Biochemical and histological effects of tetracyclines on spontaneous osteoarthritis in guinea pigs

Image Anal Stereol 2000;19:125-131Original Research Paper BIOCHEMICAL AND HISTOLOGICAL EFFECTS OF TETRACYCLINES

Department of Orthopaedics, South Hospital, Karolinska Institute, 11883 Stockholm, SwedenE-ma(Accepted May 23, 2000) Matrix metalloproteinases (MMPs) are mediators in connective tissue destruction in a variety of pathologicprocesses. Recently discovered chemically modified tetracyclines have been found to be effective inhibitorsof MMP mediated connective tissue degradation in both rheumatoid arthritis (RA) and osteoarthritis (OA).
The Hartley guinea pig model has been described with a high incidence of spontaneous OA-like changes inthe knee joint. Therefore we have studied the effect of two tetracyclines, doxycycline (Dox) and chemicallymodified tetracycline-7 (CMT-7) which have both previously been shown as potent MMP inhibitors. Wefound that prophylactic orally given CMT-7 decreases OA changes in the knee joints both in vitro and invivo in the guinea pig OA model. OA changes were most severe in the central compartment of the medialcondyle in the control group. Cartilage fibrillation and destruction, in addition to subchondral bone sclerosisand cyst formation were all less in the CMT-7 treated group compared with controls. Collagen, hyaluronanand proteoglycan content in cartilage was higher in the CMT-7 treated group compared with controls. Incontrast, OA changes were not decreased in the Dox group. These results show that tetracyclines, but not alltetracyclines, can reduce the severity of OA in the guinea pig model of spontaneous OA.
Keywords: biochemistry, cartilage, guinea pig, osteoarthritis, stereology, tetracycline.
Light microscopic studies have shown that Hartley guinea pigs develop moderate to severe A variety of matrix metalloproteinases (MMPs) destruction of the cartilage and subchondral bone especially collagenase and gelatinase, have been sclerosis between 6 and 12 months of age, implicated in the connective tissue/cartilage predominantly in the central portion of the medial degradation which characterizes osteoarthritis (OA) tibial plateau and that the changes regularly progress (Pelletier and Pelletier, 1996), also in animal models to severe OA in old age (de Bri et al., 1995; de Bri et (Greenwald et al., 1990). A large body of literature al., 1996), with typical changes that mimic human has established that certain tetracyclines (TCs) are potent in vitro and/or in vivo inhibitors of various Since the natural inter- and intra-animal MMPs (Ryan et al., 1996). Doxycycline (Dox) is variability is relatively large in normal cartilage, and especially useful in this regard, and this agent has even larger in pathological states, it is advantageous been shown to potently inhibit cartilage gelatinase to study groups rather than individuals. This can be (Cole et al., 1995), to have dramatic effects in the dog achieved by the use of stereology. In the present anterior cruciate OA model (Yu et al., 1993), and to study, we have studied two TCs which have potent reduce the excretion of collagen breakdown products inhibitory capacity against various MMPs, doxycycline in patients with rheumatoid arthritis (Greenwald et al., 1994). Chemically modified derivates which have modified tetracycline-7 (CMT-7) (see below). These been modified so as to eliminate the antimicrobial were given by mouth to a group of guinea pigs for 4 properties of the TC but preserve (and usually to 8 months and we subsequently assessed the effect enhance) its MMP inhibitory capacity. The CMTs of the compound on morphologic and biochemical have been described previously (Ryan et al., 1996).
DE BRIE E ET AL: Effects of tetracyclines on osteoartritis divided by the length of the cartilage surface tangent.
The thickness of articular cartilage and subchondral bone plate was measured perpendicular to the joint (approximately 350 gm) were used at the start of the surface; bone thickness was defined as the distance experiment. A group was immediately sacrificed as from the osteocartilaginous border to the first distal baseline controls. One group was fed regular guinea occurrence of nonosseous tissue. To control artifacts pig chow ad lib and served as untreated controls. The created by the procedure of tissue preparation, the remaining two groups were fed specially modified type of fixative, time of fixation, pH, osmolarity, diets (Purina Test Diets, Richmond In, USA) dehydration, and embedding procedures were kept containing the MMP inhibitors (see below) at a rate constant throughout the study. For statistical of 0.08% w/w. Some animals in each group was evaluation Students t-test at a rejection level of sacrificed after 4 months (age 6 months) and the p < 0.05 was used. Stereological data are presented remainder at 8 months (age 10 months).
