摘要 ?合成(cheng)材(cai)料(liao)組(zu)織(zhi)工程骨(gu)移(yi)植(zhi)(zhi)物是當前骨(gu)移(yi)植(zhi)(zhi)材(cai)料(liao)研發(fa)(fa)(fa)重(zhong)點(dian)，發(fa)(fa)(fa)展迅速，但是，目(mu)(mu)前研發(fa)(fa)(fa)的各類(lei)支(zhi)架(jia)材(cai)料(liao)性(xing)能存在(zai)缺陷(xian)，還不(bu)能完全(quan)滿足臨(lin)(lin)床要(yao)求；細胞(bao)體(ti)外篩選(xuan)、擴增(zeng)、分(fen)化(hua)和(he)為能在(zai)局部持續釋放細胞(bao)因子而采用的轉基因細胞(bao)移(yi)植(zhi)(zhi)或腺病毒轉染局部細胞(bao)等(deng)(deng)技術(shu)還存在(zai)安全(quan)隱患，回(hui)植(zhi)(zhi)體(ti)內后不(bu)能保(bao)證細胞(bao)活(huo)性(xing)，細胞(bao)骨(gu)組(zu)織(zhi)工程種植(zhi)(zhi)體(ti)構建質量、及(ji)時性(xing)，儲存、運(yun)輸等(deng)(deng)方面還不(bu)具(ju)臨(lin)(lin)床規(gui)模(mo)化(hua)應用的可行性(xing)。目(mu)(mu)前，無(wu)細胞(bao)人工合成(cheng)材(cai)料(liao)在(zai)臨(lin)(lin)床上脊柱融合、良(liang)性(xing)骨(gu)腫瘤(liu)刮除(chu)后填充等(deng)(deng)方面應用較(jiao)普遍，細胞(bao)骨(gu)組(zu)織(zhi)工程移(yi)植(zhi)(zhi)物應用僅見(jian)于(yu)散在(zai)病例報道。

關鍵詞 ?骨組織工程；支架；干細胞；細胞因子；

臨床(chuang)上(shang)嚴(yan)重(zhong)創(chuang)傷、腫瘤切(qie)除、骨(gu)(gu)(gu)感染、骨(gu)(gu)(gu)壞死等(deng)原因(yin)導致的(de)(de)(de)(de)的(de)(de)(de)(de)骨(gu)(gu)(gu)缺(que)損、骨(gu)(gu)(gu)折(zhe)不愈合(he)以及人工(gong)假體(ti)(ti)置換及翻(fan)修、脊柱(zhu)、關節融合(he)等(deng)十分常見(jian)，自體(ti)(ti)皮質(zhi)-松質(zhi)骨(gu)(gu)(gu)移(yi)植(zhi)仍是(shi)無法被替代的(de)(de)(de)(de)“金(jin)標準”，但(dan)是(shi)，存在諸(zhu)如供區新的(de)(de)(de)(de)創(chuang)傷、感染、疼痛等(deng)并發癥，特別是(shi)，來源有(you)限(xian)不能(neng)滿(man)(man)足臨床(chuang)需要，因(yin)此，同種異(yi)體(ti)(ti)骨(gu)(gu)(gu)以較(jiao)好的(de)(de)(de)(de)骨(gu)(gu)(gu)傳(chuan)導、誘導性(xing)，成為(wei)(wei)繼自體(ti)(ti)骨(gu)(gu)(gu)移(yi)植(zhi)之后的(de)(de)(de)(de)首選[1]，但(dan)是(shi)，來源仍然有(you)限(xian)，其他缺(que)陷如：新鮮(xian)異(yi)體(ti)(ti)移(yi)植(zhi)存在疾病(bing)傳(chuan)播、免疫排斥和(he)難以保存等(deng)問題；深低(di)溫或(huo)凍干(gan)處理(li)可降(jiang)低(di)免疫原性(xing)和(he)疾病(bing)傳(chuan)染，但(dan)成骨(gu)(gu)(gu)誘導性(xing)能(neng)降(jiang)低(di)；異(yi)體(ti)(ti)脫(tuo)鈣骨(gu)(gu)(gu)（demineralized bone matrix ，DBM)的(de)(de)(de)(de)骨(gu)(gu)(gu)誘導性(xing)也較(jiao)差，并受加工(gong)方法和(he)供體(ti)(ti)年(nian)齡等(deng)因(yin)素的(de)(de)(de)(de)影響等(deng)。在自體(ti)(ti)和(he)異(yi)體(ti)(ti)骨(gu)(gu)(gu)越來越不能(neng)滿(man)(man)足臨床(chuang)需要的(de)(de)(de)(de)的(de)(de)(de)(de)情(qing)況下，組織工(gong)程化骨(gu)(gu)(gu)人工(gong)合(he)成材料的(de)(de)(de)(de)研發與(yu)應用成為(wei)(wei)重(zhong)點。

1. 組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程化(hua)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)修(xiu)(xiu)復(fu)材料的(de)(de)(de)(de)臨床(chuang)(chuang)應用現(xian)狀 ?器官的(de)(de)(de)(de)組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程的(de)(de)(de)(de)一般技術(shu)路線(xian)是(shi)在(zai)體(ti)(ti)(ti)外(wai)(wai)(wai)用支(zhi)(zhi)架、種(zhong)(zhong)(zhong)(zhong)子細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)、細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)活(huo)(huo)性(xing)因子有(you)(you)機結合(he)(he)(he)，構(gou)建(jian)有(you)(you)生命(ming)的(de)(de)(de)(de)種(zhong)(zhong)(zhong)(zhong)植(zhi)(zhi)(zhi)(zhi)體(ti)(ti)(ti)，植(zhi)(zhi)(zhi)(zhi)入缺(que)損(sun)(sun)部位(wei)(wei)(wei)，以替(ti)代(dai)病損(sun)(sun)組(zu)(zu)(zu)(zu)(zu)織(zhi)器官，恢復(fu)正(zheng)常(chang)的(de)(de)(de)(de)生理(li)結構(gou)和(he)(he)功(gong)(gong)(gong)能。骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)組(zu)(zu)(zu)(zu)(zu)織(zhi)結構(gou)、生化(hua)代(dai)謝和(he)(he)生理(li)功(gong)(gong)(gong)能相對較(jiao)簡(jian)單，與(yu)軟(ruan)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)、肌(ji)腱、神(shen)經等(deng)(deng)(deng)(deng)組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程再(zai)造(zao)相比，骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程又具(ju)有(you)(you)一定的(de)(de)(de)(de)特殊(shu)性(xing)，其種(zhong)(zhong)(zhong)(zhong)植(zhi)(zhi)(zhi)(zhi)體(ti)(ti)(ti)的(de)(de)(de)(de)構(gou)建(jian)除(chu)經典的(de)(de)(de)(de)支(zhi)(zhi)架+細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)+細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)活(huo)(huo)性(xing)因子模(mo)式外(wai)(wai)(wai)，還(huan)有(you)(you)支(zhi)(zhi)架+細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)和(he)(he)支(zhi)(zhi)架+細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)活(huo)(huo)性(xing)因子等(deng)(deng)(deng)(deng)。從(cong)1965年Urist發現(xian)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)形態(tai)發生蛋白質(zhi)(zhi)（Bone Morphogenetic Protein,BMP），40年來(lai)研(yan)究者著力構(gou)建(jian)了承載(zai)BMP的(de)(de)(de)(de)緩釋系統（Drug Delivery ? 作者單位(wei)(wei)(wei)： 200025 上海交通大學(xue)(xue)醫學(xue)(xue)院瑞金(jin)醫院骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)科上海市傷(shang)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)科研(yan)究所(suo) ?基金(jin)項目(mu)： ? 上海市“登山計劃”，No.06DZ220202 System,DDS），這種(zhong)(zhong)(zhong)(zhong)支(zhi)(zhi)架+細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)活(huo)(huo)性(xing)因子植(zhi)(zhi)(zhi)(zhi)入體(ti)(ti)(ti)內后(hou)(hou)，局部持(chi)續(xu)釋放BMP，作用于(yu)隨血(xue)管和(he)(he)肉芽長(chang)入的(de)(de)(de)(de)基質(zhi)(zhi)干(gan)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)和(he)(he)血(xue)液中骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)髓干(gan)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)，誘(you)導(dao)成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)促進(jin)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)修(xiu)(xiu)復(fu)。學(xue)(xue)者將這種(zhong)(zhong)(zhong)(zhong)無(wu)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)種(zhong)(zhong)(zhong)(zhong)植(zhi)(zhi)(zhi)(zhi)體(ti)(ti)(ti)歸為廣義組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)范疇，而且后(hou)(hou)者省掉了細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)分離培養(yang)、復(fu)合(he)(he)(he)等(deng)(deng)(deng)(deng)復(fu)雜環(huan)節(jie)，不涉及細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)移植(zhi)(zhi)(zhi)(zhi)的(de)(de)(de)(de)倫理(li)學(xue)(xue)問題，生產、保存、運(yun)輸方便，更貼近臨床(chuang)(chuang)規模(mo)化(hua)應用，成(cheng)(cheng)(cheng)為骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程的(de)(de)(de)(de)重要補充。 ? ? ? 無(wu)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)人工(gong)材料普遍用于(yu)良(liang)(liang)性(xing)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)瘤(liu)刮除(chu)后(hou)(hou)填(tian)充和(he)(he)脊柱融(rong)合(he)(he)(he)等(deng)(deng)(deng)(deng) [2、3] ；細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)型組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu) 在(zai)2001年后(hou)(hou)陸(lu)續(xu)應用于(yu)臨床(chuang)(chuang)。Vacanti用羥基磷灰石(shi)支(zhi)(zhi)架（hydroxyapatite scaffolds，HAS）復(fu)合(he)(he)(he)經過體(ti)(ti)(ti)外(wai)(wai)(wai)擴(kuo)增(zeng)(zeng)的(de)(de)(de)(de)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)膜細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)后(hou)(hou)，立(li)即植(zhi)(zhi)(zhi)(zhi)入體(ti)(ti)(ti)內修(xiu)(xiu)復(fu)再(zai)造(zao)外(wai)(wai)(wai)傷(shang)性(xing)缺(que)失的(de)(de)(de)(de)拇指遠(yuan)節(jie)指骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)[4]，證(zheng)(zheng)明(ming)(ming)了此(ci)(ci)方法臨床(chuang)(chuang)有(you)(you)效性(xing)；Quarto等(deng)(deng)(deng)(deng)[5]體(ti)(ti)(ti)外(wai)(wai)(wai)培養(yang)擴(kuo)增(zeng)(zeng)自(zi)體(ti)(ti)(ti)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)髓基質(zhi)(zhi)干(gan)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)（bone mesenchymal stem cells ，BMSCs)，與(yu)HAS復(fu)合(he)(he)(he)構(gou)建(jian)細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)+支(zhi)(zhi)架移植(zhi)(zhi)(zhi)(zhi)物，立(li)刻植(zhi)(zhi)(zhi)(zhi)入4-8cm骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)干(gan)缺(que)損(sun)(sun)處，證(zheng)(zheng)明(ming)(ming)對大骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)缺(que)修(xiu)(xiu)復(fu)有(you)(you)促進(jin)作用，7年隨訪(fang)效果(guo)滿意；柴崗等(deng)(deng)(deng)(deng)[6]等(deng)(deng)(deng)(deng)用自(zi)體(ti)(ti)(ti)BMSCs復(fu)合(he)(he)(he)DBM修(xiu)(xiu)復(fu)頜(he)面部骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)缺(que)損(sun)(sun)成(cheng)(cheng)(cheng)功(gong)(gong)(gong)；楊志明(ming)(ming)等(deng)(deng)(deng)(deng)[7]用異體(ti)(ti)(ti)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)膜復(fu)合(he)(he)(he)支(zhi)(zhi)架修(xiu)(xiu)復(fu)多部位(wei)(wei)(wei)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)缺(que)損(sun)(sun)、骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)不連取得良(liang)(liang)好效果(guo)；Warnke等(deng)(deng)(deng)(deng)[8、9]利用鈦合(he)(he)(he)金(jin)網填(tian)充牛骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)松質(zhi)(zhi)顆粒，復(fu)合(he)(he)(he)自(zi)體(ti)(ti)(ti)BMSCs、BMP-7和(he)(he)Ⅰ型膠原（collagen