Elimination of tritium-labelled hyaluronic acid from normal and osteoarthritic rabbit knee joints.

The half-life of [3H]hyaluronic acid in rabbit knee joints was estimated using two methods: (i) by following the [3H]hyaluronan content of the synovial fluid after intra-articular injection and (ii) by following the 3H2O radioactivity of plasma after intra-articular injection of [3H]hyaluronan. For normal rabbits we obtained a half-life of 15.8 hours (method I) and 17.5 +/- 1.0 hours (mean +/- SEM, method II), respectively. The second method was used to estimate the kinetics of the hyaluronan elimination from normal, sham-operated, as well as from osteoarthritic rabbit knee joints (Colombo model of osteoarthritis). Four weeks after injury, during the developing phase of osteoarthritis, the half-life of hyaluronan rose significantly to 23.5 +/- 2.1 hours and returned to normal levels (17.4 +/- 2.7 hours) 12 weeks after the operation (osteoarthritis developed). At the stage of developed osteoarthritis, the clearance rates were considerably higher than in normal rabbits.

[Experimental studies of mechanically-induced articular cartilage defects following implantation of allogeneic embryonal chondrocytes in a collagen-fibrin gel in chickens]. / Experimentelle Untersuchungen mechanisch induzierter Gelenkknorpeldefekte nach Implantation allogener embryonaler Chondrozyten in einem Kollagen-Fibrin-Gel beim Huhn.

Full thickness defects (diameter 1,7 mm; depth 2,5 mm) were created mechanically in articular cartilage and subchondral bone of the condyles of tibiotarsal joints of 9-month old chickens. This full-thickness defects were repaired with cultured allogenic embryonic chick epiphyseal chondrocytes from the tibiae and femura of 10-days-old chicken embryos. The cells were embedded in a collagen-fibrinogen-matrix. Controls were similarly operated, but received either no treatment or implants the delivery substance only. Healing of the defects was observed macroscopically, histologically, histochemically and histomorphometrically after 3, 12 and 24 weeks. This graft was successfully transplanted in mechanically induced defects in 80%. The resulting hyaline cartilage was structurally reorganized according to the host pattern and under the influence of environmental conditions. The articular zone preserved it's cartilaginous phenotype, whereas the subchondral regions were transformed into bone. 12 weeks after the operation the defects in the experimental group were completely filled. In all instances in this group, there was an initial extreme increase of mitotic rate and cell number. After 24 weeks normal and subnormal values were founded. The defects in the control groups healed with fibrocartilage. Our results showed, that only the defects in the experimental group were completely filled with reparative hyaline cartilage tissue. In the present study the mixture of cultured allogenic embryonic chondrocytes and a collagen-fibrinogen-matrix was used successfully as a transplant for repairing defects in articular cartilage of chickens. Thus allogenic transplantation of cultured embryonal chondrocytes appears to be one of the most promising methods for the restoration of articular cartilage.

[Synovial volume of healthy and arthrotic human knee joints]. / Das Synovia-Volumen gesunder und arthrotischer menschlicher Kniegelenke.

The volume of synovial fluid in human knee joints was determined by the dilution method through the intra-articular injection of 35 ml 0.5% hydroxyethyl starch in saline. In 15 control knees the mean synovial volume was 6.7 +/- 2.3 ml (mean +/- SD). In patients with latent gonarthrosis we found a mean intra-articular volume of 13.6 +/- 7.4 ml (n = 21) and in activated gonarthrosis 24.2 +/- 16.3 ml (n = 34). All 3 groups differ significantly. After entire aspiration of the effusion, we found yet 13.4 +/- 4.4 ml (n = 32) synovial fluid in the knee joint. Using the dilution method, we obtained a punctate from each knee joint. The punctates can be used for biochemical investigations. Since the dilution factors of the punctates are known, the concentrations of HWM parameters in the original synovial fluid can be calculated.

[Analysis of hyaluronic acid in punctates in biochemical functional tests of joints]. / Bestimmung von Hyaluronsäure in Punktaten des biochemischen Gelenkfunktionstests.

