Alpo Pelttari

Articular cartilage superficial zone collagen birefringence reduced and cartilage thickness increased before surface fibrillation in experimental osteoarthritis

OBJECTIVES To investigate articular cartilage collagen network, thickness of birefringent cartilage zones, and glycosaminoglycan concentration in macroscopically normal looking knee joint cartilage of young beagles subjected to experimental slowly progressive osteoarthritis (OA). METHODS OA was induced by a tibial 30° valgus osteotomy in 15 female beagles at the age of 3 months. Fifteen sisters were controls. Cartilage specimens were collected seven (Group 1) and 18 months (Group 2) postoperatively. Collagen induced optical path difference and cartilage zone thickness measurements were determined from histological sections of articular cartilage with smooth and intact surface by computer assisted quantitative polarised light microscopy. Volume density of cartilage collagen fibrils was determined by image analysis from transmission electron micrographs and content of glycosaminoglycans by quantitative digital densitometry from histological sections. Results—In the superficial zone of the lateral tibial and femoral cartilage, the collagen induced optical path difference (birefringence) decreased by 19 to 71% (p < 0.05) seven months postoperatively. This suggests that severe superficial collagen fibril network deterioration took place, as 18 months postoperatively, macroscopic and microscopic OA was present in many cartilage areas. Thickness of the uncalcified cartilage increased while the superficial zone became thinner in the same sites. In operated dogs, glycosaminoglycan content first increased (Group 1) in the lateral tibial condyle and then decreased (Group 2) (p < 0.05). Conclusion—In this OA model, derangement of the superficial zone collagen network was the probable reason for birefringence reduction. This change occurred well before macroscopic OA.

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.

Electron microscopic stereological study of collagen fibrils in bovine articular cartilage: volume and surface densities are best obtained indirectly (from length densities and diameters) using isotropic uniform random sampling.

Results obtained by the indirect zonal isotropic uniform random (IUR) estimation were compared with those obtained by the direct point and interception counting methods on vertical (VS) or IUR sections in a stereological study of bovine articular cartilage collagen fibrils at the ultrastructural level. Besides comparisons between the direct and indirect estimations (direct IUR vs indirect IUR estimations) and between different sampling methods (VS vs IUR sampling), simultaneous comparison of the 2 issues took place (direct VS vs indirect IUR estimation). Using the direct VS method, articular cartilage superficial zone collagen volume fraction (Vv 41%) was 67% and fibril surface density (Sv 0.030 nm2/nm3) 15% higher (P<0.05) than values obtained by the indirect IUR method (Vv 25% and Sv 0.026 nm2/nm3). The same was observed when the direct IUR method was used: collagen volume fraction (Vv 40%) was 63% and fibril surface density (Sv 0.032 nm2/nm3) 21% higher (P<0.05) than those obtained by the indirect IUR technique. Similarly, in the deep zone of articular cartilage direct VS and direct IUR methods gave 50 and 55% higher (P<0.05) collagen fibril volume fractions (Vv 43 and 44% vs 29%) and the direct IUR method 25% higher (P<0.05) fibril surface density values (Sv 0.025 vs 0.020 nm2/nm3) than the indirect IUR estimation. On theoretical grounds, scrutiny calculations, as well as earlier reports, it is concluded that the direct VS and direct IUR methods systematically overestimated the Vv and Sv of collagen fibrils. This bias was due to the overprojection which derives from the high section thickness in relation to collagen fibril diameter. On the other hand, factors that during estimation tend to underestimate Vv and Sv, such as profile overlapping and truncation (‘fuzzy’ profiles), seemed to cause less bias. As length density (Lv) and collagen fibril diameter are minimally biased by the high relative section thickness, the indirect IUR method, based on utilisation of these estimates, is here regarded as representing a ‘gold standard’. The sensitivity of these 3 methods was also tested with cartilage from an in vitro loading experiment which caused tissue compression. In the superficial zone of articular cartilage Vv and Sv of collagen fibrils increased (P<0.05). This difference in the stereological estimates was only detected by the indirect IUR estimation but not by the direct VS or direct IUR methods. This indicated that the indirect IUR estimation was more sensitive than the direct VS or direct IUR estimations. On the basis of these observations, the indirect zonal IUR estimation can be regarded as the technique of choice in the electron microscopic stereology of cartilage collagen.

Investigation of Articular Cartilage Surface Morphology with a Semiquantitative Scanning Electron Microscopic Method

A semiquantitative scanning electron microscopic method for analysis of the articular cartilage surface morphology was developed. The method was based on a survey of large picture montages (ca. 70 X 100 cm) and classification of the cartilage surface changes at three levels. Computer technique was utilized in the analysis. The method ensured numerical expression and statistical treatment of the results. With this method we investigated the effects of physical exercise and immobilization on the articular cartilage of rabbit patella.

Ultrastructural Analysis of Human Epidermal CD44 Reveals Preferential Distribution on Plasma Membrane Domains Facing the Hyaluronan-rich Matrix Pouches

We used immunogold staining and stereology to examine the ultrastructural localization and to estimate the relative content of CD44 in different strata and cell types of normal human epidermis. We found that CD44 existed almost exclusively on the plasma membranes; only rare labeling occurred on vesicular structures within the cytoplasm. Quantitation of the immunogold particles indicated that the labeling density of melanocytes corresponded to that of basal keratinocytes, and Langerhans cells displayed a labeling density of ∼10% that of the surrounding spinous cells. Among keratinocyte strata, the highest labeling density occurred on spinous cells, suggesting upregulation of CD44 after detachment from the basement membrane. The plasma membrane distribution of CD44 was compartmentalized, with little signal on cell–cell and cell-substratum contact sites such as desmosomes, the plasma membrane domain facing the basement membrane, and the close apposition of terminally differentiating granular cells. In contrast, CD44 was abundant on plasma membrane domains facing an open intercellular space, rich in hyaluronan. This distribution is in line with a role of CD44 as a hyaluronan receptor, important in the maintenance of the intercellular space for nutritional and cell motility functions in stratified epithelia.