type-Ⅰ）后(hou)(hou)立(li)刻植(zhi)(zhi)(zhi)(zhi)入背闊(kuo)肌(ji)袋中使血(xue)管化(hua)，7W后(hou)(hou)二次移植(zhi)(zhi)(zhi)(zhi)到下(xia)頜(he)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)缺(que)損(sun)(sun)處，成(cheng)(cheng)(cheng)功(gong)(gong)(gong)再(zai)造(zao)下(xia)頜(he)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)；Kitoh等(deng)(deng)(deng)(deng)[10]報(bao)道(dao)在(zai)用牽張成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)方法來(lai)治療2例(li)軟(ruan)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)發育(yu)不良(liang)(liang)、1例(li)先天性(xing)脛骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)假關節(jie)遺留下(xia)肢(zhi)不等(deng)(deng)(deng)(deng)長(chang)（3個(ge)股(gu)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)、2個(ge)脛骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)）時(shi)，在(zai)局部注射經體(ti)(ti)(ti)外(wai)(wai)(wai)擴(kuo)增(zeng)(zeng)和(he)(he)誘(you)導(dao)向成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)樣細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)分化(hua)的(de)(de)(de)(de)自(zi)體(ti)(ti)(ti)BMSCs、混入凝血(xue)酶(mei)（thrombin）、鈣離子(calcium)的(de)(de)(de)(de)富含(han)血(xue)小(xiao)板的(de)(de)(de)(de)血(xue)漿（platelet-rich plasma ，PRP) ，可加快(kuai)成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)并安全微創；Morishita等(deng)(deng)(deng)(deng)[11]將自(zi)體(ti)(ti)(ti)BMSCs體(ti)(ti)(ti)外(wai)(wai)(wai)擴(kuo)增(zeng)(zeng)、成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)誘(you)導(dao)，在(zai)2-4d分化(hua)為成(cheng)(cheng)(cheng)纖維細(xi)(xi)(xi)(xi)(xi)胞(bao)(bao)是(shi)與(yu)HA復(fu)合(he)(he)(he)，經過4W增(zeng)(zeng)殖分化(hua)，通過檢測(ce)(ce)證(zheng)(zheng)明(ming)(ming)成(cheng)(cheng)(cheng)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)構(gou)建(jian)了體(ti)(ti)(ti)外(wai)(wai)(wai)再(zai)生骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)，并用于(yu)3例(li)良(liang)(liang)性(xing)骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)腫瘤(liu)治療，術(shu)后(hou)(hou)影像(xiang)學(xue)(xue)結果(guo)顯示，骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)修(xiu)(xiu)復(fu)速度比文獻報(bao)道(dao)的(de)(de)(de)(de)單純HA明(ming)(ming)顯加快(kuai)。.此(ci)(ci)外(wai)(wai)(wai)，組(zu)(zu)(zu)(zu)(zu)織(zhi)工(gong)程技術(shu)在(zai)口腔頜(he)面外(wai)(wai)(wai)科重建(jian)腭骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)[12]牙(ya)周組(zu)(zu)(zu)(zu)(zu)織(zhi)[7] 牙(ya)槽骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)裂(lie)成(cheng)(cheng)(cheng)型 [13] ，以及上頜(he)竇底加強 [14] 、牙(ya)槽骨(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)(gu)脊重建(jian) [15] 等(deng)(deng)(deng)(deng)都有(you)(you)報(bao)道(dao)。迄今， 臨床(chuang)(chuang)應用只(zhi)是(shi)一些(xie)零(ling)星報(bao)道(dao)和(he)(he)初步觀測(ce)(ce)，對該技術(shu)臨床(chuang)(chuang)全面評價尚(shang)缺(que)乏足夠的(de)(de)(de)(de)資(zi)料[16]

2． 規模化臨床應用面臨的主要問題和對策 ?

