Mikko J. Lammi

Epidermal Growth Factor Activates Hyaluronan Synthase 2 in Epidermal Keratinocytes and Increases Pericellular and Intracellular Hyaluronan

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3–5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitroin a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2–3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from ∼6 copies/cell in cultures before change of fresh medium, up to ∼54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of ∼21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.

Remobilization does not fully restore immobilization induced articular cartilage atrophy.

The recovery of articular cartilage from immobilization induced atrophy was studied. The right hind limbs of 29-week-old beagle dogs were immobilized for 11 weeks and then remobilized for 50 weeks. Cartilage from the immobilized knee was compared with tissue from age matched control animals. After the immobilization period, uncalcified articular cartilage glycosaminoglycan concentration was reduced by 20% to 23%, the reduction being largest (44%) in the superficial zone. The collagen fibril network showed no significant changes, but the amount of collagen crosslinks was reduced (13.5%) during immobilization. After remobilization, glycosaminoglycan concentration was restored at most sites, except for in the upper parts of uncalcified cartilage in the medial femoral and tibial condyles (9% to 17% less glycosaminoglycans than in controls). The incorporation of 35SO4 was not changed, and remobilization also did not alter the birefringence of collagen fibrils. Remobilization restored the proportion of collagen crosslinks to the control level. The changes induced by joint unloading were reversible at most sites investigated, but full restoration of articular cartilage glycosaminoglycan concentration was not obtained in all sites, even after remobilization for 50 weeks. This suggests that lengthy immobilization of a joint can cause long lasting articular cartilage proteoglycan alterations at the same time as collagen organization remains largely unchanged. Because proteoglycans exert strong influence on the biomechanical properties of cartilage, lengthy immobilization may jeopardize the well being of articular cartilage.

Speed of sound in normal and degenerated bovine articular cartilage

The unknown and variable speed of sound may impair accuracy of the acoustic measurement of cartilage properties. In this study, relationships between the speed of sound and cartilage composition, mechanical properties and degenerative state were studied in bovine knee and ankle cartilage (n = 62). Further, the effect of speed variation on the determination of cartilage thickness and stiffness with ultrasound (US) indentation was numerically simulated. The speed of sound was significantly (n = 32, p < 0.05) dependent on the cartilage water content (r = -0.800), uronic acid content (per wet weight, r = 0.886) and hydroxyproline content (per wet weight, r = 0.887, n = 28), Youngs modulus at equilibrium (r = 0.740), dynamic modulus (r = 0.905), and degenerative state (i.e., Mankin score) (r = -0.727). In addition to cartilage composition, mechanical and acoustic properties varied significantly between different anatomical locations. In US indentation, cartilage is indented with a US transducer. Deformation and thickness of tissue are calculated using a predefined speed of sound and used in determination of dynamic modulus. Based on the simulations, use of the mean speed of sound of 1627 m/s (whole material) induced a maximum error of 7.8% on cartilage thickness and of 6.2% on cartilage dynamic modulus, as determined with the US indentation technique (indenter diameter 3 mm). We believe that these errors are acceptable in clinical US indentation measurements.

Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life: a hypothesis

Regular joint loading in youth assists in the establishment and strengthening of the collagen network of articular cartilage and contributes to the prevention of osteoarthrosis later in life. A hypothesis.

Immobilisation causes longlasting matrix changes both in the immobilised and contralateral joint cartilage

