康 寧，劉 霞，曹誼林，肖 苒

(中國(guó)醫(yī)學(xué)科學(xué)院 整形外科醫(yī)院 研究中心， 北京 100144)

研究論文

單例先天性小耳殘耳軟骨細(xì)胞體外構(gòu)建人耳廓軟骨

康 寧，劉 霞，曹誼林，肖 苒*

(中國(guó)醫(yī)學(xué)科學(xué)院 整形外科醫(yī)院 研究中心， 北京 100144)

目的探討單例先天性小耳殘耳軟骨細(xì)胞體外構(gòu)建正常人大小耳廓形態(tài)組織工程軟骨的可行性。方法分離40例小耳畸形患者殘耳軟骨細(xì)胞統(tǒng)計(jì)細(xì)胞提取率；MTT法檢測(cè)細(xì)胞增殖能力、計(jì)算擴(kuò)增效率；免疫熒光和PCR檢測(cè)不同代數(shù)細(xì)胞的軟骨表型。分別應(yīng)用3例患者各自的殘耳軟骨細(xì)胞培養(yǎng)至P3～P4代，藻酸鹽凝膠包埋接種于正常人大小耳廓形態(tài)的聚羥基乙酸/聚乳酸支架，體外成軟骨誘導(dǎo)動(dòng)態(tài)培養(yǎng)10周后行組織學(xué)染色觀察。結(jié)果耳軟骨細(xì)胞提取率為(3.90±1.27)×106/g；在增殖培養(yǎng)基中細(xì)胞增殖能力明顯提高，至P4代擴(kuò)增(328.4±50.4)倍(Plt;0.05)；P3代細(xì)胞Ⅱ型膠原、蛋白聚糖表達(dá)顯著減弱，至P4代消失，Ⅰ型膠原表達(dá)增強(qiáng)；體外培養(yǎng)10周，實(shí)驗(yàn)組形成了耳廓形態(tài)的類軟骨組織，可見(jiàn)典型軟骨陷窩，番紅O、甲苯胺藍(lán)及Ⅱ型膠原染色陽(yáng)性；對(duì)照組明顯變形，未形成軟骨結(jié)構(gòu)。結(jié)論單例先天性小耳殘耳軟骨細(xì)胞經(jīng)體外擴(kuò)增和動(dòng)態(tài)誘導(dǎo)培養(yǎng)可在體外構(gòu)建正常人大小耳廓軟骨。

先天性小耳畸形；殘耳軟骨細(xì)胞；耳廓形態(tài)軟骨；組織工程

先天性小耳畸形是整形外科涉及軟骨組織缺損的常見(jiàn)疾病，發(fā)病率約1.4/萬(wàn)。目前治療多為自體肋軟骨移植或假體植入，但造成供區(qū)損傷或存在外露、感染等風(fēng)險(xiǎn)。應(yīng)用組織工程技術(shù)構(gòu)建人耳廓軟骨是組織工程向臨床應(yīng)用轉(zhuǎn)化的重要研究方向之一。殘耳軟骨組織取材簡(jiǎn)便，不損傷正常生理結(jié)構(gòu)，是一種極具應(yīng)用價(jià)值的種子細(xì)胞來(lái)源。多項(xiàng)研究表明[1-3]，應(yīng)用殘耳軟骨細(xì)胞構(gòu)建的組織工程軟骨與生理軟骨相比組織學(xué)水平未見(jiàn)明顯差異，為采用殘耳軟骨細(xì)胞進(jìn)行外耳廓重建提供了有力依據(jù)。本實(shí)驗(yàn)初步證實(shí)應(yīng)用單例患者來(lái)源的殘耳軟骨細(xì)胞體外構(gòu)建人耳廓軟骨的可行性，以為未來(lái)的臨床應(yīng)用提供理論基礎(chǔ)與技術(shù)參數(shù)。

