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ePaper created 2014-07-29, 09:08:59 | version 1.30.01
I research 08 I implants2_2014 iourandthevascularisationofanewpaste-likebone- substitutematerialcomposedof-TCP,methylcellu- lose(MC)andhyaluronicacid(HY)bymeansofhisto- logical and histomorphometrical analysis. Therefore, thecellulartissuereactiontothisnewbonesubstitute material was investigated in the subcutaneous im- plantationmodelinWistarratsduringanobservation periodof60days.Threegroupswith(a)subcutaneous implantation of pure, solid -TCP, (b) subcutaneous injectionofsodiumchloride,and(c)ashamoperated animals served as controls. _Material and methods Bonesubstitutematerial In the present study an injectable bone substitute pastemadefromcrushedpure-phase-TCP,methyl- cellulose and hyaluronic acid was investigated. The manufacturing process of sintering and crushing re- sultsinceramicparticleswithasizeof<63µm,which weremixedwithanaqueouspolymersolutioninara- tioof70wt%ceramicsand30wt%polymersolution. Studydesign With approval from the Committee on the Use of LiveAnimalsinTeachingandResearchoftheStateof Rhineland-Palatinate, Germany, 90 female, 5-week- old Wistar rats were divided in three groups and re- ceived implantation of the above mentioned in- jectable-tricalciumphosphatebonesubstitutema- terial(group1)andpure,solid-TCPgranules(group 2). For control, ten animals underwent an operation withaninjectionofsodiumchloride(group3)andan- other20animals(group4)underwentpreparationof subcutaneouspocketswithoutbiomaterialimplanta- tion.At3,10,15,30and60days,theanimalsweresac- rificedbyanoverdoseofketamineandxylazinandac- cording to a previously described method9, 36-39 the bone substitute material was explanted and processed for histological and histomorphometrical analysis. Tissuepreparation The extracted samples were fixed in 4% formalin, cutintosegmentsof4mmthickness,decalcified,de- hydrated in alcohol and embedded in paraffin. After- wardsthesampleswerecutwithamicrotomeinsec- tionsofathicknessof4µmandstainedasfollows:the firstsectionwasstainedwithhaematoxylinandeosin (H&E),thesecondsectionwithtartrate-resistantacid phosphatase (TRAP) to identify osteoclast-like cells, while the third and fourth section were used for im- munochemical staining with ED-1 antibody (for cells of the monocyte-macrophage lineage). A fifth slide was stained with Movat’s pentachrome to visualise connective tissue ingrowth within the implantation bed and a seventh slide was stained by von Kossa/Safranin-O staining for identification of cal- cium and calcium phosphates.9,36-41 Histologicalandhistomorphometricalinvestigation After staining, the sections were investigated by independent investigators with a diagnostic micro- scope (Nikon, Tokyo, Japan) and the tissue–biomate- rial interaction within the implantation bed and the peri-implant tissue was examined histomorphomet- ricallyusingtheNIS-Elementssoftware(Nikon,Tokyo, Japan). The total number of vessels and their area on each slide were determined and related to the total implantation area. Thereby, for each time point, a mean number of vessels per square millimetre and a mean total vessel area could be determined. The re- sults of the quantitative analysis were presented as mean ± standard deviation with differences consid- eredsignificantifp-valueswere<0.05(*p<0.05)and highly significant if p-values were <0.01 (**p < 0.01). _Results Alltheanimalsineachgroupsurvivedthesurgical proceduresandthepostoperativeobservationperiod without complications. No signs for severe inflam- matory response were observed. Tissuereactionto-TCPgranules Beginning on day 3, the -TCP granule group material induced penetration of phagocytes, macrophages and connective tissue fibres, resulting inapoorlyvascularisedfiberandfibroblastrichgran- ulation tissue, which had completely penetrated the implantationbedatday15.Atday30and60onlyfew remnants of the bone substitute granules were obvi- ous. The vascularisation of the implantation bed re- mained low, presenting no significant differences in Fig. 2_The tissue reaction to the triphasic bone-substitute material at day 10 after implantation: a) an overview of the total implant area by means of a total scan. (H&E-staining, total scan, 100x magnification); b) the outer region (OR, double head arrow), which was distinguishable from the inner region (IR). (red arrows: vessels; arrow heads: giant cells; H&E-staining, 200x magnification; scale bar = 100 µm); c) ingrowth of connective tissue into the outer region of the implanted paste-like bone-substitute material. (red arrows: vessels; red asterisks: polymer solution; Movat’s pentachrome staining, 400x magnification; scale bar = 100 µm); d) multinucleated giant cells within the outer region; red arrow heads: TRAP positive multinucleated giant cells; black arrow heads: giant cells without TRAP activity) (TRAP-staining, 400x magnification; scale bar = 100 µm); e) inner region of the implantation bed. (arrows: mononuclear cells; asterisks: ) aqueous polymer solution (Movat’s pentachrome staining, 200x magnification; scale bar = 100 µm). Fig. 2a Fig. 2b Fig. 2c Fig. 2d Fig. 2e