ARTICLE |
Correspondence to: Michael D. Buschmann, Biomedical Engineering Inst., Ecole Polytechnique, PO Box 6079, Station Centre-Ville, Montreal, Quebec, Canada H3C 3A7.
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Summary |
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We developed a new quantitative histochemical method for mapping aggrecan content in articular cartilage and applied it to models of cartilage degradation. Ruthenium hexaammine trichloride (RHT) forms co-precipitates with aggrecan, the main proteoglycan component of cartilage, and was previously found to be a good fixative in aiding the maintenance of chondrocyte morphology. We show that these RHTaggrecan precipitates generate a positive chemographic signal on autoradiographic emulsions, in the absence of any radioactivity in the tissue section, via a process similar to the autometallographic process used previously for localization of trace metals ions in tissues. By exploiting the inherent depth-dependence of aggrecan concentration in adult articular cartilage, we demonstrated that the density of silver grains produced by RHT-derived chemography on autoradiographic emulsions correlated with locally measured aggrecan concentration as determined by the dimethylmethylene blue assay of microdissected tissue from these different depths of cartilage. To explore the benefits of this new method in monitoring tissue degradation, cartilage explants were degraded during culture using interleukin-1 (IL-1) or digested after culture using chondroitinase and keratinase. The RHT chemographic signal derived from these samples, compared to controls, showed sensitivity to loss of aggrecan and distinguished cell-mediated loss (IL-1) from degradation due to addition of exogenous enzymes. The RHT-derived chemographic grain density represents an interesting new quantitative tool for histological analysis of cartilage in physiology and in arthritis. (J Histochem Cytochem 48:8188, 2000)
Key Words: chemography, autometallography, autoradiography, proteoglycan, aggrecan, glycosaminoglycan, chondroitin sulfate, chondrocyte, cartilage, arthritis, interleukin-1
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Introduction |
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The main proteoglycan (PG) component of cartilaginous tissues, aggrecan, plays important structural (
Histological localization methods, using immunological or genetic probes or stains, have proved to be highly useful in several studies of cartilage physiology but are usually qualitative in nature. In the area of cartilage physiology involving functional biomechanical properties of the tissue, there is also a need for spatially resolved quantitative characterization of tissue properties, motivating the development of quantitative histological methods. For example, a recently developed quantitative autoradiographic method for the spatial localization of aggrecan synthesis (
Our study describes another new method by which total glycosaminoglycan (GAG) concentration, essentially representing aggrecan, can be spatially mapped in tissue sections using techniques similar to those for the localization of PG synthesis by quantitative autoradiography. The method relies on the ability of ruthenium hexaammine trichloride (RHT) to bind to and thus retain aggrecan during fixation and to induce a positive chemographic signal when sections are processed for autoradiography. We quantified this RHT-derived chemographic grain density as a function of depth from the articular surface, using image analysis, and found that it correlated strongly with the colorimetric (DMB) evaluation of GAG (aggrecan) content in microdissected, papain-solubilized tissue from those regions. A high sensitivity of the RHT chemographic signal to tissue degradation was also found in cultured tissue explants treated with a cytokine or exogenous GAG-degrading enzymes.
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Materials and Methods |
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Tissue Isolation for Cryosections and for Fixation in the Presence of RHT
Tissue isolation was similar to a previously described procedure (/4 x 3.752 x 3 x 0.035 = 1.16 µl) taken from tissue depths of approximately 50 µm plus increments of 105 µm, from the articular surface. The other cylinder within each pair was cut to a total length of ~1.5 mm and fixed in 10 ml of 0.05 M Na-cacodylate (820670 Merck; Dietikon, Switzerland) buffer containing 2% v/v glutaraldehyde (G002, Taab Lab Equipment; Fakola, Basel, Switzerland) and 0.7% w/v RHT (ruthenium hexaammine trichloride, 2340.0100 Merck) at room temperature for 16 hr. Nonchemographic control specimens were generated by fixing similar cartilage/bone disks in 10 ml of 0.05 M Na-cacodylate buffer containing 2% v/v glutaraldehyde and 2.5% w/v CPC (N-cetylpyridinium chloride, 2340.0100 Merck).
