Biomedical subjects
Publications and source records attributed to M Tammi.
A large treadmill was constructed to allow simultaneous running and training of 10 dogs. During a period of 1 year, 10 beagles ran up to 40 km/day at a speed of 5.5 to 6.8 km/h and with an uphill inclination of 15 degrees. By the end of training, the daily running sessions lasted 7 hours. The design of the treadmill and experience of the treadmill training is given. The apparatus was reliable and convenient to use.
The distribution of proteoglycans (PGs) at 11 sites on the knee (stifle joint) cartilage of young female beagle dogs was studied following cast immobilization for 11 weeks in 90 degrees flexion and after a subsequent remobilization for 15 weeks. Immobilization induced a reduction in PG uronic acid at all sites (mean of -38%), but the greatest depletion (-64%) occurred at the anterior and posterior extremes of the femoral condyles, i.e., at locations where the immobilized cartilage lost contact to the opposing cartilage. Following remobilization, the content of uronic acid remained lower than in the age-matched controls (-18% on average), particularly at the minimum contact sites most affected by immobilization (-33%). The chondroitin-6-sulfate to chondroitin-4-sulfate ratio was reduced by immobilization in most locations (average of -14%) and returned to control values after remobilization. There was no consistent change in the percentage of aggregating PGs observed in Sephacryl S-1000 gel filtration after immobilization or remobilization. However, following remobilization, the aggregating PGs showed an enhanced proportion of the slower mobility band in agarose gel electrophoresis, indicative of a larger monomer size. In the contralateral, load-bearing knee joint, both the uronic acid content and PG monomer type distribution were identical to those observed in the experimental joint, suggesting that the state reached after the remobilization period was due to factor(s) influencing both sides. The results suggest that contact forces between articulating surfaces are required to maintain normal PG content and that the control mechanism works locally at each cartilage site. Restriction of joint mobility and loading in young animals is concluded to cause persistent changes in cartilage matrix. Furthermore, the use of the contralateral joint as the sole control in this kind of studies, although experimentally convenient, seems not to be appropriate.
A novel method is introduced for the estimation of grain numbers in autoradiographic sections of articular cartilage with an image analyzer. It is based on separation of grains from the underlying structures by gray level thresholding and determination of the percentage of total area occupied by grains in a relatively large measuring field. The mean grain size is used as a reference to calculate grain numbers per cell profile and per unit area of tissue in various zones of bovine articular cartilage labelled with 35S-sulphate in tissue culture. The results demonstrate considerable zonal differences as well as site related topographic variation in the rate of 35S-sulphate incorporation. The largest site-related variation in the grain counts was observed in the superficial zone, suggesting a delicate control of proteoglycan synthesis in this zone.
Biochemical data suggest that gingival epithelium contains hyaluronate, but there is little histochemical information about its localization. Hyaluronate was here visualized in gingival and buccal mucosa using a specific probe derived from the hyaluronate binding region of cartilage proteoglycan. Hyaluronate was found both in the gingival and buccal epithelium, but its localization was correlated with the type of keratinization. In the keratinized epithelium of gingiva, whether ortho- or parakeratotic, the intercellular spaces from basal to upper spinous layers displayed strong staining, most intense in the middle spinous cell layer. The uppermost vital cell layers as well as the cornified cell layer remained unstained. In the non-keratinized epithelium of buccal mucosa and the local non-keratinized areas of gingiva, only the basal cells and the lowermost spinous cell layers stained for hyaluronate, whereas the majority of the upper epithelium was negative. Electron microscopic examination of the basal and spinous cell layers displayed hyaluronate, both associated with the cell surface and free in the intercellular space. The subepithelial connective tissue showed positive but diffuse staining in all specimens.
The indentation stiffness of knee articular cartilage subjected to strenuous physical training (SPT: treadmill running 20 km day-1 for 15 weeks, n = 6) of young Beagles was tested and compared to that obtained from age-matched (55 weeks, n = 9) controls. The mathematical solution for the shear modulus, as determined from indentation of an elastic layer bonded to a rigid half space, was extended to small Poisson's ratios and applied to the analysis of cartilage response after a step stress (0.39 MPa) application. In these measurements with an impervious, plane-ended indenter, the equilibrium deformation was systematically greater than values predicted from the instant response by the linear biphasic theory. Therefore, the accurate determination of Poisson's ratio from the creep curves was not possible. The mean shear modulus (calculated by using the deformation at 900 s after load application and assuming a constant Poisson's ratio of 0.40 for the matrix) of canine knee articular cartilage was 0.37 MPa. While the cartilage thickness was not affected by SPT, the cartilage of the lateral tibial plateau was stiffer (13.3%, p less than 0.05) than that in controls. However, in the femoral condyles, the stiffness was at the control level or even below. Our results on cartilage structure and properties suggest that SPT, in contrast to our previous findings with moderate training, does not necessarily improve the biological properties of articular cartilage in young animals.
