[湖南師范大學(xué)第一附屬醫(yī)院(湖南省人民醫(yī)院) 關(guān)節(jié)與運(yùn)動醫(yī)學(xué)科,湖南 長沙 410000]
半月板是一種纖維軟骨結(jié)構(gòu),在關(guān)節(jié)內(nèi)傳遞負(fù)荷、營養(yǎng)軟骨及減少摩擦等,其損傷后導(dǎo)致關(guān)節(jié)內(nèi)異常負(fù)荷及炎癥的產(chǎn)生,繼發(fā)軟骨的退變,加速骨關(guān)節(jié)炎的進(jìn)展[1],成為了運(yùn)動醫(yī)學(xué)中的難題。早期的治療如半月板切除術(shù),雖能在一定程度上緩解癥狀,但導(dǎo)致軟骨的 接觸應(yīng)力大幅度增加。隨著微創(chuàng)技術(shù)的發(fā)展,關(guān)節(jié)鏡下修復(fù)半月板損傷是目前最佳的手術(shù)方式。然而,由于半月板最常見的損傷區(qū)域(白區(qū))的無脈管系統(tǒng)分布,常導(dǎo)致術(shù) 后恢復(fù)受限[2]。因此,在行手術(shù)時(shí),使用其他方法促進(jìn)半月板修復(fù)成為了熱點(diǎn)[3]。目前臨床上的方法如纖維蛋白凝塊技術(shù)[4]、滑膜移植[5-6]、藥物[7]及生長因子[8]等,效果有限,且手術(shù)難度和費(fèi)用較高。隨著基礎(chǔ)醫(yī)學(xué)的發(fā)展,間充質(zhì)干細(xì)胞(mesenchyma stem cell,MSC)得到了廣泛的研究[9],并視其為修復(fù)半月板損傷的最佳種子細(xì)胞[10]。MSC可體外復(fù)合天然生物或高分子合成支架,構(gòu)建半月板再生結(jié)構(gòu)組織[11-12];缺損處自體MSC聚集組裝移植與關(guān)節(jié)腔內(nèi)MSC注射能促進(jìn)半月板再生等[13-15]。因此,圍手術(shù)期中利用干細(xì)胞的多分化潛能成為了焦點(diǎn)[16],但現(xiàn)有的MSC治療方法存在周期長、細(xì)胞來源少、支架力學(xué)性能及生物活性差和二次手術(shù)的可能等問題[17],導(dǎo)致其在臨床上使 用受到限制。從解剖中,本團(tuán)隊(duì)發(fā)現(xiàn)膝關(guān)節(jié)毗鄰股骨髓腔,其內(nèi)有髕下脂肪墊,若通過在關(guān)節(jié)鏡下手術(shù)處理,釋放骨髓及脂肪來源 的干細(xì)胞,可減少操作流程,有望加快和簡化修復(fù)半月板損傷的進(jìn)程。本研究希望通過基礎(chǔ)實(shí)驗(yàn)及臨床研究,分析關(guān)節(jié)鏡下髁間窩微骨折術(shù)與髕下脂肪墊粉碎術(shù)對半月板損傷修復(fù)的作用機(jī)制及其療效。
DMEM/F12培養(yǎng)基、胎牛血清、甘氨酸購自Sigma公司;紅細(xì)胞裂解液、磷酸緩沖鹽溶液( p hosphate buffer saline,PBS)購自Beyotime公司;Human MSC Analysis Kit購自BD StemflowTM;二氧化碳CO2恒溫培養(yǎng)箱購自Thermo Forma公司;倒置顯微鏡、光學(xué)顯微鏡購自O(shè)lympus公司;臺式冷凍離心機(jī)購自Thermo Scientific公司。
于股骨髁間窩內(nèi)行微骨折術(shù),吸引器將孔洞流出的骨髓液吸入離心管,轉(zhuǎn)運(yùn)至實(shí)驗(yàn)室。500 g離心5 min,棄上清,紅細(xì)胞裂解液重懸,避光裂解2 min,500 g離心5 min,棄上清,PBS重懸,500 g離心5 min,棄上清,加入10%胎牛血清(fetal bovine serum,F(xiàn)BS)的DMEM/F12培養(yǎng)基重懸,轉(zhuǎn)移至培養(yǎng)瓶,于37℃+5% CO2孵箱中培養(yǎng),48 h后首次換液,每隔2 d換液,細(xì)胞匯合度達(dá)到80% 以上時(shí)傳代,對第1代實(shí)驗(yàn)細(xì)胞進(jìn)行鏡下觀察,用人間充質(zhì)干細(xì)胞試劑盒對其進(jìn)行鑒定。
