摘要 [目的] 探讨髓芯减压复合自体骨髓间充质干细胞移植治疗股骨头缺血坏死的临床效果。 [方法] 自2006年11月至2010年4月,62例93髋股骨头缺血坏死接受了髓芯减压复合自体骨髓间充质干细胞移植治疗,根据ARCO分期标准:Ⅰ期8例14髋;Ⅱ期47例68髋;Ⅲ期7例11髋。术前常规Harris评分,拍摄髋关节正位、蛙式侧位X片、CT平扫及MRI。从患者髂前上棘抽取骨髓400ml,分离浓缩中间层骨髓间充质干细胞2.2±0.3×109 L-1 ,骨坏死区用6mm空心钻行髓芯减压术后,将浓缩细胞悬液注入。[结果] 54例71髋获得12个月以上随访,术后4髋改行全髋关节置换术,其余67髋术后Harris评分明显提高(术前平均59.2分,未次随访平均88.3分),差异有统计学意义(P改变。本组Ⅰ期8例14髋;Ⅱ期47例68髋;Ⅲ期7例11髋。术前常规拍摄双髋关节正位、蛙式侧位X片、CT平扫及MRI检查,通过MRI冠状位脂肪抑制T2WI序列评价股骨头坏死区及其周围骨髓水肿情况,在坏死区周围、股骨颈、粗隆间出现边缘模糊的高信号表示骨髓水肿,根据水肿的范围分为如下3级:1级:骨髓水肿局限于股骨头区;2级:骨髓水肿局限于股骨头区及股骨颈区;3级:骨髓水肿自股骨头延伸至股骨粗隆部[1]。采用Harris髋关节功能评分标准进行评定,其中包括疼痛(满分44分),关节功能(满分47分),关节活动范围(满分5分),畸形(满分4分)。1. 2 手术方法及术后处理:患者仰卧位,术前30min静脉应用抗生素预防感染,采用气管内插管麻醉。用Gallini骨穿剌活检针从双侧髂前上棘处抽取骨髓400 ml,用10ml注射器先抽取肝素生理盐水2ml(每100ml生理盐水加入肝素12500U),再抽取骨髓至10ml,尽量注意从髂棘多点穿刺,将抽取的骨髓注射入含枸椽酸葡萄糖抗凝液的收集袋内,转送到细胞分离室,将骨髓过滤以去除脂肪及细胞碎屑,以3000r/min将骨髓梯度离心,取中间层采集骨髓间充质干细胞,细胞计数2.2±0.3×1010 L-1,单侧收集25mL ~30mL,双侧收集50mL ~60mL,每髋注入25mL ~30mL。在C臂X光透视机下,对照术前CT和MRI反复确认坏死病灶后,用1枚1.5mm导针钻入骨坏死区,从正位和蛙式位透视确认导针准确钻入骨坏死区并距股骨头软骨下骨5mm,依次用4mm、5mm及6mm空心钻钻入,用长柄刮匙和偏心钻尽量去除坏死区骨组织,用50mL注射器将浓缩的细胞悬液通过Gallini骨穿剌活检针缓慢注入股骨头内,拔除活检针同时,用明胶海绵和骨蜡封堵骨道口,尽量减少细胞流失。术中术后无肺栓塞、深静脉血栓、伤口感染及转子间骨折等并发症。术后给予红花、丹参等活血抗凝处理,在不负重情况下不限制患侧髋关节活动,2月内患肢严格禁止负重,2月后扶拐部分负重,3月后弃拐行走。术后12月内,每3月到门诊复查1次,术后12月后,每6月门诊复查1次。复查时拍摄髋关节X片,并行CT平扫及MRI检查,判断有无影像学分期进展,并通过MRI冠状位脂肪抑制T2WI序列评价股骨头坏死区及其周围骨髓水肿消退情况;观察患髋疼痛、功能受限变化等,行Harris髋关节功能评分,根据有无改行关节置换及Harris髋关节功评分决定临床成功率。1. 3 统计学方法 采用SPSS11.5统计软包进行分析。数据以均数±标准差表示,术前、未次随访Harris髋关节功能评分采用配对t检验,P
摘要: 目的 评估关节镜下保留残端股骨双隧道双束后交叉韧带(posterior cruciate ligament, PCL) 重建术的临床效果。方法2007年6月至2009年5月,对19例PCL患者行保留残端股骨双隧道双束重建,股骨双隧道位于PCL原附着区,前外侧束位于10:30(左)或1:30(右),距关节软骨7mm;后内侧束位于8:30(左)或3:30(右),距关节软骨10mm。术中保留PCL残端纤维及周围滑膜组织,移植物采用自体股薄肌和半腱肌肌腱。结果 全部病例随访12~20月,平均18个月。后抽屉实验(posterior drawer test,PDT)术前(++)不稳12例,(+++)不稳7例;术后(-)16例,(+)不稳3例。仪表盘试验(dial test)用于评估评估旋转稳定,术前不稳11例,术后全部(-)。IKDC评分术前平均(61.12±4.8)分,术后平均(92.25±4.5)分, 差异有统计学意义(P<0.05)。其中正常15例,接近正常4例。Lysholm评分由术前(54.23±5.2)提高到术后 (90.13±6.1) 分,差异有统计学意义(P<0.05)。结论 保留残端股骨双隧道双束PCL重建术可获得良好的临床结果,符合解剖重建,能较好控制胫骨后向及旋转移位。关健词:后交叉韧带;膝关节;关节镜检查;移植Arthroscopic double-bundle reconstruction of posterior cruciate ligament with double-femur-tunnel and remnant preservationAbstract [Objective] To evaluate the clinical outcomes of arthroscopic double-bundle reconstruction of posterior cruciate ligament with double-femur-tunnel and remnant preservation.[Methods] From June 2007 to May 2009, 19 patients were treated with double-bundle PCL reconstruction under arthroscopy with double-femur-tunnel and remnant preservation. The two femoral tunnels were placed on footprint, anterolateral bundle was centered at 7mm from the articular cartilage at 10:30 (left) or 1:30 (right) o'clock, while posteromedial bundle was centered at 10mm from the articular cartilage at 8:30 (left) or 3:30 (right) o'clock. Residual PCL and surrounding synovial tissue saved during operation. Hamstring tendon was used as autograft. [Results] Average follow-up was 18 months (12-20 months). Twelve patients showed PDT (++) instability and seven patients show PDT (+++) instability preoperation, while sixteen cases showed negative PDT and three cases showed (+) instability postoperation. Rotational laxity was identified by dial test results,and eleven cases showed positive dial test preoperation while all cased showed negative postoperation. IKDC score was significantly changed from 61.12±4.8 to 92.25±4.5. In IKDC evaluation fifteen patients were graded as normal and four patients were graded as nearly normal. Lysholm score was significantly increased from 54.23±5.2 to 90.13±6.1. [Conclusion] The results of this study showed that double-bundle reconstruction of posterior curciate ligament with double-femur-tunnel and remnant preservation can yield satisfied clinical outcomes . It mimics anatomic reconstruction and better stabilizes posterior and rotation tibial translation.Key words: posterior cruciate ligament; knee joint; arthroscopy;transplataion 后交叉韧带(posterior cruciate ligament, PCL)是膝关节保持后向稳定性的主要结构,提供限制胫骨后移95%的控制力[ 1 ],同时还抑制外旋和外翻,对膝关节稳定性起着非常重要的作用。近年,随着交通事故、工矿业事故及运动损伤的增多,膝关节PCL损伤也越来越多。关节镜下重建PCL已经成为普遍接受的手术,既往多采用单束重建PCL,单束PCL重建术不能完全恢复PCL功能,双束PCL重建术能更好模拟PCL前外束和后内束,疗效应该优于单束重建术[ 2 ];此外,重建术中保留PCL残端,有望促进移植PCL的血管化和本体感觉的重建。我科从2007年6月至2009年5月,采用关节镜下保留残端股骨双隧道双束后交叉韧带重建术治疗膝关节PCL断裂19例,取得了较好的疗效。1 资料与方法1.1 临床资料 2007年6月至2009年5月对19例PCL损伤患者行关节镜下保留残端股骨双隧道双束重建术,男14例,女5例;年龄27~58岁,平均年龄38岁。致伤原因:运动伤2例,车祸伤10例,重物砸伤7例。左膝11例,右膝8例。其中2例因腘血管损伤急诊行血管移植及小腿筋膜切开减压术。病史1~10周,平均3周。所有患者均经MRI检查证实为PCL断裂,5例合并半月板损伤。排除PCL胫骨止点撕脱骨折、患肢伴严重软组织损伤或骨折,无合并前交叉韧带损伤、后外侧复合体损伤及内外侧副韧带损伤。 所有患者均有受累关节疼痛、肿胀、乏力及关节不稳,90度后抽屉实验(posterior drawer test, PDT)12例为(++)不稳,7例为(+++)不稳,胫骨结节塌陷征、反向拉克曼征阳性。仪表盘试验(dial test)用于评估旋转稳定性,7例为(大于5度)不稳,4例为(大于5度)不稳,8例阴性(小于5度)。5例麦氏征阳性,内外侧扳试验阴性,前抽屉实及拉克曼征阴性。术前患肢平均IKDC评分为(61.12±4.8),平均Lysholm评分为(54.23±5.2)分。1.2手术方法1.2.1移植物的准备持续硬膜外麻醉下,患者取仰卧位,大腿固定架固定患肢,大腿根部上气囊止血带。取同侧半肌腱和股薄肌肌腱备用,于胫骨结节内侧,沿皮纹方向斜行切开2cm,逐层切开皮肤、皮下至半腱肌、股薄肌肌腱止点,钝性分离后切断胫骨侧止点,用取腱器切取,应尽量切取全长,包括胫骨止点处骨膜。根据肌腱长度可将2条肌腱对折或三折,然后用5号Ethibond缝线两端分别编织,以生理盐水纱布覆盖,较粗的一根作为前外侧(anterolateral,AL)束替代物,较细的一根替代后内侧(posteromedial, PM)束。1.2.2 关节腔检查及处理取膝前内、前外和后内侧入口,探查关节腔内所有结构,清理关节腔内积血,修整或缝合撕裂的半月板组织,用PCL剥离器分离PCL胫骨附着点周围滑膜和关节囊组织,暴露PCL胫骨及股骨附着点,不刨削PCL残端并尽量不清除或少清除PCL胫骨及股骨附着点周围滑膜组织(图1)。1.2.3 骨隧道的建立及移植物的引入及固定胫骨隧道的建立:屈膝90度,在关节镜直视下, 应用PCL胫骨导向器,入口点位于胫骨前内侧取腱切口内,胫骨结节以远10~20mm和胫骨结节以内20~30mm的交汇处,使胫骨隧道与胫骨呈50度~60度角,出口点位于胫骨平台后缘中心偏外侧,平台以下 10mm的原PCL止点处, 使用空心钻依次递增钻取胫骨隧道, 钻隧道期间用保护器置于导针尖端以防止导针及空心钻因用力过猛或意外损伤膝关节后方重要血管或神经。股骨隧道的建立:股骨隧道位于原PCL附着区,前外侧束隧道位于股骨髁间窝内髁外侧壁前上1:30 (右膝)或10:30 (左膝),距关节软骨边缘约7mm位置,骨隧道直径7mm;后内侧束隧道位于3:30(右膝)或8:30(左膝),距关节软骨10mm位置, 骨隧道直径 5mm。两隧道间距约有5mm,以防遂道间骨质塌陷入,用内向外方式建立股骨隧道(图2)。将导引线从胫骨隧道引入膝关节腔,通过股骨隧道引出,再把移植物分别引入胫骨隧道、股骨后内侧束隧道、股骨前外侧束隧道。股骨端分别用一枚微型钢板钮扣(Endobutton)拉出固定,拉紧移植物胫骨端的同时,最大限度屈伸膝关节20次,由胫骨隧道外口稍远处拧入一枚 4mm皮质骨螺钉,然后在胫骨向前的张力下,于屈膝90度和30度位分别将前外侧束和后内侧束移植物的牵引线栓桩固定于上述皮质骨螺钉,再用一枚7~8mm×25mm可吸引界面螺钉双重固定胫骨端。1.2.4 术后康复术后伸膝位可调式支具固定,冰敷,小腿下垫薄枕,麻醉清醒后即可开始踝关节主动屈伸运动及股四头肌等长收缩练习,第2周开始CPM被动屈伸练习,第8周屈伸运动基本达到正常后开始主动屈伸运动及部分负重行走,第12周可完全负重行走,6个月后可逐渐恢复运动,12个月后根据关节稳定性决定运动强度。1.2.5 统计学处理 采用SPSS13.0统计软件,数据以均数±标准差(x±s)表示,采用膝关节Lysholm和IKDC评分标准,对患者术前和末次随访时进行Lysholm和IKDC评分,采用ANOVA方差分析,并计算P值,P<0.05差异有显著性。2.结果19例均获得随访,随访时间12~20个月,平均18个月。术后胫骨结节塌陷征、反向拉克曼征阴性,PDT试验阴性16例,(+)不稳3例,仪表盘试验均阴性。患者IKDC评分由术前平均(61.12±4.8),提高到术后的(92.25±4.5)分, 差异有统计学意义(P<0.05)。其中正常15例,接近正常4例。膝关节Lysholm评分由术前平均(54.23±5.2)分,提高到术后的(90.13±6.1)分,差异有统计学意义(P<0.05);无血管神经损伤、下肢深静脉血栓和感染等并发症。膝关节伸直受限5度1例,屈曲活动正常17例,较健侧小10度2例。3.讨论3.1 PCL重建相关解剖学研究 PCL股骨、胫骨起止点、走行及分束等的解剖研究一直是PCL重建研究的热点及重点,其研究结果对临床PCL解剖重建具有重要指导意义。尸体解剖发现,PCL形成一个扇形的股骨附着点(footprint)和一个缩窄的胫骨附着点,其股骨附着点在形态和大小上变异较大,而其胫骨附着点则较为一致。PCL胫骨附着部呈一个不规则四边形,位于内、外侧半月板后角之间,关节线下方的胫骨髁间后窝处,胫骨附着处为约14度左右斜坡。股骨附着部为一宽广的扇形,位于股骨内侧髁外侧面,延伸超过髁间窝顶12点钟[3、4]。将韧带由附着区完整切下后,韧带股骨端宽(20.6±1.3)mm,中段最窄(10.4±1.0)mm,胫骨端宽(15.6±1.8)mm。 根据PCL解剖形态和不同屈膝角度的紧张状态,通常将PCL分为粗大的前外侧AL束和较细小的后内侧PM束,AL束在伸膝位松弛,中度屈膝位紧张;而PM束在完全伸膝位紧张,中度屈膝位松弛,两束在膝关节屈伸过程中张力互为补充。Edwards等[4]的研究发现,AL束的中心位于10:20±00:30,距软骨边缘7±2mm;PM的中心位于08:30±00:30,距软骨边缘10±3 mm;此外,近年的生物力学研究发现,PCL除主要限制胫骨后移外,还一定程度上影响胫骨的内、外侧移位及旋转移位[5]。3.2 PCL断裂重建方法的选择目前,PCL双束重建术是否获得优于单束重建术的临床结果仍存在争议[6],但大量比较PCL单束与双束重建对膝关节稳定性的生物力学研究发现,双束重建具有更好的膝关节稳定性。PCL单束重建通常是重建PCL粗大的AL束,其股骨隧道通常放置在股骨AL束附着处,移植物在屈膝90度拉紧固定,从而减少胫骨的后向不稳,单束重建的韧带在伸膝时承受的张力大于屈膝时承受的张力,而膝关节功能活动主要发生在70度范围内,从而可导致重建的韧带因周期性疲劳而松弛。增加PM束的PCL解剖双束重建,一般在膝关节近伸直位拉紧固定,增加了膝关节在接近伸直活动范围内的后向稳定,同时还可增加膝关节的旋转稳定,因此,PCL解剖双束重建可望获得较PCL单束重建更好的临床效果[2,7]。Markolf等[8]在尸体标本上比较了正常膝关节PCL与单束、双束重建后的PCL在膝关节0度~120度屈伸时所承受的应力,并测量了PCL单束、双束重建后膝关节的松弛度。结果发现,单束重建的AL束能很好复制正常PCL承受的应力,但在0度至30度屈伸时,关节松弛大于正常膝关节,而加入PM束的双束重建能减少膝关节在上述屈伸范围内的松弛度。临床实践中,继往多采用单束重建损伤的PCL,但术后易出现持续性的后方松弛,特别在伸膝关节时,只有大约60%左右的患膝关节功能接近正常。近年采用双束重建PCL的部分临床结果显示,近 80%甚至更高比例的患膝功能接近正常[9,10],获得了较单束重建更好的临床效果,但由于不同学者选用的移植物种类、骨隧道解剖位置、张力固定技术及术后康复方法的不同,使PCL双束重建方法的临床结果难以比较。本研究根据PCL股骨附着点宽广,胫骨附着点较缩窄的特点,并参考Edwards等[4]对AL束、PM束股骨、胫骨附着点的解剖研究成果,采用股骨双遂道双束胫骨单遂道解剖重建PCL,AL束距关节软骨7mm(浅点),PM束距关节软骨10 mm(深点)建立股骨遂道,在胫骨髁间后窝稍偏外、平台下10 mm 、PCL胫骨附着点中心建立胫骨遂道,先采用微钢板钮扣(Endobutton)固定AL束和PM束股骨端,再分别在屈膝90度和30度先后拉紧并通过栓桩和挤压螺钉双重固定,取得了与上述PCL双束重建相似的临床效果。此外,本研究所采用的手术方式较胫骨也采用双遂道双束重建PCL的简单,同时尽量不清理或少清理PCL残端周围滑膜组织,从而降低了腘窝血管及神经损伤的风险。3.3 PCL重建术中保留残端及其周围滑膜组织的意义PCL除具有稳定膝关节的生物力学功能外,还具有本体感觉功能。交叉韧带内及其周围滑膜组织内存在本体感觉感觉器,如Ruffin小体,而交叉韧带损伤后本体感觉的恢复在很大程度取决于本体感觉感觉器的残留或再生,以及神经的再生支配。韩桂全等[11]研究采用腘绳肌双束重建PCL后关节本体感觉的改变,膝关节位置觉在重建术后逐渐减退,15个月开始改善,20个月时较术前明显增强,但未恢复至正常膝关节水平。在PCL重建过程中,保留含有本体感觉感受器的 PCL残端及其周围滑膜组织,将有利于本体感觉感受器的残留或再生,以及神经的再生支配,从而加快膝关节本体感觉的恢复。此外,残留的PCL纤维及其周围滑膜组织紧邻或包裹移植物,有利于移植组织的血管化及韧带化。Gohil等[12]通过MRI检查发现,保留残端纤维的ACL重建较常规ACL重建移植物再血管化发生更早。另外,关节液可在韧带重建后渗入隧道影响腱~骨愈合,残留纤维对隧道口的封闭效应可有效防止其发生[13]。参 考 文 献:[1] KATONIS P, PAPOUTSIDAKISA, ALIGIZAKISA, et al. Mechanorecep tors of the posterior cruciate ligament [ J ]. J Int Med Res, 2008, 36 ( 3) : 387 -393.