An experimental model of osteoarthrosis (OA) induced by injection of vitamin A in knees of rabbits is presented.  This model represents a modification and improvement of a preexisting one (Boni et al., 1977;  Benazzo et al., 1982) based on a higher number of vitamin A injections. In our model, two vitamin A injections  were sufficient to induce OA without exposure of the subchondral bone. The advantages of this model  are: 1) the maintaining of articular cartilage could make the experimental joints suitable to test the reparative  efficiency of candidate intra-articular pharmacological treatments, 2) animal stress and the risk of  infections are strongly reduced in compliance with European legislation on laboratory animal welfare.

Bianco, P

Corsi, A

Grillo, M G

Lorenzini, R N

Panzini, G

Politi, L

Riminucci, M

Scandurra, R

English

Journals from University of Tartu

Scand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 31.Intra-articular injection of vitamin A:a rabbit model to study osteoarthrosisby M.G. Grillo*°, G. Panzini**°, A. Di Virgilio**, A. Corsi***, M. Riminucci***, P. Bianco***,L. Politi*, R.N. Lorenzini**, R. Scandurra*.*Dipartimento di Scienze Biochimiche, Università di Roma "La Sapienza"**Servizio Qualità e Sicurezza della Sperimentazione Animale, Istituto Superiore di Sanità, Roma.***Dipartimento di Medicina Sperimentale e Patologia, Sezione di Anatomia Patologica, Università di Roma "la Sapienza". ° These authors have equally contributed to this work.SummaryAn experimental model of osteoarthrosis (OA) induced by injection of vitamin A in knees of rabbits is pre-sented. This model represents a modification and improvement of a preexisting one (Boni et al., 1977;Benazzo et al., 1982) based on a higher number of vitamin A injections. In our model, two vitamin A injec-tions were sufficient to induce OA without exposure of the subchondral bone. The advantages of this mod-el are: 1) the maintaining of articular cartilage could make the experimental joints suitable to test the repar-ative efficiency of candidate intra-articular pharmacological treatments, 2) animal stress and the risk ofinfections are strongly reduced in compliance with European legislation on laboratory animal welfare.IntroductionOsteoarthrosis (OA) represents one of the most fre-quent articular pathologies in humans, especially inadult and elderly individuals. With the progressiveincrease of life expectancy of Western populations,it represents a Public Health. problem of increasingconcern.From a clinical point of view, OA can beclassified into acute, sub acute and chronic.Although the etiology of the various forms of OA isdifferent, the pathogenesis is common and relatedto chondrocyte injury leading to a disturbance in thesynthetic and degradative processes with the netresult of accelerated cartilage matrix breakdown bychondrocytic enzymes (Swiestra, 1983). As a con-sequence, a vicious circle develops and leads tochondrocyte apoptosis and further degradation ofthe matrix and, in the most serious cases, to thecomplete loss of cartilage articular surface withexposure of the sub-chondral bone.To date, anatomical and functional restoration ofthe integrity of the affected joints is not possible bylocal and/or systemic pharmacological treatments,but new surgical techniques, including autologouschondrocyte transplantation and osteochondralauto-transplantation (Chu et al., 1995; Gambardel-la, 1999), are now being improved to heal chondrallesions. However, local intra-articular pharmaco-logical treatments, mainly injection of drugs con-taining sodium hyaluronate (Hamburger et al.,2003) or glucosamine sulfate (Lippiello et al.,2000), have been demonstrated to protect the chon-drocytes and reduce the extensive developmentradiological evolution of OA when some articularcartilage tissue is still preserved.To test the reparative efficiency of new and alterna-tive pharmacological treatments, it would be impor-tant to generate an animal model mimicking OA inhumans in which different degrees of chondropathycan be reproduced. In order to obtain such a model,we have modified and simplified a previously pub-lished experimental model of OA in rabbits, inwhich intra-articular