The punctates of the biochemical joint function test contain hydroxyethyl starch which disturbs the assay of uronic acid and glucosamine for the estimation of hyaluronate. Therefore hyaluronate (0.03-0.75 g/l) was estimated by precipitation with Alcian blue on cellulose acetate membranes and measuring of the absorbance at 678 nm after dissolution in aqueous SDS (75 g/l). The gel-filtration HPLC separates the high-molecular-mass hyaluronate from low-molecular-mass impurities of biological samples. By comparing with a standard curve (0.5-2.5 g/l hyaluronate) the concentration can be determined. The results obtained by the two methods were significantly correlated (p less than 0.05). The Alcian blue technique is useful for rapid screening, the HPLC technique to confirm these values.

[The gradients of the inorganic components in the enamel and dentin of the deciduous teeth. II. A comparison of the concentrations of zinc, magnesium, calcium and lead]. / Gradienten anorganischer Bestandteile im Schmelz und Dentin von Milchzähnen. II. Vergleich der Konzentrationen von Zink, Magnesium, Kalzium und Blei.

The concentration of zinc, magnesium, calcium and lead in four layers of dental hard tissue was analysed in 108 deciduous teeth of children from nine different places of the G.D.R. The result was a contrary gradient from enamel to dentine of magnesium in comparison with zinc, lead and calcium.

[Plasminogen activator activity of the synovial fluid as an indicator of activation phenomena in degenerative joint diseases]. / Die Plasminogenaktivator-Aktivität der Synovialflüssigkeit als Indikator für Aktivierungsphänomene bei degenerativen Gelenkerkrankungen.

Activity of plasminogen activator in synovial fluids of patients with osteoarthritis was determined by a radial diffusion assay. Synovial fluid from human knee joints was obtained by joint lavage with 35 ml physiological saline containing 0.5% hydroxyethyl starch. Synovial fluids from contralateral, healthy knee joints served as controls. The activity of plasminogen activator in synovial fluid from activated (painful) osteoarthritis is significantly higher than in cases of latent osteoarthritis, whereas the activity of controls is significantly lower as in latent osteoarthritis. The findings are helpful for the discovery of patients with enhanced plasminogen activator levels in synovia which should be treated therapeutically.

The influence of mechanical loading on the knee-joint. Animal-experimental examinations.

A physiological maximal loading of the knee-joint of sheep is simulated by a 4-hour peak overloading of 24.5 N and a frequency of 42 impulses per minute. The reactions of articular cartilage, synovia and periarticular muscular tissue to the load were examined histologically, histochemically and biochemically. 20 hours after the loading we could observe a leucocytic influx into the joint and a metabolic hyperactivity in the examined tissue. The changes found during this acute phase are reversible as 180 days after peak overloading the parameters detected to be changed had returned to normal again.

[Gradients of inorganic components in enamel and dentin of deciduous teeth. I. Lead concentration in children with variable environmental conditions]. / Gradienten anorganischer Bestandteile im Schmelz und Dentin von Milchzähnen. I. Bleikonzentration bei Kindern mit unterschiedlichen Umweltbedingungen.

The concentration of lead in four layers of dental hard tissues was analysed in 108 deciduous teeth of children from nine places of the G.D.R.. The result was a typical gradient from enamel to dentine. The average concentration of lead corresponds with that of other industrial countries. Although regional differences were found equal exposition of lead for both urban and rural areas can be assumed.

Low molecular mass trypsin inhibitors in normal and osteoarthritic human articular cartilage.

A modified radial diffusion assay was used for the direct semiquantitative determination of low molecular mass trypsin inhibitors in small samples of human cartilage. The low molecular mass trypsin inhibitor in articular cartilage of normal human femoral heads is not distributed evenly but occurs in areas of low, medium and high content. The weight-bearing area of the femoral head belongs among the regions with low inhibitor content. The results obtained with osteoarthritic femoral heads showed that the inhibitor content in osteoarthritic cartilage is significantly lower than that in normal articular cartilage (p less than 0.1%).

