作者：付志厚 王爱民作者单位：第三军医大学附属大坪医院骨科，重庆市 400042 " n B) l }0 r
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6 C0 F+ g4 _! a; s! B6 U6 x1 u 【摘要】 观察骨髓间充质干细胞修复假体周围骨缺损及对假体界面骨整合的影响。［方法］取新西兰大白兔15只。将骨髓间充质干细胞分离、扩增、骨向诱导，体外与珊瑚羟基磷灰石复合构建组织工程骨。将动物双侧股骨髁制作0.6 cm×1.2 cm的骨缺损，分别植入钛合金植入体，左侧植入体周围骨缺损充填组织工程骨，右侧植入体周围仅充填珊瑚羟基磷灰石为对照组。于术后4周、8周和12周分别行X线检查、扫描电镜、能谱分析及脱钙骨组织学观察。［结果］X线检查示，实验组术后4周缺损区内可见部分骨痂形成，与周围骨质分界清晰；8周可见大量模糊高密度骨痂影；12周缺损区已基本被高密度骨痂所填满，与周围骨质间分界变模糊。对照组4周、8周无明显改变，12周仅边缘有少量的骨痂形成；X射线能谱分析示，不同时间点实验组较对照组内Ca、P的百分含量增高（P＜0.05），随着时间的变化两组Ca、P百分含量都呈增高的趋势。实验组钙/磷比值随时间变化有逐渐升高的趋势，8周时达高峰，随后又降低。组织学结果示实验组术后12周可见明显的新骨形成，但仍可见到未吸收的羟基磷灰石颗粒。对照组各时间点均未见骨长入。［结论］骨髓间充质干细胞骨性诱导后复合珊瑚羟基磷灰石构建组织工程骨可修复假体周围骨缺损，促进假体骨界面骨整合。 % C# |% q* i" E1 z: Y& g
【关键词】骨整合 组织工程 骨髓间充质干细胞 珊瑚羟基磷灰石 人工关节/ m$ W- m5 @- J4 _
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Effects of bone marrow stromal stem cells on bone defect around the implant and osteointegration of implant-bone interface∥FU Zhi-hou, WANG Ai-min. Department of Orthopaedics, Daping Affiliated Hospital of the Third Millitary University of PLA, Chongqing 400042, China + R7 k; V9 e9 ?7 C0 \) j 4 \+ i( w2 o5 _0 E6 O* \Abstract ［Objective］To investigate the effects of bone marrow stromal stem cells(MSCs) on the bone defect around the implant and the osteointegration of implant-bone interface.［Method］Fifteen healthy clean New Zealand rabbits were studied. MSCs were separated, cultured and induced to osteoblasts. Cancellous bone defects（0.61.2 cm）were created in bilateral femur condyle in rabbits. Titanium alloy columns (0.31.0 cm)were implanted into both defects.Tissue engineering bone was implanted around the left titanium alloy implant，but only hydroxyapatite ceramic in the right.Thebiological characteristics were evaluated by X-ray examination, SEM , energy dispersive X-ray analysis and histologic studies at 4, 8, and 12 weeks postoperatively.［Result］ X-ray examination showed the density of bone tissue around the implant was aequalis. The interspace between implant and bone was filled with lots of bone trabeculae in the experimental group at 12 weeks postoperatively. The density of bone tissue was inhomogeneous, and low-density umbra existed in the interspace in the control group. Energy dispersive X-ray analysis showed the content of the Ca and P in the experimental group was statistically higher than that in the control group at different time points（P＜0.05） With the time going by, the content of the Ca and P in both groups had a tendency to increase gradually. They showed no statistical significance between 12 and 8 weeks postoperatively（P＞0.05. The ratio of Ca to P became higher and reaching the peak level at 8 week. Histological study showed the bone defects around the implant were repaired by new mature bone in the left at 12 weeks postoperatively, while in the right no new bone was found at any time point.［Conclusion］The compounds of hydroxyapatite ceramic and osteoblasts induced by MSCs can repair the bone defect around the implant and improve the osteointegration of implant-bone interface.: z- v4 B$ ^ O, D) \ T K