[2] MCGUIRE DA, HENDRICKS SD. Comparison of anatomic versus nonanatomic placement of femoral tunnels in Achilles double-bundle posterior cruciate ligament reconstruction [ J ]. Arthroscopy, 2010, 26 (5):658-666. [3] TAJIMA G, NOZAKI M, IRIUCHISHIMA T, et al. Morphology of the tibial insertion of the posterior cruciate ligament [ J ].J Bone Joint Surg Am, 2009, 91(4):859-866. [4] EDWARDS A, BULL AM, AMIS AA. The attachments of the fiber bundles of the posterior cruciate ligament: an anatomic study [ J ].Arthroscopy, 2007, 23(3):284-290. [5] li g, papannagari r, li m, bingham j, et al. Effect of posterior cruciate ligament deficiency on in vivo translation and rotation of the knee during weightbearing flexion [ J ].Am J Sports Med, 2008, 36 (3):474-479.[6] KOHEN RB, SEKIYA JK. Single-bundle versus double-bundle posterior cruciate ligament reconstruction [ J ].Arthroscopy, 2009, 25 (12): 1470 -1477.[7] WHIDDON DR, ZEHMS CT, MILLER MD, et al. Double compared with single-bundle open inlay PCL reconstruction in a cadaver model [ J ]. J Bone Joint Surg Am, 2008, 90(9):1820-1829.[8] MARKOLF KL, FEELEY BT, JACKSON SR, et al. Biomechanical studies of double-bundle posterior cruciate ligament reconstructions [ J ].J Bone Joint Surg Am, 2006, 88 (8):1788-1794.[9] ZHAO J, XIAOQIAO H, HE Y, et al. Sandwich-style posterior cruciate ligament reconstruction [ J ].Arthroscopy, 2008, 24 (6):650-659. [10] Chen B, Gao S. Double-bundle posterior cruciate ligament reconstruction using a non-hardware suspension fixation technique and 8 strands of autogenous hamstring tendons [ J ].Arthroscopy, 2009, 25(7):777-782.[11] 韩桂全, 王韶峰, 罗文明, 等. 关节镜下半腱肌和股薄肌肌腱双束重建后交叉韧带后关节本体感觉的近期改变. 中国修复重建外科杂志[ J ]. 2008, 22(10):1181-1183. HAN GUIQUAN, WANG SHAOFENG, LUO WENMING, et al. Latest changes of joint proprioceptive function after posterior cruciate ligament reconstruction using double-bundle semitendinosus and gracilis under arthroscopy[ J ]. Chinese Journal of Reparative and Reconstructive Surgery, 2008, 22(10):1181-1183. Chinese[12] GOHIL S, ANNEAR PO, BREIDAHL W. Anterior cruciate ligament reconstruction using autologous double hamstrings: a comparison of standard versus minimal debridement techniques using MRI to assess revascularization. A randomized prospective study with a one-year follow-up [ J ]. J Bone Joint Surg Br, 2007, 89(9):1165-1171.[13] 赵金钟, 皇甫小桥. 保留并牵张残留纤维的前十字韧带束双重建术[ J ]. 中华骨科杂志, 2010, 30(6):563-567. ZHAO JZ, HUANGFU XQ. Double-bundle anterior cruciate ligament reconstruction with remnant preservation and tensioning [ J ]. Chinese Journal of Orthopaedics, 2010, 30(6):563-567. Chinese
摘要 目的:探讨壳聚糖/β-甘油磷酸钠凝胶用作骨髓间充质干细胞的载体体外构建可注射型组织工程软骨的有效性和可行性。方法:骨髓间充质干细胞与壳聚糖/β-甘油磷酸钠制成注射型细胞/凝胶复合物并行体外成软骨诱导培养,通过倒置显微镜、扫描电镜和组织学检查等观察细胞在凝胶内黏附、生长、增殖、向软骨细胞分化及形成软骨样组织等情况。结果:在体外成软骨培养条件下,壳聚糖凝胶支持骨髓间充质干细胞生长、增殖、向软骨细胞分化并形成软骨样组织。结论:骨髓间充质干细胞/壳聚糖/β-甘油磷酸钠凝胶复合物,有望用作可注射型组织工程软骨修复软骨缺损。关健词 间充质干细胞;壳聚糖/β-甘油磷酸钠;软骨;组织工程Construction of Injectable Tissue-engineering Cartilage with Mesenchymal Stem Cells and Chitosan Gels in VirtoLi Zhong, Chen geDepartment of Bone and Joint Surgery, the Affiliated Hospital of Luzhou Medical CollegeAbstract Objective: To explore the effectiveness and the feasibility of chitosan/β-glycerophosphate disodium(C/GP) gels as carriers for mesenchymal stem cells(MSCs) to construct injectable tissue-engineered cartilage in Vitro. Methods: BMSCs were incorporated into the C/GP mixture to construct injectable BMSCs-C/GP gel complex, then chondrogenesis induced cultured in vitro. The adhesion, growth, proliferation and chondrogenic differentiation of BMSCs into C/GP gel and chondrogenesis of BMSCs-C/GP gel complex were estimated by inverted phase contrast microscope, scanning electron microscope and histology. Results: C/GP gel supports the growth, proliferation and chondrogenic differentiation of BMSCs and BMSCs-C/GP gel complex can form cartilage-like tissue when chondrogenic cultured in vitro. Conclusion: BMSCs-C/GP gel complex can be used as injectable tissue-engineered cartilage to repair cartilage defects.Key words Mesenchymal stem cells(MSCs) Chitosan/β-Glycerophosphate Disodium(C/GP) Cartilage Tissue-engineering负载组织工程种子细胞的可生物降解支架材料必须具有良好的细胞相容性,种子细胞应能在支架上良好的黏附、生长和增殖等[1 ]。用猪骨髓间充质干细胞与壳聚糖/β-甘油磷酸钠混合液混匀,制成注射型细胞/凝胶复合物并行体外成软骨诱导培养,了解支架材料对骨髓间充质干细胞黏附、生长、增殖及成软骨分化等功能的影响,以便寻找适宜的注射型细胞载体,移植修复组织缺损。1 材料和方法1.1 主要试剂和材料 Percoll淋巴细胞分离液(Pharmacia公司)、胎牛血清和DMEM/F-12培养基(Hyclone)、胰蛋白酶(华美公司)、TGF-β1(深圳达科为公司)、壳聚糖(浙江玉环公司)、β-甘油磷酸钠(Fluka公司)、鼠抗人Ⅱ型胶原单克隆抗体(Medicorp公司)、生物素化羊抗鼠抗体(博士德公司)。其余均为国产分析纯。1.2方法1.2.1骨髓间充质干细胞分离培养成年健康广西巴马猪(由第三军医大学动物实验中心提供)用10mg/kg氯胺胴肌注麻醉后,常规从髂骨抽取骨髓4.5~5ml,与少量DMEM/F-12培养基在无菌条件下充分混匀,加入到含等体积密度为1.073的Percoll分离液中,以3000rpm/min分离心30min,吸取中间乳白色云雾状有核细胞层,以PBS洗涤2次。置沉淀细胞入DMEM/F-12普通培养基液(含质量分数为10%的胎牛血清,100U/ml青霉素,100μg/ml链霉素)中,计数细胞,调整细胞密度,按3×105个细胞/cm2接种培养瓶,置37℃、5%CO2、饱和湿度培养箱内培养,3天后首次全量换液,以后每2~3d换液1次。倒置显微镜逐日观察细胞形态及生长情况。细胞融合后,0.25%胰蛋白酶消化,按1:3比例进行传代培养,留取第二代细胞备用。1.2.2 2%(w/v)壳聚糖盐酸溶液、56%(w/v)β-甘油磷酸钠溶液的制备2%(w/v)壳聚糖盐酸溶液的制备:5g壳聚糖溶解于225ml0.1M稀盐酸,室温下充分搅拌混匀,高压蒸汽灭菌消毒10min,4℃冰箱保存。56%(w/v)β-甘油磷酸钠溶液的制备:28gβ-甘油磷酸钠,溶解于50ml去离子水中,0.22μm滤器过滤除菌,4℃冰箱保存。1.2.3 间充质干细胞/壳聚糖/β-甘油磷酸钠凝胶的制备冰溶中,1体积56%β-甘油磷酸钠溶液逐滴加入7体积2%壳聚糖盐酸溶液中充分搅拌混匀,加入消化收集的第2代骨髓间充质干细胞充分混合,将细胞/壳聚糖/β-甘油磷酸钠混合液接种于培养板,置37℃培养箱内静置10min待其形成凝胶后,用培养液置换3次,每次20分钟。分别加入DMEM/F-12普通培养基液和成软骨诱导培养液(含10ng/ml TGF-β1的普通培养基液),隔日换液。1.2.4 间充质干细胞在壳聚糖/β-甘油磷酸钠凝胶中的倒置显微镜观察间充质干细胞接种培养板后,用倒置显微镜动态观察细胞在支架上黏附、生长和增殖情况。1.2.5 间充质干细胞在壳聚糖/β-甘油磷酸钠凝胶中的扫描电镜观察取出体外培养1周的壳聚糖/磷酸甘油-间充质干细胞复合物,用D-Hanks缓冲液小心漂洗2次,3%戊二醛固定,梯度酒精脱水,液氮冷冻干燥,表面喷金,扫描电子显微镜观察。1.2.6 组织学及免疫组织化学观察普通培养和成软骨诱导培养的细胞-支架复合物体外培养3周,用4%多聚甲醛固定24小时,常规酒精逐级脱水,石蜡包埋,切片。行苏木素-伊红、甲苯胺蓝及Ⅱ型胶原免疫组织化学染色观察。2 结 果2.1 间充质干细胞的2维培养观察细胞接种后24h即可见部分圆形的单个核细胞贴壁,极少数贴壁细胞与成纤维样细胞形态类似,细胞为梭形。72h后细胞大部分贴壁,形态变成梭形,并有少量突起,细胞核较大,扁圆形。3d后进行首次换液,以后第2~3天换液1次,红细胞逐渐随换液而除去。5~6d细胞形成集落状,10~14d细胞汇合成单层。传代细胞贴壁较快,5~7d可长满瓶底。2.2壳聚糖/磷酸甘油凝胶复合物的物理性状所用脱乙酰度为95.11%的壳聚糖呈白色粉未状,制成的2%(w/v)壳聚糖稀盐酸溶液为高粘稠无色液体,高压蒸气灭菌器消毒10分钟后,粘稠度降低,颜色变为淡黄色;56%(w/v)磷酸甘油溶液为无色透明液体。2%(w/v)壳聚糖稀盐酸溶液和56%(w/v)磷酸甘油溶液按7:1(v /v)混合后,其混合溶液的pH值接近7.2,具有适合细胞生长的pH环境,37℃下凝固时间为5-10分钟,呈黄白色,适合用作负载细胞的可注射凝胶载体。2.3 间充质干细胞/支架复合物倒置显微镜观察刚接种的细胞呈圆形,折光性强,在支架材料内均匀分布;3-4h部分细胞开始变形,3d后大部分细胞完成伸展变形,形态为梭形,呈网状排列,小部分细胞仍呈圆形;随着培养时间的延长,支架上的细胞数量逐渐增加,但明显慢于2维培养的细胞增殖速度,增殖细胞聚集呈簇状。表明间充质干细胞能在支架材料上良好地附着、生长和增殖(见图1)。2. 4 间充质干细胞/支架复合物扫描电镜观察壳聚糖/磷酸甘油凝胶呈三维多孔状结构,凝胶内的孔隙度和孔径较小,但仍有孔洞相互连通。细胞均匀分布于支架材料的周边和中心部位,细胞大部分呈梭形,贴附生长,伸出较多突起,大小约50-80μm,少数为圆球形,周围有较多的纤维状基质相互联接呈网状(见图2)。2.5 组织学及免疫组织化学检测体外培养3周的细胞~支架复合物HE染色见,细胞数量多,分布均匀,大部分细胞呈梭形,少数细胞呈圆形,胞核蓝染,呈圆形或椭圆形,胞浆红染。复合物表面形成由多层扁平细胞组成的膜样结构。成软骨诱导细胞~支架复合物HE染色见:细胞数量多,分布均匀,大部分细胞呈梭形,胞核蓝染,胞浆红染,复合物表面形成由1-2层梭形细胞组成的膜样结构,表层膜样结构下和复合物中,由较多已形成软骨陷窝的圆形细胞聚集在一起形成“软骨岛”,软骨样细胞外基质蓝染;而未诱导细胞~支架复合物内细胞为梭形,未见形成软骨陷窝的圆形细胞,胞外基质也无异染。甲苯胺蓝染色见:成软骨诱导细胞~支架复合物形成的“软骨岛”样结构内,细胞外基质染成蓝紫色,呈强阳性;而对照组细胞外基质无异染,表明成软骨诱导细胞~支架复合物中的细胞合成软骨特异性基质——GAGs。Ⅱ型胶原免疫组化染色见:成软骨诱导细胞~支架复合物形成的“软骨岛”样结构内,细胞外基质染成棕黄色,呈阳性,“软骨岛”样结构外的细胞外基质染色呈阴性;而未诱导细胞~支架复合物内的细胞外基质染色阴性。表明成软骨诱导凝胶-细胞复合物细胞外基质内有Ⅱ型胶原阳性表达。大体观察见,随着培养时间延长,复合物表面光滑,硬度稍增加,但仍明显低于正常关节软骨。3 讨 论间充质干细胞易于分离和培养扩增,在体内外都能向成骨细胞、软骨细胞、腱细胞、脂肪细胞等方向分化。因此,间充质干细胞在组织工程、细胞治疗和基因治疗中具有广阔的应用前景[2]。间充质干细胞可来源于骨髓、脂肪、肌肉和滑膜等组织,其中,骨髓间充质干细胞可从骨髓穿剌获取,操作容易,损伤小,是目前研究得最多,也是最有可能应用于临床的间充质干细胞。骨髓间充质干细胞能在多种三维支架上黏附、生长、增殖和分化,并复合三维支架材料移植修复组织缺损[3、4]。本研究与上述研究结果相似,间充质干细胞在体外培养条件下,能在壳聚糖/β-甘油磷酸钠凝胶内良好的黏附、生长和增殖。可生物降解的三维支架材料是组织工程学研究的重要内容之一。组织工程支架材料的来源包括两大类:天然来源的材料和人工合成材料。聚乳酸、聚羟基乙酸及其共聚物等人工合成材料具有不受来源限制,容易加工成形,可根据需要调整物理、化学、生物力学和降解性能等优点,但材料的疏水性,导致细胞难于在材料表面黏附、伸展,影响了细胞的生长和增殖。此外,材料降解形成酸性产物积聚,也对细胞产生一定毒性作用;许多天然材料本身就含有与细胞外基质相似的成分和结构,因而具有良好的细胞相容性和细胞亲和性,如胶原、透明质酸和氨基葡聚糖(GAG)是软骨细胞外基质的主要成分,是用于制备软骨三维支架的良好材料。本研究中应用的壳聚糖具有良好的生物降解性和生物相容性,是海洋无脊椎动物外壳上的甲壳素脱乙酰基后的产物,来源广泛,取材料方便,其化学结构为(1,4)- 2 - 氨基 - 2 - 脱氧 -β- D - 葡聚糖,类似于软骨细胞外基质氨基葡聚糖,其降解产物在体内不积聚,无毒,无剌激,无免疫原性,无热原性、无致畸和致突变效应[5 ],广泛用于生物医用领域。壳聚糖可制成pH依赖性水凝胶,其溶解性与pH值密切相关。pH值<5时,壳聚糖完全溶解于水中形成十分黏稠的胶体,经碱化处理后,可形成凝胶。Cho等[6]以碱性盐β-甘油磷酸钠来调节壳聚糖酸溶液的pH值至中性,使壳聚糖酸溶液由pH值依赖性水凝胶变为热敏性水凝胶,壳聚糖/β-甘油磷酸钠混合物在室温下为液态,在37℃左右很快形成凝胶,从而形成了适宜于细胞载体的注射型水凝胶。本研究用骨髓间充质干细胞与壳聚糖/β-甘油磷酸钠中性溶液充分混合后在37℃形成凝胶,再将细胞~凝胶复合物体外成软骨诱导三维培养3周,结果发现,细胞能在支架材料上良好的附着、生长、增殖和向软骨细胞分化,骨髓间充质干细胞-壳聚糖/β-甘油磷酸钠凝胶复合物能形成软骨样组织。以上结果表明,壳聚糖/磷酸甘油复合物具有与骨髓间充质干细胞良好的细胞相容性,且骨髓间充质干细胞能在壳聚糖/磷酸甘油复合物内良好的黏附、生长、增殖和均匀分布。成软骨诱导壳聚糖/磷酸甘油-骨髓间充质干细胞复合物体外培养3周,可逐步形成有一定机械强度,表面光滑的组织工程软骨,并能保持复合物的初始形态。表明壳聚糖/磷酸甘油复合物支持骨髓间充质干细胞生长、增殖和成软骨分化,有望用作负载骨髓间充质干细胞的注射型凝胶载体微创修复关节软骨缺损。参 考 文 献1 李忠,杨柳,陈光兴,等.胶原凝胶复合CPPf/PLLA支架与软骨细胞的相容性研究.四川医学,2005;26 (6):591 2 杨思远,石应康,伍长学等.流体剪应力作用于间充质干细胞对其诱导内皮分化全抗氧化能力的影响.生物医学医学工程学杂志,2008;25 (3):6163 Hu J, Feng K, Liu X, Ma PX. Chondrogenic and osteogenic differentiations of human bone marrow-derived mesenchymal stem cells on a nanofibrous scaffold with designed pore network.Biomaterials; 2009,30(28):50614 Bosnakovski D, Mizunl M, Kim G, et al. Chondrogenic dedifferentiation of bovine bone marrow mesenchymal stem cells (MSCs) in different hydrogels: influence of collagen typeⅡextracellular matrix on MSC chondrogenesis. Biotechnol Bioeng, 2006; 93 (6 ): 11525 王征, 刘万顺, 韩宝芹等. 不同分子量羧甲基壳聚糖的制备及其对皮肤成纤维细胞和角质形成细胞生长的影响. 生物医学医学工程学杂志, 2007, 24 (2): 340 6 Cho J, Heuzey MC, Begin A, et al. Effect of urea on solution behavior and heat-induced gelation of chitosan-β-glycerophosphate. Carbohydrate Polymers, 2006; 63 (4): 507