[Risk of hemorrhage in the knee joint following intraarticular injections]. / Das Blutungsrisiko in das Kniegelenk nach intraartikulären Injektionen.

The relative and absolute blood contents in the knee joint after intraarticular injections were determined using a biochemical joint function test. Normally, only minimal amounts of blood leak into the joint cavity, but in a few cases greater quantities were found, unnoticed at the time of injection. Intraarticular injections should be done only by a specialist.

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay.

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.

[Detection of low molecular weight trypsin inhibitors in small samples of cartilage, cartilage extracts and synovial fluid by gel diffusion]. / Nachweis von niedermolekularen Trypsininhibitoren in kleinen Knorpelproben, in Knorpelextrakten und Gelenkflüssigkeit mittels Geldiffusion.

The radial diffusion assay is a very useful method for detection of low amounts of proteinase inhibitors in biological materials. The determination of low molecular weight (LMW) inhibitors in the presence of high molecular weight inhibitors is possible by the combination of radial diffusion and ultrafiltration. Using this method LMW trypsin inhibitors could be demonstrated in human articular cartilage, but not in human synovial fluid. In cow, pig and sheep a LMW trypsin inhibitor could be found in both the articular cartilage and in the synovial fluid. On the other hand, a LMW trypsin inhibitor could not be found neither in the canine cartilage nor in the canine synovial fluid. The method allows also the direct determination of LMW trypsin inhibitors in cartilage extracts in the presence of 4 M guanidinium hydrochloride or 6 M urea. Therefore, the method is recommended for direct determination of LMW inhibitors by column chromatographic separations of inhibitors.

Investigations for the development of a biochemical joint function test.

The reversible and thus potentially curable, early stage of degenerative joint diseases, so consequential for both the individual and the economy, has so far not been diagnosable objectively. Intraarticular functional tests aim at detecting latent metabolic changes in the joint in order to find out early changes in risked persons. The present animal experiments on osteoarthrosis showed early changes of the glycogen metabolism and the swelling behaviour of the articular cartilage. Therefore, the diagnostic value of intraarticular injections of depot glucose was tested in the form of metabolizable hydroxyethyl starch and the non-metabolizable dextran, which were administered as non-buffered solutions. 35 ml of 0.5% solutions of hydroxyethyl starch or dextran in isotonic saline were injected intraarticularly to unselected patients before knee operations. By means of punctures of the joint 10 min, 1 h, 5 h, and 24 h after the injection, about 3 ml fluid samples were taken for biochemical, biophysical and cytological examinations. The examination produced, for the first time, evidence on the volume and composition of the synovia of osteoarthrosis patients from whom no synovial fluid can be obtained. The analysis of the punctates after loading permits of grouping the patients according to osteoarthrotic changes and various reaction patterns of the synovial marginal tissue with regard to disposition to exudation.

[Sulfated macromolecules in connective tissue and their metabolism]. / Sulfatierte Makromoleküle des Bindegewebes und ihr Stoffwechsel.

Sulfated macromolecules are one of the major constituents of the extracellular matrix of connective tissue. The degree of sulfation is responsible for the physico-chemical properties of the extracellular matrix. Especially the fixed charged groups contribute to the reversible compressibility of the cartilage. The sulfation may play also a role in the regulation of metabolic pathways. The derivatives of sulfate are formed by the help of specific sulfotransferases, transferring the "active" sulfate group from PAPS to the macromolecules. Too low an activity of certain sulfotransferases is responsible for some types of spondyloepiphyseal dysplasia, and too low a concentration of PAPS leads to dwarfism. The sulfate groups of the macromolecules are degraded by specific sulfatases. A genetically caused deficiency in one of these enzymes (mucopolysaccharidoses) is frequently associated with skeletal deformities. The amount and composition of proteoglycans is altered in the ontogenesis. Similar alterations are associated with ageing and arthrotic processes, being difficult to differentiate. The characterization of the alterations in degenerative joint diseases, especially, is important for ethiopathogenetic studies. The most widely used methods for the determination of sulfated macromolecules and particularly analytical parameters of the sulfate metabolism are described.