Two tetracycline (TC) compounds were used (CollaGenex Pharmaceuticals, Inc., Newtown PA,USA): doxycycline (Dox), and CMT-7 (12α deoxy, Chondroitin sulfates (CS) (grade II preparation) and a high molecular weight hyaluronan (HA)standard were obtained from Sigma (St. Louis, MO, Since the protocol required daily administration USA) and Pharmacia AB (Healon®, Uppsala, of compounds for up to 8 months, it was elected to Sweden) respectively. The aggrecan standard from incorporate the drugs into the diet. Previous work had chondrosarcoma was a generous gift from Prof. B.
established that a daily dose of 20 mg/kg was an Caterson (Cardiff, U.K). The chondroitinase AC, affective inhibitor of in vivo MMP activity chondroitinase ABC, chondroitinase-6-sulfate, chondroitinase-4-sulfate and disaccharide standardswere obtained from Sigma (St. Louis, MO, USA).
For chemical analysis of matrix components, 3 The specimens were fixed in a neutral-buffered animals per group were used. The tibial articular 4% formalin, decalcified for 5-7 days in 40% formic cartilage was divided into two peripheral areas acid, cut into the medial and lateral plateaus, and covered by the medial and lateral menisci and the embedded in paraffin wax. With random start, 4-6 two corresponding central uncovered areas, pooling histological sections were cut through each central material from the various animals. All analyses were portion of the plateau with a constant interval of 125 µm, and stained with hematoxylin and eosin.
The tissue specimens were dissected, weighed From these sections, volume densities (Vv) of and immediately frozen at -20°C, cut into 20 µm cartilage and bone were measured by point- and thick slices, using a cryostat, lyophilized at -50°C for 24 hrs, wherefore the dry weight was determined.
plateau) in a projection light microscope (Reichert- The Proteoglycans (PG) were extracted with 4 M Jung, Germany) at a final magnification of x50. In this microscope, the image is projected onto a screen, containing 0.01 M EDTA, 0.05 M sodium acetate on which the transparent grid (lattice) used for counting points and line intersections is attached.
5 mM benzanhydrochloride, 5 mM N-ethylmaleimide Cysts were defined as cavities larger than 100 µm (the last mainly to prevent disulfide exchange). The devoid of marrow cells, osteophytes as extra-articular extractions were performed at 4°C for 18 hrs, using osteocartilaginous tissue. Reference volume for 2x40 ml per mg dry tissue. The nonextracted residues cartilage, bone, and cysts was the entire epiphysis, were digested with papain and aliquots of these i.e. the area bordered proximally by articular digests were hydrolyzed in 6 M HCI for 18 h and cartilage, anteriorly and posteriorly by cortical bone, analyzed for their hydroxyproline contents. Large and and distally by the physeal remnant, osteophytes small (PG) were separated electrophoretically. Ethanol excluded. Cartilage fibrillation was measured by precipitates of the extracts were dissolved in a SDS- intersection counting with a cycloid grid for vertical containing electrophoresis buffer and run in 1.2% sections (Baddeley et al., 1986) of the traced length agarose gels at 90 V for 1.5 h. The gels were stained of the contour of the cartilage surface and was with toluidine blue, scanning the distribution of PGs using a Shimadzu Dual-Wavelength Chromato- compartment of the medial condyle. Also horizontal Scanner Model CS-930. The aggregability of the PG separation of the tidemark was observed in areas monomers was monitored by also incubating with adjacent to cartilage destruction. The underlying HA before electrophoresis and comparing the trabecular structure of the subchondral bone plate mobility with that of preparations in which the HA- was disturbed, and was replaced by an increased binding regions had been reduced. High-performance bone formation or bone sclerosis, and subsequently liquid chromatography (HPLC) was used to quantify less bone marrow cavities, in addition to a thick the contents of CS and HA and to characterize the subchondal bone plate. Cysts were frequently sulfation pattern. Aliquots were incubated with encountered in the sclerotic bone. Adjacent to the chondroitinase AC and chondroitinase ABC, and the destructed cartilage, a transitional zone with sulfation pattern was monitored by separating the fibrillation of the cartilage surface containing fewer delta-disaccharides obtained by HPLC, using chondrocytes, and separation of the uncalcified and external standards. The total amounts of CS and HA calcified cartilage at the tidemark level was noticed.