GAG Content vs Tissue Depth Using Dimethylmethylene Blue (DMB) Spectrophotometry of Solubilized Cryosections
Each group of three 35-µm-thick cryostat sections was digested in 0.5 ml of papain [0.125 mg/ml (Sigma, St Louis, MO) in 0.1 M Na2HPO4, 0.01 M Na2-EDTA, and 0.01 M L-cysteine-HCl, pH 6.5] for 16 hr at 60C. Duplicates of 5, 10, and 15 µl volumes of each digest were transferred to 96-well flat-bottomed microtiter plates (Flow Laboratories 76-381-04; Allschwil, Switzerland). A total of 250 µl of DMB dye solution [46 µM 1,9 dimethylmethylene blue chloride #03610 (Polysciences; Warrington, PA) in 0.04 M NaCl + 0.04 M L-glycine #4201.1000 (Merck) pH 3] was added to each well with a multipipette and the absorbance read at 530 nm within 1 min of adding the DMB solution (
Embedding, Sectioning, and Detection of RHT-derived Chemographic Signal Using Autoradiographic Emulsion, and Quantitation Using Computerized Image Analysis
Immediately after fixation (1 day post isolation), 1.5-mm-thick cartilage/bone disks were washed four times over 2 hr in 0.1 M Na-cacodylate buffer, pH 7.4, and transferred to 70% ethanol. Because we wished to measure the density of RHT-derived chemographic signal as a function of depth within cartilage explants, we obtained vertical sections along the axis of disks. The fixed disks were axially bisected with a razor blade. Disk halves were dehydrated in a graded series of ethanol concentrations (80, 90, 96, 100%) over a total period of 5 hr and were then placed once more in 100% ethanol overnight. On the second day after isolation, specimens were placed in 1,2 propylene oxide (PPO, Merck) for 1 hr and then embedded in EPON 812 (Flukka; Buchs, Switzerland) in the following stages: 1:3 EPON:PPO for 24 hr; 1:1 EPON:PPO with 0.6% v/v N-benzyldimethylamine (BDMA from Flukka) for 24 hr; 3:1 EPON:PPO with 0.6% BDMA for 3 days; pure EPON with 0.6% BDMA for 3 days; pure EPON with 1.2% BDMA for 5 days at 60C. Embedded disk halves were trimmed to remove most of the bone and semithin 1-µm sections, obtained with an ultramicrotome (Ultracut S; Leica, Vienna, Austria), were taken from the cut face of the disk halves and were therefore rectangular in shape, with a width equal to the disk diameter (~4 mm) and a height equal to the trimmed disk thickness (~1.2 mm). Sections were placed on gelatin-coated glass slides (Superfrost Plus; Menzel Glases, Haska, Bern, Switzerland).
To detect and quantify the RHT-derived chemographic signal, we followed in detail a procedure developed previously for quantitative autoradiographic analysis of sections of radiolabeled cartilage (
Models of Cartilage Degradation Involving Loss of Aggrecan: Explant Culture with Interleukin-1 and Digestion with Chondroitinase and Keratinase
A full-thickness mature bovine articular cartilage tissue explant system was employed ( (200-LA; R&D Systems, Minneapolis, MN). The third disk (digested) from each group was placed in culture without IL-1 but was digested at the end of culture in 0.25 ml of Tris-buffered saline (0.1 M Tris + 0.1 M NaCl) containing 200 U/ml of chondroitinase ABC (100332-1 Seikagaku; PDI BioScience Aurora, Ontario, Canada) and 200 U/ml of keratanase (100810-1, Seikagaku) and 0.01% BSA, three times for 24 hr each at 37C. These nine disks were fixed and processed for RHT chemography as described above. Because signal intensity in the degraded disks (IL-1 and digested) was above background in the deeper regions of the cartilage only, grain counting was confined to these areas for both control and degraded disks.
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Results |
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Grain Density on Nonradioactive Sections Fixed in the Presence of RHT Is Due to RHT-derived Positive Chemography and Correlates with Local Aggrecan Concentration
Unlabeled, nonradioactive semithin plastic sections of cartilage that were fixed in the presence of the metal-containing trivalent cationic agent RHT, displayed a significant and spatially variable silver grain density when processed in a manner typically used for autoradiography of radioactively labeled specimens (Figure 1). The silver grains on RHT-fixed specimens were due to the presence of RHT in the fixative because similar specimens processed in the same manner, but with the non-metal-containing monovalent cationic agent cetylpyridinium chloride replacing RHT in the fixative, produced a negligible background silver grain density (not shown, but <1/4 the grain density seen in Figure 1B). The RHT-derived chemographic signal appeared to be stronger in deeper regions of the cartilage, consistent with a higher density of RHTPG precipitates in those deeper regions, where PG density is known to be higher (Figure 1).
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Both the RHT-derived chemographic grain density and the equivalent chondroitin sulfate concentration from DMB-assayed papain-solubilized tissue sections increased monotonically with tissue depth (Figure 2) and were therefore correlated as a function of distance from the articular surface. The grain density was approximately eight times higher in the deep zone compared to the superficial zone (Figure 2A). The equivalent chondroitin sulfate content was approximately four times higher in the deep zone compared to the superficial zone (Figure 2B). The ratio of grain density to equivalent chondroitin sulfate content, calculated using the 10 matched cartilage/bone cylinder pairs, was dependent on the distance from the articular surface (Figure 2C). The ratio varied linearly with depth and was approximately doubled in the deepest zone compared to the most superficial zone (Figure 2C).