摘 要: 目的 探讨临床症状、体征和影像学表现不典型的膝关节“隐匿性”滑膜软骨瘤病的关节镜诊断和治疗效果。方法2006年月11月至2009年10月我科关节镜诊治4例“隐匿性”膝关节滑膜软骨瘤病,临床表现为膝关节肿胀、疼痛,X线平片和磁共振检查未发现游离体,关节镜检查确诊,并行关节镜下滑膜软骨瘤摘除和病变滑膜切除术。 结果 膝关节“隐匿性”滑膜软骨瘤病表现为:大量微小软骨游离体存在于关节腔、或少量微小软骨游离体局限于滑膜组织内。4例患者均经病理检查证实,全部获得随访,平均随访14个月,所有病例术后功能良好,未见复发,仅1例伴软骨损伤患者残留膝关节轻度疼痛。结论 膝关节“隐匿性”滑膜软骨瘤病临床表现不典型,X平片和磁共振检查常不能确定诊断,关节镜是该病唯一可靠的诊断手段和有效治疗方法。关键词 滑膜软骨瘤病 膝关节 关节镜 滑膜化生Arthroscopic diagnosis and treatment of occult synovial chondrmatosis of the knee Abstract: Objective The purpose of this study was to determine the arthroscopic diagnosis and treatment outcomes of occult synovial chondromatosis with atypical clinical symptoms, signs, or imagings. Methods From November 2006 to October 2009, 4 cases of occult synovial chondromatosis of the knee were diagnosed and treated with arthroscopy. Clinical picture was characterized by pain and swelling of the involved joint, while the characteristic X-radiograph and magnetic resonance imaging (MRI) findings were negative. All cases were diagnosed and treated with loose body removal and partial synovectomy by arthoscopy. Results Occults synovial chondromatosis of the knee demonstrated a large number of minute loose cartilaginous bodies in joint or a few minute cartilaginous bodies under the surface of the synovial membrane and confirmed by histological examination. Average follow-up was 14 months. All of the patients showed good outcomes, no patients required revision surgery. One case suffered from pain of the involving joint postoperation due to cartilage degeneration. Conclusions The clinical symptoms and signs of occult synovial chondromatosis of the knee are atypical, and conventional radiology and MRI often delay diagnosis. The definitive diagnosis was made on the basis of arthroscopic findings and arthroscopy debridement surgery is the effective treatment for occult synovial chondromatosis.Key word synovial chondromatosis; knee joint; arthroscopy; synovial metaplasy滑膜软骨瘤病(Synovial Chondrometosis, SC)由Ambroise Paré于1558年首先描述,以前又称为:滑膜骨软骨瘤病(Synovial osteochondromatosis),滑膜软骨化生不良(Synovial Chondrometaplasy),滑膜软骨病 (Synovial Chondrsis) 等,是一种少见的滑膜组织良性病变[1,2]。主要根据临床表现及X线平片和磁共振检查发现关节腔内存在游离体而得到诊断[3]。我科从2006年月11月至2009年11月共收治膝关节滑膜软骨瘤病患者25例,其中4例临床表现不典型, X线平片和磁共振检查也未能发现膝关节腔内存在游离体,通过关节镜得到诊断和治疗,并通过病理学检查证实,笔者称其为“隐匿性”滑膜软骨瘤病,现报告如下。1 临床资料1.1 一般资料本组病人4例,4个膝关节;男2例,女2例,年龄12~40岁,平均27岁;右膝2例,左膝2例;病史2月~12个月,平均9.5个月;临床表现:3例有轻微膝关节外伤史,所有患者主要表现为膝关节反复肿胀、疼痛,伴膝关节周围广泛压痛及股内侧肌萎缩,关节屈伸活动基本不受限,无关节内结节或肿块,也无关节绞锁及弹响;3例出现关节线压痛及McMurray试验阳性者,术前误诊为半月板撕裂。膝关节X线片未见关节内游离体钙化影,关节间隙不狭窄;膝关节磁共振(MRI)检查表现为关节腔大量积液,与通常滑膜软骨瘤病在MRI所有扫描序列都表现为灶状低信号区不同,4例患者均未表现出灶状低信号区的游离体钙化影,也无骨髓水肿及关节边缘骨侵蚀。实验室检查:血常规正常,血沉、C-反应蛋白、类风湿因子、抗“O”及血尿酸无异常升高。抽取关节液作检查:大体观察:关节液量多,呈黄色、稍清稀,无明显浑浊;关节液常规检查:细胞数及分类无异常;关节液行革兰氏染色涂片查细菌、结核PCR及细菌培养均阴性。1.2 手术方法珠网膜下腔阻滞麻醉下平卧,大腿固定架固定患肢,气囊止血带下手术,采用美国Smith & Nephew公司直径4.0mm的30°关节镜系统和电动刨削系统及美国杰西公司Arthocare射频消融刀。常规先建立膝前内、前外入口,从关节镜镜鞘引流出关节液,关节液行大体观察及送实验室检查(包括关节液常规、结核PCR、革兰氏染色涂片查细菌及细菌培养和药敏试验)。用关节镜按髌上囊、外侧间沟、外侧间室、髁间窝、内侧间室、内侧间沟依次检查关节腔内各个部分;必要时再建立后内侧入口和后外侧入口分别检查膝关节后内侧间室和后外侧间室。3例患者表现为,膝关节腔内有大量(约数千枚)直径约2~3mm大小均匀的软骨性游离体(图1),将关节镜镜鞘置入关节腔内,用关节灌洗液尽量将游离体从关节镜鞘内冲洗出,关节腔内剩余游离体用游离体抓钳或髓核钳取尽,取病变滑膜组织及游离体送病理检查,同时用刨刀和Arthocare射频消融刀行增生、充血和水肿的病变滑膜组织全切术,其中1例病史超过1年的40岁患者,根据Outerbridge软骨损伤分级法,髌股关节和外侧关节间室出现Ⅲ~Ⅳ度软骨损伤,用射频消融刀修整,使之表面尽可能平整光滑;另1例病史2月的青年患者,关节镜下表现为关节腔内无游离体,约2cm×1cm的髌上囊滑膜组织稍充血,其内隐约可见白色直径约1mm大小均匀的软骨样“米粒体”数枚,软骨样“米粒体”被包裹在滑膜内,与滑膜组织无蒂相连,取此处含软骨样“米粒体”的充血滑膜组织送病检(图2),仅切除局部充血滑膜组织。术后关节腔内注入吗啡一支,置入负压引流管于术后12小时后开放,局部加压包扎,伸膝位支具固定,术后膝关节冰敷,根据引流量的多少,术后24~48小时内拨除引流管。术后第二天行膝关节主动伸屈、股四头肌训练和直腿抬高锻炼,拨除引流管后即可下地行走。2 结果本组4例,术后均经病理检查证实,全部病例均获随访,平均随访时间14个月(6~30月),所有随访病例未见复发。除1例伴软骨损伤患者术后膝关节仍诉轻中度疼痛外,所有随访病例膝关节肿胀及疼痛症状逐渐消失,关节功能均超过术前水平。治疗前以及最后1次随访时进行Lysholm膝关节评分,治疗前评分平均67.25分(52~75),治疗后平均93.00分(80~100),由于病例数量较少,未行统计学处理。3讨论滑膜软骨瘤病是来源于关节滑膜组织软骨化生的一种良性滑膜病变,其病因尚不清楚,以关节滑膜组织及关节腔内形成软骨结节或游离体为其特征[4,5]。原发性滑膜软骨瘤病形成的软骨结节大小和形状相似,可附着于关节滑膜或从滑膜脱离而游离于关节内。本病好发于40~60岁,主要累及膝、肩、髋和踝等大关节[6、7 ],儿童极少发病。可因游离体机械卡压或长期磨损关节出现退变性关节炎而出现临床症状,质硬游离体在关节腔内移动伴关节绞锁是其典型的临床表现,非特异性临床表现还有关节肿胀、疼痛、压痛、活动障碍及骨擦音等。滑膜软骨瘤病的影像学表现与软骨结节是否发生钙化或骨化密切相关,若软骨结节已发生骨化,则可从X线平片或CT扫描上清楚发现多发的骨化软骨小体,大约70%~95%的原发滑膜软骨瘤病病例可通过放射学检查发现分布于关节腔的多发性骨化软骨小体[8],若软骨性结节未钙化,X线平片或CT扫描不能显示。由于MRI检查具有良好的软组织分辨率,是诊断甚至定性某些关节滑膜病变的良好选择,未骨化的软骨结节也能在MRI上清楚显示[9、10]。根据软骨结节骨化程度可有多种MRI表现,绝大多数滑膜软骨瘤病表现为,在所有MRI扫描序列上为灶状低信号区;还可表现为外周呈低信号环,中央呈脂肪样高信号等。此外,MRI还可显示滑膜增厚、软骨损伤、骨侵蚀、骨髓水肿及关节腔积液量等。虽然MRI扫描具有良好的软组织分辨率,但由于本组病例中的软骨结节太小(直径约1mm)或仅有极少量的微小软骨结节包裹在滑膜组织内,可能超过了临床常用的1.5T磁共振仪的分辨率,导致MRI影像上未能显示软骨结节。典型的膝关节滑膜软骨瘤病表现为关节肿痛、关节内有可游走的质硬的游离体、关节绞锁、打软腿,甚至出现膝关节活动范围受限。游离体直径往往达到数毫米甚至更大,结合X平片和MRI检查可获得诊断。本组病例由于软骨结节太小或仅有极少量的微小软骨结节包裹在滑膜组织内,临床症状不典型,仅表现为膝关节肿痛,不能扪到游离体,也无关节绞锁及关节活动受限。关节液检查虽能排除细菌、结核感染等炎性关节液,实验室检查又排除了常见的类风湿性关节等非炎性关节炎,同时X平片和MRI检查也不能明确诊断,最终通过膝关节腔关节镜探查获得诊断和治疗,并通过病理检查证实为滑膜软骨瘤病。 Mi l g r a m等曾提出本病的分期理论,将本病分为三期;I 期为活动性滑膜内病变,光镜下可发现滑膜内软骨化生,肉眼观察正常;I I期为过渡性滑膜病变合并滑膜软骨瘤及游离体,肉眼观察可见带蒂的软骨或骨软骨小体悬垂于滑膜组织,但未脱落;Ⅲ期滑膜病变静止,形成多个由软骨或骨软骨组织构成的游离体。由于缺乏研究滑膜软骨瘤病发生、发展的技术方法,很难真正了解滑膜软骨瘤病的自然病史,本研究中发现的非典型病例是滑膜软骨瘤病的早期病变?还是此病的特殊类型?尚需进一步研究证实,我们暂称之为“隐匿性”滑膜软骨瘤病。对于临床上无明显外伤史的膝关节肿痛患者,排除了常见的细菌性、结核性炎性关节炎和痛风性关节炎、骨关节炎及类风湿关节炎等非炎性关节炎、X线片和MRI检查未发现异常者,要考虑到患滑膜软骨瘤病的可能,经减少活动量、关节液穿剌抽吸、对症止痛等保守治疗3个月以上效果欠佳者,可考虑行关节镜探查术。一方面,关节镜探查具有创伤小、康复快的优点,关节腔探查术一般不会造成关节内结构的损伤[11];另一方面,若关节腔探查发现关节内病变,可作出关节镜下初步诊断,同时即可用关节镜治疗相应病变,并可取病变组织作病理学检查以验证关节镜诊断,做到临床表现、影像学、关节镜下大体解剖观、病理学检查结合以确定诊断[12]。若保守治疗时间过长,软骨小体可磨损关节软骨造成不可逆的软骨损害。本组病例中一例患者病程较长(12个月),出现关髌股关节和外侧关节间室出现Ⅲ~Ⅳ度软骨损伤,术后残留轻中度关节疼痛。本组病例中有3例病人术前出现关节线压痛及McMurray试验阳性者,术前误诊为半月板撕裂,可能与软骨小体积聚于膝关节腔内或外侧沟,并剌激滑膜组织产生炎症反应有关[13、14]。一旦诊断为滑膜软骨瘤病,一般应积极手术治疗,取除游离体和切除病变滑膜组织是治疗本病的有效治疗方法,取出游离体,可防止发生关节软骨磨损及退变,同时在关节镜下行局限性滑膜切除,彻底清除异常的滑膜组织,可有效防止复发[15]。熟练掌握关节镜下操作技术,可到达膝关节腔的各个部位,特别是膝后方关节腔,行关节游离体取出,并行部分滑膜切除,是治疗本病的首选方法。根据本组病例的随访结果,关节镜术后膝关节功能可很快恢复到术前水平,并且未见临床复发,但如果出现关节软骨损伤,则可后遗关节疼痛,因此,宜尽早行关节镜手术治疗。 参考文献:1. 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关节镜下半月板部分切除制备骨关节炎动物模型李 忠1,杨 柳1,戴 刚1,陈光兴1,鲁晓波2,阳运康2,张卫东1,张洪鑫1,王晓宇1 (1第三军医大学附属西南医院关节外科中心,重庆 400038;2泸州医学院附属医院骨科,泸州 646000)提要:目的 探讨经关节镜建立猪双膝骨关节炎动物模型及用磁共振成像(MRI)监测骨关节炎骨软骨损害的有效性和可行性。方法 4只小型猪经关节镜切除双膝外侧部分半月板,术后12周,行大体、组织学和MRI观察与切除半月板相对股骨髁的骨软骨变化。结果 大体观察发现,局限性软骨表面纤维化,股骨髁间切迹骨赘形成;组织学发现,软骨细胞数量和蛋白多糖含量减少、成簇排列细胞数量增加;MRI发现,软骨厚度变薄和骨软骨信号强度改变。结论 经关节镜半月板部分切除能有效建立猪双膝关节骨关节炎动物模型,MRI能灵敏监测到骨关节炎动物模型的骨软骨变化。Establishment animal models of osteoarthritis by partial meniscectomy arthroscopilyLI Zhong1, YANG Liu1, DAI Gang1, CHENG Guang-xing1, LU Xiao-bo2, YANG Yun-kang2, ZHANG Wei-dong1, ZHONG Hong-xin1,WANG Xiao-yu (1Centre for Joint Surgery, Southwest Hospital, the Third Military Medical University, Chongqing 400038, 2Department of Orthopaedics, Affiliated hospital of Luzhou medical collage, Luzhou 646000,China )Abstract: Objective To explore the effectiveness and the feasibility of establishment of osteoarthritis animal model by arthroscopy and monitor the osteoarthritis lesion of bone and cartilage by magnetic resonance imagine (MRI) in bilateral knee joint of mini-pigs. Methods Bilateral partial menisci resections were performed on four mini-pigs (8 joints) by arthroscopy. Changes of bone and cartilage contacted with the resected menisci were analyzed by gross observation, histology, and MRI at twelve post-surgery. Results Gross observation showed focal fibrillation on articular cartilage surfaces and significant osteophyte formation at lateral intercondylar notches of the distal femur. Histology revealed the loss of cellularity and proteoglycan and increase in the numbers of chondrocytes of condrocytes clustering. MRI showed the cartilage thickness were decreased and signal intensity of bone and cartilage were changed. Conclusion osteoarthritis animal models in bilateral knee joint of mini-pigs can be established effectively by partial meniscectomy arthroscopily. Changes of bone and cartilage of osteoarthritis animal models can be monitored sensitively by MRI.Key words: Arthroscopy; Osteoarthritis; Animal model; Magnetic resonance imagine骨关节炎(Osteoarthritis, OA)是医学临床最常见的骨关节病患。目前常用的治疗方法都存在一定缺陷,如使用非类固醇类抗炎药物、关节内注射透明质酸钠等保守治疗,难以有效控制病变进展;而针对晚期患者的人工关节置换手术尚存在术后感染、远期假体松动等并发症[1]。OA动物模型在研究骨关节炎发生、发展、有效防治药物及治疗方法中具有重要作用[2、3]。开放性外科手术切除膝半月板是常用的诱发动物OA的方法,但开放手术创伤大,会对实验结果产生一定影响;此外,OA动物模型骨软骨病变的观察主要采用有创的组织形态学及免疫组织化学的方法。本研究探讨采用微创手术方法建立猪双膝OA动物模型及用MRI无创检查监测OA模型骨软骨变化的有效性和可行性。1 材料与方法1.1材料成年小型猪4只,雌性,体重22-33kg,由第三军医大学实验动物中心提供;美国Smith & Nephew公司超三晶数字化关节镜系统,采用直径2.7mm的30°关节镜及电动刨削系统,意大利Arthocare低温等离子刀,意大利ESAOTE公司0.5T的Artoascan磁共振(MRI);日本奥林巴斯BX41光学显微镜。1.2 实验方法1.2.1 麻醉方法 实验猪按序编为1-4号,术前30min阿托品0.02mg/kg肌注,氯胺胴20mg/kg肌注诱导麻醉后,气管内插管。采用戊巴比妥钠(20-30mg/kg)静脉复合麻醉,戊巴比妥钠首次用总量的1/3-1/4,每次加量为首量的1/3-1/4,缓慢推注。术毕自主呼吸、角膜反射恢复后拔除气管内导管。术中静滴抗生素(青霉素960万单位,链霉素1.0g)防止感染。1.2.2 手术方法 膝关节备皮、碘复消毒后,铺无菌治疗巾。建立前外侧、前内侧关节镜入口:屈膝60°,接近髌腱内缘、内侧半月板前角上方2mm处,经髁间窝插入关节镜建立前内侧入口,进行关节腔检查、摄影。在前外侧局部皮肤光亮区插入穿剌针头,证实针头在关节腔内后,经髁间窝建立前外侧入口。经前内侧关节镜入口用低温等离子刀将前中1/3交界处的外侧半月板由游离缘汽化到滑膜缘,使外侧半月板完全切断,产生约5mm半月板缺损,关节腔止血、灌洗,拔除关节镜器械,缝合伤口。另一侧膝关节采用相同的方法在相同部位产生同样大小的半月板缺损。术后肌注青霉素320万单位/只,链霉素1.0g/只,每日2次,共5天。动物肢体不固定,任其自由活动,常规饮食饲养。1.2.3 观察指标1.2.3.1 术后一般情况 术后动物肢体活动、饮食量及伤口愈合情况。1.2.3.2 MRI检查 术后12周,处死动物后立即行双膝关节MRI检查,选用矢状面SE T1, TSE T2, GE-STIR, 3D GE T1 序列。