经典孔隙结构钛合金内置物在兔肱骨近端大结节处骨长入的初步组织学研究

Preliminary histological study on bone-ingrowth Characteristics of titanium metal of classical pore structure in the greater tuberosity of proximal humerus in rabbits

目的建立兔肱骨近端生物型内置物植入模型,进行随时间演变内置物骨长入情况的组织学研究。方法建立肱骨近端经典孔隙结构即正六面体孔隙结构的钛合金内置物植入模型,将15只骨骼成熟雄兔随机分为3组,分别于建模后3、6、12周对肱骨近端内置物植入部分进行取材、切片及甲苯胺蓝染色,观察随着时间的演变内置物中骨长入的情况。结果在对15个样本进行定性观察后发现随着时间的进展,内置物空隙内的骨质在不断的增长,骨长入面积在不断的扩大。组织学定量分析发现3周与6周雄兔的骨长入面积百分比差异无统计学意义(P>0.05),而3周与12周以及6周与12周相比差异均有统计学意义(P<0.05),且总体在12周内,骨长入面积百分比与时间成对数关系。结论随着时间的推移,兔肱骨近端长入生物型钛合金内置物的骨质不断增加,且在12周内,骨长入面积百分比与时间成对数关系,骨长入速度逐渐变慢。

Background The postoperative nonunion of greater tuberosity is the mostcommon complication after the treatment of complex proximal humeral fracture with traditional shoulderprosthesis replacement,which severely affects shoulder joint function and reduces operative effect.Currently,the porous-coated prosthesis applied in clinic has several defects,such as low porosity,interfacial shear force between coating and prosthetic frame space,galvanic effect,etc.,which can affectbone ingrowth and reduce biological fixation strength.However,the titanium alloy biological implantwith classical regular hexahedral pore structure made by electron beam melting can overcome theshortcomings of traditional coated prosthesis and further impro ve the effect of bone ingrowth theoretically.In this study,the New Zealand rabbit was used for experiment.The classic pore structure,namely thetitanium alloy biological implant with regular hexahedral pore structure,was implanted into greatertuberosity.With the help of histological research,the purpose of this study includes the confirmations ofwhether the titanium alloy of classical pore structure has the potential of bone ingrowth over time;what arethe histological characteristics over time.This study can provide theoretical basis and evidential supportfor the application of titanium alloy biological implant in the field of shoulder surgery and the design ofartificial shoulder prosthesis for enhancing the healing of gre ater tuberosity.Methods(1)Design:animalmodeling observation.Time and location:it was completed in the animal laboratory of our hospital from May2015to November2016.Materials:15adult male New Zealand rabbits of4months old(random allocation with threerabbits in each group).The body weight was2.41-2.70kg,and the rabbits were raised in single cage and fedwith standard diet.All rabbits were observed preoperatively for1week to confirm healthy.The disposal of animalduring the experimental process is in conformity with the standard of medical ethics.(2)Methods.Establishmentof rabbit proximal humeral biological implant model:The rabbit was given the intramuscular injection ofmianmixin II(0.2ml/kg)for general anesthesia.Afterward,the rabbit was placed in left lateral decubitus.Skinpreparation,cleaning,regular disinfection and draping were carried out preoperatively.A2-cm longitudinalincision was made in the shoulder,and the deltoid muscle was bluntly split to expose greater tuberosity.A2.0Kirschner wire was drilled through the center of humeral shaft at the insertion of greater tuberosity,which had anangle of120°with the long axis of distal humerus.The titanium alloy screw with classic pore structure was insertedthrough bone tunnel to establish the model of proximal humeral titanium alloy biological implant.Without activehemorrhage,the wound was closed layer by layer.(3)Management after modeling:the rabbits were awakenednaturally and fed regularly.They were kept in cage(65cm×40cm×40cm)for activity restriction throughoutthe experiment,and the affected limb was immobilized.Penicillin(4×105U)was injected intramuscularly toprevent infection for the first3days after modeling,and the samples were collected randomly and respectively atthe3rd,6th and12th week.The bone sample from humeral head to its distal end of3cm was kept for use,and allthe muscle and soft tissue were removed.The bone sample was fixed with neutral buffered formalin for48hoursand then dehydrated with gradient alcohol of70%,80%and90%for7days and100%for twice and2days pertime.Afterward,the samples were infiltrated with Technovit7200VLC UV-curable resin for1month to completephotocurable embedding.The slices were made by EXAKT400CS/AW micro grinding system,which were cutparallel to humeral shaft with anatomical axis as the center.With about1mm set aside in both lateral and medialsides,the bone tissue was cut into slices of approximately2mm in thickness for toluidine blue staining.Underoptical microscope(Nikon Eclipse90i;Nikon Instruments,Inc.,Melville,NY,USA),the image acquisitionwas performed by4times magnification.The measurement of bone in growth area was processed and analyzed byImage-Pro Plus software(Media Cybernetics,Silver Spring,MD,USA).A rectangle that contained the lengtha of the screw and its width b was made in the sagittal section of the slice,and the total area S0=a×b.Therectangular area S1with screws removed was calculated as well as the toluidine blue stained area Six,and thedefinition of bone in growth area S%=Sx/S1×100%.All the histological measurements were performed by thesame operator.(4)Major observation indexes.①Qualitative observation:general situation of bone ingrowth ofbiological implant with time.②Quantitative measurement:changes of bone in growth area percentage with time.(5)Statistical analysis.SPSS20.0software(SPSS/PC Inc.,Chicago,IL)was used for statistical analysis.The datawas expressed as mean value±standard division,and a P value<0.05was regarded as statistically significant.The ANOVA variance analysis was performed to evaluate the difference of bone in growth area percentages in the3experimental groups at3-time points.Results(1)Quantitative analysis of experimental animals.All rabbitswere in good health after operation,and they all began to take food independently on the same day of modeling.Nowound infection or unpredicted death occurred during observation.In the process of modeling,the local anatomicalstructure of rabbit shoulder joint was similar to that of human being’s,and the anatomical position of surgicalapproach was relatively constant.The establishment of biological implant model of greater tuberosity was highlyrepeatable.(2)General change of biological implant bone ingrowth with time.Under the microscopic view ofhistological staining,the number of chondrocyte in screw gap showed enhancing trend with expanding range ofdistribution based on the3-time points of3rd,6th and12th months.(3)Changes of bone ingrowth percentagewith time.The histological data showed that the difference of bone ingrowth percentage among3groups wasstatistically significant,and the percentage of bone in growth area of12th week increased remarkably comparedto those of3rd week(P<0.05)and6th week(P<0.05).However,there was no statistical difference betweenthe bone in growth area percentages of3rd week and6th week(P>0.05).Meanwhile,we made the histogramand line chart of bone in growth area percentage of3-time points.The percentage of bone in growth area and itscorresponding time point were used for function fitting,and it was discovered that the bone ingrowth and time werein logarithmic relationship within3months.S%=13.706ln(T)+5.3095;R2=0.9868.Conclusion In thisstudy,the histological sections confirmed that the bone in growth area of titanium alloy biological implant withclassic pore structure in rabbit proximal humerus increases over time.Within12weeks of modeling,the bone in growth area and time were in exponential relationship,and the growth rate slowed down gradually.The results ofthis study suggest that it is necessary to strictly follow the rehabilitation protocol of active functional exercise aftershoulder arthroplasty to reduce the occurrence of postoperative complication in clinic.