cartilage the perichondrium is capable of producing cells from its inner surface which would differentiate into chondrocytes. In articular cartilage, which lacks a ...
1. Exp. Path. (I990), 71, 395-402
Altered chondrocytic oxidative metabolism during the restoration of depleted intercellular matrix F. Boussidan and A.M. Nahir H. Schussheim Rheumatology Research Laboratory, Faculty of Medicine, Technion-Israel Institute of Technology, and B. Shine Department of Rheumatology, Rambam Medical Center, Haifa, Israel
Received for publication June I989 Accepted for publication 30 November I989 i
Summary. The depletion of proteoglycans (PGs), induced by a single intravenous injection of a useful model for studying the response of chondrocytes in vivo to injury. The present study concentrated on the activity of enzymes related to the synthesis of PGs, either directly, with uridine diphosphoglucose dehydrogenase (UDPGD), or indirectly, through the general oxidative metabolism of the chondrocytes. Most of the enzymes showed diminished activity on day 2; in some, there was little change in activity, whereas in others there was marked increase in activity over the following days. Thus, on day 9 the activities of glucose-6phosphate dehydrogenase and of glyceraldehyde-3-phosphate dehydrogenase were twice the original (day o) values, and those of succinate dehydrogenase and of UDPGD were one and a half times greater than the original activities. Such increased enzymatic activity preceded the increase in PG content, which by day I4 reached up to 80% of the initial value. Both the increased activity and the replenishment of the PG content were inhibited when hydrocortisone (io mg/kg) was injected. papain, is
Keywords: chondrocyte oxidative metabolism, proteoglycan depletion and synthesis, quantitative cytochemistry, microdensitometry
Cartilage is difficult to heal. In non-articular cartilage the perichondrium is capable of producing cells from its inner surface which would differentiate into chondrocytes. In articular cartilage, which lacks a perichondrium, repair is either intrinsic from the chondrocytes themselves or extrinsic from the underlying subchondral bone. Intrinsic repair is usually very poor, although some proliferation of cells and increased 35S incorporation by these cells have been reported (Stockwell I9 79). The cartilage formed by extrinsic repair differs from normal articular
cartilage in its biochemical and biomechanical properties and is more prone to early degenerative processes (Furukawa et al. I980). A model of complete healing of damaged cartilage, mediated by chondrocytes themselves, was described by Thomas (I956). A single intravenous injection of papain into young rabbits leads to a rapid depletion of proteoglycans (PGs) from the cartilage matrix, manifested by drooping of the previously erect ears. Within 8-Io days, without any further intervention, PG content
Correspondence: Dr A.M. Nahir, H. Schussheim Rheumatology Research Laboratory, Faculty of Medicine, Technion-Israel Institute of Technology, POB 9649, 3I096 Haifa, Israel.
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F. Boussidan & A.M. Nahir returns almost to its initial level and the ears cular injection of I0 mg/kg hydrocortisone, become erect again. When repeated injec- starting 6 h after injection of the papain. tions of cortisone are given, the matrix remains depleted of its PGs and the ears Methods remain drooped. It should be stressed that this sequence of The tissues were chilled immediately at events, either with or without cortisone, - 70°C in pre-cooled n-hexane and stored at occurs in all types of cartilaginous tissue in - 70°C until use. All enzymatic studies were the rabbit and is not limited to the auricular done within I 5 days of chilling the tissue. All cartilage; consequently, the results almost punches of the same rabbit were tested on certainly apply to other cartilaginous tissues the same day, using the same reagents and as well. conditions. Such intense intrinsic healing with PG Sections (I 8 pm) were cut on a Bright's synthesis presents a unique opportunity to cryostat using a knife pre-cooled to - 70°C look for dynamic changes in the activity of and with an automatic motor-drive to enzymes linked with either energy produc- ensure constant thickness (Chayen et al. tion or PG synthesis. For the demonstration I973b). Incubation was carried out at 370C of PG synthesis, the cytochemical method of in an oxygen-free atmosphere. The enzymes McGarry and Gahan (I985) for uridine studied were: uridine diphosphoglucose diphosphoglucose dehydrogenase activity in dehydrogenase (UDPGD), glucose-6-phosplant tissues was applied to these tissues. phate dehydrogenase (G6PD), glyceraldeThe auricular cartilage was chosen for this hyde-3-phosphate dehydrogenase (GAPD), study, not only because of its convenience lactate dehydrogenase (LDH), succinate but also because it allowed the development dehydrogenase (SDH), and ,B-hydroxyacyl of the changes in the cartilage, from