injections of vitamin A wereused at different times and doses (Boni et al., 1977;Benazzo et al., 1982; Lapadula et al., 1995). Weobtained OA after 2 injections and sacrifice of theanimals 28 days after the second dose of the drug.In these animals, however, articular cartilage, whichwas never completely lost, could be responsive to3Scand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 314.pharmacological treatments. In addition, the induc-tion of OA after the admi-nistration of two injec-tions of the drug has the significant advantage ofreducing the animal stress and the risk of infectionderiving from experimental procedures. Moreoverthis model reduces the suffering of the animalswhen compared to instability models of OA .Materials and MethodsIn this study 14 male New Zealand rabbits (CharlesRiver Italy, Calco, Italy), weighing between 2.8 and3.3 Kg, were used. The animals were housed andtreated according to the European Directive86/609/EEC. They were singly housed in steel wirebottomed cages, fed a rabbit standard pellet diet(Mucedola, Settimo Milanese, Italy) and givenwater ad libitum. The rabbits were anaesthetisedwith Ketamine 35mg/kg im (Ketavet, FarmaceuticiGellini, Aprilia Italy) and xylazine 5mg/kg im(Rompun, Bayer, Milano, Italy). After tricotomy,the right knees were infiltrated with 1 ml of liposol-uble vitamin A (Arovite-Roche) at the same dose(300,000 I.U. in 1 ml of peanut oil containing alphatocopherol 6.92 mg/ml) previously used by Benaz-zo et al. No alteration of the cartilage was inducedin the left knees. Rabbits were divided into 7groups (2 animals each) based on the number ofintra-articular injection of vitamin A and time ofsacrifice (table 1). The animals were sacrified withan intravenous injection of mebenzonium iodide(Tanax) (Hoechst Roussel Vet, Milano, Italy); con-trol and experimental knees were exposed throughinterior parapatellar incision, the patellar ligamentwas then incised and the capsulotomy was per-formed. After incision of the collateral and cruciateligaments, the articular surface was exposed. Kneeswere removed by distal femur and proximal tibiaosteotomies and immediately fixed in 4% formald-heyde (freshly made from paraformaldheyde) in0.1M phosphate buffer pH7.2. After removal of thesurrounding soft tissues, femur and tibia were sep-arated. A 5 mm thick section was obtained from theexternal condyle and undecalcified for glycol-methacrylate (GMA) embedding as previouslydescribed (Bianco et al., 1984). Five mm thick sec-tions obtained from the intercondyloid fossa andthe tibia plateau were decalcified in 10% EDTA inphosphate buffer at pH 7 and routinely embeddedin paraffin. GMA sections were stained with MayGrunwald-Giemsa (MGG); paraffin sections werestained with either haematoxylin and eosin oralcian blue.Table 1. Time and number of intra-articular vitamin A injections. GROUP 1 2 3 4 5 6 7 TIME (day)0 FIRST  vitamin A injection 7 Sacrifice   12   Sacrifice SECOND  vitamin A injection 19   Sacrifice THIRD  vitamin A injection 26   Sacrifice       33   Sacrifice     40   Sacrifice Sacrifice Scand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 315.Results As previously described in diverse animal species(Boni et al., 1977; Adams et al., 1982; Benazzo etal., 1982; Kimura et al., 1994; Lapadula et al.,1995), we were able to induce OA in the experi-mental knees of the rabbits by intra-articular injec-tions of vitamin A. Gross changes of articular carti-lage were not observed in  the control knees, (fig1A) nor in the experimental i.e. treated knees of theanimals included in group 1 and 2 (fig 1B). In theexperimental knees of the rabbits from group 3 to 5the articular cartilage was less shiny than in thecontrol knees and  hyperemic areas were alsodetected (Fig 1C). Small abrasions on the surface ofthe articular cartilage of experimental knees ofthose rabbits  in groups 6-7 and reduction in theBC DFigure 1. Macroscopic views of rabbit knees.(A) control knee: undamaged cartilage which appears shiny.(B) experimental knee of rabbit from group 2 at day 12 after the first vitamin A injection. The cartilageappears macroscopically less shiny.                                                                                                  (C) experimental knee of rabbit from group 3 at day 7 after the second vitamin A injection. The cartilageappears less shiny macroscopically, and it is associated to hyperemic areas.(D) experimental knee of rabbit from group 7 at day 28 after the second vitamin A injection. The cartilageappears less shiny and less thick macroscopically; hyperemic and abrasion areas are also present.AScand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 316.Figure 2. Histology of articular cartilage of experimental (a-c) and control (d-f) knee of rabbit from group3 at day 7 after the second vitamin A injection. Reduced staining of the extracellular cartilagineous matrixwith alcian blue (b) and foamy appearance of chondrocyte for vacuolization of the cytoplasm (c) are evi-dent at different degree.Figure 3. Articular cartilage of experimental (a-d) and control (e-h) knee of rabbit from group 7 at day 28after the second vitamin A injection. In experimental knee (a), chondrocyte cellularity is not homogeneousfor the occurrence of hypercellular and hypocellular areas, and articular surface is irregular as proved bythe presence of partially detached portion of the cartilage matrix. Apoptotic bodies (arrow and inset in b)and empty lacunae (c, d), and osteoclasts (d, arrows) are evident in the more superficial area of articularcartilage and at the junction of cartilage with subchondral bone, respectively. All these changes were pre-dominantly observed in the knees of the rabbits included in group 5, 6 and 7.Scand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 317.thickness of articular cartilage were noted (fig 1D).Histological changes were detected in the experi-mental knees of the animals included in groupsfrom 3 to 7 (Figures 2, 3 and 4). The absence of his-tological changes in the articular cartilage of theanimals included in groups 1 and 2, accords wellwith previously published data by which, after onedose of vitamin A, morphological changes are evi-dent only by ultrastructural analysis (Lapadula etal., 1995). As previously described (Boni et al.,1977; Benazzo et al., 1982; Lapadula et al., 1995),these changes consisted of a foamy appearance ofthe chondrocyte cytoplasm, reduction in proteogly-can content within the cartilage matrix (as provedby the reduced staining of the cartilage matrix withAlcian Blue), irregular cartilage surface, and vari-able chondrocyte density. Variability in chondrocytedensity, with coexistence of hypercellular andhypocellular areas, was particularly pronounced inthe knee of the rabbits in groups 5 to 7, in whichintra-lacunar apoptotic bodies and many emptychondrocyte lacunae and osteoclasts were detectedin the superficial articular cartilage layer and at thejunction between articular cartilage and subchon-dral bone, respectively. In the experimental knee ofthe animals included in the groups 6 and 7, areas ofmarked reduction in the thickness of the articularcartilage and focal new bone formation within thesubchondral tissue were also observed. DiscussionThe aim of our study was to create a simple OA ani-mal model, which may be useful to analyse the effi-ciency of pharmacological treatments, while previ-ous studies (Benazzo et al., 1982) were aimed atstudying the pathogenesis of vitamin A induced OA.Moreover our model, when compared to the insta-bility OA model, greatly reduces the animal suffer-ing according to Directive 86/609/EEC.Based on our data, our model of OA in rabbitsinduced by intra-articular vitamin A injection is ofsome interest at least for two reasons. First, asdemonstrated in the experimental knees of the rab-bits included in group 3 and 7, it was possible toinduce OA histological changes after two injectionsof Vitamin A, compared to previous work in whichrabbits received more vitamin A injections (Benazzoet al., 1982). The reduction of injections represents aFigure 4. Transmitted (a, c) and polarized (b, d) light views of articular cartilage and adjacent subchondralbone of experimental (a, b) and control (c, d) knees of rabbit from group 7 at day 28 after the second vita-min A injection. Please note in experimental knee the reduction in the thickness of articular cartilage (a)and the subchondral new bone formation with well established haversian’systems (b). These changes werelimited to the knee of the animals from groups 6 and 7.Scand. J. Lab. Anim. Sci. No. 1. 