2.1 ?目(mu)前制(zhi)約骨(gu)組(zu)(zu)織工程(cheng)臨(lin)(lin)床(chuang)規模化(hua)應(ying)用(yong)(yong)(yong)的(de)(de)(de)(de)是基(ji)(ji)(ji)(ji)礎研(yan)(yan)究(jiu)(jiu)的(de)(de)(de)(de)各個環(huan)節存(cun)(cun)在(zai)(zai)(zai)的(de)(de)(de)(de)技(ji)(ji)術(shu)瓶頸。 ? ? 首(shou)先(xian)，目(mu)前開發(fa)(fa)的(de)(de)(de)(de)單(dan)一(yi)(yi)支架材料(liao)各有(you)(you)優缺點，如(ru)(ru)天然(ran)膠(jiao)原和(he)纖維蛋(dan)白(bai)(bai)(bai)凝膠(jiao)組(zu)(zu)織相(xiang)容性(xing)(xing)(xing)(xing)較好，但(dan)機械性(xing)(xing)(xing)(xing)能(neng)差；可(ke)降解是聚乳酸(suan)等(deng)(deng)人(ren)工材料(liao)優點，但(dan)親水(shui)性(xing)(xing)(xing)(xing)及組(zu)(zu)織相(xiang)容性(xing)(xing)(xing)(xing)差；以磷酸(suan)為(wei)基(ji)(ji)(ji)(ji)礎的(de)(de)(de)(de)骨(gu)水(shui)泥、陶瓷或生(sheng)(sheng)(sheng)物(wu)活性(xing)(xing)(xing)(xing)玻璃(li)等(deng)(deng)材料(liao)的(de)(de)(de)(de)生(sheng)(sheng)(sheng)物(wu)相(xiang)容性(xing)(xing)(xing)(xing)及骨(gu)傳(chuan)導性(xing)(xing)(xing)(xing)較好，但(dan)力(li)學性(xing)(xing)(xing)(xing)能(neng)差、易碎裂而不(bu)能(neng)滿足(zu)(zu)臨(lin)(lin)床(chuang)要(yao)求(qiu)。復(fu)合(he)材料(liao)性(xing)(xing)(xing)(xing)能(neng)一(yi)(yi)定程(cheng)度(du)上(shang)可(ke)產(chan)生(sheng)(sheng)(sheng)優勢互補，但(dan)距臨(lin)(lin)床(chuang)要(yao)求(qiu)存(cun)(cun)在(zai)(zai)(zai)差距。目(mu)前臨(lin)(lin)床(chuang)應(ying)用(yong)(yong)(yong)的(de)(de)(de)(de)主(zhu)(zhu)要(yao)是羥基(ji)(ji)(ji)(ji)磷灰石。其(qi)次(ci)，細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)選(xuan)擇(ze)、培養(yang)等(deng)(deng)環(huan)節存(cun)(cun)在(zai)(zai)(zai)安(an)(an)全(quan)隱患。自(zi)體(ti)(ti)(ti)(ti)BMSCs的(de)(de)(de)(de)應(ying)用(yong)(yong)(yong)最貼近臨(lin)(lin)床(chuang)，但(dan)干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)在(zai)(zai)(zai)骨(gu)髓中(zhong)(zhong)含量極低(di)（0..003—0.015%） [17] ,必須先(xian)獲(huo)(huo) 取并在(zai)(zai)(zai)體(ti)(ti)(ti)(ti)外(wai)(wai)擴(kuo)增(zeng)(zeng)，然(ran)而，研(yan)(yan)究(jiu)(jiu)表明最大24-40次(ci)倍增(zeng)(zeng)就會進(jin)入老化(hua)或生(sheng)(sheng)(sheng)長(chang)停(ting)滯期，從而限(xian)制(zhi)了(le)臨(lin)(lin)床(chuang)應(ying)用(yong)(yong)(yong)[18]。基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)工程(cheng)方(fang)法異(yi)(yi)(yi)位表達(da)端粒(li)酶反(fan)轉錄酶，可(ke)以消除(chu)(chu)因(yin)(yin)(yin)多次(ci)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)分(fen)裂引起染(ran)(ran)色體(ti)(ti)(ti)(ti)端粒(li)的(de)(de)(de)(de)縮(suo)短，細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)可(ke)無限(xian)分(fen)裂，從而無限(xian)延長(chang)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)壽命(ming) [19] ，但(dan)出(chu)(chu)于安(an)(an)全(quan)考慮只限(xian)于實(shi) 驗室(shi)。