在股骨髁間窩前方髕下脂肪墊處,刨削刀將一小塊脂肪墊打碎成1.0 mm×1.0 mm大小,吸引器將脂肪小塊連同周圍的液體吸入離心管,轉(zhuǎn)運(yùn)至實(shí)驗(yàn)室。PBS反復(fù)清洗,100μm及40μm濾網(wǎng)過濾,400 g離心5 min,棄上清,紅細(xì)胞裂解液重懸,避光裂解10 min,400 g離心5 min,加入10% FBS的DMEM/ F12培養(yǎng)基重懸,轉(zhuǎn)移至培養(yǎng)瓶,于37℃+5% CO2孵箱中培養(yǎng),48 h后首次換液,每隔2 d換液,細(xì)胞匯合度達(dá)到80%以上時(shí)傳代,對第1代實(shí)驗(yàn)細(xì)胞進(jìn)行鏡下觀察,用人間充質(zhì)干細(xì)胞試劑盒對其進(jìn)行鑒定。
回顧性分析2014年1月-2016年1月診療的單側(cè)單純半月板損傷并行手術(shù)的患者,共計(jì)75例(75膝),病例均來自本院同一治療組的住院患者。納入標(biāo)準(zhǔn):年齡18~35歲,無嚴(yán)重肝、腎、心及造血系統(tǒng)疾病等;既往無膝關(guān)節(jié)手術(shù)史;符合下述診斷標(biāo)準(zhǔn)者;簽署知情同意書者。排除標(biāo)準(zhǔn):患有韌帶損傷、髕骨軟骨軟化、軟骨損傷、游離體及滑膜皺襞綜合征等其他關(guān)節(jié)紊亂疾病;精神狀態(tài)欠佳或不配合者;既往有膝關(guān)節(jié)手術(shù)史;患有嚴(yán)重肝、腎、心及造血系統(tǒng)疾病等;骨腫瘤;未簽知情同意書者。按照關(guān)節(jié)鏡中手術(shù)處理方式不同,將75例患者分為髁間窩微骨折術(shù)組(A組25例)、髕下脂肪墊粉碎術(shù)組(B組25例)及對照組(C組25例)。3組患者的年齡、體質(zhì)指數(shù)(body mass index,BMI)、半月板損傷類型及性別構(gòu)成比等方面分布相近,差異無統(tǒng)計(jì)學(xué)意義(P>0.05),具有可比性。見表1。
表1 各組患者一般資料比較Table 1 Comparison of general data among the three groups
1.5.1 癥狀急性受傷時(shí)膝關(guān)節(jié)內(nèi)可有撕裂聲;上下樓時(shí)有打軟腿及錯(cuò)動感;患肢有乏力不穩(wěn)感;運(yùn)動能力明顯下降。
1.5.2 體征急性期間可有關(guān)節(jié)腫脹;關(guān)節(jié)間隙壓痛;McMurry(+),Apley(+);過伸、過屈(+)。
1.5.3 檢驗(yàn)結(jié)果急性損傷可有紅細(xì)胞沉降率(erythrocyte sedimentation rate,ESR)、C反應(yīng)蛋白(C reactive protein,CRP)及白細(xì)胞數(shù)目增多;陳舊性損傷中血生化檢查一般無明顯異常。
1.5.4 影像學(xué)檢查X片及CT一般無明顯異常,偶見關(guān)節(jié)間隙增寬;磁共振成像(magnetic resonance imaging,MRI)提示半月板損傷(多為Ⅲ度信號,通向上下關(guān)節(jié)腔表面)影像。
A組:在行半月板成型縫合術(shù)后,用微骨折器械,于髁間窩處鉆出小孔,使一部分骨髓和血液從孔中滲出。B組:在行半月板成型縫合術(shù)后,在髁間窩前方髕下脂肪墊處,用刨削刀將一小塊脂肪墊(約0.5 cm×0.5 cm)充分反復(fù)打碎,成1.0 mm×1.0 mm大小。C組:空白對照組,行常規(guī)的半月板縫合術(shù)后,并未進(jìn)行其他特殊處理。
常規(guī)手術(shù)方式:麻醉滿意后,患者取仰臥位,取常規(guī)膝前內(nèi)外側(cè)入路長約0.5 cm,置入關(guān)節(jié)鏡,探查可見半月板損傷,清理半月板白區(qū)撕裂組織,紅白區(qū)采用Fast-fix進(jìn)行縫合,修整半月板邊緣,注入玻璃酸鈉,彈力繃帶加壓包扎。術(shù)中具體過程見圖1。
所有患者手術(shù)前及隨訪時(shí)均采用Lysholm、視覺模擬評分法(visual analogue scale,VAS)評分 及不良事件(關(guān)節(jié)腫脹及膝前區(qū)疼痛)評估。
應(yīng)用SPSS 21.0統(tǒng)計(jì)學(xué)軟件進(jìn)行處理。檢驗(yàn)數(shù)據(jù)差值是否符合正態(tài)分布和滿足組間方差齊性,如果滿足,采用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,組間采用單因素方差分析與配對t檢驗(yàn);若不滿足,采用秩和檢驗(yàn),計(jì)數(shù)資料的比較采用χ2檢驗(yàn)。以P<0.05為差異有統(tǒng)計(jì)學(xué)意義。
