白茅苷对去卵巢大鼠骨髓间充质干细胞功能和骨量的影响

目的观察白茅苷治疗去卵巢大鼠骨髓间充质干细胞功能和骨量的影响并初步探索可能机制。方法通过双侧去卵巢建立骨质疏松大鼠模型;随后随机分为假手术组(Sham)、去卵巢组(OVX)以及白茅苷组(BMG),每组10只;其中BMG组去卵巢大鼠每天给予白茅苷(20 mg/kg)灌胃治疗;待12周治疗结束后分离培养各组大鼠骨髓间充质干细胞(BMSCs),使用碱性磷酸酶(ALP)和茜素红(ARS)染色并使用蛋白质印迹检测BMP-2、Runx2、OPN、OCN、ALP和Col1蛋白表达;进一步使用Micro-CT和骨生物力学检测观察治疗效果。结果 OVX组大鼠BMSCs向成骨细胞分化后ALP和ARS染色阳性面积以及BMP-2、Runx2、OPN、OCN、ALP和Col1表达较Sham组明显降低(P0.05);而经过BMG治疗,BMG组大鼠BMSCs向成骨细胞分化后ALP和ARS染色阳性面积以及BMP-2、Runx2、OPN、OCN、ALP和Col1表达较OVX组明显增加(P0.05)。OVX组股骨最大载荷和弹性模量、BMD、BV/TV、Tb.N和Tb.Th较Sham组明显降低,而Tb.Sp则明显升高(P0.05)。BMG组左侧股骨最大载荷和弹性模量、BMD、BV/TV、Tb.N和Tb.Th均明显高于OVX组(P0.05),而Tb.Sp明显低于OVX组(P0.05)。结论白茅苷通过促进BMSCs诱导成骨分化来减少去卵巢大鼠骨骨密度、骨量和骨强度下降。     

脂肪干细胞向腱细胞分化促进腹正中切口愈合的实验研究

目的探讨脂肪干细胞(ASCs)向腱细胞分化在腹正中切口愈合中的作用。方法体外分离培养大鼠ASCs,利用骨形态发生蛋白-12(BMP-12)诱导其向腱细胞分化。20只大鼠随机分为两组,建立腹正中切口愈合模型,实验组将分化的ASCs涂抹至白线切口两缘,对照组未实施ASCs干预。1个月后利用生物力学测试及Masson染色评价切口愈合效果。结果成功分离大鼠ASCs,BMP-12诱导其向腱细胞分化,体外证实其特异性标志物Scx表达增加,I型胶原纤维(Collagen I)表达明显增多。实验组切口愈合后所能承受的最大拉力为(4.2±0.4)N,显著高于对照组(3.63±0.46)N,两组比较差异有统计学意义(P0.05);Masson染色提示实验组胶原纤维生成增多、致密,排列秩序显著改善。结论 ASCs向腱细胞分化可增加Collagen I的生成,促进腹正中切口的愈合。     

甲减患者治疗前后部分骨代谢指标的变化

目的探讨甲状腺激素替代治疗前后甲减患者血清骨保护素(0PG),血清骨钙素(OCN)的变化。方法选择健康者15例和甲减患者30例,其中临床甲减(oHT)和亚临床甲减(sHT)各15例。所有甲减患者采用左旋甲状腺素(L-T4)补充治疗至甲状腺功能在正常范围内。血清0PG、OCN采用酶联免疫法测定。结果 L-T4治疗前oHT和sHT患者血清OPG水平分别为3.60±0.38ng/ml和3.51±0.32ng/ml,明显高于对照组(2.68±0.48ng/ml,P=0.000);血清OCN水平分别为3.36±0.87ng/ml和3.42±0.75ng/ml,明显低于对照组(4.79±0.87ng/ml,P=0.000);多元回归分析结果显示,oHT和sHT患者血清OPG均与TSH呈正相关(r=0.580,P=0.023;r=0.934,P=0.000),血清OCN均与TSH呈负相关(r=-0.964,P=0.000;r=-0.825,P=0.000)。治疗至甲状腺功能正常后,两组血清OPG水平明显降低(分别为2.77±0.37ng/ml和2.92±0.31ng/ml,P=0.000),接近对照组水平;血清OCN水平明显升高(分别为4.63±0.70ng/ml和4.62±0.70ng/ml,P=0.000),接近对照组水平。结论甲减患者L-T4治疗后骨代谢紊乱可能得到改善。     

