Histopathological Study on the Influence of AD DIEE Biophytomodulators on Cartilage Regeneration in a Leporidae ModelCristian CRECAN.1,DanielaOROS1,AncuDINC2,FlaviuTBRAN1,LiviuOANA11 FacultyofVeterinaryMedicine,UASMVCluj,cristi_crecan@yahoo.com,2 Centerforbiosynergicresearchandstudy DincAncuBucharest

BulletinUASVMVeterinaryMedicine71(1)/2014,43-48PrintISSN1843-5270;ElectronicISSN1843-5378

AbstractArticular cartilage regeneration was and is one of the most controversial topics in human and veterinary orthopedic surgery research. Studies on vascularization and joint nutrition represented crucial steps in this research. The purpose of this study is the histological evaluation of articular cartilage regeneration using AD-DIEE biophytomodulators in surgically induced chondral defects. Identical chondral defects were created in 33 common breed rabbits of both genders, aged 8 months. The test subjects were divided into 4 groups: a group that was treated with AD-DIEE, biophytomodulators; a reference group left untreated and 2 reference groups treated with hyaluronic acid which represents the standard treatment at this moment. Cartilage samples for histological study were collected 14, 27 and 60 days post-operatory, prepared with Hematoxylin-Eosin and examined with an optical microscope. Results showed a fibrous tissue of the cartilage defect in every one of the samples, conjunctive fibrous tissue in different developmental stages that filled up the defects cavities. We have found more advanced healing processes in the group treated with biophytomodulators as compared to the untreated reference group, a better chondrocytic organization, the lack of chondral fissures as well as chondral matrix normochromia.

Keywords: rabbits, biophytomodulators, cartilage, hyaluronic acid

INTRODUCTIONJoint cartilage is one of themost used com-

ponentsofsynovialjoints,beingthecontactlayerbetweenthetwoendsofbone,ithasavariabilityof individual and species and it is varying inthickness, cell density and composition of theextracellularmatrix.(Bruckneret al.,1988,1995,2006). All synovial joints contain joint cartilagecomposedof thesamecomponentsand fulfillingthesamerole.(Duncanet al.,1987)Thepropertiesthatmakeitspecialanduniqueareitsresistancetopressure,highcapacityforcedistributionwhichisexertedonthe jointendsandwearresistance.(Mackay-Smithet al,1962)Cartilageiscomposed

ofchondrocytesembeddedinanorganizedstruc-ture, the extracellularmatrix composed of colla-gen,proteoglycans,otherproteins,ionsandwater.(McIlwraithet al,1982).

Between the extracellular matrix and chon-drocytes there are varied interactions, enablingnormaloperationofcartilageandmaintenanceoftheintegrityofthecartilagetissue.

Posttraumatic repair and regeneration ofadultjointcartilageareminimal,thetissuehavingthe ability to fix itself through the restitutio adintegrum, structural alterations are irreversibleonce theyhaveappearedoncartilaginous tissue.(NilssonG.et al,1999)

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CRECAN et alNumerous studies have been conducted

to better understand the processes leading tocartilagedegradationandalteredcompositionandprocessesrelatedtotherepairandregenerationofcartilagetorestoreitsfunction.(McIlwraithet al.,1987)

MATERIALS AND METHODSThe study included a total of 33 rabbits of

commonbreed,agedeightmonths,withthesameorigin,whohavehadthesamegrowingconditionsand care before and throughout the experiment.We used materials and specific surgical instru-ments: scalpels, scissors, hemostatic forceps, ne-edle holders, sterile fields, absorbable and nonabsorbablesuturematerials,enrofloxacin5%(5%EnroxilKRKA),dentalbur,disposablesyringesand needles, saline, hyaluronic acid 10mg /ml(Hyalgan,FidiaFarmaceutici),specificreactivefor histological processing. The animals in thestudyhavehadarticularcartilagelesionsinducedinthestiflejoint, inthepassageoftrochleaeandlateralfemoralcondylearea(Figure1.).Cartilage

defectswereidentical,withalengthof4.4mmandadepthof0.2mm(Fig.2.).