1 材料與方法

1.1 樣本來(lái)源

經(jīng)本院倫理委員會(huì)同意，樣本取自Ⅲ期耳廓再造術(shù)中廢棄的殘耳軟骨，患者年齡7～25歲，共40例，均已知情同意。

1.2 主要試劑和因子

堿性成纖維細(xì)胞生長(zhǎng)因子(basic fibroblast growth factor, b-FGF)(Peprotech公司)，轉(zhuǎn)化生長(zhǎng)因子β1(transforming growth factor-β1, TGF-β1)(Peprotech公司)，胰島素鐵硒傳遞蛋白(insulin-transferrin-selenium, ITS)(Sigma公司)，維生素C，地塞米松，噻唑藍(lán)[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, Sigma公司]，兔抗人Ⅱ型膠原多克隆抗體、羅丹明-羊抗兔IgG(中杉金橋公司)。

1.3 殘耳軟骨細(xì)胞的分離培養(yǎng)和細(xì)胞提取率

將剝離軟骨膜的殘耳軟骨稱重記錄。切成2 mm×2 mm×2 mm小塊以0.25%胰蛋白酶消化20 min，0.2% Ⅳ型膠原酶37 ℃搖床消化8～12 h，200目濾網(wǎng)過(guò)濾，2 000r/min離心8 min，錐蟲藍(lán)染色檢查細(xì)胞活力并計(jì)數(shù)，計(jì)算初始細(xì)胞提取率。以 1.5×104個(gè)/cm2的密度接種，分別以普通培養(yǎng)基(DMEM-LG, 10%胎牛血清, 100 U/mL青霉素，0.1 mg/mL鏈霉素)和增殖培養(yǎng)基(普通培養(yǎng)基加入10 ng/mL bFGF)培養(yǎng)，4 d傳代1次。

1.4 細(xì)胞增殖能力檢測(cè)

1.4.1 MTT比色法繪制生長(zhǎng)曲線：取普通培養(yǎng)基培養(yǎng)的P1代及增殖培養(yǎng)基培養(yǎng)的P1～P4代殘耳軟骨細(xì)胞，按細(xì)胞1 000個(gè)/孔接種96孔板，加MTT溶液(5 g/L，pH 7.4)37 ℃4 h后以DMSO溶解結(jié)晶，490 nm波長(zhǎng)處測(cè)定吸光度值。

1.4.2 細(xì)胞增殖倍數(shù)計(jì)算：在普通和增殖培養(yǎng)基培養(yǎng)下，分別于原代接種及第4代末行細(xì)胞計(jì)數(shù)。細(xì)胞增殖倍數(shù)：P=M最終/ M初始。

1.5 軟骨細(xì)胞表型檢測(cè)

取增殖培養(yǎng)基培養(yǎng)的原代、P3、P4代殘耳軟骨細(xì)胞，分別進(jìn)行番紅O、Ⅱ型膠原免疫熒光染色，及Ⅱ型膠原(Type Ⅱ collagen, COLⅡ)、蛋白聚糖(Aggrecan, ACAN)、Ⅰ型膠原 (Type Ⅰ collagen, COL Ⅰ)PCR檢測(cè)，引物如下：18S F:5′-TGAGAAA CGGCTACCACATC-3′，R:5′-TCCCAAGATCCAACT ACGAG-3′;COL Ⅱ F:5′-AGGTCACAGAGGTTATCC AG-3′，R:5′-GTCCGTCCTCTTTCACCAG-3′;ACAN F:5′-CGGCGAAGCAGTACACATC-3′，R:5′-TGGTGT GAGGACGTATGGC-3′;COL Ⅰ F:5′-TGGGATGGAG GGAGTTTAC-3′，R:5′-ATGGCTGCACGAGTCACAC-3′。

1.6 耳廓形態(tài)支架的制備

依據(jù)成人外耳廓CT掃描數(shù)據(jù)，應(yīng)用快速成型技術(shù)制作外耳廓陽(yáng)模(圖1A, B, D)，倒制硅膠陰模(圖1C)。取400 mg無(wú)紡聚羥基乙酸(Polyglycolic acid, PGA)(組織工程國(guó)家工程研究中心)均勻嵌入陰模，以陽(yáng)模壓制，滴加0.5%聚乳酸(Polylactic acid, PLA)(Sigma公司)二氯甲烷溶液塑型成寬3 cm，長(zhǎng)5.5 cm的耳廓支架(圖1E)。

