Xenogenic demineralized bone matrix, Inżynieria chemiczna i procesowa, Publikacje, Inżynieria Bioprocesów i ...
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//-->J Orthopaed Traumatol (2008) 9:73–80DOI 10.1007/s10195-008-0006-6ORIGINAL ARTICLEXenogenic demineralized bone matrix and fresh autogenouscortical bone effects on experimental bone healing: radiological,histopathological and biomechanical evaluationA. S. BighamÆS. N. DehghaniÆZ. ShafieiÆS. Torabi NezhadReceived: 4 February 2008 / Accepted: 7 April 2008 / Published online: 10 May 2008ÓSpringer-Verlag 2008AbstractBackgroundBone grafting is used to enhance healing inosteotomies, arthrodesis, and multifragmentary fracturesand to replace bony loss resulting from neoplasia or cysts.They are source of osteoprogenitor cells and induce boneformation and provide mechanical support for vascular andbone ingrowth. Autografts are used commonly but quantityof harvested bone is limited. The aim of this study is toevaluate autograft and new xenogenic bovine demineral-ized bone matrix (DBM) effects on bone healing process.Materials and methodsTwenty male White New Zealandrabbits were used in this study. In group I (n=10) thedefect was filled by xenogenic DBM and in autograft groupthe defect was filled by fresh autogenous cortical graft andfixed by cercelage wire. Radiological, histopathologicaland biomechanical evaluations were performed blindly andresults scored and analyzed statistically.ResultsStatistical tests did not reveal any significantdifferences between two groups on the 14th postoperativeday radiographically (P[0.05). There was a significantdifference for union on 28th and 42nd postoperative daysand for remodeling at on the 56th postoperative dayradiologically (P\0.05). Statistical tests did not supportany significant differences between two groups for radio-logical bone formation (P[0.05). Histopathological andbiomechanical evaluation revealed no significant differ-ences between two groups.ConclusionsThe results of this study indicate that satis-factory healing occurred in rabbit radius defect filled withxenogenic bovine DBM. Complications were not identifiedand healing was faster, same as in cortical autogenousgrafting.KeywordsXenogenic DBMÁAutogenous cortical boneÁBone healingÁRabbitIntroductionBone grafting is used to enhance healing in delayedunions, nonunions, ostoectomies, arthrodesis, multifrag-mentary fractures and to replace bony loss resulting fromneoplasia or cysts [1]. Autogenous bone graft is commonlyused and is the standard to which allografts and graftsubstitutes are compared [2–7]. They may providea source of osteoprogenitor cells (osteogenesis), induceformation of osteoprogenitor cells from surrounding tis-sues (osteoinduction), and provide mechanical supportfor vascular and bone ingrowth (osteoconduction) [8].Though autogenous bone grafts have been clinicallyeffective, the additional surgical time required to harvestan autogenous graft, the morbidity associated with itscollection, and the limited availability of autogenous bonein some patients, have encouraged the search of suitablebone graft substitutes [5,9–11].Therefore, the use ofvarious bone graft substitutes including autografts, allo-grafts, xenografts, polymers, ceramics and some metalshave been employed to promote bone reunion [12,13].A. S. Bigham (&)Department of Veterinary Surgery and Radiology,Faculty of Veterinary Medicine, Shahrekord University,P. O. Box: 115, Shahrekord, Irane-mail: dr.bigham@gmail.comS. N. DehghaniÁZ. ShafieiDepartment of Surgery, School of Veterinary Medicine,Shiraz University, Shiraz, IranS. Torabi NezhadCollege of Medicine, University of Medical Science, Shiraz, Iran12374J Orthopaed Traumatol (2008) 9:73–80Allogenic, demineralized bone matrix (DBM) has beenused for several decades in human surgery for the treat-ment of nonunions, osteomyelitis and large defectsresulting from benign tumor removal [14]. The process ofdemineralization with hydrochloric acid destroys, but alsodecreases antigenic stimulation and may enhance therelease of bone morphogenic protein (BMP) [15]. BMPsstimulate local undifferentiated mesenchymal cells totransform into osteoblasts (osteoinduction), and the col-lagenous framework of the DBM particles allows formigration of tissue into the site (osteoconduction).Extensive research continues to identify the differentBMPs that might be osteoinductive, and these are beingreadied for clinical application [16–19]. Beyond their rolein osteoinduction, certain BMPs and DBM have shownpromise in aiding repair of osteochondral defects [20,21].Advantages of DBM over other substitutes include inher-ent osteoinductive capacity (unlike tricalcium phosphateand hydroxyapatite) and availability in large amounts. Theaim of study reported here was to compare the effects ofxenogenic bovine DBM and fresh cortical autogenousbone on the healing of bone defects in rabbits.and then ashed at 600°C for 24 h. These samples werethen dissolved in 0.6 mol/l nitric acid and analyzed byatomic absorption spectrophotometry to determine percentcalcium per