具体参数:T1-weighted:TR/TE(ms)= 560-600/18-26;T2-weighted:TR/TE(ms)=2600-3000/80-100; GE-STIR:反转时间75ms,TR/TE(ms)=1500/16;3D GE T1 TR/TE(ms)= 50/16,翻转角度65度。FOV=180*180, Matrix=256*192, 激励次数2,层厚4mm,层间距0.4mm,3D序列层厚0.8mm。观察外侧半月板对应股骨外髁骨软骨形态和信号变化。1.2.3.3 大体观察 显露膝关节,肉眼观察关节内有无粘连、滑膜有无充血水肿、外侧半月板对应股骨外髁骨软骨形态及骨赘形成情况。1.2.3.4 组织学检查 截取外侧半月板对应病变区股骨外髁骨软骨组织,新配的4%多聚甲醛固定48小时,0.5M的EDTA脱钙3周,每周更换脱钙液2次。常规酒精逐级脱水,石腊包埋,切片,行HE染色观察组织结构与细胞形态,甲苯胺蓝染色观察细胞外基质蛋白多糖的形成。2 结果2.1 一般情况 术后动物活动量及饮食较前减少,麻醉清醒后即可站立、行走,轻微跛行,双后肢均匀负重;术后一周缝合伤口均一期愈合,动物四肢活动和饮食恢复正常;到术后12周无实验动物死亡,也未再出现四肢活动受限及跛行。2.2 MRI检查 外侧半月板形态不规则,相应侧股骨髁前方及远端软骨形态、信号不规则改变:局部信号变薄,连续性中断,可见长T1、T2液性信号填充;梯度回波序列见局部软骨信号不规则降低,脂肪抑制像可见软骨下骨局部信号增高,提示充血、水肿可能;3D梯度回波序列能较为清晰的识别软骨及软骨下骨的信号改变,包括局部软骨、软骨下骨信号不规则降低,近端可见低信号带环绕,提示硬化可能,并可对面积、深度进行较为准确的评估(图1)。2.3 大体观察 关节内无积液及粘连,滑膜组织无明显充血水肿;外侧半月板对应股骨外髁软骨粗糙、不透明、失去光泽;股骨外髁靠近髁间切迹处骨赘形成。内侧股骨髁软骨光滑、透明,无骨赘形成(图2)。2.4 组织学观察 HE染色见,局限性局部软骨变薄,表层纤维化,表层细胞数量明显减少甚至消失,中层细胞呈特征性簇状排列(图3);组织学上较难显示软骨下骨的变化。甲苯胺蓝(TB)染色见,病变区细胞外基质染色较淡,尤以表层软骨明显,提示病变区细胞外基质蛋白多糖含量减少。 术后12周,4只猪双后肢8个膝关节的MRI表现、大体及组织学观察均出现类似改变。3 讨论 骨关节炎动物模型主要分为两类:一类是通过基因改造或人工筛选获得的自发性骨关节炎动物模型;另一类是通过关节固定、关节腔注射致软骨降解物质或人工造成关节负荷异常等方法诱导关节退性变。半月板部分或完全切除是建立骨关节炎动物模型的常用方法,半月板切除后,相应胫股关节负荷传导紊乱,压应力集中于极小的区域,导致关节软骨发生退行性变,软骨表面粗糙、纤维化、甚至完全缺失。在上述半月板切除建模时,大多采用开放手术切除一侧后肢的半月板组织,诱导一侧肢体出现骨关节炎[4]。开放手术具有视野清楚、操作方便的优点,但手术创伤大、出血多,关节内积血及创伤性滑膜炎可影响软骨、滑膜的生化代谢,从而影响实验结果。本实验经关节镜切除部分半月板组织,成功诱导了双膝骨关节炎损害的特征性表现,包括:(1)局限的股骨髁和胫骨平台关节软骨磨损;(2)关节周围骨赘形成;(3)软骨下松质骨结构的改变。我们选用股骨髁用于组织学和磁共振成像监测。关节镜下微创手术建立双膝骨关节炎模型有以下优点:(1)手术创伤小,出血少,大大降低了关节内积血和创伤性滑膜炎对实验结果的影响,有助于减少实验本身造成的误差;(2)关节镜辅助下,可在不同时间点对病变骨软骨组织细针穿剌活检,无需处死动物,即可实现对同一动物病变组织的动态监测;(3)双膝关节同时建立骨关节炎模型,一侧膝关节用于实验性操作,另一侧膝关节用于对照,允许每一个动物用作自身对照,避免单膝骨关节模型不同动物间变异对实验结果的影响。此外,步态分析发现,单膝骨关节炎模型伤肢跛行,负荷减少,而双膝骨关节炎模型双侧肢体负荷均衡,从而可排除单肢骨关节炎模型负荷不均衡对实验结果的影响。骨关节炎是生物学和力学因素相互作用下,使关节软骨细胞、细胞外基质和软骨下骨合成与降解的正常进行失去平衡的结果。近来研究发现,骨关节炎的软骨下骨转换(turnover)速度加快及骨矿化密度增加与骨关节发生及严重程度相关,提示软骨下骨密度和结构的改变会对关节软骨退变产生重要影响。动物实验也证实,骨吸收抑制剂可有效抑制软骨下骨硬化和骨赘形成,且具有一定的软骨保护作用[5]。目前,软骨下骨改变在骨关节炎发生、发展中的作用尚不清楚[6]。用无创检测方法动态监测骨关炎发生、发展过程中骨软骨变化,特别是软骨下骨变化,对判断软骨下骨改变在骨关节炎发病过程中的作用及筛选合适的改变病程的药物具有重要作用。本研究应用0.5T的磁共振,选用梯度回波序列、脂肪抑制回波序列和3D梯度回波序列,清楚显示了骨关节炎软骨和软骨下骨损伤的特征性表现,具有灵敏度高、显示清楚和无创的优点。应用3D梯度回波序列甚至可对骨软骨损害面积、深度进行较为准确的评估,以用于损害严重程度的判断。磁共振成像显示的软骨损害与大体及组织形态学观察到的结果较一致,而磁共振成像还能清楚显示组织学难以显示的软骨下骨损害。综上所述,经关节镜微创手段能有效建立猪双膝关节骨关节炎动物模型,并具有创伤小、模型制备可靠、重复性好的优点,有利于减少实验误差;磁共振成像能灵敏、无创监测骨关节炎动物模型骨和软骨的特征性损害,尤其对软骨下骨损害的显示具有明显的优势。参考文献:1. 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BACKGROUND: Chitosan-disodium β-glycerol phosphate (C/GP) gel has been shown to be compatible with the entrapment of viable chondrocytes, and bone marrow mesenchymal stem cells (BMSCs) are considered to be the potential cells used in tissue engineering. This experiment is aimed to observe the cytocompatibility of BMSCs with C/GP gel.OBJECTIVE: To study the effect of C/GP gel on the growth, proliferation and chondrogenic differentiation in vitro cultured BMSCs and explore a new carrier for the application of cartilage tissue engineeringDESIGN: Completely randomized controlled experiment. SETTING: Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College; Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA. MATERIALS: The experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006. Six adult female mini-pigss were employed. C/GP gel is prepared by mixture the HCl solution of chitosan with salt solution of β-glycerol phosphate, allowed gel at 37℃ in incubator for about 5 to 10 minutes.METHODS: ① BMSCs culture: 4–6 ml of bone marrow harvested from the posterior superior iliac crest were plated at 20×106/ 100mm dish and then grown for 14 days in complete media, consisting of DMEM/F-12 supplemented with 10% fetal bovine serum. Cells were harvested and re-seeded for subculture. ②BMSCs differentiation assays: Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures under osteogenic conditions. Chondrogenic differentiation was evaluated by histology for toluidine blue and immunohistochemistry for type II collagen in cultures under chondrogenic conditions. ③ In vitro assays, expanded BMSCs were suspended in C/GP solution and allowed gel at 37℃ in incubator for about 5 to 10 minutes, then cultured under chondrogenic conditions for 3 weeks. Cells attached to and viability in C/GP gel was monitored with the aid of an inverted light microscope. Chondrogenic differentiation of cell in C/GP gel were assessed by histological and immunohistochemistry. The cell proliferated was monitored by MTT after 2,5,8 days seeding. MAIN OUTCOME MEASURES:① Characterization of mini-pigs’BMSCs. ② BMSCs attached to and viability in C/GP gel. ③ Chondrogenic differentiation of BMSCs in C/GP gel. ④ BMSCs proliferated in C/GP gel. RESULTS:① Characterization of mini-pigs’ BMSCs: Cultured BMSCs showed fibroblastic morphology and were able to differentiate to chondrocytes or osteogenic cells under chondrgenic or osteogenic cultured condition respectively. ② BMSCs attached to and viability in C/GP gel: BMSCs attached to and remained>90% viable in C/GP gels immediately post-casting, and throughout the 21 day, using MTT staining. ③ Chondrogenic differentiation of BMSCs in C/GP gel: During 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. ④ BMSCs proliferated in C/GP gel: Chondrogenic induced BMSCs cultured in C/GP gels continued to proliferate. There is a significant difference among the values of optical density in the cells-gel constructs compared to the controls without cells after 2, 5, and 8 days of culture (P<0.05).CONCLUSION: It is confirmed that C/GP gel shows good cytocompatibility with BMSCs and contributes to the growth, proliferation and chondrogenic differentiation for BMSCs in vitro culture. C/GP gel can be a potential cell-carrier for tissue engineering of articular cartilage. INTRODUCTIONThe biological repair of articular cartilage defects remains a difficult challenge due to a limited intrinsic repair response of cartilage. Tissue engineering has proved to be one of the most promising approaches for cartilage repair[1,2]. A three-dimensional porous scaffold plays an important role in the tissue engineering of cartilage. Chitosan is a polymer of glucosamine and N-acetyl glucosamine derived by the partial depolymerization and deacetylation of chitin extracted from the shells of crustaceans. It can be induced to form hydrogels, either by self-association or by covalent cross-linking, and has been shown to be compatible with the entrapment of viable chondrocytes[3-5]. The cationic nature of the chitosan gel makes it an attractive scaffold to facilitate the entrapment of type II collagen and the highly anionic glycosaminoglyean (GAGs) produced by chondrocytes or cells induced by Bone marrow mesenchymal stem cells (BMSCs).BMSCs are considered to be the potential cells used in tissue engineering of articular cartilage[6,7].In the present work, BMSCs from the mini-pigss were encapsulated in chitosan-based gels and maintained in tissue culture, in order to explore chitosan -gelatin as a potential cell-carrier for tissue engineering of articular cartilage,the cytocompatibility of BMSCs with chitosan-gelatin was studied.MATERIALS AND METHODSMaterialsAfter approval by the Animal Experimentation Committee of the Third Military Medical University, the experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006. Six adult female mini-pigs were employed. The animals were provided from Experimental Animal Center of Third Military Medical University (Qualified No: F99017) and bred with standard forage. The experiment required DMEM/F-12 (Gibco, USA), fetal bovine serum (Gibco, USA), disodium β-glycerol phosphate (Sigma, USA), recombinant human transforming growth factor β1(Oncogene Science,Cambridge, MA ), Chitosan (with a 90% DDA and average molecular weight 216 kDa, provided by Yuhuan Company, China), Bio-rad 450 microplate spectrophotometer (Bio company , USA).MethodsBone marrow Mesenchymal stem cell isolation and culture: After approval by the Animal Experimentation Committee at the author’s institution, BMSCs isolation from 6 adult female mini-pigs (average weight 13.5 ± 0.4 kg) was done by cell attachment to tissue culture plastic as previously described[8]. Briefly, marrow was harvested from the posterior superior iliac crest via an 18-gauge needle, collecting 4-6 ml of bone marrow into 3000 units of heparin. The aspirates were washed twice with phosphate-buffered saline and plated at 20×106/ 100-mm dish and then grown for 14 days in complete media (CM), consisting of DMEM/F-12 supplemented with 10% fetal bovine serum and antibiotics (penicillin G, 100 U/ml; streptomycin, 0.1 mg/ml; amphotericin B, 0.25 mg/ml). On day 14, the cells were harvested and re-seeded in T-175 flasks at 1×106 cells/flask and grown to confluence. In all in vitro and in vivo assays, BMSCs cultures were used only up to the third-passage.In vitro BMSCs differentiation assays: Assessment of osteogenic differentiation