骨碎补总黄酮对iPS-MSCs成骨分化的影响

摘要：目的 研究骨碎补总黄酮对诱导多能干细胞来源的间充质干细胞(iPS-MSCs)成骨分化的影响。方法 将骨碎补总黄酮稀释为15.63、31.25、62.5、125、250 μg/mL的6个浓度,加入细胞培养基中,其中不含骨碎补总黄酮的培养基作为对照组,将iPS-MSCs分别接种于不同浓度的培养基中。采用CCK8法检测各组iPS-MSCs增殖情况,ALP活性检测及茜素红染色法检测各组成骨分化情况,RT-PCR检测各组OCN、Collagen Ⅰ mRNA表达情况。结果 CCK8法检测显示125、250 μg/mL 浓度的骨碎补总黄酮抑制iPS-MSCs生长,因此选用62.5 μg/mL浓度以下研究iPS-MSCs的分化情况。在成骨诱导培养基培养后,3个处理组ALP活性均高于空白对照组,并伴有茜素红观察钙化结节相应增多,差异均具有统计学意义(P＜0.01)。除此之外,RT-PCR结果显示3个浓度组的 OCN、Collagen Ⅰ mRNA表达量均高于空白对照组,差异具有统计学意义(P＜0.01)。结论 骨碎补总黄酮能够促进iPS-MSCs成骨分化,其作用机制可能与促成骨关键因子OCN和collagen Ⅰ的表达有关。     

骨髓间充质干细胞对肌腱移植物在骨隧道内愈合效果及示踪的实验研究

[目的]观察骨髓间充质干细胞对肌腱移植物在骨隧道内愈合的影响,并为干细胞的标记示踪提供实验依据.[方法]采用贴壁分离筛选法获取大鼠的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs),用超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)和DiI(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)对其标记.39只8周龄雄性SD大鼠随机分为两组,实验组21只,对照组18只.实验组在骨隧道内腱-骨结合面注入含双标的BMSCs和Pluronic F-127载体,对照组只注入载体,术后2、4、8周进行生物力学评估愈合效果,实验组2、4、8周组织冰冻切片荧光显微镜和手术后即刻、3、7 d行7.0 T MR示踪移植的BMSCs.[结果]SPIO、DiI能有效标记干细胞.实验组2、4、8周荧光显微镜观察在腱骨界面有DiI标记的阳性细胞存在.7.0 T MR未能在实验组腱骨界面观察到明显信号改变.生物力学最大拔出载荷实验组和对照组在术后2周无统计学差异,在4、8周实验组显著高于对照组(P<0.05).[结论]移植的骨髓间充质干细胞可以促进骨隧道内腱骨结合部的早期愈合,DiI能有效标记示踪干细胞,SHO能有效标记干细胞,但在该实验模型中7.0T MR可能不能活体示踪磁粒子标记的干细胞.     