in these digests were determined following a further Only a few of the guinea pigs in the control group digestion with chondroitinase-4- and -6-sulfatases.
exhibited osteophytes at the joint margins, while the The non-sulfated delta-disaccharides obtained from doxycycline- and CMT-7- treated animals showed no the respective GAGs were separated by ion osteophytes. In the CMT-7-treated group, all guinea pigs had a milder form of OA, including fibrillationof the cartilage surface, but excluding overtdestruction of the cartilage. Moreover, no cysts were encountered, and the trabecular structure of thesubchondral bone was preserved and the subchondal No signs of OA were discernable in the four bone plate was thinner. In addition, there were no animals that were immediately sacrificed at two signs of calcified cartilage eburnation or horizontal months of age. In addition no OA changes were separation of the tidemark. The peripheral compartment found macroscopically or on histological examination of the medial condyle showed no signs of OA. No in the control, Dox and CMT-7 groups.
evidence of OA was observed in the central At 10 months, all animals in the control and Dox (meniscus non-covered) condyles in neither group.
groups had developed advanced OA lesions in thecentral (meniscus non-covered) part of the medial condyle, while the peripheral (meniscus covered) part of the medial condyle was virtually non-affected by medial/lateral condyle since OA changes occurred OA. In addition, the lateral condyle showed no only in the medial condyle, the lateral condyle thus macroscopic signs of OA. The lesions included serving as an internal control. The CMT-7-treated cartilage destruction and occasional eburnation of the guinea pigs had lower Vv of bone, but higher Vv underlying calcified cartilage and subchondral bone.
cartilage compared to the Dox group, however not The CMT-7-treated guinea pigs showed only mild reaching statistical significance compared to the signs of OA, including surface fibrillation but control group (Table 1). There were no differences without cartilage destruction and eburnation in the between the Dox group and the control group.
medial condyle, while the lateral condyle was Cartilage fibrillation was lower in the CMT-7-treated guinea pigs compared to both the Dox and controlgroups. In contrast there were no differences between the Dox group and the control group. Also the The guinea pigs in the control and Dox groups thickness of the cartilage was higher, and the exhibited cartilage destruction, thereby exposing the thickness of the subchondral bone plate was lower in calcified cartilage and occasionally the subchondral the CMT-7-treated guinea pigs compared to both Dox bone in the central (non-meniscus covered) DE BRIE E ET AL: Effects of tetracyclines on osteoartritis Table 1. Guinea pigs treated with doxycycline (Dox) and chemically modified tetracycline-7 (CMT-7) arecompared with untreated controls. a = p < 0.05 between CMT-7 group and control group, b = p < 0.05 between CMT-7 group and doxycycline group. Volume densities ofbone (Vvbone) and cartilage (Vvcartilage), the thickness of articular cartilage (Thcart) and subchondral bone (Thbone) in addition tocartilage fibrillation (fibrill) were measured in the central medial and lateral condyles separately. The ratios medial/lateral condyle aregiven as means (SD).
contained considerably lower levels of PGs than theircentral counterparts.