干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)從體(ti)(ti)(ti)(ti)內(nei)(nei)到體(ti)(ti)(ti)(ti)外(wai)(wai)，細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)會出(chu)(chu)現(xian)(xian)行(xing)為(wei)變(bian)(bian)異(yi)(yi)(yi)與轉型(xing)[20]，體(ti)(ti)(ti)(ti)外(wai)(wai)擴(kuo)增(zeng)(zeng)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)輸入體(ti)(ti)(ti)(ti)內(nei)(nei)后也(ye)(ye)會異(yi)(yi)(yi)常(chang)生(sheng)(sheng)(sheng)長(chang) [21] ，一(yi)(yi)項研(yan)(yan)究(jiu)(jiu)顯示(shi)鼠(shu)類(lei)的(de)(de)(de)(de)骨(gu)髓基(ji)(ji)(ji)(ji)質干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)經過(guo)體(ti)(ti)(ti)(ti)外(wai)(wai)長(chang)期培養(yang)后導致(zhi)染(ran)(ran)色體(ti)(ti)(ti)(ti)積累 損傷(shang)(shang)，最后出(chu)(chu)現(xian)(xian)惡(e)變(bian)(bian)[22]；細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)體(ti)(ti)(ti)(ti)外(wai)(wai)培養(yang)常(chang)規在(zai)(zai)(zai)培養(yang)液中(zhong)(zhong)加胎(tai)牛(niu)(niu)血清(qing)，不(bu)可(ke)避(bi)免(mian)(mian)(mian)會傳(chuan)染(ran)(ran)疾病如(ru)(ru)瘋牛(niu)(niu)病[23]或由異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)蛋(dan)白(bai)(bai)(bai)觸發(fa)(fa)免(mian)(mian)(mian)疫(yi)(yi)(yi)反(fan)應(ying)[24]。此外(wai)(wai)，也(ye)(ye)有(you)(you)研(yan)(yan)究(jiu)(jiu)者立足(zu)(zu)自(zi)體(ti)(ti)(ti)(ti)成(cheng)體(ti)(ti)(ti)(ti)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)，利用(yong)(yong)(yong)轉基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)技(ji)(ji)術(shu)，將4種(zhong)(zhong)基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)[25]OCT4、NANOG、SOX2、LIN28或OCT3/4、SOX2、C-MYC和(he)KLF4 [26] 轉入到人(ren)的(de)(de)(de)(de)體(ti)(ti)(ti)(ti)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)中(zhong)(zhong)，得到類(lei)似于胚胎(tai)干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)的(de)(de)(de)(de)多能(neng)干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)，增(zeng)(zeng)強(qiang)增(zeng)(zeng)殖分(fen)化(hua)能(neng)力(li)， 滿足(zu)(zu)治療需要(yao)，但(dan)潛在(zai)(zai)(zai)致(zhi)瘤風險只限(xian)于實(shi)驗室(shi)。自(zi)體(ti)(ti)(ti)(ti)來(lai)源(yuan)的(de)(de)(de)(de)種(zhong)(zhong)子(zi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)不(bu)能(neng)用(yong)(yong)(yong)來(lai)儲(chu)備(bei)建(jian)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)庫(ku)，急需時方(fang)可(ke)著(zhu)手(shou)準(zhun)備(bei)，體(ti)(ti)(ti)(ti)外(wai)(wai)擴(kuo)增(zeng)(zeng)時間長(chang)，患者不(bu)得不(bu)長(chang)時間等(deng)(deng)待。同種(zhong)(zhong)異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)可(ke)作(zuo)為(wei)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)庫(ku)種(zhong)(zhong)子(zi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)預先(xian)儲(chu)備(bei)，但(dan)是，除(chu)(chu)了(le)遇到與自(zi)體(ti)(ti)(ti)(ti)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)相(xiang)同培養(yang)問題外(wai)(wai)，涉及更(geng)為(wei)復(fu)雜的(de)(de)(de)(de)免(mian)(mian)(mian)疫(yi)(yi)(yi)原性(xing)(xing)(xing)(xing)問題。