OBJECTIVE The capacity of articular cartilage matrix to recover during 50 weeks of remobilisation after an atrophy caused by 11 weeks of immobilisation of the knee (stifle) joint in 90° flexion starting at the age of 29 weeks, was studied in young beagle dogs. METHODS Proteoglycan concentration (uronic acid) and synthesis ([35S]sulphate incorporation) were determined in six and three knee joint surface locations, respectively. Proteoglycans extracted from the cartilages were characterised by chemical determinations, gel filtration, and western blotting for chondroitin sulphate epitope 3B3. RESULTS The proteoglycan concentrations that were reduced in all sample sites immediately after the immobilisation, remained 14-28% lower than controls after 50 weeks of remobilisation in the patella, the summit of medial femoral condyle, and the superior femoropatellar surface. In the contralateral joint, there was a 49% increase of proteoglycans in the inferior femoropatellar surface after remobilisation, while a 34% decrease was simultaneously noticed on the summit of the medial femoral condyle. Total proteoglycan synthesis was not significantly changed after immobilisation or 50 weeks’ remobilisation in the treated or contralateral joint, compared with age matched controls. The chondroitin 6- to 4- sulphate ratio was reduced by immobilisation both in the radioactively labelled and the total tissue proteoglycans. In the remobilised joint, this ratio was restored in femur, while in tibia it remained at a level lower than controls. Neither immobilisation nor remobilisation induced epitopes recognised by the monoclonal antibody 3B3 on native (undigested) proteoglycans. CONCLUSION These results show that the depletion of proteoglycans observed after 11 weeks of immobilisation was not completely restored in certain surface sites after 50 weeks of remobilisation. The significant changes that developed in the contralateral joint during the remobilisation period give further support to the idea that a permanent alteration of matrix metabolism results even from a temporary modification of loading pattern in immature joints.

Stress–relaxation of human patellar articular cartilage in unconfined compression: Prediction of mechanical response by tissue composition and structure

Mechanical properties of articular cartilage are controlled by tissue composition and structure. Cartilage function is sensitively altered during tissue degeneration, in osteoarthritis (OA). However, mechanical properties of the tissue cannot be determined non-invasively. In the present study, we evaluate the feasibility to predict, without mechanical testing, the stress-relaxation response of human articular cartilage under unconfined compression. This is carried out by combining microscopic and biochemical analyses with composition-based mathematical modeling. Cartilage samples from five cadaver patellae were mechanically tested under unconfined compression. Depth-dependent collagen content and fibril orientation, as well as proteoglycan and water content were derived by combining Fourier transform infrared imaging, biochemical analyses and polarized light microscopy. Finite element models were constructed for each sample in unconfined compression geometry. First, composition-based fibril-reinforced poroviscoelastic swelling models, including composition and structure obtained from microscopical and biochemical analyses were fitted to experimental stress-relaxation responses of three samples. Subsequently, optimized values of model constants, as well as compositional and structural parameters were implemented in the models of two additional samples to validate the optimization. Theoretical stress-relaxation curves agreed with the experimental tests (R=0.95-0.99). Using the optimized values of mechanical parameters, as well as composition and structure of additional samples, we were able to predict their mechanical behavior in unconfined compression, without mechanical testing (R=0.98). Our results suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing.

Densitometric assay of nanogram quantities of proteoglycans precipitated on nitrocellulose membrane with Safranin O

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.

Ultrasound indentation of normal and spontaneously degenerated bovine articular cartilage

OBJECTIVE We have previously developed a handheld ultrasound indentation instrument for the diagnosis of cartilage degeneration. The instrument has been demonstrated to be capable of quantifying mechanical and acoustic properties of enzymatically degraded and normal bovine articular cartilage in vitro and in situ. The aim of this study was to investigate the sensitivity of the instrument to distinguish between normal and spontaneously degenerated (e.g., in osteoarthrosis) articular cartilage in vitro. DESIGN Thirty articular cartilage samples were prepared from the bovine lateral patellae: 19 patellae with different degenerative stages and 11 patellae with visually normal appearance. Cartilage thickness, stiffness (dynamic modulus) and ultrasound reflection from the cartilage surface were measured with the handheld instrument. Subsequently, biomechanical, histological and biochemical reference measurements were conducted. RESULTS Reproducibility of the measurements with the ultrasound indentation instrument was good. Standardized coefficient of variation was < or =6.1% for thickness, dynamic modulus and reflection coefficient. Linear correlation between the dynamic modulus, measured with the ultrasound indentation instrument, and the reference dynamic modulus was high (r=0.993, n=30, P<0.05). Ultrasound reflection coefficient, as determined from the cartilage surface, showed high linear correlations (typically r(2)>0.64, n=30, P<0.05) with the cartilage composition and histological or mechanical properties. The instrument was superior compared to visual evaluation in detecting tissue degeneration. CONCLUSION This study indicates that the ultrasound indentation technique and instrument may significantly improve the early diagnosis of cartilage degeneration. The results revealed that visual evaluation is insensitive for estimating the structural and mechanical properties of articular cartilage at the initial stages of degeneration.