! Q; H U& d+ J2 ^3 Y4 mKey words：osteointegration;tissue engineering;bone marrow stromal stem cell;coral hydroxyapatite ceramic;arthroplasty ; z" `+ U2 r/ k/ _2 T , I, V) Z: o8 Z人工关节置换技术被认为是20世纪骨科最成功的治疗方法之一。人工假体的出现解决了许多从前不能实现的难题，但其也不是毫无瑕疵的，如感染、松动等，其中假体无菌性松动是导致人工关节置换术后中远期失败的主要原因。大量的分析研究表明假体-骨组织界面微动磨损是导致人工关节松动的一个主要因素。文献报告假体材料、表面特性及周围微环境等均可影响生物性假体周围骨长入。本文应用骨髓间充质干细胞(bone marrow stromal stem cell，MSCs)骨向诱导后复合珊瑚羟基磷灰石构建组织工程骨修复假体周围骨缺损，观察骨髓间充质干细胞对假体骨界面骨整合的影响。 ' ]) N2 }+ _) q* t4 ]$ E _2 }5 q+ s " C$ Y% D$ G% n1材料与方法 $ G( Y- |; U; b# j/ t; @8 ~: r% p$ C) i6 D- U* K% a) t; \
1.1实验动物 , x: S) t% e. Z3 q : X$ Z2 r7 U5 [% z9 A$ l5～7月龄健康清洁级新西兰大白兔(济南军区总医院动物中心，编号：SCKX 204057)15只，5个月月龄，体重2.5～2.9 kg，雌雄不限，分笼、颗粒干饲料喂养。饮用自来水。* Q1 i# K. w( e% q( b, X
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1.2材料 6 [ x2 A2 K+ o) H: t; p/ i7 t8 G2 Q! c# x m# H4 l
珊瑚羟基磷灰石为颗粒状5P10R（直径1～4 mm），孔径大小400～500 μm，空隙率30%～70%，由北京意华健科贸有限公司提供。钛合金植入体为Ti-6Al-4V，直径3 mm,由北京百慕高科股份有限公司提供。$ ~4 ]% M) K0 f; `, ^
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1.3.1骨髓MSCs的分离、培养与骨向诱导 ! _; m, y; Q, N0 W. {& M# H ^ - T/ F: B/ S3 J$ ]* w( I, P2 _无菌条件下于兔股骨大转子下穿刺，抽取骨髓约5 ml后缓慢注入含10 ml淋巴细胞分离液的离心管中；2 500 r/min离心15 min,吸取界面层细胞,用D-Hanks平衡盐溶液洗2次,2 500 r/min离心10 min；将细胞悬液按4105/mL接种于75 ml培养瓶内,按贴壁筛选法分离骨髓MSCs。7 d后细胞生长状态良好，已铺满瓶底的50%后按1∶3比例传代。取第4代骨髓MSCs将基础培养液更换为成骨诱导培养液（10-8mol/L地塞米松 10 mmol/L甘油磷酸钠 50 mg/L维生素C， pH7.3 0）。置37℃、 5%CO2、饱和湿度孵箱中培养。每3 d换1次液。第12 d行ALP染色、茜素红染色及I型胶原免疫组化染色，鉴定成骨细胞。- {( _) f I' k# T
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1.3.2体外组织工程骨的构建 4 X% E( v" f( e3 n4 F" h! A- r- E - w' d, J2 u$ b3 [将骨髓MSCs密度调整到106/ml，先加100 μl成骨诱导培养液于24孔培养板底，放入珊瑚羟基磷灰石颗粒，向材料表面加100 μl成骨细胞悬液，放置37℃、5%CO2、饱和湿度孵箱中培养2 h，翻转后另一面再加100 μl成骨细胞悬液，培养2 h，加入成骨诱导培养液盖过材料，放置37℃、5%CO2、饱和湿度孵箱中孵育，次日改换成骨诱导培养液，孵育1周。 q b, I8 V5 E, i D9 P. I, x2 ?- n: g' m, m
1.3.3动物模型的建立6 V0 k- C# K4 U" } p% ?; K
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速眠宁耳静脉麻醉后，无菌条件下取兔左侧膝关节外侧切口，显露股骨外髁，切去骨膜。用直径0.6 cm环锯由外髁向内髁方向制作一0.6 cm1.2 cm的松质骨穿通性骨缺损，植入钛合金植入体0.3 cm1.0 cm，周围用组织工程骨填塞、压实，关闭切口。同法制作右侧股骨髁骨缺损，钛合金植入体周围仅植入珊瑚羟基磷灰石作为对照。 ; L6 _. ~/ Q" W$ ~7 C* v. f ! H. K, a; m" \0 |5 Q6 |& l# ^1.4观察指标 2 f4 g; ^! i1 {& }: W/ I, f' l: d4 _1 A3 k$ W9 F4 P; u
1.4.1一般情况：包括观察动物进食、切口愈合情况。' Q; f3 d' f' @+ K9 L' [) g
. A& s8 i6 M4 R" {# _1 i1.4.2放射学观察：于术后4、8、12周拍摄双膝关节正侧位X线片，了解骨缺损修复、骨小梁形成情况。. i) p0 l$ `4 e: S- z ^( y9 [$ M
- M8 Q+ i# P. N' D+ r* A- ^1.4.3扫描电镜及能谱分析：术后4、8、12周分别处死5只动物，取植入体及周围骨组织，喷镀后制成扫描电镜标本，采用JSM-5800LV型扫描电镜，行扫描电镜观察及能谱分析。 . i) _2 M* w2 S 4 W1 {4 f5 m+ ?, h' g1.4.4组织学观察：切取股骨髁骨标本，用10%中性福尔马林固定，脱钙，树脂包埋，切片后HE染色，普通光镜下观察假体周围新生骨情况。 ; a$ B; V6 Q, E: W . X$ T( `0 i- g+ s# E# x P3 N1.5统计学分析：所有数据以x-±s表示，对照组与实验组的组间T检验，采用SPSS 12.0统计软件进行数据处理。' U* f5 A% c- a# G
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术后7 d动物可自由活动，未出现切口红肿、渗血渗液等情况，进食正常。 6 D! C5 w6 r2 E' j. \( V7 U ; S' q" _( }0 b, F* K P2.2X线观察5 S, ^# s7 l3 n* D J
1 Z7 \; q$ f7 n) w 【参考文献】9 y& H, E4 J/ p9 N
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［8］ Guglielmotti MB, Renou S, Cabrini RL. Evaluation of bone tissue on metallic implants by energy-dispersive X-ray analysis: an experimental study［J］. Implant Dent J, 1999, 8:303-309.作者: 榴榴莲 时间: 2015-5-27 17:22