兔自体骨膜包裹同种异体肌腱移植对腱-骨愈合的影响

目的 探讨兔同种异体肌腱表面包裹自体骨膜植入骨隧道对移植肌腱与骨隧道之间界面愈合的影响. 方法 健康4～5月龄新西兰白兔20只,体重2.5～3.0 kg,随机取一侧后肢作为实验组,另一侧后肢作为对照组.以同种异体肌腱在兔胫骨干骺端建立腱-骨固定模型,实验组骨隧道肌腱表面包裹自体骨膜,骨膜生发层朝向骨隧道;对照组骨隧道肌腱表面不包裹自体骨膜.分别于术后4、8周取标本作腱.骨界面的组织学检查(n=2),并进行最大拔出负荷的生物力学测试(n=8). 结果 术后4、8周标本大体观察,实验组骨隧道口周围新生骨较多;对照组新生骨少或不明显.组织学观察:术后4周实验组骨膜生发层内有大量间充质细胞增生,出现明显的膜内成骨,沿新生骨小梁周围可见大量骨母细胞呈栅形排列,新生骨小梁与骨膜延续:对照组腱-骨界面间无明显新生骨形成,有疏松结缔组织填充,腱-骨间连接较疏松.术后8周实验组新生骨和肌腱与骨隧道连接紧密,无间隙,新生骨量多,新生骨排列比较规则、整齐,腱-骨界面可见潮线形成,类似于正常腱-骨附着结构,骨膜内成纤维细胞增殖活跃,骨膜与肌腱间有较多纤维连接;对照组腱-骨界面局部有新生骨形成,排列紊乱,腱-骨间可见较多胶原纤维连接.术后4、8周,实验组最大拉出或拉断强度分别为(35.03±1.21)N/cm和(42.36±1.31)N/cm,对照组分别为(26.14 ±6.13)N/cm和(31.63±6.87)N/cm,两组比较差异均有统计学意义(P<0.05). 结论 兔自体骨膜包裹同种异体肌腱移植可缩短腱-骨间成骨时间,提高愈合强度,加速腱-骨间愈合.     

胰岛素样生长因子-1基因转染骨髓间质干细胞移植对糖尿病大鼠骨折愈合的影响

目的 观察胰岛素样生长因子-1(IGF-1)基因转染的骨髓间质干细胞(BMSCs)移植对糖尿病大鼠骨折愈合的影响,探讨糖尿病骨折的基因疗法.方法 Wistar雄性6周龄大鼠50只,随机分为对照组、实验组,链脲佐菌素诱导为糖尿病模型后均造成右侧胫骨骨折,体外高糖环境下Ad-IGF-1转染BMSCs,将相应组别的BMSCs移植于骨折局部.于1、2、3、4、6周摄X线,取局部骨痂行苏木素-伊红(HE)染色,并免疫组织化学检测骨痂中IGF-1,酶联免疫吸附试验(ELISA)检测血清IGF-1.结果 第4周骨痂IGF-1灰度值:实验组为103±5,对照组为109±4(P＜0.05).第4周血清IGF-1浓度:实验组为(668.80±8.07) ng/L,对照组为(569.20±9.31) ng/L(P ＜0.01).结论 IGF-1基因转染的BMSCs移植能促进糖尿病大鼠的骨折愈合.     

骨髓间充质干细胞移植对大鼠小肠缺血再灌注损伤的影响

目的 探讨大鼠骨髓间充质干细胞( BMSC)移植对其小肠缺血再灌注损伤的影响.方法 获取健康Wistar大鼠骨髓,体外提取、培养BMSC,收获传代培养的第3代细胞并鉴定其表型.在健康Wistar大鼠肠黏膜下注射皮肤黑色素瘤细胞,并用底物荧光素法示踪细胞.另外制作Wistar大鼠肠道缺血再灌注模型,将模型大鼠分为2组:实验组大鼠在肠黏膜下注射BMSC悬液1 ml(含5×106个细胞);对照组大鼠在肠黏膜下注射等量生理盐水.分别于术后0、2、6、24、72和120h处死大鼠,留取血清和小肠组织样本.采用酶联免疫吸附试验检测血清二胺氧化酶( DAO)、D-乳酸和肿瘤坏死因子α(TNF-α),用光镜和透射电镜观察肠组织,蛋白质印迹法和免疫组织化学法检测紧密连接蛋白-1(ZO-1).结果 体外成功分离培养出BMSC.经肠黏膜移植后的皮肤黑色素瘤细胞定植在肠道.实验组大鼠小肠病理改变较对照组轻,小肠黏膜屏障更完好.术后6h实验组DAO为(11.36±1.89) IU/ml,对照组为(14.27±2.09) IU/ml(P＜0.05);24 h时实验组DAO为(5.04±1.04)IU/ml,对照组为(7.35±1.46) IU/ml(P＜0.05).术后6 h实验组D-乳酸为(1.57±0.25) mmol/L,对照组为(1.93±0.19)mmol/L(P＜0.05);术后24 h实验组D-乳酸为(1.09±0.13)mmol/L,对照组为(1.41±0.07) mmol/L(P＜0.01).术后6h实验组TNF-α为(266.09±8.84)ng/L,对照组为(286.81±11.54) ng/L (P＜0.01);术后24 h实验组TNF-α为(190.39±4.24) ng/L,对照组为(218.49±15.51 )ng/L(P＜0.01).实验组ZO-1的表达高于对照组.结论 肠黏膜下移植BMSC可以减轻小肠缺血再灌注损伤.     