glycosaminoglycan (GAG) found in the articular Overall, the CS of unmineralized cartilage cartilage. Total proteoglycan (PG) content, expressed showed a predominance of 6-sulfated disaccharides as CS-derived uronic acid, was higher in the medial (Table 3), with a considerable proportion of 4- central condyle in the CMT-7-treated group sulfated disaccharides (Table 3), leaving a minimal compared to the control group. No differences were amount of non-sulfated disaccharides per chain. No observed in the Dox group (Table 2). The amounts of oversulfated disaccharides were found. However, the both large and small PGs were also relatively higher sulfation pattern varied in the various areas of the in the central medial condyle in the CMT-7 group tibial articular cartilage. We observed increased (Table 2). The highest PG concentration levels were amounts of both 6S and 4S, predominantly in the found centrally in the medial condyle, while lower central medial condyle in the CMT-7 group levels were found in the lateral condyles. The two compared with the Dox and control groups.
articular cartilage fractions representing tissue However, the ratio of 6S/4S was constant in the covered by the menisci (peripheral compartment) different groups and areas (Table 3).
Table 2. Guinea pigs treated with doxycycline (Dox) and chemically modified tetracycline-7 (CMT-7) arecompared with untreated controls. The amounts of chondroitin sulphate, large proteoglycans are given for the central and peripheral compartments of the medial andlateral condyles, respectively (µg CS-derived uronic acid per mg dry weight cartilage).
Table 3. Guinea pigs treated with doxycycline (Dox) and chemically modified tetracycline-7 (CMT-7) arecompared with untreated controls. The amounts of proteoglycans sulphated in 6 and 4 position in addition to the 6/4 sulphate ratio are given for the central and peripheralcompartments of the medial and lateral condyles, respectively (µg CS-derived uronic acid per mg dry weight cartilage).
The CS was extracted from the uncalcified tissue Collagen content, expressed as hydroxyproline, in the form of PGs which, by electrophoresis, could was higher in the medial central condyle in the CMT- be separated into one smaller, faster-moving band 7-treated group compared to the control group, while and two dominating closely migrating but distinct no obvious difference was observed between the Dox bands with lower mobility, representing a larger group and the control group (Table 4).
molecular size. The faster moving band migrated The tissue contained a minor component of slightly slower than the free CS chains, while the hyaluronan (HA). The concentration was higher in mobility of the large PG was similar to that of the central medial condyle in the CMT-7-treated chondrosarcoma aggrecan. The mobility of the PG group, compared to the control group (Table 4). No populations remaining in different fractions was differences were observed between the Dox group similar in all three groups. Furthermore, the ratios of and the control group. However, the ratios of large large to small PGs in the uncalcified tissue fractions PG/HA were similar in all three groups. The were very similar in the three groups.
aggregability was the same in all three groups.
Table 4. Guinea pigs treated with doxycycline (Dox) and chemically modified tetracycline-7 (CMT-7) arecompared with untreated controls. The amounts of hyaluronan and hydroxyproline are given for the central and peripheral compartments of the medial and lateralcondyles, respectively (µg hyaluronan and hydroxyproline per mg dry weight cartilage).
destruction in a variety of pathologic processes,including rheumatoid arthritis and OA. The CMTs are potent inhibitors of several classes of matrix pathologic breakdown of the joint extracellular metalloproteinases, preventing collagen breakdown.
matrix in OA. TCs have been found to be effective We chose the Hartley guinea pigs, because of a inhibitors of MMP-mediated connective tissue previously reported high incidence of OA-like DE BRIE E ET AL: Effects of tetracyclines on osteoartritis changes in the proximal tibia (Bendele and Hulman, effective, perhaps because the uptake after oral 1988). The advantages of spontaneous OA models administration might have been impaired in contrast are evident. In many surgically induced models, it is difficult to control post-operative changes. It is known Naturally, these results have to be confirmed by a that the increased cytokine levels induced by trauma, study with a larger sample size, and the uptake of the and the subsequent joint inflammation, may have a compounds has to be elucidated. Moreover, not only direct effect on the development of OA, in contrast to the prophylactic effect, but also the therapeutic spontaneous OA models, where the intensity of effects of CMT-7 and Dox have to be evaluated.
inflammation is low. Moreover, surgically induced OAmodels incompletely reproduce the slowly progressive In conclusion, prophylactic CMT-7 given orally course of primary OA. In secondary OA, the lesions decreases OA changes in the knee joints both in vitro develop rapidly, thereby differing from the slowly and in vivo in the Guinea pig OA model. In contrast, progressive disease process in primary OA.