1.7 旋轉(zhuǎn)式培養(yǎng)裝置的制備

在250 mL離心瓶側(cè)壁嫁接15 mL離心管，接口密封，固定于垂直旋轉(zhuǎn)攪拌儀側(cè)翼，調(diào)轉(zhuǎn)速為6r/min(圖1H)。

1.8 細(xì)胞-材料復(fù)合物的體外培養(yǎng)及組織學(xué)檢測(cè)

將0414、0.692和 0.785 g 3例小耳患者的殘耳軟骨細(xì)胞分別擴(kuò)增至P4、P3、P3代，以細(xì)胞濃度80×106個(gè)/mL重懸于1.2%藻酸鹽溶液，各自接種支架后浸入102 mmol/L的CaCl2溶液，37 ℃放置3 min，凝膠形成后(圖1F)放入旋轉(zhuǎn)培養(yǎng)裝置，加誘導(dǎo)培養(yǎng)基(含10% 胎牛血清，10 ng/mL TGF-β1,0.22 μmol/L維生素C,10-8mol/L地塞米松，1×ITS，100 U/mL青霉素，0.1 mg/mL鏈霉素)動(dòng)態(tài)培養(yǎng)，設(shè)為實(shí)驗(yàn)組。其余1例0.925 g殘耳來(lái)源細(xì)胞擴(kuò)增至P3代，不使用藻酸鹽凝膠重懸(圖1G)，直接以普通培養(yǎng)基培養(yǎng)作對(duì)照，體外培養(yǎng)10周后取材行HE、番紅O、甲苯胺藍(lán)和Ⅱ型膠原免疫組化染色。

1.9 掃描電鏡檢測(cè)

將實(shí)驗(yàn)組、對(duì)照組體外培養(yǎng)24 h的復(fù)合物以2.5%戊二醛固定、乙醇梯度脫水、冷凍干燥后行掃描電鏡檢測(cè)。

1.10 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 殘耳軟骨細(xì)胞提取率

40例殘耳軟骨組織重1～1.5 g不等，細(xì)胞提取率為(3.90±1.27)×106/g。

2.2 殘耳軟骨細(xì)胞的增殖能力和增殖倍數(shù)

增殖培養(yǎng)基中P1～P4代細(xì)胞增殖能力無(wú)明顯差別，第5天后顯著高于普通培養(yǎng)基培養(yǎng)的P1代細(xì)胞(Plt;0.05)(圖2A)；殘耳軟骨細(xì)胞在普通培養(yǎng)基中傳至P4代可擴(kuò)增(107.36±18.32)倍，增殖培養(yǎng)基中顯著增至(328.4±50.4)倍(Plt;0.05)(圖2B)。

A, B.data model of human ear shape obtained by computer assistont design; C.silicon negative model; D.resin positive model; E.adult human ear-sized and shaped PGA/PLA scaffold; F.polymerization of cell-scaffold complex embedded in alginate gel; G.complex without alginate gel set as control; H.modified rotating culture devicep

圖1細(xì)胞材料復(fù)合物的體外培養(yǎng)
Fig1Invitrocultureofcell-scaffoldcomplex

2.3 軟骨細(xì)胞表型的變化

P0代殘耳軟骨細(xì)胞呈多角形，番紅O、Ⅱ型膠原免疫熒光染色均為強(qiáng)陽(yáng)性(圖3A)，至P3代細(xì)胞呈長(zhǎng)梭形，陽(yáng)性著色明顯減弱(圖3B)，至P4代基本消失(圖3C)。COL Ⅱ、ACAN的mRNA表達(dá)情況與細(xì)胞水平一致，COL Ⅰ表達(dá)隨傳代逐漸增強(qiáng)(圖3D)。

2.4 掃描電鏡檢測(cè)結(jié)果

藻酸鹽凝膠包埋的殘耳軟骨細(xì)胞呈球狀，懸浮在支架當(dāng)中(圖4A, B)；對(duì)照組細(xì)胞外形長(zhǎng)而扁平，黏附在支架上(圖4C, D)。