was done in monolayer culture for 18 days and measured using increased alkaline phosphatase activity and calcium mineralization as markers. Briefly, monolayers of BMSCs were grown in medium containing 100 nm dexamethasone, 10mm disodium β-glycerol phosphate, and 50 mm ascorbic acid 2- phosphate. Alkaline phosphatase activity was detected qualitatively using naphthol AS-MX phosphate as a substrate and calcium deposition was determined using von Kossa staining[9,10]. In chondrogenic induction, BMSCs were cultured in pellet format as described previously[10,11]. Briefly, 200,000 cells were placed in a 15ml conical polypropylene tube, washed with incomplete chondrogenic media (ICM), consisting of DMEM/F-12 supplemented with 1 mM sodium pyruvate, o.1 mmol/L ascorbic acid-2-photophate, 100 nM dexamethasone,1% ITS+, and antibiotics, and resuspended in 0.5ml complete chondrogenic media (CCM) (ICM containing 10ng/mL recombinant human transforming growth factor β1. Cells were centrifuged at 500g for 5 minutes at 20℃. The pellets were maintained in culture with 1 pellet/tube and 0.5 ml CCM/tube. Media was changed every 2-3 days. Chondrogenic pellets were harvested at 21 days, then cut into frozen sections, and evaluated by histology for toluidine blue and immunohistochemistry for type II collagen. Preparation of auto-gelling chitosan solution: Auto-gelling chitosan solution was prepared with mixture of solutions of chitosan and disodium β-glycerol phosphate[12]. Briefly, 200mg of Chitosan (with a 90% DDA and average molecular weight 216 kDa) was dissolved in 9ml of HCL solution (0.1M). To the resulting solution, 560mg of disodium β-glycerol phosphate dissolved in 1ml of distilled water, was carefully added drop by drop to obtain a clear and homogeneous liquid solution. The pH value of the final solution before heating was about 7.15. Sterile formations were obtained by regular liquid autoclaving of chitosan solutions, 0.22μm filtration of GP solutions, and sterile preparation of C/GP solutions to be used.Encapsulation and chondrogenic induction of BMSCs: BMSCs were suspended in C/GP solution at an initial concentration of 5×106/ml and the mixture was poured into the well of the 96-well polypropylene culture plate, allowed to gel into discs at 37℃. The discs were then cultured in 0.1ml CCM/well. Medium was changed every 2-3 days. Gel constructs without cells were carried through identical culture conditions as controls with cells. Cell viability was determined using MTT staining. Discs were harvested at 21 days for analysis.Histologic and immunohistochemical analysis: Cultured pellets and discs were harvested and fixed in 4% paraformaldehyde and 1% glutaraldehyde in 100mM sodium cacodylate buffer, at pH 7.2. For histological assessment, samples were embedded in LR-White acrylic resin, and semithin (1 mm) sections were cut on an ultramicrotome and stained with toluidine blue. For immunohistochemistry, 5-mm-thick paraffin sections of samples fixed as above. Type II collagen was detected using monoclonal antibody AF5710. Endogenous peroxidase activity was blocked by 15-minute incubation in 0.3% H2O2 in methanol. Sections were then pretreated with 40 mU/ml chondroitinase ABC in 0.1M Tris–acetate, pH 7.6, containing 1% (weight/volume) bovine serum albumin (BSA) for 30 minutes at 37°C for optimal antigen retrieval. Residual enzyme was removed with two 5-minute washes with Tris buffered saline (TBS) and 10% volume/volume normal goat serum was placed on the section for 1 hour at room temperature to block nonspecific background staining. Primary antibody was diluted to 10μg/ml with TBS containing 1% BSA immediately prior to use and placed on the sections for 2 hours at room temperature. After washing with TBS to remove residual primary antibody, reactivity was detected using biotinylated goat anti-rabbit secondary antibody and streptavidin–peroxidase. Peroxidase activity was visualized using diaminobenzidine as substrate. The sections were counterstained with hematoxylin. Negative controls consisted of the same treatment with the omission of the primary antibody in the first incubation.Colorimetric MTT-based assay: After culturing the cells in discs for a period of 2, 5, and 8 days, the culture medium was discarded from the wells, 50μl of MTT was added to each well and incubated for 4 hours at 37℃. After the formation of formazan precipitate, the supernatant was carefully aspirated from the wells without disturbing the formazan crystals. The crystals were dissolved in 200μl/well dimethyl sulphoxide (DMSO). The absorbance was measured within two hours of dissolution as the optical density (OD) at 570nm using microplate spectrophotometer. Gel constructs without cells were carried through identical culture conditions as constructs with cells. The designer of the experiment is Yang Liu, which is performed and assessed by Li Zhong and Wang Fu-You separately. They all have the relative certifications. Statistical analysis: Statistical analysis was performed by Jin Xu-Hong. All data were expressed as Means±SD, and the results were analyzed with t-test. P<0.05 indicated significant difference. RESULTSCharacterization of mini-pigs BMSCsBMSCs were isolated from bone marrow of adult female mini-pigs and expanded to form confluent cultures of adherent cells with a fibroblastic morphology (Figure 1). Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures maintained for 18 days under osteogenic conditions. All BMSCs preparations evaluated in this study were capable of elaborating a calcium-rich matrix when compared with control cells incubated in media without osteogenic supplements. To analyze whether BMSCs are able to differentiate to chondrocytes and form cartilage, we used the micromass pellet culture technique, which express differentiated markers, such as type II collagen and GAGs. Upon culture in micromass pellets for 21days, the majority of BMSCs differentiated to chondrocytes and formed cartilage tissue, which contained type II collagen and GAGs.Histologic and immunohistochemical analysisDuring 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. BMSCs remained>90% viable in the C/GP gels immediately post-casting, and throughout the 21 day, using MTT staining (Fig. 2). Cells cultured in chitosan accumulated pericellular sulfated GAG-containing matrix, as measured by toluidine blue staining. Chitosan scaffold was still present after 21 days in culture. The histological appearance of chitosan gel cultured from 1 to 21 days without cells was similar to that of chitosan gel with cells, indicating that the gel did not biodegrade during 3 weeks in culture. Chondrogenic induced BMSCs in chitosan cultures produced type II collagen, as reflected by type II collagen immunohistochemistry assay.Colorimetric MTT-based assayThe cell proliferation was evaluated with the MTT colorimetric assay (Table 1). Chondrogenic induced BMSCs cultured in the C/GP gels continued to proliferate. There is a significant difference among the viability of cells after 2, 5, and 8 days of culture in the gels compared to the controls (P<0.05), but no significant difference among the controls (P>0.05). Table 1 MTT colorimetric assay (n=6, ±S,OD)Group2days5days8daysBMSCs-C/GP gels0.209±0.039a0.522±0.023a0.687±0.025aC/GP gels (Control)0.192±0.0160.189±0.0140.188±0.014aP<0.05 vs control;DISCUSSIONDevelopments in therapeutic strategies for cartilage repair have increasingly focused on the promising technology of tissue engineering, proposing to use BMSCs or other cell types to regenerate articular cartilage in situ[13,14]. The method of cell implantation within the articular joint poses special challenges, including three-dimensional porous scaffold to be used as cell-delivery vehicles must meet stringent biocompatibility requirements in that the material should be non-toxic, non-immunogenic, and biodegrade without generating toxic by-products or debris that could subsequently injure the joint. The biomaterial must either integrate in a mechanically and physiologically harmonious manner with the repair tissue, or degrade without leaving gaps or fissures in the tissue[15]. Thus, one function of a three-dimensional porous scaffold acting as a cell carrier for cartilage repair is to serve as temporary scaffolding that permits the accumulation of extracellular matrix by delivered cells, while leading to congruent attachment with the perimeter of the cartilage defect and the subchondral bone[16].The ideal cell carrier substance should be the one that most closely resemble the natural environment in the articular cartilage[17]. Glycosaminoglycans (GAGs) are the major components of extracellular matrix of articular cartilage. Proteoglycans and hyaluronic acid, two of the major macromolecules found in articular