gram dry weight (% Ca:DW) [23,24].Demineralization was considered adequate when sampleswere no longer visible radiographically and when calciumcontent was less than 1% [25]. After demineralization, allbone pieces were rinsed in sterile water and placed inphosphate buffer overnight. The bone pieces were thenrinsed and the pH was adjusted to 7.3. They were placedin ethanol, the ethanol was allowed to evaporate overnight,and the pieces were packaged aseptically and stored at4°C.Preparation of fresh cortical autogenous bone graftFresh autogenous cortical bone was harvested at the time ofsurgery during the creation of radius bone defect. Then allsoft tissues were removed from the harvested bone andused as a fresh autogenous cortical bone graft.Surgical techniqueAnimals were anaesthetized with ketamine (40 mg/kg, IM)and xylazine (5 mg/kg, IM). The left forelimb was shavedand prepared aseptically with povidone iodine and the limbdraped with sterile drapes. An incision was made directlyover the radius; which was exposed by dissection ofsurrounding muscles. Then an osteoperiosteal segmentaldefect was created on the middle portion of each radius atleast twice as long as the diameter of the diaphysis forcreation of nonunion model [26]. The created defects werefilled in ten rabbits (group I) with DBM (20 mg/defect) andin other ten rabbits (group II) with same harvested segmentof cortical bone and fixed by cercelage wire for preventionof segment dislocation in the grafted area.Postoperative evaluationRadiological evaluationRadiographs of each forelimb were taken postoperativelyon 1st day and at the 2nd, 4th, 6th and 8th weeks toevaluate bone formation, union and remodeling of thedefect. Results were scored using a modified Lane andSandhu scoring system [27] (Table1).Histopathological evaluationEight weeks after operation the rabbits were euthanizedpharmacologically for histopathological and biomechanicalevaluation. Histopathological evaluation was carried out onMaterials and methodsAnimalsTwenty male New Zealand Albino rabbits 12 months oldand weighing 3.0±0.5 kg were used in this study. Theresearch protocol for this experiment was approved by theShiraz University research committee.Preparation of bovine demineralized bone matrixDemineralized bone matrix, prepared from the midshafts ofthe long bones of a 2-year-old Holstein cow, were collectedfrom the local slaughterhouse. All bones were collectedaseptically, and the soft tissues were removed beforestorage at-70°C.The bones were later cleared of fasciaand cut into 1-cm pieces with a Stryker saw under saline(0.9% NaCl) solution lavage. Bone pieces were stored at-70°Cuntil further use. The pieces were then thawed in200-proof ethanol and air-dried. All bones were milled(Universal Mill A-20; Tekmer Co, Cincinnati, OH, USA)and placed through a sieve to collect 2- to 4-mm pieces.The pieces were then decalcified in 0.6 mol/l HCL at 4°Cfor 8 days under constant agitation.Demineralization was evaluated with radiography andcalcium analysis [22]. Density loss of xenogenic demin-eralized bone matrix was evaluated radiographically. Also,random samples of DBM were dried at 95°C, weighed,123J Orthopaed Traumatol (2008) 9:73–80Table 1Modified Lane and Sandhu radiological scoring systemBone formationNo evidence of bone formationBone formation occupying 25% of defectBone formation occupying 50% of defectBone formation occupying 75% of defectBone formation occupying 100% of defectUnion (proximal and distal evaluated separately)NonunionPossible unionRadiographic unionTotal point possible per categoryBone formationProximal unionDistal unionRemodelingMaximum Score42221012123475Table 2Lane and Sandhu histopathological scoring system modifiedby Heiple et al. [28]Union (proximal and distal evaluated separately)No evidence of unionFibrous unionOsteochondral unionBone unionComplete organization of shaftCancellous boneNo osseous cellular activityEarly apposition of new boneActive apposition of new boneReorganizing cancellous boneComplete reorganization of cancellous boneCortical boneNonEarly appearanceFormation under wayMostly reorganizedCompletely formedMarrowNone is resected areaBeginning to appearPresent in more than half of the defectComplete colonization by red marrowMature fatty marrowTotal points possible per categoryProximal unionDistal unionCancellous boneCortexMarrowMaximum score444442012341221012341234five rabbits of each group randomly. Left forelimb wereharvested and dissected free of soft tissues. Sagittal sectionsthat contained the defect site were cut with a slow-speedsaw. Each slice was then fixed in 10% formalin. The for-malin-fixed bone samples were decalcified in 15% bufferedformic acid solution and processed for routine histologicalexamination. Two 5-micron thick sections were cut fromthe centers of each specimen and were stained with hema-toxylin and eosin. The sections were individually evaluatedand scored by pathologist blinded to the treatment. Scoringsystem was according to lane and Sandhu modified scoringsystem by Hieple et al 1987 (Table2)[28].Biomechanical evaluationMechanical