Dox did not have any effect on the OA changes.
A preliminary report of some of the data was biochemical OA-changes in guinea pigs were less presented at the Xth International Congress for severe after 8 months of per oral treatment with Stereology, Melbourne, Australia, 1-4 November CMT-7. The CMT-7 treated animals showed only mild fibrillation compared to the severe OA changesfound in the control group including both cartilagedestruction and subchondral bone sclerosis. These changes were most pronounced in the central medial Baddeley AJ, Gundersen HJG, Cruz-Orive LM (1986).
condyle which is most commonly affected by OA (de Estimation of surface area from vertical sections. J Bri et al., 1995). Stereological parameters, allowing for quantitative data and comparisons between Bendele AM, Hulman JF (1988). Spontaneous cartilage groups, showed lower Vv of both bone and cysts, in degeneration in guinea pigs. Arthritis Rheum 31:561-5.
addition to a thinner subchondral bone plate, Brandt K (1994). Insights into the natural history of indicating less bone involvement. In contrast, Vv osteoarthritis provided by the cruciate-deficient dog.
cartilage was higher, and the cartilage was thicker suggesting less destruction of the articular surface.
Cole AA, Yi W, Kuettner K, Golub LM, Greenwald RA Also fibrillation of the cartilage was lower in this (1995). The effects of chemically modified group. Biochemically, total PG content (both small tetracyclines on cartilage degradation in chicken tibial and large PGs), HA and collagen content in cartilage explants. Trans Orthop Res Soc 20:337.
was higher in the CMT-7 treated animals compared De Bri E, Reinholt FP, Svensson O (1995). Primary to the OA affected control group suggesting a better osteoarthrosis in guinea pigs: A stereological study. J preservation of articular cartilage. No differences were observed in the sulfation pattern with regards to De Bri E, Jönsson K, Reinholt FP, Svensson O (1996).
the 6S/4S ratio, although some studies have reported Focal destruction and remodelling in guinea pig differently (Roughley and White, 1980). However the arthrosis. Acta Orthop Scan 67(5):498-504.
CMT-7 group had higher levels of both 6S and 4S. In Greenwald RA, Golub LM, Ramamurthy NS, McNamara T OA, the function of the joint cartilage is impaired due (1990). Direct detection of collagenase and gelatinase to matrix destruction, where proteoglycan content is in periarticular tissue from adjuvant arthritis rats:inhibition by tetracyclines and potential amelioration decreased proportionally to the severity of OA (Venn of bone destruction. Trans Orthop Res Soc 15:270.
and Maroudas, 1977). Interestingly there were no Greenwald RA, Moak SA, Golub LM (1994). Low-dose differences in OA changes between the Dox and the doxycycline (LDD) inhibits pyridinoline (PYD) excretion in selected patients with rheumatoid arthritis. In Inhibition biochemically in contrast to earlier findings. Brandt of Matrix Metalloproteinases: Therapeutic Potential, (1994) showed that in surgically induced OA in the dog model, prophylactic administered Dox ameliorated Pelletier JM, Pelletier JP (1996). Wanted - the collagenase the induced pathologic changes. Active and latent responsible for the destruction of the collagen network collagenase and gelatinase levels in extracts of the in human cartilage. Br J Rheum 35:818-20.
Roughley PJ, White RJ (1980). Age-related changes in the administration. However, in our hands, Dox was not structure of proteoglycan subunits from human articular cartilage. J Biol Chem 255:217 24.
Ryan ME, Ramamurthy NS, Golub LM (1996). Matrix Yu LP, Smith GN, Brandt KD, Myers SL, O'Connor BL, metalloproteinases and their inhibition in periodontal Brandt DA (1992). Reduction of the severity of canine treatment. Curr Opin Peridont 3:85-96.
osteoarthritis by prophylactic treatment with oral Venn MF, Maroudas A (1997). Chemical composition and doxycycline. Arthritis Rheum 35:1150-9.
swelling of normal and osteoarthritic femoral headcartilage. I. Chemical composition. Ann Rheum Dis



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