The levels and types of proteoglycans in articular cartilage of the knees of young beagle dogs were studied after 15 weeks of running exercise, at 4 km/day. Running increased the levels of proteoglycans in the cartilage of the patella, the superior patellofemoral groove, and the summit of the medial condyle of the femur, all of which are considered contact sites subject to enhanced loading caused by running. The elevated content of uronic acid at the femoral sites proved to be due to proteoglycans that were unable to aggregate with hyaluronic acid. There was no change in the content of aggregating proteoglycans. Analysis of chondroitinase AC-derived disaccharides at the same sites showed an increase in chondroitin-6-sulfate content as compared with chondroitin-4-sulfate levels. We believe that this modulation of the proteoglycan matrix reflects enhanced tissue maturation and physiologic adjustment to higher local contact pressures.
The restoration of the biomechanical properties of articular cartilage was studied after 15 weeks of remobilization of the knee joint in beagles previously immobilized with a cast for 11 weeks. The shear moduli were determined with an indentation creep test immediately after load application and at equilibrium at six predefined test points of femoral, tibial, and patellar cartilages. Permeability of the cartilage was estimated from the creep measurements. The values were compared with nontreated, age-matched (55 weeks) controls and with cartilage collected immediately after immobilization. Remobilization reduced the high creep rates created by immobilization and shifted the depressed equilibrium shear moduli towards those of the controls. However, in the femoral condylar cartilage, the equilibrium shear modulus remained at lower level (p less than 0.05) and permeability at higher level (p less than 0.05) as compared with the controls. We conclude that articular cartilage, showing signs of atrophy after long-term immobilization, was capable of restoring its biomechanical properties during remobilization. This repair was not, however, completed in all parts of the knee joint by the end of the observation period.
An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.
Retinoic acid (RA) has been shown to retard the differentiation of epidermal keratinocytes by several morphologic and biochemical criteria. In this study, the epidermal content and localization of hyaluronate (HA), as well as its synthesis and disappearance in human skin organ culture, were characterized to test the idea that some of the RA influences on epidermal differentiation are associated with keratinocyte HA metabolism. RA stimulated the incorporation of 3H-glucosamine into HA by up to 60% at concentrations between 50 nM and 5 microM, while pulse-chase experiments revealed little change in its disappearance rate from epidermis. After 5 d in culture, the chemically quantified HA was more than doubled in the treated epidermis. The accumulation of HA was substantiated by light and electron microscopy with a specific probe prepared from the HA binding region of cartilage proteoglycan. The staining was particularly enhanced between the upper spinous cell layers, where the terminal differentiation into corneocytes normally takes place. A patchy, discontinuous staining was also seen in stratum granulosum and corneum layers, which are not stained at all in control cultures. The present study demonstrates that RA leads to an accumulation of HA in the superficial layers of epidermis by stimulating its synthesis in keratinocytes. This may account for the delay in terminal differentiation, and the weakened cohesion of the keratinocytes previously observed both in vivo and vitro.
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Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.
Lymphocyte interactions with high endothelial venules (HEV) are important to the in vivo migration of normal and neoplastic lymphocyte populations. We have previously described an 85- to 95-kDa lymphocyte surface glycoprotein(s) defined by mAb Hermes-1, that is involved in the recognition of HEV by human lymphocytes: antibodies against distinct epitopes of the Hermes-1 Ag differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial HEV. Here we characterize further the Hermes-1-defined glycoproteins. No well defined differences were observed between the Hermes-1 Ag immunoprecipitated from PBL and from mucosa- vs lymph HEV-specific cell lines. The Ag is an acidic (isoelectric point = 4.2) sulfated molecule bearing both O-linked and (3,4) N-linked oligosaccharide side chains. A subset of the Hermes-1-immunoprecipitated species is modified by covalent linkage to chondroitin sulfate, yielding a Mr of approximately 180 to 200 kDa. Pulse-chase labeling reveals a major precursor of 76 kDa that appears to be processed either to the 85- to 95-kDa form or, by addition of chondroitin sulfate, to a 180- to 200-kDa form. The potential role of these structural modifications, and particularly of chondroitin sulfate, in the function of the putative adhesion molecules is discussed.
Proteoglycan (PG) and glycosaminoglycan (GAG) samples corresponding to a minimum of 10 ng of uronic acid were reliably quantified as precipitates with the cationic dye Safranin O, collected by vacuum-aided filtration onto a cellulose acetate/nitrate membrane in a standard 96-well dot assay apparatus. The reflectances of the precipitation dots were measured by automatic densitometric scanning of the membrane sheets. Standard GAGs produced reflectance values which were related to the number of anionic groups per unit disaccharide; hyaluronate and keratan sulfate gave lower values while heparin yielded values higher than those of chondroitin sulfates. The presence of 8 M urea, 1% Triton X-100, 30% sucrose, 0.02% NaN3, or mixtures of proteinase inhibitors and various buffers did not markedly influence the reflectances, while 4 M guanidinium chloride and 3 M CsCl reduced the sensitivity of the assay to 30-50 ng. Samples containing sodium dodecyl sulfate (SDS) were not applicable because SDS precipitated with Safranin O. Proteins showed virtually no response, while nucleic acids gave significant although smaller reflectances than GAGs. Owing to its marked sensitivity and convenience the method is particularly suitable for the detection of PGs during their preparative purification and fractionation as well as in various analytical assays.
