protein: hexuronate ratio. These data are interpreted to indicate that the inhibitory R2 fraction was degraded or otherwise inactivated at the zone of provisional calcification and that this inhibitor participates in the physiological mechanism that regulates endochondral calcification.

Determination of sedimentation coefficient distributions for cartilage proteoglycans

Abstract Sedimentation coefficient distributions of widely polydisperse proteoglycan preparations were made using a previously developed transport sedimentation methodology. Boundary stability was improved by centrifuging samples in a preformed CsCl density gradient (0.016 g/cm 4 ). The results were compared with the distributions obtained with an interferometric analytical centrifugation method. When these two techniques were applied to analyze A1 and A1–D1 proteoglycan preparations, results were in substantial agreement with respect to the mean sedimentation coefficients of the peaks, average S value, sedimentation coefficient distribution, skewness, proportion of monomer and aggregates, and linearity of the plot ln( s ) versus C extrapolations to zero concentration. The lower solute concentration compatible with the transport (velocity gradient) method makes this technique particularly suitable for studying the details of proteoglycan distribution of molecular sizes, especially for aggregates.

Ultracentrifugal studies in capillary cells: I. Determination of sedimentation coefficients

Abstract A technique has been developed which is based on transport method equations and allows determination of sedimentation coefficients using less than 5 ng of a biological active principle. Glass capillaries of 0.30 mm inside diameter and 3.0 mm length are used as sedimentation cells. About 200 nl of pure or impure solutions with concentration as low as 0.05 mg/ml are ultracentrifuged in a swinging-bucket rotor with a conventional preparative ultracentrifuge. The capillary microcells are easily sectioned after centrifugation through a plane previously marked on the glass surface. The technique was successfully extended to the use of larger glass capillaries, up to 1.25 mm inside diameter, and plastic centrifuge tubes of 5.0 mm diameter and 0.40 ml capacity. The method has been experimentally verified with proteins and protein-polysaccharides of known sedimentation constants.

Ultracentrifugal studies in capillary cells: II. Sedimentation equilibrium molecular weight determinations

Abstract An equilibrium centrifugation technique has been designed that allows molecular weight determination at the nanogram level. Microcapillary columns of 0.30 mm to 1.25 mm diameter and 3.0 mm to 8.0 mm length are used. After equilibrium is attained the cell is fractionated, producing a centripetal segment length 3.0 times or greater than the centrifugal segment. The corresponding solute concentration in the homogenized upper cell section is determined. This value, in connection with an approximate equation allows evaluation of the parameter σ = sω 2 D . Subsequent use of Svedbergs formula yields the molecular weight of the centrifuged substance. Adsorption effects, diffusion perturbations, concentration limits, reproducibility of working conditions, and extension of the technique to macro volumes were studied.

A sectorial centrifuge cell for swinging bucket rotors--application to a velocity gradient centrifugation methodology.

A sectorial cell of 1.55 ml capacity, designed to be used in swinging bucket rotors, is introduced and applied to boundary sedimentation studies. The cell, made of polycarbonate by injection molding, represents an improvement over previously existing models in terms of resistance and attainable speeds. Its use has been extended to polydispersity determination by evaluation of the s value distribution function g(s) = dC/ds. The latter can be corrected to standard conditions (water at 20 degrees C), by using newly derived equations in connection with a cesium sulfate linear density and viscosity gradient which is introduced for boundary stabilization. The cell performance is illustrated with the centrifugal characterization of a polydisperse hyaluronate solution and a paucidisperse proteoglycan A1 preparation. In the latter case a distinctive distribution of aggregates into two polydisperse families of molecules, hitherto not reported in the literature but previously observed with cylindrical cells in this laboratory, has been clearly confirmed. Analysis of plateau dilution during centrifugation indicated absence of artifacts.

Unipolar Induction Revisited: New Experiments and the “Edge Effect” Theory

A brief historical review is made of the 180-year-old debate on Faradays unipolar inductor. By introducing two convenient modifications of Faradays original experiment of 1832, pertinent answers are experimentally found to the most debated problems: 1) Can Faradays law be used? Yes; 2) Do the magnetic field lines rotate when the magnet rotates? No. 3) Can the seat of induction be unambiguously determined? Yes. 4) Is there a fundamental difference between rotational and translational motional induction? Yes: the “edge effect”, whereby a negative v×B field appears whenever a magnetic edge moves perpendicularly to itself. An additional experiment is presented to verify the theory. Finally, 5) Can Relativity Theory be applied? The Special Theory, no; the General one, yes.

Structural Changes of Sulfated Proteoglycans of the Growth Cartilage of Rats during Endochondral Calcification

Publisher Summary The chapter focuses on structural changes of sulfated proteoglycan of the growth cartilage of rats during endochondral calcification changes, as reflected through their sedimentation properties. Previous findings have suggested a regulatory capacity of proteoglycan aggregates in cartilage mineralization and a role of lysozyme as a biochemical cell mediator to regulate the size of these macromolecules. Lysozyme could probably exert this regulation by influencing the mol. wt. of hyaluronic acid or other structural characteristics of this substance essential to the formation of large aggregates. Samples of cartilage fluid were obtained from the expanded growth cartilage of two groups of rachitic rats: (1) phosphate and vitamin D deficient and (2) phosphonate treated. Samples were also obtained from animals undergoing 72–76 h of healing from rickets: Group A, after restoring phosphate and vitamin D in the diet and Group B, interrupting the addition of EHDP after a 10- day treatment. Concentrations of phosphate and hexuronate and cartilage lysozyme activity were determined. Centrifugal properties of proteoglycan were studied by use of the transport methodology, which has been recently modified as a gradient-velocity centrifugation technique.

The Edge Effect in a Rectilinear Motional Induction Experiment

Modification of a previously described rotational unipolar induction experiment leads to a rectilinear version of the same, in which the induction of an electromotive force can be obtained independently of the relative motion between magnet and conductor, even when both are moving in a straight line. This experiment seems to contradict the usual relativistic canon that motional electromagnetic induction always requires relative motion between conductors and magnets. The theoretical and practical consequences of the new experiment are discussed.

Treatment of Osteoarthritis with Tiaprofenic Acid and Indomethacin

SummaryCage-matched control and experimental greyhounds were sacrificed 12 weeks after production of instability in the right knee, due to anterior cruciate ligament cut. Eroded osteoarthritis and normal articular cartilage were used for the analyses. In these tissue samples, significant histological protection against cartilage breakdown was afforded by orally administered tiaprofenic acid 5 mg/kg bodyweight but not by orally administered indomethacin 1 mg/kg bodyweight. Tiaprofenic acid caused preservation of fast sedimenting proteoglycan aggregates, as well as retention of hyaluronate content and favourable proteoglycan aggregate S value levels. Biochemical data on indomethacin remain to be reported.