For surgery, the animals were subjected togeneral anesthesia using ketamine 35 mg / kgandxylazine4mg/kg.Theskinwas incisedonthesideofthestiflejointwherethejointcapsuleandsidefemoral-patellarligamentwereexposed.The capsule was incised longitudinally with theexpression of synovial fluid and of the chondralsurface.

Tohighlighttheplaceofchoiceforperformingthe defect, the patella was sprained medially.Chondral defects have been performed bymeansof adentaldrill of0,2/4,4mm.Surgicalreconstruction was performed on two layers:thejointcapsulewassuturedcontinuouslyusingpolyglycolic acid 3.0 (Fig. 3.) and the skin wassutured in separate pointswith 2.0 surgical silk(Fig.4.).Therabbitswererandomlydivided intofourgroups:groupM(control)BFgroup(treatedwith biofitomodulators), group H (hyaluronicacid- treated control), HS group (treated withhyaluronicacidinableedingenvironment).

Fig. 1 The site of the defect

Fig. 3 Suturingthejointcapsule

Fig. 2 Removalofthecartilage Fig. 4Skinsuture

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AnimalsintheBFgrouphadthreeDIEEbio-fitomodulators applied, protected with tape andfixedbya3-pointsutureintheskinwithnonabsor-bablematerials(Fig.5),securedwithaprotectivedressing in the upper cervical area. Protectivedressing has been applied to all the individualsubjectstoinducethesamelevelofstress.AnimalsintheHSgrouphad5mghyaluronicacidinjectedintra-articularimmediatelyaftersurgery(Fig.6.).

Animals in group H have been injected 5mg of hyaluronic acid five days after surgery,according to the manufacturers indications. Allindividuals were subjected to antibiotic therapy,enrofloxacin25mg/animal,whichwereweighed;temperature was taken, and the animals wereclinicallyevaluatedfor7dayspost-operatively.Allanimalswerehoused,caredforandfedidentically.Evaluationof theresultswasdonebyharvestingthe lesion area after animal euthanasia, by thelaws in force,every14,27and60days fromtheoccurrence of defects. In the case of the groupH, sampling was made 5 days later, so that theperiodoftimeafterthetreatmentapplicationwas

identical. Theharvestedpartswere fixed for24hoursin10%formalin,anddecalcifiedwith20%trichloroaceticacid,washedwithtapwaterfor24hours, dehydrated in alcohol, clearedwith butylalcohol(n-butanol)andincludedinparaffinat57C.Sectionswithathicknessof5mm,stainedwithhematoxylin-eosinmethodwereexaminedbylightmicroscopy (Olympus BX41 camera equippedwithDP25and imageprocessingsoftwareCELLB-Olympus)(CRECAN,2012).

RESULTS AND DISCUSSIONS Control group. Inallhistopathologicalsamples

in this group we identified a connective matrixof similar size. Localized mucosal conjunctiveperilesionaltissue,(Fig.8).cartilagecellsarrangedinchondrones,multiplecartilaginouscracks(Fig.7),earlycartilaginousmetaplasia(Fig.10).

Reparativeprocessesthatareunderwaycanbeobserved,but inarelativelyearlystage, largenumbers of active fibroblasts and relatively thincollagenfibers(Fig.9).

HistopathologicalStudyontheInfluenceofADDIEEBiophytomodulatorsonCartilageRegeneration

Fig. 7. ChondralcracksFig.8 Perilesionalabundantconjunctive

matrixwithmucoidcells

Fig. 5 Securingthebiophytomodulators Fig. 6 Injectinghialuronicacid

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(Figure11), fullyoccupying thedeeppartof thedefect,inthedefectinterioronecanobserveadultfibrous tissue with some chondroid metaplasiastarted (Fig. 15) from themarginal areas to thecenterofthedefect(Fig.16).

Defectsize ismuchsmallercompared to theuntreatedcontrolgroup(Fig.12).