骨碎补总黄酮对去卵巢大鼠下颌骨显微结构及最大载荷的影响

目的应用Micro-CT和骨生物力学技术,探讨骨碎补总黄酮对去卵巢大鼠的下颌骨显微结构及最大载荷的影响。方法40只3月龄雌性SD大鼠,随机分为5组:假手术组(sham operation group,Sham)、去卵巢模型组(ovariectomized group,OVX)、骨碎补总黄酮高剂量组(high-dose drynaria total flavonoids group,GB-H)、骨碎补总黄酮低剂量组(low-dose drynaria total flavonoids group,GB-L)和戊酸雌二醇组(Estradiol Valerate group,EV),建模成功后连续给药12 w。实验结束后,取下颌骨进行显微CT扫描及三维重建,然后进行最大载荷测量。结果与Sham组相比,OVX组大鼠下颌骨微结构:骨密度、相对骨体积、骨小梁厚度、骨小梁数目明显减小(P0.05),骨表面积体积比、骨小梁间隙明显增高(P0.05);下颌骨的最大载荷明显减少(P0.05)。与OVX组相比,EV组大鼠下颌骨微结构获得良好修复,最大载荷也明显修复。骨碎补总黄酮低剂量组相对骨体积、骨小梁数目较OVX组显著升高(P0.05),骨小梁间隙显著减小(P0.05)。骨碎补总黄酮高剂量组疗效优于低剂量组,大鼠下颌骨的相对骨体积、骨表面积体积比、骨小梁间隙、骨小梁数目以及下颌骨骨密度均得到一定程度修复,下颌骨最大载荷也较OVX组显著增加(P0.05)。结论骨碎补总黄酮能够修复去卵巢大鼠下颌骨微结构,提高下颌骨骨密度和最大载荷,这将可能为颌骨骨质疏松的防治提供一种新的途径。     

Comparison of tendon-bone healing between a newly developed ultrasound device and the conventional metallic drill in a rabbit MCL reconstruction model

BackgroundLigament reconstructive surgeries demand tunnel creation using an over-drilling technique, though this technique has some problems such as metallic particle liberation or difficulties in tunnel creation other than circular cross-section. Recently, a new ultrasound (US) device for bone excavation to overcome these problems was developed. This study aimed to compare the tendon-bone healing in tunnels created using the new US device to that created using the conventional drill in a rabbit model.MethodsA total of 72 rabbits underwent a reconstruction for the anterior half of the medial collateral ligament (MCL) using a half of the patellar tendon. For the femoral tunnel creation, a new US device was used in 36 rabbits (US group), while a conventional metallic drill was used for the remaining 36 rabbits (DR group). At 4, 8, and 12 weeks postoperatively, biomechanical (n = 10) and histological (n = 2) evaluations were performed.ResultsThe ultimate failure load was almost equivalent between the US and DR groups at each period (US/DR; 4 weeks, 50.0 ± 12.8 N/43.4 ± 18.9 N, p = 0.62; 8 weeks, 78.6 ± 11.5 N/77.3 ± 29.9 N, p = 0.92; and 12 weeks: 98.9 ± 33.5 N/102.2 ± 38.3 N, p = 0.80). Pull-out failure from the femoral tunnel was only observed in two rabbits in the US group and one rabbit in the DR group at 4 weeks postoperatively. At 8 and 12 weeks, all specimens had a mid-substance tear. The collagen fiber continuity between tendon and bone occurred 8 weeks postoperatively in both groups and no histological difference was recognized throughout the evaluation period.ConclusionsThe tunnels created using the new US device and the conventional drill had equivalent biomechanical and histological features in tendon-bone healing. The bone excavation technology by the new US device may be applicable in ligament reconstructive surgeries.     