2.5 復(fù)合物體外培養(yǎng)10周檢測(cè)結(jié)果

實(shí)驗(yàn)組復(fù)合物形成耳廓形態(tài)的類軟骨組織(圖5A, B)，觸之有彈性，HE染色見(jiàn)軟骨陷窩，有少量未降解的藻酸鹽凝膠(圖5C)，番紅O、甲苯胺藍(lán)與Ⅱ型膠原染色均呈陽(yáng)性(圖5C1～4)。對(duì)照組復(fù)合物變形，新生組織薄弱(圖5D)，HE染色示體外4周細(xì)胞系長(zhǎng)且不規(guī)則，無(wú)陷窩結(jié)構(gòu)，PGA尚未降解，體外10周時(shí)有部分壞死細(xì)胞(圖5E)。

3 討論

先天性小耳患者殘耳軟骨是構(gòu)建組織工程耳廓軟骨重要的種子細(xì)胞來(lái)源。但殘耳軟骨大小不一，提取的細(xì)胞量不定，若以構(gòu)建正常人大小耳廓軟骨為目的，殘耳軟骨細(xì)胞需經(jīng)歷大量體外擴(kuò)增。因此，明確殘耳軟骨細(xì)胞提取率、 增殖效率及其表型隨傳代的變化情況，是應(yīng)用自體殘耳軟骨細(xì)胞進(jìn)行臨床個(gè)體化耳廓再造的重要基礎(chǔ)。

A.growth curve of P1-P4 cells with or without b-FGF during 10 days; B.amplification folds of cells expanded until P4 with or without b-FGF;*Plt;0.05 between chondrocytes cultured with b-FGF and without b-FGF;#Plt;0.05 between P1 with b-FGF and other groups

圖2殘耳軟骨細(xì)胞的增殖能力
Fig2Proliferationofhumanremnantearchondrocytes

A.safranin O and Col Ⅱ staining of P0 chondrocytes; B.safranin O and Col Ⅱ staining of P3 chondrocytes; C.safranin O and Col Ⅱ staining of P4 chondrocytes; D.gene expressions of COLⅡ, ACAN, and COLI in P0, P3, and P4 chondrocytes

圖3軟骨細(xì)胞表型隨代數(shù)的變化
Fig3Chondrocyticphenotypealteringofremnantearchondrocyteswithpassages(×200)

A, B.SEM of experimental group after seeding 24 hrs; C, D.SEM of control group without alginate gel after seeding 24 hrs圖4 掃描電鏡觀察細(xì)胞材料復(fù)合物Fig 4 SEM pictures of cell-scaffold complex (A, C:×2 000; B, D:×5 000)

A.gross view of neo-tissue in experimental group; B.gross view of neo-tissue in experimental group from up and down; C.HE staining; C1～4.amplifications of HE staining, safranin O staining, toludine Blue staining, and immunohistochemical staining of Col Ⅱ; D.gross view of neo-tissue in control group; E.HE staining at 4 weeks (black arrow: undegraded PGA fibers) and 10 weeksinvitro

圖5體外培養(yǎng)10周大體觀及組織學(xué)
Fig5Grossviewandhistologyofneo-tissuesconstructed10weeksinvitro(C:×100;C1～4:×400;E:×200)