研(yan)(yan)究(jiu)(jiu)發(fa)(fa)現(xian)(xian)基(ji)(ji)(ji)(ji)質干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)具有(you)(you)免(mian)(mian)(mian)疫(yi)(yi)(yi)調(diao)(diao)節作(zuo)用(yong)(yong)(yong)[27]，包括促進(jin)I型(xing)輔助T細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)向(xiang)Ⅱ型(xing)轉變(bian)(bian)、下調(diao)(diao)NK細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)釋放干(gan)(gan)(gan)擾(rao)素--γ(IFN-γ)、和(he)B細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)合(he)成(cheng)抗體(ti)(ti)(ti)(ti)，并且，為(wei)避(bi)免(mian)(mian)(mian)牛(niu)(niu)血清(qing)培養(yang)帶(dai)來(lai)風險，用(yong)(yong)(yong)人(ren)冰(bing)凍血漿 [28] 或血小板裂解液 [29] 代替(ti)牛(niu)(niu)血清(qing)，發(fa)(fa)現(xian)(xian)不(bu)僅可(ke)提高增(zeng)(zeng)值(zhi)率，而且不(bu)改(gai)變(bian)(bian) 形態學、免(mian)(mian)(mian)疫(yi)(yi)(yi)表型(xing)和(he)分(fen)化(hua)能(neng)力(li),也(ye)(ye)沒有(you)(you)基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)異(yi)(yi)(yi)常(chang)[30、31]、并且保(bao)持(chi)了(le)免(mian)(mian)(mian)疫(yi)(yi)(yi)修飾，抑制(zhi)異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)蛋(dan)白(bai)(bai)(bai)導致(zhi)淋巴細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)增(zeng)(zeng)殖[32]，對裸鼠(shu)不(bu)具致(zhi)瘤性(xing)(xing)(xing)(xing)[33]，這為(wei)用(yong)(yong)(yong)異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)基(ji)(ji)(ji)(ji)質干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)作(zuo)為(wei)種(zhong)(zhong)子(zi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)建(jian)庫(ku)提供支持(chi)；另外(wai)(wai)，研(yan)(yan)究(jiu)(jiu)發(fa)(fa)現(xian)(xian)CTLA4-Ig（cytotoxic T lymphocyte-associated antigen4-Ig）是一(yi)(yi)種(zhong)(zhong)融合(he)免(mian)(mian)(mian)疫(yi)(yi)(yi)球蛋(dan)白(bai)(bai)(bai)，可(ke)以選(xuan)擇(ze)性(xing)(xing)(xing)(xing)地(di)阻(zu)斷CD28與B7的(de)(de)(de)(de)信號傳(chuan)導通(tong)路，阻(zu)斷T細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)殺傷(shang)(shang)作(zuo)用(yong)(yong)(yong)和(he)產(chan)生(sheng)(sheng)(sheng)免(mian)(mian)(mian)疫(yi)(yi)(yi)耐受(shou)。將CTLA4-Ig基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)轉到異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)MSCs，改(gai)變(bian)(bian)移(yi)(yi)植細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)的(de)(de)(de)(de)性(xing)(xing)(xing)(xing)狀，逃避(bi)或阻(zu)止(zhi)宿主(zhu)(zhu)識別，降低(di)異(yi)(yi)(yi)體(ti)(ti)(ti)(ti)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)免(mian)(mian)(mian)疫(yi)(yi)(yi)原性(xing)(xing)(xing)(xing)，以此構建(jian)組(zu)(zu)織工程(cheng)骨(gu)成(cheng)功實(shi)現(xian)(xian)了(le)異(yi)(yi)(yi)位成(cheng)骨(gu) [34] ；除(chu)(chu)了(le)干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao) 