Fig. 9 Conjunctivematrixwithactivefibroblasts

Fig. 11Abundantfibrouspannusandcondroidalmetaplasia

Fig. 13 Chondrocytesarrangementincords

Fig. 10Cellsarrangedinchondrons,hyperchromematrix

Fig. 12 Lesionoutbreaks,hyperchromematrix

Fig. 14Basophiliccellsarrangedinnests,matrix

Biophytomodulator treated group. Nearthe hypercellular defect, layers of chondrocytesare arranged in nests up in the vicinity of thedefectzone,thechondroidmatrixnearthedefectis intensely basophilic (Fig. 14), hyperchrome,pne cannotice the lackof cartilage fissure lines,thenumberofchondrocytesindifferentstagesofnecrosis is very small, abundant fibrous pannus

CRECAN et al

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Group H and HS. Following the histologicalexamination, a well-developed chondrocyte ma-trixwasobserved(Fig.18),surfaceirregularities,fragmentationof fibroustissue, fociofneovascu-larisation (Fig. 17), mucous conjunctive tissueand cartilaginous metaplasia similar to the BFgroup (Fig.20). The defect cavitywas filledwithcartilaginous tissue (hyaline) (Fig.19) withchondrocytesarrangedintheformofdisorganizedgroups with arrangement in the form of cords

Fig. 17 Fragmentationoffibroustissue,outbreaksofneovascularisation

Fig. 19 Defectcavityfilledwithhyalinecartilage

Fig.21 Condrocyte matrix surface, welldevelopedirregularities

Fig. 18 Condrocytematrixwelldeveloped

Fig. 20Fragmentationoffibroustissue,connectivebasedmucosaltissueandcartilaginous

metaplasiasimilartotheBFgroup.

Fig. 22Island-shapedbonematrix,deepbonemetaplasia.

(Fig.22)thechondroidmatrixhadheterochromia(hypochromicaspectinmostareas,hyperchromeon the surface), fibrillar. It contains islandshape bone matrix in the deep third with focalbone metaplasia (Fig. 22). Few crack lines withhorizontalarrangementbetweenthebaseofjointcartilagesubchindralbonewithfewchon-drocytesinnecrosis,arrangedintheformofgroupsintheimmediatevicinityofthedefect.

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CONCLUSIONSFollowingthisstudyweconcludethefollowing:

The experimental model used was found to beusefulforthestudyofchondraldefectsinsynovialjoints.Reparativeprocesses in thegroup treatedwith biofitomodulators were identified in moreadvanced developmental stages compared withtheuntreatedcontrolgroup.

In the groupH andHS reparative processeswere similar to those present in group BF andmore advanced than in group M. The resultsobtainedinthisstudyareencouragingforfurtherresearchontheeffectofADbiofitomodulatorilosinjointtherapy.

REFERENCES1. Bruckner P.; Mendler, M.; Steinmann, B.; Huber, S.; and

Winterhalter, K. H.: (1988). The structure of humancollagen type IX and its organization in fetal and infantcartilagefibrils.J.Biol.Chem.;263:16911-16917

2. Buckwalter, J. A. (1995): Should bone, soft-tissue, andjoint injuries be treatedwith rest or activity. J. Orthop.Res.;13:155-156

3. ByersP.D., BrownR.A.(2006):Cellcolumnsinarticularcartilagephysesquestioned:areview; OsteoArthritisandCartilage;14,3-12

4. Duncan H., Jundt J., Riddle J.M., Pitchford W.,Christopherson T., (1987) The tibial subchondral plate.Ascanningelectronmicroscopicstudy; JBoneJointSurgAm.;69:1212-1220

5. Callender GR, Kelser RA. Degenerative arthritis: acomparison of the pathological changes in man andequines.AmJPathol1938;38:253.

6. Mackay-SmithMP.Pathogenesisandpathologyofequineosteoarthritis.(1962)JAmVetMedAssoc;141:1246.

7. McIlwraith CW. (1982). Current concepts in equinedegenerativejointdisease.JAmVetMedAssoc;180:239e50.

8. Nilsson G, Olsson SC. (1973) Radiologic and patho-anatomicchangesinthedistaljointsandphalangesoftheStandardbredhorse.ActaVetScand;44(Suppl1):1.

9. McIlwraithCW,YovichJV,MartinGS.(1987).Arthroscopicsurgeryforthetreatmentofosteochondralchipfracturesintheequinecarpus.JAmVetMedAssoc;191:531e40.

10.CrecanC,DanielaOros,A.Dinc,P.Bolf,L.Oana,Lucia

Bel,C.Petean,C.Ober.(2012).StimulationofReparativeProcessesinCartilageDefectsinRabbitsUsingAD(DIEE)TypeBiophytomodulators.

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