cartilage, both contain GAGs. GAGs play a critical role in regulating expression of chondrocytic phenotype and in supporting chondrogenesis both in vitro and in vivo. Given the importance of GAGs in stimulating the chondrogenesis, the use of biomaterial comprising GAGs or GAGs analogs as cell carrier for articular repair was considered. One such candidate is chitosan[3,18]. Chitosan includes N-acetylglycosamine and structurally resembles GAGs present in articular cartilage and has a bioactivity. Chitosan is degradable and the degradation kinetics appears to be inversely related to the degree of deacetylation. Highly deacetylated forms exhibit the lower degradation rates, whereas with lower levels of deacetylation, degradation appears more rapidly. The degradation products are oligosaccharides of variable length. These materials have been found to evoke a minimal immunoreaction. In the short term, a significant accumulation of neutrophils in the vicinity of the implant is of often seen, but which dissipates rabidly and a chronic inflammatory response does not develop. The most important characteristic of chitosan as a biomaterial for tissue engineering is its biocompatibility and bioactivity. Lu et al[19] have demonstrated that chitosan solution injected into the knee articular cavity of rats causes a significant increase of the density of chondrocyte in the knee articular cartilage, suggestion that the chitosan could be potentially beneficial to the wound healing of articular cartilage. Francis Suh et al[3] have demonstrated the chondrocytes cultured on chondroitin 4-sulfate (CSA) augmented chitosan hydrogels maintain many morphological characteristic and functional features of chondrocytes in normal cartilage. The predominant collagen produced by chondrocytes culture on CSA-chitosan is type II. For chondrocytes cultured on the control polystyrene surface, the predominant collagen produced by chondrocytes is typeⅠ. Chenite et al[12] have demonstrated that C/GP gels can deliver active bone protein (BP) leading to de novo cartilage and bone formation in an ectopic site. They also implanted isolated bovine articular chondrocytes embedded in C/GP gels subcutaneously in athymic mice and demonstrated that the implanted chondrocytes secreted matrix characteristic of normal cartilage and expressed cartilage specific genes such as type II collagen and the large aggregating proteoglycan aggrecan. It showed C/GP gels can be particularly well for the delivery of sensitive biological materials such as BP and chondrocytes.Bone marrow mesenchymal stem cells were regarded as the potential for tissue engineering of cartilage because they had the advantage of being easily harvested and power ability of proliferation and multipotent[20]. In this experiment, chondrogenic induced BMSCs embedded in C/GP gel were cultured. The biocompatibility of the biomaterial and its effect on the proliferation and chondrogenesis were analyzed to explore this biomaterial as a potential BMSCs carrier for tissue engineering. C/GP gel demonstrated good biocompatibility and bioactivity when BMSCs were cultured with them. Chondrogenic induced BMSCs continued to proliferate and secreted matrix characteristic of normal cartilage such as type II and GAGs.The results presented here showed that C/GP gel maintains the bioactivity feature of chitosan and even enhance the proliferation and chondrogenic differentiation of in vitro cultured BMSCs. It has the potential to be used as a carrier in delivery of BMSCs for chondral defect repair in tissue engineering. REFERENCES1 Li Z, Yang L, Cheng GX, et al. Detectable effects of green fluorescent protein on the construction of tissue-engineered articular cartilage. Zhongguo Linchuang Kongfu 2005;9(30):84-62 Bai T,Shu J,Wang J,et al. Experimental research on repair of rabbit articular cartilage deffects with composite of autologous cell-carriers. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2008;22(4):487-91 PMID: 18575455 3 Francis Suh JK, Matthew HWT. 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[摘要] 目的 探讨桡骨远端截骨治疗桡骨远端骨折畸形愈合的临床疗效。方法从2003年1月至2006年8月,对12例桡骨远端骨折畸形愈合患者,背侧入路行桡骨远端截骨、自体髂骨移植、“T”型钢板螺钉内固定。结果 随访10-21月,平均14.6个月。X线片示桡骨远端畸形矫正良好,根据改良的Mcbridge腕关节功能评分标准,术后腕关节功能明显改善,与术前腕关节功能评分相比,差异具有统计学意义(P<0.05)。结论 桡骨远端截骨是治疗桡骨远端骨折畸形愈合的有效治疗方法。[关健词] 桡骨截骨矫形;桡骨远端骨折;畸形愈合;Corrective osteotomy for distal radius fracture malunions [Abstract] Objective To study the clinical efficacy of radial corrective osteotomy in treating distal radius malunion. Methods From January 2003 to August 2006, 12 patients with distal radius malunion were treated with radial corrective osteotomy, autologous bone graft and T shape plate internal fixation through dorsal approach. Results All cases were followed up for 10 to 21 months and the average time was 14.6 months. The X-ray pictures showed that distal radius deformities were corrected properly. The functions of wrist were improved posteoperation and significant difference compared to peroperation according to modified Mcbide grading system. Conclusion Radial corrective osteotomy is one of the effective methods to treat distal radius malunion. [Key words] radial corrective osteotomy; distal radius fracture; malunion; 桡骨远端骨折是临床最常见的骨折之一,约占急诊骨折患者的1/6,多见于老年人桡骨远端骨质疏松者。近年来,虽然桡骨远端骨折的治疗方法不断取行进展,但桡骨远端骨折畸形愈合仍然是其最常见的并发症之一[1]。我们从2003年1月至2006年8月采用桡骨远端截骨治疗桡骨远端骨折畸形愈合共12例,术后效果满意,现报告如下。1 临床资料1.1 一般资料:本组12例,男8例,女4例,年龄24-72(平均39.6)岁;摔伤10例,压砸伤2例;Colles骨折9例,Smith骨折3例;左侧7例,右侧5例;所有患者入院前都在当地医院经过手法复位,石膏或小夹板外固定治疗;受伤至到我院手术治疗时间47 -232d,平均65d。术前X线片检查桡骨远端关节面:尺偏角:2°-15°,平均8.5°;掌倾角:负2°-负15°,平均负5.2°;桡骨茎突缩短移位:0.8-2.1cm,平均1.5cm。所有患者都存在不同程度腕关活动障碍:背伸:12°-52°,平均31.2°;掌屈18°-49°,平均27.4 °;尺偏:11°-28°,平均17.5°;桡偏:10°-22°,平均12.4°;前臂旋前:35°-76°,平均47.3°;前臂旋后:29°-68°,平均42.4°;8例患者存在外观畸形;6例患者存在抓握无力;2例患者存在腕关节活动时疼痛。根据改良的Mcbridge腕关节功能评分标准[2]:腕关节功能12-23分,平均18.4分。评分标准:患者主诉的疼痛、功能、握力的满意度及对生活的影响;临床检查腕关节的活动度,握力和外观有无畸形;X线片测量掌倾角、尺偏角及桡骨短缩程度。1.2治疗:患者平卧位,患肢外展置于手术侧台上,臂丛神经阻滞或全身麻醉,准备同侧髂嵴,铺单。如果应用臂丛神经阻滞,则在髂嵴处加用局麻。取桡骨远端背侧入路,沿Lister结节的尺侧做10cm纵行切口,不切开桡腕背侧韧带,纵行切开第3伸肌腱鞘,从桡骨远端游离软组织,向两侧分开,第4伸肌腱鞘向尺侧牵开,第2伸肌腱鞘向桡侧牵开,显露出桡骨远端。在桡骨远端平面把一根细胞克氏针插入桡腕关节,在关节近侧约5mm,垂直于桡骨背侧皮质插入第二根克氏针,垂直于近侧骨折块插入第三根克氏针,第二、第三根克氏针的夹角就是预期矫正的角度。在关节面近侧2-3cm,用摆锯在原骨折处做平行于关节面的截骨,不切断掌侧皮质,撑开截骨处至第二和第三根克氏针平行,以保证矫正畸形。从髂嵴切取单皮质楔形骨块,修整成适当大小,置入桡骨截骨撑开处,用克氏针暂时固定,C-臂X光机透视,确定掌倾角、尺偏角和桡骨长度矫正满意后,在桡骨远端背侧放入一块斜T形钢板,序列打入螺钉,桡骨远端至少固定2枚螺钉。修复伸肌支持带,放置负压引流,逐层缝合皮下及皮肤,石膏托固定腕关节于中立位。1.3术后处理:术后早期开始伸、屈腕和手指肌肉的等长收缩锻炼,第二天拨除引流管, 14d拆线。拆线后更换短臂石膏管型,腕关节制动4-6周,术后1、2和4周复查X线片,检查桡骨对位情况。拆除短臂石膏管型后,更换可拆卸的腕关节夹板2-4周,每天将夹板拆卸后配合中药薰蒸各方向活动腕关节数次,以利关节功能恢复,7-10周骨折愈合后拆除夹板,加强腕关节功能锻炼,特别是旋转功能锻炼。1.4 结果:12例全部获利随访,随访时间10-21月,平均14.6个月。术后X线复查结果:X线片显示骨折全部愈合,平均愈合时间为8周。X线测量尺偏角16°-25°,平均20.3°;掌倾角6°-15°,平均10.4°;桡骨茎突短缩均≤2m;腕关节活动度:背伸:25°-60°,平均47.3°;掌屈38°-56°,平均47.6 °;尺偏:21°-38°,平均31.5°;桡偏:15°-27°,平均22.8°;前臂旋前:61°-86°,平均77.3°;前臂旋后:57°-84°,平均68.9°,腕关节关节活动范围较术前都有明显改善。随访最后一次的改良Mcbridge腕关节功能评分:腕关节功能1-11分,平均4.8分。统计学分析:手术前和手术后改良Mcbridge腕关节功能评分比较,差异有显著性,P<0.05。所有病例都无感染、钢板松动等并发症。3 讨论桡骨远端骨折畸形愈合在临床骨科比较常见,大约5%的患者发生早期和晚期再移位而出现畸形愈合。桡骨远端骨折畸形愈合通常由于跌倒时手伸地,造成尺偏角、掌倾角丢失和桡骨短缩等,畸形愈合可分以下三类:一类为造成桡腕关节和/或桡尺关节关节面不平整的经关节面骨折畸形愈合;另一类为造成干骺端成角、短缩、旋转及横向移位的关节外骨折畸形愈合;第三类为上述两类的混合。临床及生物力学研究发现,桡骨远端骨折畸形可引起桡腕关节、腕中关节及下尺桡关节解剖及生物力学改变。如桡骨短缩和桡腕关节面背倾导致通过尺骨的应力显著增加,关节面从10°掌倾变为45°背倾,尺侧承受的负荷从21%增加到67%;桡骨短缩2.5mm,尺骨轴向负荷则从18.4%增加到41.9%;关节背倾还可引起腕中关节近排腕骨向背侧旋转、腕中关节动力性不稳及腕关节固定性畸形等;而下尺桡关节畸形可引起关节接触面减小、尺桡背侧韧带断裂、三角纤维软骨复合体紧张等。大多数桡骨远端畸形愈合患者会出现腕关节乏力、活动受限和疼痛等临床症状,但并非所有患者都会出现腕关节功能改变,因此,手术指征为有临床症状的桡骨远端畸形愈合,而不仅是放射学上出现桡骨远端畸形愈合。有学者比较了伤后8周和40周行桡骨远端骨折截骨矫形的治疗效果,结果发现两者的腕关节功能和放射学结果无明显差异,但早期手术矫形组整个治疗时间缩短及恢复工作时间更快,因此认为,只要局部软组织条件许可,桡骨远端骨折畸形愈合应尽可能早地行远端截骨矫形术[3]。大多数桡骨远端骨折的标准治疗方法是手法复位外固定, 虽然Colles认为桡骨远端骨折后遗畸形与功能受限无关,随着近年来对桡骨远端骨折类型及其畸形愈合对功能影响认识的提高,以及手术器械和手术技术的改进,大多数学者倾向于采用更积极的方法来处理此类骨折。对于简单的干骺端骨折及移位较小的关节内骨折,仍然首选手法复位外固定,若手法复位失败或骨折再移位则需手术治疗,因此,对于采用手法复位外固定的新鲜桡骨远端骨折,一方面,要注意复位后的骨折是否达到了功能复位的标准,普遍接受的功能复位标准如下:(1)掌倾角的改变不超过10°,即中立位或向掌倾20°;(2)尺偏角改变小于5°;(3)桡骨短缩小于2mm;(4)关节内骨折,关节阶梯不超过2mm。另一方面,即使骨折已达到功能复位标准,在复位后2-3周要每周复查X线片,以密切观察骨折复位的维持情况。在最初2-3周内缓慢移位比早期一次性显著移位多见。无论早期移位或缓慢发生的再移位都应重新复位,并以经皮穿针外固定或内固定来维持复位,而不应再用石膏或小夹板外固定。对于无法复位的不稳定骨折及关节内骨折,不要强求手法复位,而需手术切开复位。不稳定骨折的判断:背侧倾斜超过20°;桡骨短缩5 mm以上;严重干骺端粉碎骨折;关节面阶梯超过2mm;掌、背侧缘粉碎骨折超过背侧至掌侧距离的50%;伴有尺骨骨折;严重骨质疏松等。同时需考虑患者的年龄、功能要求及全身情况。桡骨远端骨折手术和非手术治疗后都容易出现骨折畸形愈合[4]。手术矫正伴有疼痛、功能受限或无力的桡骨远端骨折畸形愈合,已为大多数学者所接受,许多研究已证实,截骨矫正手术能改善腕关节功能、前臂旋转、握力及疼痛[1]。目前桡骨远端截骨手术方式繁多,尚无一种被广泛接受的有效手术方法[4-6]。多数学者采用桡骨远端截骨结合髂骨移植矫正桡骨远端解剖形态及力线;以缓解腕关节症状及恢复其功能。与上述研究相似,本研究分析了桡骨远端背侧截骨、自体髂骨移植、钢板螺钉内固定治疗桡骨远端畸形愈合的临床效果,术后:X线片见桡骨远端畸形矫正良好,腕关节活动范围及功能较术前明显改善,手术前、后腕关节功能评分差异有显著性,表明此治疗方法效果良好,是一种有效的治疗方法。参 考 文 献[1] Prommersberger KJ. Corrective osteotomy of the distal radius through volar approach. [J]. Techniques in hand and upper extremity Surgery, 2004, 8 (2): 70-77.[2] Femandez DL, Jupiter JB. Fractures of the distal radius: a practical approach to management [M], New York: Springer-Verlag, 1996, 145-151.[3] Jupiter JB, Ring D. A comperison of early and late reconstruction of malunited fractures of the distal end of the radius[J]. Bone Joint Surg Am. 1996,78:739–748.[4] Brunelli GA. Dome-shaped osteotomy for distal radius fracture malunions[J]. Techniques in hand and upper extremity Surgery, 2003, 7 (2): 75-79.[5] Wada T, Tsuji H, Iba K, et al. Simultaneous radial closing wedge and ulnar shortening osteotomy for distal radius malunion[J]. Techniques in hand and upper extremity Surgery, 2005, 9 (4): 188-194.[6] Shin EK, Jones NF. Temporary fixation with the agee-wristjack during correctional osteotoyies for malunionsand nonunions of the distal radius[J]. Techniques in hand and upper extremity Surgery, 2005, 9 (1): 21-28.