外(wai)(wai)，有(you)(you)研(yan)(yan)究(jiu)(jiu)證(zheng)明胎(tai)兒骨(gu)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)在(zai)(zai)(zai)免(mian)(mian)(mian)疫(yi)(yi)(yi)原性(xing)(xing)(xing)(xing)和(he)成(cheng)骨(gu)性(xing)(xing)(xing)(xing)能(neng)上(shang)優于骨(gu)髓干(gan)(gan)(gan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)[35]，少量來(lai)源(yuan)就可(ke)滿足(zu)(zu)種(zhong)(zhong)子(zi)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)建(jian)庫(ku)需要(yao)；以上(shang)發(fa)(fa)現(xian)(xian)有(you)(you)待于長(chang)期觀測論證(zheng)。第(di)三，目(mu)前構建(jian)的(de)(de)(de)(de)緩釋系統對成(cheng)骨(gu)因(yin)(yin)(yin)子(zi)緩釋不(bu)能(neng)完(wan)全(quan)模擬體(ti)(ti)(ti)(ti)內(nei)(nei)過(guo)程(cheng)。常(chang)用(yong)(yong)(yong)的(de)(de)(de)(de)一(yi)(yi)些(xie)基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)技(ji)(ji)術(shu)手(shou)段如(ru)(ru)轉基(ji)(ji)(ji)(ji)因(yin)(yin)(yin)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)移(yi)(yi)植或腺病毒轉染(ran)(ran)局(ju)部細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)在(zai)(zai)(zai)局(ju)部生(sheng)(sheng)(sheng)產(chan)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)因(yin)(yin)(yin)子(zi)，有(you)(you)導致(zhi)免(mian)(mian)(mian)疫(yi)(yi)(yi)反(fan)應(ying)、傳(chuan)染(ran)(ran)疾病、細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)惡(e)變(bian)(bian)之嫌[22、23、24、35]。 。第(di)四，種(zhong)(zhong)植體(ti)(ti)(ti)(ti)在(zai)(zai)(zai)體(ti)(ti)(ti)(ti)內(nei)(nei)外(wai)(wai)構建(jian)過(guo)程(cheng)中(zhong)(zhong)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)生(sheng)(sheng)(sheng)存(cun)(cun)狀態不(bu)確定，因(yin)(yin)(yin)為(wei)，細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)生(sheng)(sheng)(sheng)存(cun)(cun)要(yao)求(qiu)穩定外(wai)(wai)環(huan)境，包括溫度(du)、PH值(zhi)和(he)營養(yang)和(he)氧氣(qi)供應(ying) [37] ，體(ti)(ti)(ti)(ti)內(nei)(nei)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)和(he)毛(mao)細(xi)(xi)(xi)(xi)血管距離(li)20-200μm，細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)通(tong)過(guo)彌散(san) 獲(huo)(huo)取營養(yang)和(he)氧氣(qi)[38]，在(zai)(zai)(zai)體(ti)(ti)(ti)(ti)外(wai)(wai)，100-200μm距離(li)也(ye)(ye)可(ke)實(shi)現(xian)(xian)[39],細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)在(zai)(zai)(zai)體(ti)(ti)(ti)(ti)外(wai)(wai)三維結構中(zhong)(zhong)，營養(yang)和(he)氧氣(qi)獲(huo)(huo)取困難，復(fu)合(he)細(xi)(xi)(xi)(xi)胞(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)(bao)的(de)(de)(de)(de)結構質量逐(zhu)漸下降[40、41、42、43]；在(zai)(zai)(zai)靜態培養(yang)條件下發(fa)(fa)現(xian)(xian)三維支架