三七总皂苷促进腱骨愈合的实验研究

目的：研究三七总皂苷对腱骨愈合的影响。方法：取20只成年新西兰大白兔,随机分为PNS组及空白对照组,每组10只。两组动物均切断双侧膝关节前交叉韧带,以趾长伸肌腱重建前交叉韧带,制作腱骨愈合模型。PNS组骨隧道内注射三七总皂苷注射液,干预腱骨愈合。空白对照组不予干预措施。术后4周及8周每组各处死5只新西兰大白兔,获取腱骨愈合标本。通过肉眼、组织学切片观察两组腱骨愈合的变化及差异。结果：顺利完成所有手术,20只大白兔无死亡及关节感染。大体观察,两组动物4周时腱骨界面为纤维连接,8周时腱骨间隙已不容易辨认。组织学观察,PNS组腱骨界面细胞成熟度更高,sharphy纤维生长更加密集,间质钙化程度更高,新骨形成量更多。8周时界面组织形态分类,两组分布差异有统计学意义。结论：三七总皂苷能够促进腱骨界面细胞成熟,促进腱骨界面胶原生长,促进新骨形成,加快腱骨愈合速度,提高腱骨愈合质量。但本研究尚未对各项指标进行定量测量,三七总皂苷促进腱骨愈合的详细机制尚未完全明确。     

金葡素促进兔前交叉韧带重建后腱骨愈合的实验研究

目的 :探讨兔膝关节前交叉韧带(ACL)重建后,金黄色葡萄球菌滤液制剂(金葡素)对移植肌腱与骨隧道愈合的作用。方法:将3月龄新西兰大白兔24只(体重平均2.56 kg,雌雄不限)随机分为实验组和空白对照组,每组12只。采用兔自体趾长伸肌腱建立膝前交叉韧带重建后的腱骨愈合模型。实验组分别于术中及术后第2天向胫骨端腱骨界面处缓慢均匀注射金葡素0.1 ml,对照组以相同时间和方法注射等量0.9%Na Cl注射液。术后双膝不予制动,以青霉素注射液80万U/d连续肌肉注射3 d。分别于术后4、8和12周各处死动物8只,完整获取腱骨愈合标本并分别进行组织学分析,肉眼观察判断ACL重建大体情况,苏木精-伊红染色进行Yamakado腱骨界面组织形态分型评价,苦味酸-天狼猩红染色观察腱骨界面胶原纤维情况,血管内皮生长因子免疫组化染色定量评价腱骨界面再血管化情况,甲苯胺蓝染色定量评价腱骨界面新骨生成情况。结果:大体观察显示全部动物术后伤口Ⅰ期愈合,关节功能恢复良好,未见感染、死亡及金葡素注射不良反应。腱骨界面形态学分型结果显示术后各时间点实验组腱骨界面愈合质量均优于对照组(P0.05))。苦味酸-天狼猩红染色显示实验组胶原纤维术后4周生成少,8周大量生成,12周Sharpey纤维有序排列,集结成束,各时间点胶原纤维生成情况均优于对照组。基于血管内皮生长因子免疫组化染色和甲苯胺蓝染色的定量分析显示实验组术后4、8和12周腱骨界面新生血管面积和新骨生成面积均高于对照组(P0.05)。结论:金葡素能够在葡萄球菌肠毒素C等多种明确成分的协同作用下,通过局部启动无菌性炎症反应,加快腱骨界面血管生成和血液供应,激活骨细胞和纤维细胞大量增殖,有效促进兔ACL重建后腱骨愈合,有望成为促进ACL重建后腱骨愈合的新的临床方法。     

Biomechanical comparison of new Achilles tendon rupture repair technique the “Giftbox” versus the Krackow technique in New Zealand white rabbits: An experimental animal study