[摘要] 目的 探讨软组织松解结合近侧趾间关节成形术治疗僵硬性锤状趾的临床效果。方法 从2005年2月至2008年6月,8例保守治疗效果不满意的僵硬性锤状趾畸形患者采用软组织松解、近侧趾间关节成形术,在治疗前及最后一随访时根据美国矫形外科协会足踝功能评分标准(AOFAS)评估其疗效。结果 术后平均随访17.6月,所有患者锤状趾畸形矫正满意,近侧趾间关节残余动度平均为15度.。AOFAS评分:术前平均为43.5分,术后平均为81.4分,差异有统计学意义(P<0.01)。结论 软组织松解结合近侧趾间关节成形术治疗僵硬性锤状趾畸形,临床效果满意,畸形无复发,是保守治疗无效的僵硬性锤状趾畸形的有效治疗方法。[关节词] 锤状趾;关节成形术;畸形;外科治疗Proximal interphalangeal arthroplasty for the treatment of rigid hammer toe deformity. [Abstract] Objective The purpose of the present study is the evaluation of clinical results of correction of rigid hammer-toe deformity with soft-tissue release and proximal interphalangeal arthroplasty. Methods Between February 2005 and June 2008, 8 patients with hammer-toe deformity underwent soft-tissue release and proximal interphalangeal arthroplasty after failed conservative treatment. The American Orthopaedic Foot and Ankle Society Scores (AOFAS) were adopted for evaluation before surgery and at the last follow up. Reaults The mean follow up period was 17.6 months. All patients were satisfied with the procedure. There was about 15 degrees residual motion between proximal interphalangeal joint. The median preop AOFAS score was 43.5, and the median postop AOFAS score was 81.4. Significant difference was found between preop and postop evaluation (P<0.01). Conclusion It is an effective method for the treatment of rigid hammer-toe deformity with soft-tissue release and proximal interphalangeal arthroplasty after failed conservative treatment and shows good results with no recurrence of deformity. [Key Words] hammer toe; arthroplasty; deformity; surgery锤状趾是第2~4趾中的一趾或多趾的近节趾间关节的异常屈曲畸形,中节趾骨屈曲于近节趾骨上,多发生于第2趾,此种屈曲畸形可以是僵硬性的,即无法将畸形被动纠正到中和位.也可能是非僵硬性的,能被动纠正畸形。近年来随着对前足解剖结构及功能特点的深入研究,对前足畸形的认识及治疗有了很大进展。我科于2005年2月至2008年6月,采用软组织松解、近侧趾间关节成形术治疗僵硬性锤状趾8例,疗效满意,现报告如下:1 临床资料1.1 一般资料:本组8例,发5例,男3例,年龄24~58岁,平均42.6岁。病程1~9年。第2趾锤状趾伴拇外翻2例,第2、3趾锤状趾伴拇外翻1例,2~4趾锤状趾5例。病因:穿鞋不适5例,骨筋膜室综合征后出现锤状趾2例,胸腰段脊髓损伤致小腿肌肉瘫痪后锤状趾1例。所有畸形都不能用手法矫正,保守治疗效果不佳。都表现为近节趾间关节背侧疼痛性“鸡眼”,趾间疼痛性胼胝,穿鞋困难。1.2 手术方法:本组8例病人入院拍完善术前检查,排除糖尿病、类风湿性关节炎,报标准的正、侧和斜位X片,术前患足有真菌感染者行抗真菌治疗。病人仰卧位,蛛网膜下腔阴滞或硬膜外阻滞麻醉。在近节趾间关节背侧行椭圆形切口直到骨面,除多余的皮肤组织,分开伸趾肌腱,切开背侧关节囊,松解侧副韧带,切除近节趾骨头至近侧趾间关节完全没有张力,不切除中节趾骨基底关节面,如仍不能伸直近节趾间关节,可经皮在远节趾骨基底行趾长屈肌腱切断术,即可矫正近节趾间关节屈曲畸形,用1枚1mm克氏针由中节趾骨基底水平穿出趾尖,再逆行将克氏针固定近节趾骨,C-臂X光机透视见近节、中节趾骨对位、对线良好,即可冲洗并缝合伤口。修剪并重叠缝合近节趾间关节背侧关节囊组织,短缩缝合伸趾肌腱,修剪近节趾间关节背侧椭圆形皮肤切口,缝合皮肤及皮下组织。石膏托固定前足于中和位。术后静脉应用抗生素7天,4周后去除克氏针,逐渐下地行走。1.3 8例病人都获得随访,术后无伤口感染、克氏针断裂。经8~24个月临床随访(平均17.6月),近节趾间关节畸形矫正满意,残余动度平均约15度,畸形无明显丢失,所有病人都对治疗结果表示满意。根据美国矫形外科协会足踝功能评分标准(AOFAS),治疗前评分平均为43.5分,最后一次随访时评分平均为81.4分,并对手术前后病人AOFAS评分以统计学方法进行X2检验,差异有统计学意义(P<0.01)。2 讨 论锤状趾是指由于各种原因引起的足趾趾间关节和跖趾关节在水平面、冠状面或矢状面的畸形,以及由此引起的一系列症状。是前足最常见的畸形之一,其发生随着年龄的增长而增多。畸形可以是一个平面的,也可是多个平面的。畸形可以累及前足一个或多个足趾,但以外侧足趾更为多见[1]。发锤状趾病原因甚多,有机械性和神经肌肉性等多种因素。机械性因素主要是由于长年穿足前端过窄的鞋子所致,故其在女性中的发病率较男性高,男女比可达1:5。长期穿尖头高跟鞋可使跖趾关节过伸,进而使 跖趾关节压力过高,导致跖盘和双侧副韧带病变而产生锤状趾畸形。机械性因素还包括包括急性创伤损伤了跖趾关节的支持韧带和关节囊,导致跖趾关节不稳定;拇趾畸形如拇外翻也可传递压力至第二趾,使拇趾与第二趾重叠,产生锤状趾畸形。神经肌肉疾病引起相关肌肉失衡也可引起锤状趾畸形,如进行性神经性腓骨肌萎缩、脑瘫、脊髓损伤、脊髓发育不良、多发性硬化、筋膜间室综合征、糖尿病相关的神经疾病等。可见,锤状趾的发生与机械性和神经肌肉性所致近侧趾间关节在矢状面上的力线不稳有关。正常情况下近侧趾间关节稍屈曲或处于中和位本组病例中,而锤状趾形成后则出现近侧趾间关节严重屈曲畸形。本组8例病人中,机械性因素所致锤状趾5例,神经肌肉性因素所致锤状3例,男性患者比例偏高,可能与本组病例数量少及含有机械性因素所致锤状趾有关。疼痛是锤状趾最常见的症状。有3个部位最容易发生疼痛:①近节趾间关节背侧疼痛的硬鸡眼,这是因为近节趾间屈曲畸形,背侧皮肤受鞋顶部挤压摩擦所致。②足趾末端鸡眼疼痛。③跖骨头下形成疼痛性胼胝,这是因为近节趾骨基部向背侧半脱位之故。临床医师除了正确诊断锤状趾外,还应判断这些畸形是僵硬性和非僵硬性,同时还要评估畸形的严重程度和所有的前足异常,如拇外翻等[2]。非僵硬性畸形可以通过患者正确的站立而纠正,而僵硬性畸形不能通过中和位而被纠正。根据畸形程度锤状趾可分为三度:①轻度畸形,即跖趾关节或近侧趾间关节无僵硬性挛缩,畸形随负重的增加而增加。②中度畸形,即近侧趾间关节出现僵硬性跖屈挛缩,但跖趾关节未出现背伸挛缩。③重度畸形,即近侧趾间关节出现僵硬性跖屈挛缩,同时伴跖趾关节背侧挛缩。重度畸形通常们有跖趾关节半脱位或完全脱位[3]。锤状趾的治疗:非手术方法治疗锤状趾效果不佳,尤其是僵硬性畸形,大多数需要手术治疗。尽管手术可以恢复足趾的对线,但常常带来足趾的僵硬,畸形复发率也较高。以僵硬换取疼痛是否能得到患者的认同,术前需要与患者充分的沟通。锤状趾的手术适应证包括无法缓解的疼痛、穿鞋障碍、锤状趾伴拇外翻引起的重叠趾畸形、锤状趾导致溃疡、跖趾关节脱位等。手术禁忌包括足趾血管功能障碍,活动性感染和溃疡、控制不佳的糖尿病及患者对手术不积极等。锤状趾畸形的手术方式,包括软组织手术和骨与关节手术两类。软组织手术适用于有症状的非僵硬性锤状趾,常用的软组织矫正术有经皮屈趾长肌腱切断术、Girdleston-Taylor屈肌至伸肌转移术。对僵硬性锤状趾畸形,须加行骨与关节手术畸形才可能矫正。近侧趾间关节融合术是最常用的骨与关节矫形术式,近年,开发了多种内固定方法用于此关节融合术,效果尚满意[4、5],但术后常发生足趾僵硬。本组病例在软组织松解基础上,采用近侧趾骨头切除,保留中节趾骨基底面的关节软骨,克氏针临时固定4周,术后近节趾间关节形成大约15度的残余动度,可有效缓解足趾僵硬带来的不适症状,平均随访17.6月,临床效果满意,根据美国矫形外科协会足踝功能评分标准(AOFAS),治疗前评分平均为43.5分,最后一次随访时评分平均为81.4分,差异有统计学意义(P<0.01)。Dhukaram等[6]采用跖趾关节软组织可松解术和近侧趾间关节成形术治疗了84例锤状趾圆畸形,83%的患者对该术式表示满意,以美国矫形外科协会足踝功能评分标准(AOFAS),平均为83%分,功能评分与本组病例结果相近,但本组病例数量较少,随访时间短,其临床结果还有待更大样本的长期随访研究。参考文献:[1] Konkel KF, Menger AG, Retzlaff SA. Hammer toe correction using an absorbable intramedullary pin[J]. Foot Ankle Int. 2007, 28(8):916-20.[2] 范清宇,唐农轩主译.临床骨科学[M].第2版.西安:世界图书出版西安公司,2004,949-95.[3] 汪文,俞光荣.锤趾研究进展[J].国际骨科学杂志,2006,27(6):370-372. [4] Pietrzak WS, Lessek TP, Perns SV. A bioabsorbable fixation implant for use in proximal interphalangeal joint (hammer toe) arthrodesis: Biomechanical testing in a synthetic bone substrate[J]. J Foot Ankle Surg. 2006,45(5):288-94.[5] Caterini R, Farsetti P, Tarantino U, et al. Arthrodesis of the toe joints with an intramedullary cannulated screw for correction of hammertoe deformity[J]. Foot Ankle Int. 2005,26(12):1101.[6] Dhukaram V, Hossain S, Sampath J, et al. Correction of hammer toe with an extended release of the metatarsophalangeal joint[J].J Bone Joint Surg Br.2002,84(7)986-990.
肩关节 急性疼痛性钙化性肌腱炎的关节镜治疗 李忠1 杨洪彬1 叶里子1 王远辉1 王治1 鲁晓波1 戴刚2 杨柳2 (1.泸州医学院附属医院骨关节科 646000;2.第三军医大学西南医院关节外科中心 400038)摘 要: 目的 探讨关节镜治疗急性疼痛性肩关节钙化性肌腱炎的临床效果。方法 8例保守治疗效果不满意的急性疼痛性肩袖钙化性肌腱炎患者根据Grtner放射学分类:I型2例,II型2例,III型4例;所有病例采用关节镜下钙化沉积物清除术,其中2例加行肩峰成形术,在治疗前及最后一随访时行Constant-Murley评分评估其疗效。结果 6例术后钙化沉积物消失, II型1例和III型1例有钙化物残留。8例术后平均随访16.2月,肩关节疼痛及活动明显改善,Constant-Murley评分:术前平均为22.2分,术后平均为88.5分,差异有统计学意义(P<0.01)。结论 关节镜下清除钙化沉积物,具有疼痛缓解明显、关节功能恢复快、可同时处理肩关节内病变等优点,是保守治疗无效的急性疼痛性肩关节钙化性肌腱炎的有效治疗方法。关键词: 关节镜治疗;钙化性肌肌腱炎;肩关节Arthroscopic Treatment of Acutely Painful Calcifying Tendonitis of ShoulderLI Zhong, YANG Hong-bing, YE Li-zhi, et al.(1.Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College, Luzhou, 646000;2.Center for Joint Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, China)Abstract: Objective The purpose of the present study is the evaluation of clinical results of arthroscopic treatment of acutely painful calcifying tendonitis of shoulder. Methods 8 patients with acutely pain of rotator cuff calcifying tendonitis of the shoulder were evaluated according to the Grtner radiological classification revealed 2 type I deposits, 2 type II and 4 type III deposits. All patients underwent arthroscopic resection of calcific deposits after failed conservative treatment via posterior approach. 3 of them had additional acromioplasty. Constant-Murley score were adopted for evaluation before surgery and at the last follow up. Reaults Postoperative no calcific deposits were seen in 6 patients, 1 type II and 1 type III deposits. The mean follow up period was 16.2 months. There was greater improvement in the range of motion and pain relief of the shoulder. The average preop Constant-Murley score was 22.2, and the average postop Constant-Murley score was 88.5. Significant difference was found between preop and postop evaluation (P<0.01). Conclusion Arthroscopic resection of calcific deposits shows promise for pain relief and functional restoration of calcific tendinitis with negligible complications. It is an effective method for the treatment of calcifying tendinitis after failed conservative treatment. Key Words: Arthroscopic treatment; Calcifying tendonitis; Shoulder joint 肩袖内的钙化性沉积物可引起肌腱炎,是肩关节疼痛的常见原因,本病最常发生于女性,发病的平均年龄是30-50岁,据报道肩关节钙化性肌腱炎的发病率介于2.7% 到23.6%之间。急性肩关节钙化性肌腱炎主要表现为突发肩关节剧烈疼痛及活动受限,保守治疗效果不佳者可行手术治疗,继往多采用开放性手术治疗,肩关节镜下钙化灶清除术具有创伤小、恢复快等优点,已成为治疗钙化性肌腱炎的最佳治疗方法[1]。我科自2006年11月至2008年6月收治急性肩关节钙化性肌腱炎8例,经短期保守治疗无效后采用关节镜下钙化灶清除术,术后效果满意,现报告如下。1 临床资料与方法1.1 一般资料:本组共8例,女性7例,男性1例;年龄45~59岁,平均48.7岁。左侧3例,右侧5例。5例钙化灶位于冈上肌腱内,2例位于冈下肌腱内,1例同时涉及冈上肌腱和冈下肌腱。Grtner分型:Ⅲ型4例,Ⅰ型和Ⅱ型各2例。所有患者继往都无肩关节疼痛及活动受限病史,本次发病表现为突发肩关节剧烈疼痛及活动明显受限,肩关节活动时疼痛加重明显,局部压痛明显,3例患者有明显夜间痛。所患者都经肩关节X线平片、CT和MRI确定诊断,常规肩关节X线平包括内旋位和外旋位的前后位片、肩胛出口位片和腋位片;肩关节MRI扫描包括斜冠状位、斜矢状位及轴位T1、T2加权序列和脂肪抑制序列。所有患者经口服塞来昔布、患肩制动、局部冰敷及肩峰下滑囊封闭等保守治疗效果不满意,即选择关节镜下手术治疗。疼痛发作至手术时间为2-15天,平均8.4天。手术采用钙化灶清除术,其中2例还附加肩峰成形术。本组病例均采用美国Smith & Nephew公司直径4.0mm的30°关节镜系统及电动刨削系统,意大利Arthocare射频消融刀。1.2 治疗方法:患者采用气管内插管全麻,取沙滩椅半坐位,背部抬高约70°,在常规消毒铺巾前,用标记笔在皮肤上标记肩关节的骨性标记志,包括肩峰、肩锁关节、喙突以及所计划的入口部位。将0.1%的肾上腺素溶液2ml加入3L关节镜生理盐水冲洗液中,未使用关节镜泵。