bending test was performed on radial-healeddefect of the left forelimb of five rabbits of each group bybiomechanical testing machine (Shimatzo, Japan). Duringthe test, the bone ends were placed between two jaws in thetesting machine and the load exerted at the grafting areauntil the failure. The forces, which were needed to breakthe bones were recorded. Data derived from mechanicaltesting were expressed as the mean±SEM (standard errormean) for each group.Statistical analysisThe radiological and histopathological data were comparedby Kruskal–Wallis, non-parametric ANOVA, whenP-val-ues were found to be less than 0.05, then pair wise groupcomparisons were performed by Mann–WhitneyUtest. Thebiomechanical data was compared by a Student’st-test(SPSS 15.00).ResultsThere was no intraoperative and postoperative death duringthe study. None of the rabbits sustained a fracture of theradius.Radiographic findingsThere was 25% bone formation in some rabbits in group Iand group II on 14th postoperative day. Although there wasunion in some rabbits of group I, there was no evidence ofunion in group II. Remodeling was not found in eithergroup. Statistical tests did not support any significantdifference (Table3,P[0.05) (Fig.1).There was 50–75% bone formation in some rabbits ofgroup I and 0–25% bone formation in some rabbits of groupII on 28th postoperative day. Although there was some union12376Table 3Radiological findingsat 2nd weekJ Orthopaed Traumatol (2008) 9:73–80PaGroup II (n=10)0 (0–1)0 (0–1)0 (0–0)0 (0–0)0.110.361.0001.000Median (min–max)Group I (n=10)Bone formationProximal unionDistal unionRemodeling0 (0–0)0 (0–0)0 (0–0)0 (0–0)aKruskal–Wallis non-parametric ANOVAFig. 1Radiographs of forelimbon 14th postoperative day.(a Xenogenic DBM.bautograft)in most rabbits of group II, remodeling was not seen in allrabbits of either groups. There was a statistically significantdifference only for union at the 28th postoperative day in theradiological signs of bone healing (P\0.05). When pair-wise group comparisons were performed by Mann–WhitneyUtest, group II was found to be superior to group I (Table4,P=0.008 andP=0.03) (Fig.2).There was 75–100% bone formation in all rabbits ingroup I and 50–75% bone formation in all rabbits of groupII on 42nd postoperative day. Although there was someunion in all rabbits of both groups and some remodeling ingroup I. There was a statistically significant difference onlyfor union at the 42nd postoperative day in the radiologicalTable 4Radiological findings at 4th weekMedian (min–max)Group I (n=10)Bone formationProximal unionDistal unionRemodelingabcsigns of bone healing (P\0.05). When pairwise groupcomparisons were performed by Mann–WhitneyUtest,group II was found to be superior to group I (Table5,P=0.01) (Fig.3).There was 100% bone formation and union in group Iand 75–100% bone formation and some union in group IIon 56th postoperative day. There were 25–50% pointsremodeling in the two groups. Group II was statisticallysuperior to group I only in terms of radiological callusremodeling (P\0.05). When pairwise group comparisonswere performed with Mann–WhitneyUtest, the group IIwas superior to group I (Table6,P\0.03) (Fig.4).Histopathological findingsHistopathologically there was no statistically significantdifference between the groups in terms of cancellous andcortical bone, union and marrow formation. None of thegrafted materials elicited a significant inflammatory reac-tion. In the group II the chondroblastic differentiation zonewas observed (Table7,P[0.05) (Fig.5).Biomechanical findingsThere was no statistically significant difference betweentwo groups in terms of biomechanical bending test(Table8,P[0.05).PaGroup II (n=10)1 (1–1)1 (1–1)b1 (0–1)0 (0–0)c1 (1–1)0 (0–0)0 (0–0)0 (0–0)0.0060.0040.0061.000SignificantP-valuesare presented in bold faceKruskal–Wallis non-parametric ANOVAP=0.008 (compared with group I by Mann–WhitneyUtest)P=0.03 (compared with group I by Mann–WhitneyUtest)123J Orthopaed Traumatol (2008) 9:73–80Fig. 2Radiographs of forelimbon 28th postoperative day.(a Xenogenic DBM,bautograft)77Table 5Radiological findings at 6th weekMedian (min–max)Group I (n=10)Bone formationProximal unionDistal unionRemodelingaPaGroup II (n=10)2 (1–2)2 (1–2)2 (1–2)1 (0–1)b2 (1–3)1 (0–1)1 (0–1)1 (0–1)0.110.0080.010.17SignificantPvalues are presented in bold faceKruskal–Wallis non-parametric ANOVAP=0.01 (compared with group I by Mann–WhitneyUtest)bFig. 3Radiographs of forelimbon 42nd postoperative day.(a Xenogenic DBM,bautograft)DiscussionIn this study a radius defect model was created tocompare healing of bovine DBM implant as a newxenograft and fresh autogenous cortical bone graft in therabbit model. This model has been reported previouslysuitable because there was no need for internal orexternal fixation that can influence the healing process[29]. The osteoperiosteal segemental defect was createdin middle portion of radius at least twice as long as thediameter of diaphysis to produce nonunion model andprevent spontaneous healing [26].123
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