The local influences of physical exercise on thickness and glycosaminoglycan (GAG) content of canine articular cartilage were measured by microspectrophotometry of Safranin O- and periodic acid-Schiff (PAS)-stained tissue sections. Female Beagle dogs were housed in individual cages (bottom 0.9 x 1.2 m) and divided into runner (n = 6) and control (n = 8) groups. The training program started at the age of 15 weeks. During the subsequent 10 weeks, the dogs were accustomed to running on a treadmill inclined 15 degrees uphill. Thereafter, the dogs ran 1 h daily, 5 days a week, at a speed of 4 km/h for 15 weeks. At the age of 40 weeks, the dogs were killed, and the samples for histology were taken from 11 different anatomical locations of the right knee (stifle) joint. The thickness of the uncalcified cartilage increased 19-23% on the lateral condyle and patellar surface of the femur, whereas the enhancement was smaller in other parts of the trained cartilage. The calcified cartilage did not show thickness alterations. Total GAGs were augmented by 28% in the summits on the femoral condyles, more on the medial than lateral side. The increased GAGs appeared to be predominantly chondroitin sulphates and were localized in the intermediate, deep, and even in the calcified zones, whereas the superficial zone did not show changes. There was a concomitant increase of non-GAG oligosaccharides in the intermediate and deep zones, but not in the calcified cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)
Proteoglycans (PGs) and collagen were quantified in the knee articular cartilages of femoral and tibial medial condyles following 1-8 weeks of moderate running exercise of 4- to 6-month-old rabbits. The total content of PGs extractable with 4 mol/l guanidium chloride was elevated in the weight-bearing cartilage of the tibial medial condyle, while their concentration, expressed as uronic acid per wet weight, and collagen remained unchanged. The content of glucosamine (GlcN) and its ratio to galactosamine (GalN) was elevated in femoral cartilage PGs purified by centrifugation in dissociative CsCl gradients, indicating an increase in keratan sulfate. After 8 weeks of running, the chondroitin sulfate chains of PGs from tibial medial condyle contained less unsulfated disaccharide units. The content of chondroitin sulfate was elevated in the nonextractable residue of the tibial medial condyle as indicated by uronic acid and GaN assays. The content of nonextractable GlcN was increased even more, both in tibial medial and femoral cartilages. Moderate running thus increased (1) keratan sulfate-rich PGs, (2) the degree of sulfation of the chondroitin sulfate chains, and (3) nonextractable PGs; these modulatory alterations probably enhance the stability and elastic stiffness of the PG matrix.
Hyaluronate is actively synthesized by cultured epidermis and dermis, but no direct histological data have been available about its localization in normal human skin. A hyaluronate-specific biotinylated probe, prepared from the hyaluronate binding region of cartilage proteoglycan, was applied to human skin sections and visualized using the biotin-avidin-peroxidase system. The specificity of this staining was confirmed by hyaluronidase predigestion and by hyaluronate-derived oligosaccharides added to the staining solution. All dermis showed diffuse binding of the probe, but the highest staining intensity was observed in the epidermal intercellular spaces. The stainability extended from basal cells to the middle layers of the epidermis, whereas the granular layer and stratum corneum were completely negative. Also, the basal side of basal cells (basement membrane) did not bind the hyaluronate probe. The abundance of hyaluronate on surfaces and intercellular spaces of the spinous cells is suggested to have an important role in the physiology of human epidermis.
Effects of long-term physical training (treadmill running 4 km/day for 15 weeks) and immobilization (casting of the limb for 11 weeks) on the blood flow, bone marrow uptake of radio-colloid, histomorphology as well as elemental composition of the proximal canine femur were investigated. In the runners both bone marrow and mean bone perfusions measured with a 133Xe washout method were increased by 53.5% (P less than 0.05) and 54.1% (P less than 0.05), respectively, as compared with the age-matched controls. Bone marrow uptake of [99mTc]tincolloid and volumetric fractions of trabecular bone and bone marrow were not significantly altered after training or immobilization. The amount of zinc measured by atomic absorption spectrophotometry was, however, significantly higher (P less than 0.01) after immobilization as compared with the controls. Consequently, long-term physical training is suggested to stimulate bone metabolism by enhancing its blood perfusion even in the resting state, whereas the increase of zinc content was considered to be linked to the obvious bone resorption caused by immobilization.
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