圖1 術(shù)中具體過程Fig.1 Intraoperative specific process
圖2 髁間窩微骨折體外細(xì)胞培養(yǎng)鏡下圖片F(xiàn)ig.2 Micrograph of cells released by fossa intercondyloidea of micro-fracture
2.1.1 髁間窩微骨折術(shù)體外實(shí)驗(yàn)在體外培養(yǎng)8~10 d后可見培養(yǎng)瓶中細(xì)胞類似 成纖維細(xì)胞,生長迅速,長梭狀,呈旋渦狀分布,細(xì)胞界限清楚,排列緊密,密度較高,具有干細(xì)胞的特性,見圖2。將第1代細(xì)胞用人干細(xì)胞分析試劑盒進(jìn)行鑒定,可見樣本細(xì)胞表面CD90、CD105、CD73及CD44表達(dá)比例高(>95%),符合干細(xì)胞的定義標(biāo)準(zhǔn),考慮其為骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)[18],見圖3。
圖3 髁間窩微骨折體外細(xì)胞干細(xì)胞試劑盒鑒定結(jié)果Fig.3 Human MSC Analysis Kit results of cells released by fossa intercondyloidea of micro-fracture
2.1.2 髕下脂肪墊粉 碎術(shù)體外實(shí)驗(yàn)在體外培養(yǎng)8~10 d后可見培養(yǎng)瓶中絕大多 數(shù)細(xì)胞類似成纖維細(xì)胞,含有少量雜質(zhì)及壞死細(xì)胞,呈長梭狀,呈平行排列,網(wǎng)狀輻射狀生長,細(xì)胞界限清楚,排列規(guī)則,具有干細(xì)胞的特征,見圖4。將第1代細(xì)胞用人干細(xì)胞分析試劑盒進(jìn)行鑒定,可見樣本細(xì)胞表面CD90、CD105及CD73表達(dá)比例高(>95%),CD44相對較少,符合干細(xì)胞的定義標(biāo)準(zhǔn),考慮其為髕下脂肪墊來源干細(xì)胞(infrapatellar fat pad-derived stem cells,IPFPSCs)[18],見圖5。
圖4 髕下脂肪墊粉碎體外細(xì)胞鏡下圖片F(xiàn)ig.4 Micrograph of cells released by comminution of subcutaneous fat pad
所有患者均獲平均10個(gè)月(8~11個(gè)月)隨訪,無神經(jīng)血管損傷、感染等并發(fā)癥。術(shù)前,A、B、C組的Lysholm及VAS評分的組間差異無統(tǒng)計(jì)學(xué)意義(P>0.05),不良事件大致相同。術(shù)后末次隨訪中,各組末次隨訪的結(jié)果較治療前均有明顯改善,差異有統(tǒng)計(jì)學(xué)意義(P<0.05);A組與C組、B組與C組間的Lysholm及VAS評分差異具有統(tǒng)計(jì)學(xué)意義(P<0.05);A組與B組間的差異無統(tǒng)計(jì)學(xué)意義(P>0.05),見表2。B組的不良事件早期發(fā)生率高于A組及C組,末次隨訪時(shí)無明顯差異,見表3。半月板損傷修復(fù)術(shù)后情況見圖6。
圖5 髕下脂肪墊粉碎體外細(xì)胞干細(xì)胞試劑盒鑒定結(jié)果Fig.5 Human MSC Analysis Kit results of cells released by comminution of subcutaneous fat pad
表2 各組治療前后Lysholm和VAS評分比較 (分,±s)Table 2 Comparison of Lysholm and VAS score among the three groups (score,±s)
表2 各組治療前后Lysholm和VAS評分比較 (分,±s)Table 2 Comparison of Lysholm and VAS score among the three groups (score,±s)
注:1)A組與C組比較,差異有統(tǒng)計(jì)學(xué)意義(P <0.05);2)B組與C組比較,差異有統(tǒng)計(jì)學(xué)意義(P <0.05)