IntroductionTo compare the strength between the Achilles tendons repaired with the “Giftbox” and the Krackow techniques in New Zealand white rabbits post six weeks of tendon healing.Materials and MethodsEight rabbits were randomized into Giftbox and Krackow groups. Tenotomy was performed on the Achilles tendon of one side of the lower limb and repaired with the respective techniques. The contralateral limb served as control. Subjects were euthanized six weeks post-operative, and both repaired and control Achilles tendons were harvested for biomechanical tensile test.ResultsThe means of maximum load to rupture and tenacity in the Giftbox group (156.89 ± 38.49 N and 159.98 ± 39.25 gf/tex) were significantly different than Krackow's (103.55 ± 27.48 N and 104.91 ± 26.96 gf/tex, both p = 0.043).ConclusionThe tendons repaired with Giftbox technique were biomechanically stronger than those repaired with Krackow technique after six weeks of tendon healing.     

Enveloping the tendon graft with periosteum to enhance tendon-bone healing in a bone tunnel: A biomechanical and histologic study in rabbits

Purpose: Fixing and incorporating the tendon graft within the bone tunnel is a major concern when using grafts for ligament reconstruction. The periosteum contains multipotent stem cells and has the potential to form osteogenic and chondrogenic tissues. This study uses histologic and biomechanical analyses to examine the effect of periosteum on tendon-bone healing within a bone tunnel. Type of Study: Experimental study in an animal model. Methods: In this study, 36 adult New Zealand White rabbits were used. The long digitorum extensor tendon was transplanted into a bone tunnel of the proximal tibia. The periosteum from the proximal tibia was sutured on the surface of the tendon portion. The tendon was pulled through a drill-hole in the proximal tibia and attached to the medial aspect of the tibia. Histologic examination of the tendon-bone interface and biomechanical test for maximal pullout load were evaluated at 4, 8, and 12 weeks after operation. Results: Histologic analysis of the tendon-bone interface showed a fibrous layer formed between the tendon and the bone by the periosteum. This layer became progressively integrated with the tendon and bone surface during the healing process. At 4 weeks, the cancellous bone lining in the bone tunnel was interdigitated with the fibrous interface tissue. At 8 weeks, progressive new bone grew into the interface fibrous layer. At 12 weeks, collagen fibers anchored to the bone and organization with fibrocartilage formation developed between the tendon and bone. Biomechanical testing revealed higher maximal pullout strength in the periosteum-enveloped group at all time points, with a statistically significant difference at 8 and 12 weeks. The periosteum-treated group had a higher interface strength-to-length ratio and significant increase at 8 weeks and 12 weeks. Conclusions: The histologic and biomechanical studies demonstrated that, if periosteum was sutured on the tendon that was transplanted within a bone tunnel, it resulted in a superior healing process and better healed strength. When doing ligament reconstruction with a tendon graft, the periosteum can be sutured to the graft to enhance tendon-bone healing.Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 19, No 3 (March), 2003: pp 290–296     

The Detrimental Effects of Systemic Ibuprofen Delivery on Tendon Healing Are Time-Dependent

Background

Current clinical treatment after tendon repairs often includes prescribing NSAIDs to limit pain and inflammation. The negative influence of NSAIDs on bone repair is well documented, but their effects on tendon healing are less clear. While NSAIDs may be detrimental to early tendon healing, some evidence suggests that they may improve healing if administered later in the repair process.

Questions/purposes

We asked whether the biomechanical and histologic effects of systemic ibuprofen administration on tendon healing are influenced by either immediate or delayed drug administration.

Methods

After bilateral supraspinatus detachment and repair surgeries, rats were divided into groups and given ibuprofen orally for either Days 0 to 7 (early) or Days 8 to 14 (delayed) after surgery; a control group did not receive ibuprofen. Healing was evaluated at 1, 2, and 4 weeks postsurgery through biomechanical testing and histologic assessment.