2004. Vol. 318.significant advantage since it lowers both the animalstress, and the risk of infection deriving from exper-imental procedures. The comparison of morphologi-cal changes observed in the articular cartilage of theexperimental knees of the animals included in group3 (two injections and sacrifice 1 week after the sec-ond one), 6 (three injections and sacrifice threeweeks after the last one), and 7 (two injections andsacrifice four weeks after the last one) indicates thatthe time of exposure to vitamin A, more than thenumber of doses, plays a critical role in determiningthe OA severity. As a matter of fact, the most severehistological changes, including significant reductionin articular cartilage thickness and focal subchondralnew bone formation, were detected in the experi-mental knee from the animals included both in group6 and 7, but not in group 3. Second, these changes,even when severe as in the knees of the animalsincluded in group 6 and 7, did not include the com-plete loss of articular cartilage with exposure of thesubchondral bone. The importance of maintainingcartilage tissue over subchondral bone has beenproved to be a necessary condition for the local intra-articular pharmacological treatment of OA. Thus, ourexperimental model, by which marked OA withoutcomplete loss of the articular cartilage was inducedin the knee of rabbits by intra-articular injections oftwo doses of vitamin A and sacrifice 4 weeks later,represents a reversible model of severe OA andoffers the possibility to test the reparative efficiencyof candidate pharmacological treatments. AcknowledgementsThe authors wish to thank Mr. Luciano Strangio forhelping in carrying out the experimental procedureson rabbits.ReferencesAdams ME & MEJ Billingham: Animal models ofdegenerative joint disease. Current topics ofPathology.1982, 71, 265-297.Benazzo F, G Cetta, E Finardi, A Parbellini, E Cov-oni & L Ceciliani: Artrosi Sperimentale da vita-mina A nel coniglio e possibilità terapeutichecon acido jaluronico. Giornale Italiano di Orto-pedia. 1982, 393-420.Bianco P, A Ponzi & E Bonucci: Basic and "special"stains for plastic sections in bone marrowhistopathology, with reference to May-Grun-wald Giemsa and enzyme histochemistry. BasicAppl Histochem. 1984, 28, 265-279.Boni M, L Lenzi, P Berlanda , C Balduini, G Aure-li, M Rizzotti & F Mascarello: Experimentalarthrosis from intraarticular vitamin A injectionin the rabbit. Morphological and biochemicalstudy.Consideration on the pathogenesis ofhuman arthrosis in relation of the experimentalmodel. J.Orthop.Traumatol. 1977, 3, 5-26.Chu CR, RD Coutts &  M Yoshioka: Articular carti-lage repair using allogenetic perichondrocytte-seeded biodegradable porous polylactic acid(PLA): A tissue engeneering study. J. BiomedMaterRes. 1995, 29, 1147-1154.Gambardella RA: Autogenous osteocondral graft-ing: A multicenter review of clinical results,AANA, Vancouver, BC; Canada 1999Hamburger MI, S Lakhanpal, PA Mooar & D Oster:Intra-articular hyaluronans: A review of pro-duct-specific safety profiles. Semin ArthritisRheum. 2003, 32, 296-309.Kimura Y: Morphological changes in the knee jointof rat by intra-articular injection of vitamin A.Nippon Seikeigeka Gakkai Zasshi 1994, 68,572-584.Lapadula G, B Nico, F P Cantatore, R La Canna, LRoncali & V Pipitone: Early ultrastructuralchanges of articular cartilage and synovialmembrane in experimental vitamin A-inducedosteorathritis. Journal of Rheumatology. 1995,22, 1913-21. Lippiello L, J Woodward, R Karpman & TA Ham-mad: In vivo chondroprotection and metabolicsynergy of glucosamine and chondroitin sul-fate. Clin Orthop. 2000, 381, 229-40. Swiestra BA: Experimental arthrosis. Early changesin synovial morphology and lysosomial enzymeactivities. 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Intra-articular injection of vitamin A: a rabbit model to study osteoarthrosis

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Intra-articular injection of vitamin A: a rabbit model to study osteoarthrosis

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