(1)首先探查盂肱关节:触摸肩关节后方的软点部位,约位于肩峰后外侧角的下方2cm和内侧1cm处。用18号腰穿针朝向喙突向前穿入关节内,将50ml生理盐水注入关节内,皮肤切口之后,将关节镜鞘管和钝性穿剌芯放入盂肱关节腔内,液体经鞘管返流得到证实。仔细探查盂肱关节,发现关节内所有病变并正确处理,本组2例关节软骨损伤者用射频消融刀行了关节软骨修整术,1例关节软骨缺损行了微骨折手术,未发现SLAP损伤。仔细探查肩袖下表面,尤其是怀疑存在钙化性沉积物的部位,发现可疑隆起、充血和损伤区域,本组有2例在受累的肩袖关节面发现存在“草莓样”炎性充血水肿滑膜,即可判断为钙化灶的准确位置,为了在滑囊侧确定病变部位,从肩峰外侧缘穿入腰穿针,并使针头穿过肩袖的钙化沉积物,一旦针头定位准确,将一根PDS缝线经针穿入,观察到进入关节面侧时,去除针头,将缝线保留于肌腱内,若此前已建立肩关节前方入口,可用抓物钳将关节内的缝线端拉出。(2)探查肩峰下间隙:在完成盂肱关节内的操作之后,将关节镜鞘管及钝性穿剌芯在后方改变方向,进入肩峰下间隙,清除肩峰下间隙的大部分滑膜组织,确保获得完整视野。用18号腰穿针剌入肩峰下间隙,在直视下建立外侧入口,通常位于肩峰外侧缘前方和中1/3交界处。2例已用标记线定位钙化灶部位者,则可准确定位并在滑囊侧进行钙化灶清理;另外6例未能用标记线定位者,则将关节镜从各个入口置入并旋转上肢,观察位于滑囊侧钙化灶部位,其中有3例钙化灶已穿破肩袖滑囊侧者定位较容易,另外3例则根据术前X线片和术中C臂X光机透视定位。用探针、刮匙和小直径刨削器进行清除,清除钙化灶时释放的钙质呈牙膏状,若释放的钙质呈絮状飘入肩峰下间隙,形成“暴风雪”样征象。钙化灶清除后探查肩袖肌腱的缺损情况,本组患者钙化灶清除后肩袖缺损较小,未行肩袖修补术。(3)探查肩峰下表面:肩胛出口位片显示为Ⅲ型肩峰及肩锁关节退变,可同时在关节镜下发现肩峰下骨赘,则行肩峰下减压术,以预防术后发生撞击症。术毕用生理盐水彻底冲洗肩峰下滑囊,以尽量除去残留的钙质。术后康复:术后早期使用颈腕带,局部冰敷,常规使用镇痛泵和口服非甾体类消炎止痛药,康复计划与肩峰下成形术基本相同,允许患者术后早期开始被动活动,术后2周鼓励进行主动活动。2 结 果本组8例,所有患者术后疼痛即明显好转或消失,平均住院时间为7(5-10)d,术后全部获得随访,随访7-25个月,平均16.2个月。术后X线片显示8例中6例钙化点完全消失(图1,2);II型1例和III型1例有钙化物残留,术后3月复查X线片显示残留钙化物消失。治疗前以及最后1次随访时进行Constant-Murley评分,治疗前评分平均22.2分(18-42),治疗后平均88.5分(78-95),并对手术前后病人Constant-Murley评分以统计学方法进行X2检验,差异有统计学意义(P<0.01)。3 讨 论钙质沉积于关节周围组织内可导致钙化性肌腱炎而引发疼痛,尽管钙质可沉积于身体的其他部位,肩袖肌腱仍然是常见的受累部位。肩关节钙化性肌腱炎临床表现差别很大,可偶然经放射学检查发现,而无任何症状,也可表现为急性疼痛和功能障碍。钙化性肌腱炎的病因不明,大多数学者将其分为两期:慢性期(形成期)和急性期(吸收期),典型的形成和吸收过程并非一成不变,在同一时间内各个独立病灶可能处于不同的形成和吸收阶段,甚至同一病灶也可能处于不同的形成和吸收过程。Neer认为钙化性肌腱炎的疼痛可能有4种机制。(1)钙质沉积物对组织的化学剌激;(2)钙质沉积物吸收水分,引起肿胀,加大肌腱内压力;(3)肩峰下滑囊对钙化物发生反应性增生,导致滑囊炎或撞击;(4)由于肩关节活动减少引发粘连性关节囊炎。与肌肉骨骼系统的其他疾病不同,在钙化性肌腱炎形成的某段时期内(慢性期)可表现为症状轻微或无症状,随着进入吸收期(急性期),通常出现急性起病的疼痛。本组8例急性起病的钙化性肌腱炎病人可能发生于疾病的吸收期,病变部位内血管和细胞成分的增加可导致肌腱内压力升高,相似于无菌性脓肿,吸收时的巨噬细胞和多核巨细胞浸润释放的炎性因子也可引起疼痛。急性钙化性肌腱炎有如下临床特点:(1)起病急,疼痛非常剧烈,患者常能指出具体的发病时间,部分患者有明显夜间痛;(2)肩关节通常无法耐受活动,各个方向活动明显受限;(3)受累区域常有压痛;(4)保守治疗效果常常不理想。根据临床表现及X线片和MRI可确定钙化性肌腱炎的诊断,必须除外肩关节疼痛的其他原因,因为钙质沉积物可在放射学检查中偶然发现。X线片和MRI影像学检查可用于钙化性沉积物的诊断和定位。盂肱关节后前位片可较好显示冈上肌腱内的钙化灶;内旋位盂肱关节后前位片可用于确定冈下肌腱内的钙化灶;肩胛出口位有有助于前后方向上确定冈上肌腱内的钙化灶,以及发现位于后方冈下肌腱内的沉积物,此外,可判断肩峰形态及是否存在肩峰下骨赘;腋位片可用于发现肩胛下肌腱内的钙化灶。沉积物在T1加权像表现为低信号强度,T2加权像上可表现为围绕钙化物病灶周围的高信号强度条状信号,提示存在水肿,可通过分析两个互相垂直的图像确定其部位,此外MRI还可发现有无肩袖撕裂和肩峰下撞击。急性钙化性肌腱炎的治疗:首先行非手术治疗,非手术治疗方法包括口服非甾体类消炎止痛药、患肩制动、局部冰敷、肩峰下滑囊封闭及穿剌冲洗等,若非手术治疗未能有效缓解疼痛,即可考虑手术治疗[2]。继往常采用开放性手术治疗,手术创伤大,恢复慢,近年来随着肩关节镜技术的开展,肩关节镜下清除钙化灶,必要时行肩峰下减压及肩袖修补术,已成为治疗肩关节钙化性肌腱炎的首选手术治疗方法。关节镜下治疗肩关节钙化肌腱炎有如下优点:包括损伤小、功能恢复快、住院时间短、外形美观等优点,还可一并处理盂肱关节及肩峰下间隙内存在的病变。研究发现,仅50%的病例对非手术治疗满意,而对非手术治疗效果欠佳病例行手术治疗,大多可获得满意临床疗效。对于清除钙化灶后是否进行肩峰成形术目前仍存在争议。近年的研究证实,清除钙质同时行肩峰下减压术并不能改善肩关节功能,但对于存在肩峰形态改变的患者,例如骨赘形成和Ⅲ型肩峰等,具有肩峰下减压的手术指征[3]。本组病例未常规行肩峰下减压术,仅对2例肩峰形态改变病例行了肩峰下减压术。大部分学者认为手术治疗钙化性肩袖肌腱炎时应将钙化灶彻底清除, 在清除巨大病灶时如肩袖连续性遭到破坏,需行肩袖修补术[4]。但本研究及其他文献发现少量钙质残留并不影响治疗效果,而且术后数月内可自行吸收。因此,钙化灶特别巨大时, 手术中彻底去除病灶会导致肩袖的完整性破坏,巨大的钙化灶清除导致的肩袖缺损有时会使修补术变得极为困难,是彻底切除病灶还是尽量保留肩袖的完整性仍然是个值得探讨的问题[5]。有学者对15例巨大的肩关节钙化性肌腱炎病人分别采用保留关节面侧肩袖完整, 允许少量钙化灶残留和彻底清除钙化灶并缝合破坏后肩袖的两种方法行关节镜下治疗,结果发现病人手术效果无明显差异,但保留肩袖组具有手术方式简单、花费少、创伤小、恢复快等优点[6],但上述研究临床病例数量较少,还需要大量临床研究加以验证。参 考 文 献1 Lorbach O, Kusma M, Pape D, et al. 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李忠*杨洪彬* 王远辉* 王治* 阳运康* 鲁晓波* 杨柳**[摘要] 目的 介绍关节镜辅助下清创、自体髂骨移植治疗长骨骨折萎缩型不愈合,特别是伴有严重皮肤软组织损伤的萎缩型不愈合的初步经验。 方法 13例萎缩型长骨骨折不愈合,男8例,女5例,伴严重皮肤软组织损伤7例。 X-线监视下建立两个关节镜入口,关节镜直视下骨折端清创、自体松质骨移植治疗。 结果 骨折端手术平均出血量为15ml,平均手术时间为65min。除取髂骨区出现并发症外,植骨区都无严重的术中和术后并发症,所有骨折都获得无痛和稳定的骨性愈合,关节活动范围无进一步丢失。骨折愈合时间3~7个月,平均为5.2个月。 结论 关节镜辅助下植骨治疗长骨萎缩型不愈合,创伤小,愈合率高,特别适用于伴有严重皮肤软组织损伤者。[关键词] 不愈合; 关节镜治疗; 骨折骨折是临床骨科最常见的疾病之一。尽管骨折治疗的理念、治疗手段不断更新,手术技术也不断提高,但骨折不愈合率仍高达2.5~46%[1]。骨折不愈合可导致关节僵直、肢体短缩及功能障碍,给患者带来极大痛苦。特别是骨折局部伴有严重皮肤软组织损伤的萎缩型骨折不愈合患者,再次手术治疗难度大,并发症多且恢复时间长,手术效果也不理想[2]。因此,寻找有效治疗骨折不愈合,特别是伴有严重皮肤软组织损伤的萎缩型骨折不愈合的方法,是临床骨科医师经常要面对的难题。自2006年8月至2008年4月,我科共采用关节镜辅助下骨折端清创、自体骨移植治疗长骨萎缩型骨折不愈合13例,其中7例伴有较严重皮肤软组织损伤,术后效果满意。现报告如下。1 临床资料与方法1.一般资料:本组男8例,女5例;年龄21~49岁,平均32岁。交通伤8例,坠落伤3例,砸伤2例。开放性骨折按Crustilo-Anderson法进行分类,胫骨干Ⅲ度开放性骨折7例,都使用外固定器固定,其中6例合并皮瓣转位;胫骨干Ⅰ度开放性骨折1例,闭合性骨折1例,均用加压钢板固定;肱骨干Ⅲ度开放性骨折2例用外固定器固定,合并皮瓣转位1例;肱骨干闭合性骨折1例用加压钢板固定;股骨干闭合性骨折交锁髓内针固定后骨折不愈合,行交锁髓内针动力化术后骨折仍不愈合1例。骨折病史1.2~3.1年,平均1.8年;入院前手术1~4次,平均2.2次。本次手术距最近一次手术时间5~21个月,平均9.4个月。入院时,胫骨骨折加压钢板固定螺钉断裂1例,外固定器固定针道感染3例,固定针感染伴松动2例。所有患者术前骨折部位均有压痛,但无假关节活动,也不伴感染;X线片发现骨折端有明显缝隙,无骨痂生长,骨折端硬化,为萎缩型骨折不愈合。本组患者均采用美国Smith & Nephew公司直径4.0mm的30°关节镜系统及电动刨削系统,意大利Arthocare低温等离子刀。2.手术方法:患者仰卧位,术前30min静脉应用抗生素预防感染,采用臂丛或硬膜外阻滞麻醉。术前用C形臂X线机透视骨折不愈合部位并做好标记,以确保正确建立关节镜通道及定位骨折不愈合处,若手术部位允许安放止血带,则术中尽可能使用驱血带和止血带。若外固定器妨碍了关节镜操作,保留外固定针,拆除部分外固定器连接装置后行皮肤消毒。关节镜通道的建立:为避免损伤邻近神经血管组织,远离神经血管结构建立1cm皮肤穿剌口,止血钳钝性分离至皮下组织,钝性带鞘关节镜套针穿剌入骨皮质,用一窄骨膜剥离起子在骨质表面推开软组织,产生一个利于关节镜和器械操作的骨膜下间隙,置入关节镜后,在局部皮肤光亮区插入穿剌针头,证实针头在骨膜下间隙后,按上述方法建立另一通道。液体灌注系统悬吊于手术部位上方1m内,以预防因过高液体灌注压可能引发的骨筋膜间隔综合征。对于股骨干骨折不愈合患者,在骨折不愈合处大腿外侧各建立一个关节镜通道,两个通道相距至少5cm,以利于关节镜和器械的操作;对于肱骨干骨折不愈合者,在骨折不愈合处上臂肌肉的外缘建立两个通道,一个通道位于骨折不愈合处,另一个通道距骨折部位近端或远端3cm处;对于胫骨干骨折不愈合者,以骨折不愈合处为中心,距其远、近端3cm在胫骨前外侧经胫前肌建立两个通道。骨折不愈合断端清创:建立的一个关节镜通道用于置入关节镜,另一个通道用于器械操作,关节镜和器械可在两个通道间互换,以便于骨折端清创及骨移植。关节镜置入后可见骨折不愈合处的纤维瘢痕组织,再次用C形臂X线机透视证实,关节镜及器械置于骨折不愈合处。将白色的不愈合组织与黄色的骨组织分辨开,用电动刨刀刨削、低温等离子刀汽化及刮匙刮除骨折不愈合处纤维组织,用磨钻及刮匙等清除骨折端硬化骨至剩余骨床出血,暴露两侧骨折端及骨髓腔。自体髂骨移植:沿髂嵴切开皮肤、皮下组织3~4cm,将髂骨外板软组织从骨膜下分离,保留骼骨内板,取骼骨外板及松质骨剪成米粒样大小备用。将一次性使用的硬质塑料吸引管剪至合适长度,在关节镜直视下,尽量吸尽局部冲洗液及血液,用塑料管及探针把米粒样自体骨输送到骨折不愈合部位,移植骨的范围包括骨折端间及骨折近、远端1cm。植骨后不进行冲洗,也不放置引流,局部用弹力绷带加压包扎,安放好术前拆除的外固定连接装置。3.术后处理和随访术后静脉应用抗生素1d,外固定针道每天用体积分数为75%酒精消毒4次。术后患者能耐受取髂骨处疼痛后即可下地行走,下床时间2~10d,平均6d,不限制骨折邻近关节主、被动活动,术后1周和1、3、5、7月复查X线正侧片至骨折愈合。2 结 果本组13例平均手术时间55min,取髂骨术出血量20~60 ml,骨折端清创植骨术出血量10~20ml。1例出现股外侧皮神经支配区大腿前外侧麻木,2例出现取髂骨区疼痛。2例术后行走过程中出现外固定针感染伴松动,感染控制后,拔除外固定针,用羟基磷灰石喷涂的外固定针改换穿针部位重新固定,骨折邻近关节活动范围无进一步丢失。临床愈合标准为:患者恢复无痛的完全活动,X-线片显示骨折端有连续骨痂通过,骨折线消失[2]。本组患者全部获得骨性愈合,愈合时间3~7个月,平均5.2个月(表1)。典 型 病 例例1:女, 30岁, 2年前因车祸伤致右股骨中段横形骨折,行了切开复位、交锁髓内钉内固定术,术后14月X-线平片显示骨折端存缝隙,无骨痂形成,诊断为萎缩型骨折不愈合,行了交锁髓内钉动力化手术,二次术后10月X-线平片提示,骨折端间隙明显,无骨痂形成,仍无骨折端骨性连接趋势,遂来我科求治。采用关节镜辅助下植骨治疗,C形臂X线机透视证实骨折不愈合处,关节镜辅助下行骨折端清创及植骨,术后不限制患肢活动,植骨术后4月X-线片显示骨折端有连续骨痂通过,骨折线消失,骨折达到骨性愈合(图1),已恢复正常生活和轻体力劳动。例2:男,28岁。15月前因车祸伤致左胫腓骨中下段Ⅲ°开放性骨折,在外院急诊行了清创,二期骨折复位钢板螺钉内固定术。术后伤口感染、钢板螺钉及骨质外露,经局部伤口换药无明显好转,遂行钢板螺钉取除、局部清创、皮瓣转位和外固定器固定术,术后伤口换药后逐渐愈合,但第二次术后10个月复查X-线平片见,骨折间隙明显,无骨痂生长。采用关节镜辅助下植骨治疗,术后不限制患肢活动,但术后2月出现2枚外固定针感染伴松动,感染控制后,拔除松动外固定针,用羟基磷灰石喷涂的外固定针改换穿针部位重新固定,植骨术后7月复查X-线片显示,骨折已骨性愈合(图2)。讨 论骨折不愈合是骨折手术后最常见的严重并发症之一,特别是对萎缩型骨折不愈合的治疗更为棘手[3],其治疗关键在于骨折断端间彻底清创,补充成骨性细胞和骨诱导物质,重新启动骨折愈合过程,促进骨折愈合,达到恢复患肢功能的目的[4]。目前已有多种非手术或手术方法用于治疗萎缩型骨折不愈合,非手术治疗方法有高能震波治疗、低强度脉冲超声治疗和脉冲电磁场治疗等。高能震动波治疗骨折不愈合已有20年历史,但其治疗萎缩型骨折不愈合或骨断端间隙大于5mm的骨折不愈合患者效果较差[5]。Jingushi等[6]用低强度脉冲超声治疗72例长骨骨折不愈合或延迟愈合患者获得了75%的骨折愈合率,并发现骨折愈合率和距最近一次手术时间的长短有显著的相关性,建议低强度脉冲超声治疗应在最近一次手术6个月内开始使用。虽然上述非手术方法治疗骨折不愈合获得了一定成功,但其适应证有限,且对骨断端间隙较大或病程较长的萎缩型骨折不愈合治疗效果较差。目前临床主要采用手术方法治疗萎缩型骨折不愈合。传统的开放植骨术仍是治疗萎缩型骨折不愈合的“金标准”,即通过开放手术暴露骨折断端,用刮匙、咬骨钳等彻底清除断端间纤维组织及硬化骨组织,将断端骨质粗糙化或去皮质化,再取自体髂骨移植于清创后的骨折断端间及其周围。近来,有人采用骨折断端开放清创后不植骨,而仅用Ilizarov外固定架加压固定断端的方法治疗骨折不愈合,也取得了较好的临床效果[7]。但上述两种开放手术治疗方法仍存在一定缺陷,特别是对于骨折局部伴有严重皮肤软组织损伤的患者,在发生骨折不愈合前,往往已经历了多次清创、换药、植皮或皮瓣转位等才将伤口闭合,导致骨折端周围皮肤软组织血液供应不足、瘢痕形成、抗感染能力差及生物学成骨能力不足,使传统开放手术进行骨折端清创及植骨手术存在较大风险,术后出现局部皮肤软组织感染、坏死、伤口愈合不良、骨外露等治疗失败的机会大大增加。因此,探寻有效治疗伴严重皮肤软组织损伤的萎缩型骨折不愈合的微创或无创治疗方法,成为人们研究的难点和热点,经皮自体骨髓移植或经皮植骨即是对此的有益尝试。经皮自体骨髓移植是在X线透视下行骨折端清创后,行多次自体骨髓移植,但由于骨折端清理不彻底,存在骨髓移植空间有限及移植骨髓不能较好局限于骨折端等缺陷。Kettunen等[8]采用X线透视下经皮行骨折端清创植骨治疗41例胫骨骨折不愈合或延迟愈合,其中37例在术后13周获得骨性愈合。该方法对局部皮肤软组织干扰小,但不能在直视下进行骨折断端清创,导致骨折端清创不彻底是其不足之处。与Johnson 等[9]的研究结果相似,笔者采用关节镜直视下骨折断端清创、自体髂骨移植治疗长骨骨折不愈合13例,特别是伴有严重皮肤软组织损伤7例,全部获得骨性愈合,骨折线消失时间平均为5.2个月,植骨区无术中及术后并发症,获得满意临床效果。本方法具有如下优点:(1)关节镜电子图像放大系统提高了可视程度,在直视下通过微创的方法彻底清理骨折断端纤维瘢痕组织和硬化骨,达到与开放手术清理骨折端相似的效果,并可提供足够空间用于足量骨的精确移植。(2)对局部组织干扰小,大大减小了对皮肤软组织的损伤和血循环破坏,对伴有严重皮肤软组织损伤的难治疗性萎缩型骨折不愈合的治疗具有明显优势。(3)可早期活动邻近关节,利于关节功能恢复。(4)骨折愈合率高。(5)住院时间短,花费少,并发症少。适应证:关节镜辅助下清创植骨适用于骨折端稳定的长骨骨折延迟愈合和不愈合患者治疗,特别适用于伴有严重皮肤软组织损伤者,也适用于骨折间隙较大的骨折不愈合的治疗。骨折端不稳定和感染是其相对禁忌证。 取自体髂骨用作移植材料存在一定供区并发症,如血肿、感染、疼痛和股外侧皮神经损伤等。本组中3例出现供区并发症,1例股外侧皮神经支配区大腿前外侧麻木,2例现取髂骨区疼痛,供区术后疼痛限制了患者早期下床活动。由于自体髂骨移植存在上述并发症,寻找其他移植材料替代自体髂骨成为研究的又一热点,如使用骨髓、富含血小板血浆、同种异体骨、磷酸钙骨水泥和骨形态发生蛋白等[1、10]。此外,细胞、生长因子和各种载体复合移植还有望进一步加速骨折不愈合治疗进程,促进患肢功能更快恢复[11、12]。参 考 文 献1 Sen MK, Miclau T. 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Ilizarov frame fixation without bone graft for atrophic humeral shaft nonunion: 28 patients with a minimum 2-year follow-up. J Orthop Trauma, 2007, 21 (8): 549-556.8 Kettunen J, Mkel EA, Turunen V, et al. Percutaneous bone grafting in the treatment of the delayed union and non-union of tibial fractures. Injury, 2002; 33 (3): 239-245.9 Johnson LL, Morrison KM, Wood DL. The application of arthroscopic principles to bone grafting of delayed union of long bone fractures. Arthroscopy, 2000,16 (3):279-289.10 Nishikawa S, Toh S, Tsukahara S, et al. Endoscopic treatment of delayed unions and nonunions with calcium phosphate paste. Arthroscopy, 2003, 19 (4):436-439.11 Kujala S, Raatikainen T, Ryhnen J, et al. Composite implant of native bovine bone morphogenetic protein (BMP), collagen carrier and biocoral in the treatment of resistant ulnar nonunions: report of five preliminary cases. Arch Orthop Trauma Surg, 2004, 124 (1):26-30.12 Shen HC, Peng H, Usas A, et al. 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