Results

Biomechanical evaluation resulted in decreased stiffness and modulus at 4 weeks postsurgery for early ibuprofen delivery (mean ± SD [95% CI]: 10.8 ± 6.4 N/mm [6.7–14.8] and 8.9 ± 5.9 MPa [5.4–12.3]) when compared to control repair (20.4 ± 8.6 N/mm [16.3–24.5] and 15.7 ± 7.5 MPa [12.3–19.2]) (p = 0.003 and 0.013); however, there were no differences between the delayed ibuprofen group (18.1 ± 7.4 N/mm [14.2–22.1] and 11.5 ± 5.6 MPa [8.2–14.9]) and the control group. Histology confirmed mechanical results with reduced fiber reorganization over time in the early ibuprofen group.

Conclusions

Early administration of ibuprofen in the postoperative period was detrimental to tendon healing, while delayed administration did not affect tendon healing.

Clinical Relevance

Historically, clinicians have often prescribed ibuprofen after tendon repair, but this study suggests that the timing of ibuprofen administration is critical to adequate tendon healing. This research necessitates future clinical studies investigating the use of ibuprofen for pain control after rotator cuff repair and other tendon injuries.     

跟腱断裂术后即刻功能锻炼的临床研究

目的:通过与跟腱断裂术后2周进行功能锻炼及下地部分负重行走的比较,探讨跟腱断裂术后即刻功能锻炼及下地部分负重行走对患肢功能及跟腱再断裂的影响。方法:将2012年3月至2013年3月收治的64例闭合性跟腱断裂手术患者分为两。治疗组34例,男18例,女16例;年龄(41.4±7.6)岁;于术后第2天即行功能锻炼及佩戴支具下地部分负重行走。对照组30例,男16例,女14例;年龄(39.9±7.6)岁;术后予患肢短腿石膏跖屈位固定2周,2周后行功能锻炼及佩戴支具下地部分负重行走。两组患者由同一组医生采用相同的手术方式处理,观察并比较两组患者AOFAS踝关节功能评分、跟腱再断裂及伤口并发症情况。结果 :术后2个月AOFAS评分治疗组74.3±3.9,对照组71.7±4.2,治疗组高于对照组;术后1年治疗组93.3±3.9,对照组92.0±4.1,两组比较差异无统计学意义。术后治疗组无跟腱再断裂,对照组1例发生跟腱再断裂;术后治疗组2例发生伤口并发症,对照组1例发生并发症,差异无统计学意义。结论:跟腱断裂术后患者即刻功能锻炼,踝关节AOFAS功能评分优于固定2周后再行功能锻炼,同时不增加术后跟腱再断裂率及并发症发生率,有利于患肢功能恢复。     

Gene and protein expressions of bone marrow mesenchymal stem cells in a bone tunnel for tendon-bone healing

Purpose

Bone marrow mesenchymal stem cells (BMSCs) injected around tendon grafts can enhance tendon-bone healing and promote fibrocartilage formation. To understand gene and protein expressions of cells during tendon-bone healing, auto-BMSCs were implanted into a bone tunnel in anterior cruciate ligament reconstruction in a rabbit model.

Methods

BMSCs were harvested from New Zealand white rabbits. By an anterior cruciate ligament reconstruction model, 1 × 10⁶ BMSCs in 0.35 mL of fibrin glue was injected into bone tunnel as Fibrin-BMSC group. Only fibrin glue (Fibrin group) was injected as control. Three chondrogenesis genes and proteins, including Sox 9, collagen Type II (COII), aggrecan, and three osteogenesis genes and proteins, including Runx2, collagen type I (COI), and osteocalcin, between Fibrin-BMSC and Fibrin group were compared by real-time polymerase chain reaction assay and immunohistochemical assay postoperation.

Results

In real-time polymerase chain reaction assay, Sox9, COII, aggrecan, COI, and osteocalcin expressions upregulated and Runx2 downregulated were determined in Fibrin-BMSC group at 1 week. COII, aggrecan upregulated, and Runx2 and osteocalcin downregulated were determined at 4 weeks. In immunohistochemical assay, only Sox9, COII, and aggrecan proteins in only Fibrin-BMSC group were observed at 4 weeks. The protein expression as same as gene expression was obtained in a bone tunnel.