t值 P值治療前 末次隨訪 治療前 末次隨訪A組(n =25) 55.16±5.09 90.16±2.801) 30.15 0.001 2.24±0.93 0.64±0.571) 7.37 0.001 B組(n =25) 54.96±5.22 90.92±2.342) 31.44 0.0 01 2.12±1.01 0.68±0.632) 6.04 0.001 C組(n =25) 54.16±4.88 87.20±3.07 28.66 0.001 2.24±1.05 1.08±0.57 4.85 0.001 F值 0.273 12.75 0.12 4.26 P值 0.792 0.001 0.887 0.018 Lysholm評分VAS評分組別t值 P值
表3 各組治療前后不良事件發(fā)生例數(shù)比較 例Table 3 Comparison of adverse event rate among the three groups n
圖6 半月板損傷修復(fù)術(shù)后Fig.6 Repaired meniscus injury
半月板損傷后增加關(guān)節(jié)內(nèi)接觸壓力,導(dǎo)致關(guān)節(jié)功能障礙[19]。近年來,隨著基礎(chǔ)醫(yī)學(xué)的深入,通過再生醫(yī)學(xué)修復(fù)損傷的半月板得到了關(guān)注[20-21],并認(rèn)為最佳的修復(fù)方法通過下列途徑:①細(xì)胞通過黏附于機(jī)械支架并形成新的組織形態(tài);②選擇合適的種子細(xì)胞進(jìn)行誘導(dǎo)分化,來促進(jìn)組織的愈合及新基質(zhì)的合成;③添加某種生長因子或?qū)ふ夷艽碳ぜ?xì)胞分化及基因表達(dá)的信號[22-24]。
目前,研究者們希望使用MSCs治療半月板白區(qū)損傷。HORIE等[25]研究發(fā)現(xiàn)將滑膜干細(xì)胞移植黏附于半月板白區(qū)受損部位,最終可分化成類似于天然半月板成纖維軟骨的細(xì)胞;HATSUSHIKA等[26]通過組織學(xué)和MRI檢查發(fā)現(xiàn)滑膜干細(xì)胞使半月板缺損處滑膜早期填充,可有效保留更多的半月板;DUYGULU等[27]通過電鏡觀察到自體BMSCs可使半月板缺損處新生血管、纖維軟骨細(xì)胞及軟骨斑塊增多。因此,MSC被認(rèn)為是修復(fù)半月板損傷的最佳種子細(xì)胞[28]。
臨床中常用微骨折術(shù)修復(fù)關(guān)節(jié)軟骨損傷,其機(jī)制是利用從骨髓腔流出的BMSC填充于缺損處,利用其多分化功能,最終可形成纖維軟骨[29]。由此,本研究設(shè)想其是否同樣可以利用來修復(fù)半月板損傷。相比常規(guī)在軟骨上的微骨折術(shù),選擇髁間窩處微骨折,在釋放干細(xì)胞的基礎(chǔ)上,可避免造成新的軟骨損傷、能減少疼痛的發(fā)生率及康復(fù)難度?;A(chǔ)實(shí)驗(yàn)可證實(shí)術(shù)中釋放的骨髓液中含有BMSC;臨床隨訪可見Lysholm評分及VAS評分較對照組改善。因此,髁間窩微骨折術(shù)從理論、基礎(chǔ)和臨床應(yīng)用中證實(shí)其對半月板損傷修復(fù)有良好的效果,這與HOWARTH等研究是相同的[30]。
半月板損傷后,髕下脂肪墊常發(fā)生水腫,導(dǎo)致膝前區(qū)疼痛。在術(shù)中,脂肪墊需修整,減少其病理炎癥的發(fā)展。研究表明:髕下脂肪墊靠近滑膜面,所處環(huán)境與關(guān)節(jié)腔類似,具有同源性[31],其干細(xì)胞為脂肪來源,具有多分化能力[32-33]。由此,本研究探索性地提出了髕下脂肪墊粉碎術(shù),在緩解病理炎癥的基礎(chǔ)上,釋放脂肪干細(xì)胞至關(guān)節(jié)內(nèi)?;A(chǔ)實(shí)驗(yàn)表明粉碎后的脂肪墊懸液中含有IPFPSC。臨床隨訪提示該技術(shù)的術(shù)后評分優(yōu)于對照組,與髁間窩微骨折術(shù)的效果相當(dāng),這與NISHIMUTA及HINDLE等研究較為相似[32-34]。然而,本研究中發(fā)現(xiàn),髕下脂肪墊粉碎術(shù)的早期不良事件發(fā)生率較高,遠(yuǎn)期則無明顯差異,可能與脂肪墊的出血及無菌炎癥反應(yīng)增加有關(guān)[35]。綜上所述,關(guān)節(jié)下髁間窩微骨折術(shù)與髕下脂肪墊粉碎術(shù)能在一定程度上促進(jìn)半月板損傷的修復(fù),并具有獨(dú)特的優(yōu)勢。至于其具體的詳細(xì)作用機(jī)制及修復(fù)的遠(yuǎn)期效果,還有待進(jìn)一步研究。
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