Altered Golgi apparatus in hydrostatically loaded articular cartilage chondrocytes.

OBJECTIVES: Articular cartilage proteoglycan content is controlled by joint loading. This study aimed to elucidate the role of hydrostatic pressure in this regulation. METHODS: Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure, applied continuously or cyclically at 0.125 or 0.05 Hz. The Golgi apparatus was visualised either by a fluorochrome coupled wheat germ agglutinin or by transmission electron microscopy. Proteoglycan synthesis was studied by the incorporation of sulphur-35 labelled sulphate. RESULTS: After 30 MPa continuous hydrostatic pressure, the Golgi apparatus was observed in a compact form with a concomitant decrease in proteoglycan synthesis. The normal stacked appearance of the Golgi apparatus was no more visible in the electron microscopy preparation of the pressurised chondrocytes. This effect was reversible and was also noticed after 15 MPa continuous load, though to a minor extent. Cyclic pressures (5-30 MPa) caused no apparent change in the Golgi apparatus. The shape of some cells changed to a more retracted form after 30 MPa continuous pressure. Nocodazole, which causes disassembly of the microtubules, blocked the compacting influence of pressurisation on the Golgi apparatus, and reduced proteoglycan synthesis to about half of the control level. CONCLUSIONS: The packing of the Golgi apparatus is dependent on microtubules and may contribute to the inhibition of proteoglycan synthesis observed in articular cartilage subjected to high hydrostatic pressure.

Biomechanical, biochemical and structural correlations in immature and mature rabbit articular cartilage.

OBJECTIVE The structure and composition of articular cartilage change during development and growth. These changes lead to alterations in the mechanical properties of cartilage. In the present study, biomechanical, biochemical and structural relationships of articular cartilage during growth and maturation of rabbits are investigated. DESIGN Articular cartilage specimens from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits were collected from seven age-groups; 0 days (n=29), 11 days (n=30), 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24) and 18 months (n=19). The samples underwent mechanical testing under creep indentation. From the mechanical response, instantaneous and equilibrium moduli were determined. Biochemical analyses of tissue collagen, hydroxylysylpyridinoline (HP) and pentosidine (PEN) cross-links in full thickness cartilage samples were conducted. Proteoglycans were investigated depth-wise from the tissue sections by measuring the optical density of Safranin-O-stained samples. Furthermore, depth-wise collagen architecture of articular cartilage was analyzed with polarized light microscopy. Finite element analyses of the samples from different age-groups were conducted to reveal tensile and compressive properties of the fibril network and the matrix of articular cartilage, respectively. RESULTS Tissue thickness decreased from approximately 3 to approximately 0.5mm until the age of 3 months, while the instantaneous modulus increased with age prior to peak at 4-6 weeks. A lower equilibrium modulus was observed before 3-month-age, after which the equilibrium modulus continued to increase. Collagen fibril orientation angle and parallelism index were inversely related to the instantaneous modulus, tensile fibril modulus and tissue thickness. Collagen content and cross-linking were positively related to the equilibrium compressive properties of the tissue. CONCLUSIONS During maturation, significant modulation of tissue structure, composition and mechanical properties takes place. Importantly, the present study provides insight into the mechanical, chemical and structural interactions that lead to functional properties of mature articular cartilage.