Conclusion

Auto-BMSCs promoted COII and aggrecan expression and reduced Runx2 and osteocalcin expression in a bone tunnel. It demonstrated that these cells could enhance fibrocartilage formation because of higher chondrogenesis expression during tendon-bone healing.     

骨形态发生蛋白诱导移植物成骨化促进腱－骨愈合

目的探讨骨形态发生蛋白(BMP)诱导肌腱移植物成骨化对肌腱－骨隧道愈合(简称腱－骨)的影响。 方法取健康成年新西兰兔26只建立腱－骨愈合模型,实验组采用BMP处理移植物及腱－骨界面,对照组仅采用生理盐水处理。术后12周时研究对象进行大体解剖观察、组织形态学检测及CT扫描,采用t检验比较各组之间腱－骨愈合情况。 结果12周时,实验组移植肌腱质地变硬,与宿主骨组织紧密连接、局部相互融合。苏木精－伊红染色(HE)和Masson染色显示实验组腱－骨界面以纤维软骨及Sharpey’s纤维连接为主。肌腱内部及界面之间均可见新生骨组织形成,部分区域新生骨与宿主骨组织形成骨性连接。而对照组移植物肌腱形态结构无变化,腱－骨界面间隙明显,以纤维结缔组织连接为主、未见新生骨形成。组织学评分结果显示BMP处理组腱－骨愈合更加成熟(6.6±0.8) vs( 2.7±0.7),(t＝13.361,P <0.01)。CT扫描检测结果显示实验组肌腱移植区域骨密度(191±17),明显高于对照组(11±4)(t＝31.591,P <0.01),提示肌腱移植区域发生成骨化。 结论BMP能够诱导肌腱移植物成骨化并促进腱－骨愈合。     

Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study

BACKGROUND: The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits. METHODS: The anterior cruciate ligaments of adult New Zealand White rabbits were replaced with autologous double-bundle semitendinosus tendon grafts. The semitendinosus tendon grafts had been infected in vitro with adenovirus-luciferase, adenovirus-LacZ (AdLacZ), or adenovirus-BMP-2 (AdBMP-2); untreated grafts served as controls. The grafts were examined histologically at two, four, six, and eight weeks after surgery. In additional experiments, the structural properties of the femur-anterior cruciate ligament graft-tibia complexes, from animals killed eight weeks postoperatively, were determined from uniaxial tests. The stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. RESULTS: Genetically engineered semitendinosus tendon grafts expressed reporter genes as well as BMP-2 in vitro. The AdLacZ-infected grafts showed two different histological patterns of transduction. Intra-articularly, infected cells were mostly aligned along the surface, and they decreased in number between two and eight weeks after surgery. In the intra-tunnel portions of the grafts, the number of infected cells did not decrease during the observation period. Moreover, a high number of transduced cells was found in the deeper layers of the tendons. In the control group, granulation-type tissue at the tendon-bone interface showed progressive reorganization into a dense connective tissue, and a later establishment of fibers resembling Sharpey fibers. In the specimens with an AdBMP-2-infected anterior cruciate ligament graft, a broad zone of newly formed matrix resembling chondro-osteoid had formed at the tendon-bone interface at four weeks after surgery. This area was increased at six weeks, showing a transition from bone to mineralized cartilage and nonmineralized fibrocartilage. In addition, in the AdBMP-2-treated specimens, the tendon-bone interface in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness (29.0 +/- 7.1 N/mm compared with 16.7 +/- 8.3 N/mm) and the ultimate load to failure (108.8 +/- 50.8 N compared with 45.0 +/- 18.0 N) were significantly enhanced in the specimens with an AdBMP-2-transduced graft when compared with the control values (p < 0.05). CONCLUSION: This study demonstrates that BMP-2 gene transfer significantly improves the integration of semitendinosus tendon grafts in bone tunnels after reconstruction of the anterior cruciate ligament in rabbits. Clinical Relevance: Novel technologies including gene therapy and tissue engineering, such as those described in this study, may provide useful therapeutic procedures to enhance biological healing after reconstruction of the anterior cruciate ligament.