Collagenase-Induced Changes in Articular Cartilage as Detected by Electron-Microscopic Stereology, Quantitative Polarized Light Microscopy and Biochemical Assays

The purpose of this study was to compare the ability of electron-microscopic (EM) stereology with quantitative polarized light microscopy (PLM) and biochemical collagen (hydroxyproline) and crosslink (pyridinoline) analyses to detect changes in the superficial collagen network of bovine articular cartilage after digestion in vitro with purified bacterial (Clostridium histolyticum) collagenase (30 U/ml) for 24 and 48 h. Collagen volume (VV) and surface (SV) densities of the uppermost third of the superficial zone were estimated indirectly from zonal isotropic uniform random sections using collagen length density (LV) and average collagen fibril diameter, or its average second power. Collagenase digestion caused a significant decrease in fibril diameter (64 to 62%), VV (89 to 95%) and SV (64 to 86%) after incubation for 24 and 48 h. Collagen LV remained unchanged after 24 h incubation but decreased 63% after 48 h. Collagen concentration per dry weight, assayed biochemically from the whole superficial zone, decreased also significantly (29 to 60%) after 24 and 48 h digestions, respectively. The pyridinoline concentration per dry weight of the superficial zone decreased (31 to 57%) whereas the pyridinoline concentration per collagen remained unchanged. PLM revealed that the birefringence of the uppermost third of the superficial zone was decreased by 36% after digestion for 24 h though the total birefringence of the whole zone was not reduced. However, after 48 h, the birefringence of the whole superficial zone was significantly reduced (76%). All of the techniques compared in this study could detect collagen network degradation in bovine articular cartilage but the EM stereological technique was more sensitive at detecting the changes than PLM or biochemical assays.

Effect of locally administered zoledronic acid on injury-induced intramembranous bone regeneration and osseointegration of a titanium implant in rats

BackgroundIntramedullary implantation causes injury-induced stimulation of intramembranous bone regeneration. Intramedullary bone injury along with stress shielding may induce periimplant bone loss and cause early aseptic loosening of an implant. The aim of this study was to determine the effect of locally administered zoledronic acid on periimplant bone and injury-induced stimulation of intramembranous bone regeneration in a rat model.MethodsA total of 28 male rats had a titanium implant inserted into their right femur. During the operation, the medullary canal was lavaged using 20 μM zoledronic acid (Zometa 4 mg/5 ml) or sodium chloride. Follow-up times were 4 and 12 weeks, with each follow-up group consisting of seven rats. The femurs with the titanium implants in situ were harvested, and three microscope sections were cut from each femur. The sections were photographed and analyzed with the Analysis computer program.ResultsBetween 4 and 12 weeks, the length of fluorescence bone contact increased significantly in both groups (control 15.7% SD and zoledronic acid 18.8% SD), although the difference between the groups was not significant. Periimplant bone volume (thickness) was increased in the 4-week zoledronic acid group compared to the controls (±13.4%, P = 0.002) but at 12 weeks the groups no longer differed from each other.ConclusionsOur results suggest that zoledronic acid may prevent injury-induced bone loss near an intramedullary implant by inhibiting bone resorption shortly after implantation. This may provide better periimplant bone stock during the early postoperative period.

Early bone growth on the surface of titanium implants in rat femur is enhanced by an amorphous diamond coating

Background and purpose Amorphous diamond (AD) is a durable and compatible biomaterial for joint prostheses. Knowledge regarding bone growth on AD-coated implants and their early-stage osseointegration is poor. We investigated bone growth on AD-coated cementless intramedullary implants implanted in rats. Titanium was chosen as a reference due to its well-known performance. Materials and methods We placed AD-coated and non-coated titanium implants (Ra ≈ 0.2 μm) into the femoral bone marrow of 25 rats. The animals were divided in 2 groups according to implant coating and they were killed after 4 or 12 weeks. The osseointegration of the implants was examined from hard tissue specimens by measuring the new bone formation on their surface. Results 4 weeks after the operation, the thickness of new bone in the AD-coated group was greater than that in the non-coated group (15.3 (SD 7.1) μm vs. 7.6 (SD 6.0) μm). 12 weeks after the operation, the thickness of new bone was similar in the non-coated group and in the AD-coated group. Interpretation We conclude that AD coating of femoral implants can enhance bone ongrowth in rats in the acute, early stage after the operation and might be an improvement over earlier coatings.

The relative thickness of intracellular membranes in epithelial cells of the ventral lobe of the rat prostate

SynopsisThe relative thickness of intracellular membranes of epithelial cells in the ventral lobe of the rat prostrate was measured by a densitometric method. Glutaraldehyde perfusion followed by ruthenium tetroxide immersion fixation appeared to be the most suitable method for membrane thickness measurements. By thickness, the membranes could be roughly subdivided into three groups. The inner and outer membranes of the mitochondrion made up the thinnest membranes of the cell. The second group of membranes consisted of the membranes of the rough-surfaced endoplasmic reticulum and the Golgi apparatus, the different faces of the latter organelle, and the Golgi vesicles. The thickest group of membranes included those of the cell membrane, secretory granules, condensing vacuoles, lysosomes, autophagic vacuoles and multivesicular bodies. The differences in thickness of the membranes are probably due to the varying protein/lipid ratio, and the qualities and proportions of the different lipids in the membranes.

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate.

SynopsisA densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.