its dehydrogenase (HOAD). The medium and depletion to its restoration, to be studied in incubation times for each enzyme are sumthe same animal. marized in Table i. All reactions were linear with time. In pilot studies of UDPGD reaction in this tissue, the reaction was NAD and substrateMaterials and methods dependent and was specifically inhibited by uridine disphosphoglucuronate (McGarry & Animals Gahan I985). The conditions in Table i Six young albino rabbits (common European were found to be optimal, and under these rabbits) weighing 850-950 g were studied. conditions the reaction was linear with time One millilitre of I% crude papain (Sigma over the range 0-30 min. Chemical Company, St Louis, Missouri, USA) The critical electrolyte/alcian blue method dissolved in sterile normal saline was filtered (Scott & Dorling I965) was used with two and then injected intravenously. Serial different concentrations of MgCl2 (0.02 5 and punch biopsies (8 mm diameter) were taken 0.5 M) to study the PG content. For this under anaesthesia from both ears of each procedure, the sections were cut at 2 Pm. rabbit on days o (before injection), 2, 6, 9 Sections from each block were also stained and I4. Special attention was taken to with haematoxylin and eosin for general ensure that the punches were from the same histology. horizontal line (that is, the same distance from the base of the ear) and to avoid the thickened cartilage at the margins ofthe ears Source of materials (Fig. i). Another six rabbits were treated n-Hexane, low in aromatic hydrocarbons, identically, but were given a daily intramus- was obtained from BDH Chemicals, Poole,
Chrondrocyte metabolism during matrix restoration
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Chrondrocyte metabolism during matrix restoration 399 UK; polyvinyl alcohol, grade G04/I40, from expressed as MIE/MIEo ± s.e.m., that is, Wacker Chemicals Ltd, Walton-on-Thames, Surrey, UK; NAD, NADP, and fructose-i-6disphosphate, from Boehringer, Mannheim, FRG; and all other materials from Sigma Chemical Company, St Louis, Missouri, USA.
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Measurements
Results
Measurements of the formazan precipitate within the cells were made at 585 nm using a Vickers M85 scanning and integrating microdensitometer with a 40-power objective, an A4 mask covering one cell, and a flying spot i. The results are expressed as the mean integrated extinction (MIE x iooo) per cell per minute. Each result is the mean± s.e.m. of 20 individual measurements of 20 cells of two duplicate sections. The activity of a substrate-free control section was subtracted from the activity found in the corresponding experimental section. The initial enzymatic activity varied in each animal. In order to express the relative changes, the enzymatic activities were expressed relative to the activity recorded on
Changes induced by the injection of papain Two days after the injection of papain, the alcian blue staining in the matrix of the ears was depressed by as much as 36% as measured with 0.025 M MgCl2, and by 65% as measured with 0.5 M MgCl2. In all rabbits, the depletion of staining with the higher concentration of MgCl2 was apparently maximal by the second day, and remained at this level for the next 4 days; with the lower concentration of MgCl2, which allowed staining of all acidic moieties, the maximal depletion occurred apparently only after 6 days. From the sixth day the amount of staining with both concentrations of MgCl2 increased; by the I4th day it had reached 80% of the initial value as measured in the presence of o.s M MgCl2, and 72% as measured in the presence of 0.025 M MgCl2 (Fig. 2). In the biopsies taken before the injection of
day o (i.e. MIEO). PG content was measured by alcian blue staining at 56o nm using a B6 mask and the same flying spot i. These results are
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Fig. 2. Changes in alcian blue staining of rabbit ears following a single intravenous papain injection. Results are expressed as the mean integrated extinction/mean integrated extinction on day o. MgCl2: 0, 0.025 M; 0, 0.5 M.
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Fig. 3. Relative enzyme activities of chondrocytes ofrabbit ears following a single intravenous injection of papain. MIE, mean integrated extinction per cell per minute; MIEO, mean integrated extinction per cell per min on day o. 0, G6PD; 0, GAPD; A, LDH; *, SDH; 0, UDPGD; A, HOAD.
papain, the activities of the various enzymes, measured as MIE x I000 per minute of reaction time, were as follows: LDH, I02±8; G6PD, 33±6; GAPD, 31±5; SDH, II±I.5; HOAD, 4.2 ± I.2; and UDPGD, 9 ± I.2. The activities of these enzymes, following the injection of papain in six rabbits, are shown in Fig. 3. Three enzymes, G6PD, HOAD, and UDPGD, showed diminished activity on the second day. The activity of UDPGD decreased to as low as 40% of its initial value; those of G6PD and HOAD decreased to around 70% of their initial values (P