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The RHT-derived Chemographic Signal Was a Sensitive Indicator of Aggrecan Loss in both IL-1-degraded Tissue Explants and Explants Exposed to Exogenous Chondroitinase and Keratinase
Exposure of tissue explants to IL-1 over 11 days in culture or digestion with exogenous GAG-degrading enzymes resulted in greatly reduced grain density, reflecting the loss of aggrecan in these two models of cartilage degradation (Figure 3). When grains were counted, grain density in the degraded cartilage was found to be many-fold (x510) less than in the control tissue explants (Figure 4). Exposure to IL-1
over 11 days in culture reduced grain density to background levels (14 ± 2 µm-2 x 10-3 ; mean ± SEM, n = 3) in the upper two thirds of the cartilage. Grain density was only slightly above background in the lower third of the IL-1-treated cartilage (deep radial zone) but significantly higher than background (~fourfold) for the disks treated with exogenous GAG-degrading enzymes (Figure 4).
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Discussion |
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This study investigated the use of RHT-derived chemographic signal as a quantitative histochemical indicator of aggrecan concentration in cartilage. RHT is a small (MW = 309), soluble trivalent cationic agent which has been used previously to aid in the retention of aggrecan, and thereby of chondrocyte morphology, in cartilage tissue during fixation and further processing, via the formation of stable RHTaggrecan precipitates (
The physicochemical process involved in the chemographic generation of silver grains by RHT in tissue sections is similar to that used in the techniques of autometallography developed previously for the detection of trace levels of metal ions (e.g., gold, silver, mercury, zinc) in tissues (
The quantitative ratio of RHT-derived chemographic grain density to equivalent chondroitin sulfate concentration by DMB of papain-solubilized tissue pieces depended slightly (within a factor of 2) on distance from the articular surface. If chondroitin sulfate (CS) were the only reactive polyanion in the tissue for RHT and in the papain-solubilized samples for DMB, one would expect the above ratio to be constant, independent of depth or of other factors. However, even in that case, the RHTCS binding in the tissue would take place under much denser conditions (25,000-fold higher CS concentration) than the DMBCS binding in the microplate assay. Therefore, one hypothesis explaining the systematic variation of the RHT/DMB ratio (Figure 2C) is that higher concentrations of GAGs in the deeper regions bind proportionately more RHT in the tissue than DMB at the low concentrations in the solution assay. Therefore, RHTGAG binding in tissue sections may display a cooperative character not present in DMBGAG binding in dilute solution. Other factors that could influence the RHT/DMB ratio include the presence of other species of reactive polyanions (keratan sulfate, dermatan sulfate, and hyaluronan) in addition to our standard, chondroitin sulfate, in the tissue sections and papain digests. The concentrations of these GAGs also vary with distance from the articular surface (
An important observation in our study was the high sensitivity of the RHT chemographic signal to the effects of matrix degradation. We chose two in vitro models of cartilage degradation to explore the use of this new technique to monitor aggrecan loss in articular cartilage. Exposure of tissue explants to 5 ng/ml of IL-1 reduced grain densities by an order of magnitude compared to controls throughout the entire thickness of the cartilage and to background levels in all but the deep radial zone (Figure 3 and Figure 4). Estimation of GAG content using the DMB method on tissue treated similarly with IL-1 revealed a five- to tenfold reduction of aggrecan compared to controls (down to 36 µg/µl), and an ELISA method showed no detectable change of total collagen II content, again confirming the role of aggrecan in generating the RHT chemographic signal. We can also glean from this data and our measurements of background grain density in many samples that the minimim GAG concentration required to produce a detectable RHT chemographic signal is in the range of 510 µg/µl. Interestingly, our second method of inducing aggrecan loss by directly digesting the tissue explants with GAG-degrading enzymes was able to deplete most of the cartilage of aggrecan to the levels of the IL-1-treated tissue except in the deep radial zone, where grain densities remained significantly higher than those produced by IL-1 (Figure 4). We hypothesize that the exogenously added enzymes are sterically blocked from penetrating the deeper cartilage regions where extracellular matrix is most dense, whereas the action of IL-1 on chondrocytes in upregulating the synthesis of matrix-degrading enzymes within those regions is not impeded. This distinction further highlights the potential of the new technique.
In summary, we have developed a quantiative histochemical method to estimate aggrecan concentration in cartilage tissue sections using the chemographic signal produced when ruthenium hexaammine trichloride is included in the fixative. In addition to providing a new tool in the analysis of pathologies such as osteoarthritis, this method can be used to localize functional anisotropic and spatially heterogeneous biomechanical properties of cartilage. For example, cartilage explants compressed under defined but variable loading conditions were fixed in the compressed state and processed for RHT-derived chemography (
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Acknowledgments |
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Supported by the AO/ASIF Foundation (93-G46), Switzerland, the Medical Research Council of Canada, and the Arthritis Society of Canada.
We thank Dr Thomas Quinn, Pirmin Schmid, and Anne Légaré for performance of some of the technical analyses.
Received for publication May 13, 1999; accepted September 8, 1999.
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