本實(shí)驗(yàn)從大量殘耳樣本中首次明確了殘耳軟骨組織的初始細(xì)胞提取率。bFGF可刺激間充質(zhì)干細(xì)胞與軟骨細(xì)胞的增殖和成熟[4]，本實(shí)驗(yàn)證實(shí)應(yīng)用bFGF可在短期內(nèi)獲得充足的軟骨細(xì)胞量，同時(shí)證實(shí)細(xì)胞擴(kuò)增至P3代COL Ⅱ和ACAN表達(dá)已明顯減弱，COL Ⅰ表達(dá)持續(xù)增強(qiáng)，說(shuō)明軟骨細(xì)胞已發(fā)生去分化。因此, 按取1 g殘耳軟骨計(jì)算，細(xì)胞擴(kuò)增到P3或P4代雖可滿足構(gòu)建正常人大小耳廓軟骨的細(xì)胞量，但需進(jìn)行表型誘導(dǎo)再分化才能形成軟骨組織。文獻(xiàn)表明3D培養(yǎng)可誘導(dǎo)軟骨表型再分化[5-7]。電鏡結(jié)果證實(shí)藻酸鹽凝膠包埋的軟骨細(xì)胞呈球形，而球形結(jié)構(gòu)利于軟骨表型的穩(wěn)定[8]，此外藻酸鹽凝膠也有助于維持構(gòu)建物的特定形態(tài)[9]。旋轉(zhuǎn)培養(yǎng)裝置可使構(gòu)建物在持續(xù)動(dòng)態(tài)旋轉(zhuǎn)的環(huán)境中生長(zhǎng)，不僅利于軟骨表型再分化，更便于營(yíng)養(yǎng)物質(zhì)滲透及代謝交換[10]。

探索耳廓軟骨的體外構(gòu)建是軟骨組織工程產(chǎn)業(yè)化發(fā)展的必然需求。構(gòu)建復(fù)雜形態(tài)、大體積的軟骨組織對(duì)細(xì)胞接種和營(yíng)養(yǎng)滲透提出了更高要求。本實(shí)驗(yàn)結(jié)果證實(shí)應(yīng)用單例小耳畸形患者來(lái)源的殘耳軟骨細(xì)胞，綜合藻酸鹽凝膠、生長(zhǎng)因子及旋轉(zhuǎn)培養(yǎng)條件進(jìn)行再分化誘導(dǎo)，可在體外構(gòu)建正常人大小耳廓形態(tài)的類軟骨組織。下一步尚需體內(nèi)實(shí)驗(yàn)進(jìn)一步證實(shí)其穩(wěn)定性。

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Generation of full-sized and ear-shaped cartilagewith passaged remnant ear chondrocytes from microtia individual

KANG Ning, LIU Xia, CAO Yi-lin, XIAO Ran*

(Research Center of Plastic Surgery Hospital, CAMS amp; PUMC, Beijing 100144, China)

ObjectiveTo test the feasibility ofinvitrogeneration of a full-sized and ear-shaped cartilage with remnant ear chondrocytes derived from individual microtia patient.MethodsThe initial cell yield of remnant ear chondrocytes was analyzed from 40 cases of microtia. Proliferation was tested by MTS and expansion efficiency was calculated. The chondrocytic phenotype altering after continuous passages was characterized by immunofluorence staining and PCR. P3～P4chondrocytes were seeded onto the PGA/PLA scaffold with the shape and full size of adult human ear. The complex was cultured with a redifferentiation system composed of chondrogenic factors, alginate gel, and a rotating culture device.ResultsThe initial cell yield from remnant ear tissue was(3.90±1.27)×106/g. The proliferative ability of remnant ear chondrocytes from P1～P4passages was enhanced by adding bFGF, and the amplification of the cells expanded to P4could reach around(328.4±50.4)folds. However, the COLⅡ and ACAN expressions gradually declined with passages and became negative in P4 chondrocytes whereas COLI expression showed stronger. The neo-cartilage in the experimental group maintained the ear shape well and

formed cartilaginous structure with positive staining of Safranin O, Toludine Blue, and COL Ⅱ, while the control group failed to form cartilage tissue and only showed fibrous structure.ConclusionsA full-sized and ear-shaped cartilage can be engineeredinvitroby the passaged remnant ear chondrocytes derived from individual microtia under a redifferentiation culture system.

microtia; remnant ear chondrocyte; ear-shaped cartilage; tissue engineering

2013-09-26

2013-11-01

國(guó)家自然科學(xué)基金(31300801)；北京市科技計(jì)劃項(xiàng)目(D090800046609003)

*通信作者(correspondingauthor)： xiaoran@pumc.edu.com

1001-6325(2014)05-0583-06

R 318

A