CN106834119A - 轴型血管化组织瓣培育器 - Google Patents
轴型血管化组织瓣培育器 Download PDFInfo
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Abstract
本发明涉及一种轴型血管化组织瓣培育器,应用于外科医学。所述轴型血管化组织瓣培育器,利用移出体外的在患者体内构建的含完整血管系统的血管化包膜、或者含轴心血管的血管化轴型包膜瓣,由体外人工灌流系统供养含完整血管系统的血管化包膜或轴型包膜瓣,将血管化包膜或轴型包膜瓣轴心血管的动静脉断端分别与人工灌流系统的管道连接,灌流系统将人造血液灌注至血管化包膜或包膜瓣,并将包膜瓣的代谢产物及时运走,成为组织培育器。
Description
技术领域
本发明涉及一种轴型血管化组织瓣培育器,应用于外科医学。
背景技术
自体组织瓣的移植是修复重建外科常用的治疗手段,特别是覆盖血运条件不佳的创面如放射性溃疡、肌腱或骨质裸露区,或进行器官再造。然而,该方法会造成机体供瓣区损伤,而且转移的轴型皮瓣外观臃肿,有碍美观。近年来,一些研究用机体肌肉、骨膜、肉膜等天然血管载体作为“组织培育器”,植入物在其内孵化成血管化组织,其微血管网与“组织培育器”的天然供养血管相连通,即所孵化组织与“组织培育器”共同构成轴型组织瓣,可进行岛状或游离移植以修复组织器官缺损。相关研究进展总结如下:
以轴型肌肉瓣为“组织培育器”
2013年,Hidekazu等[1]重塑SD大鼠股动静脉周围的血管网,1周后将股动静脉连同其周围的肌肉一起切下,铺设于体外组织培养器内,形成血管床,股动静脉分别与模拟心脏的人工灌流系统连接,利用人工血液对股部肌肉血管床进行营养灌注,并及时运走其代谢产物;同时将温度敏感培养皿内收获的3张鼠共培养心肌细胞片叠加,放置于体外有血液灌流的肌肉血管床上,3天后,细胞片内即有微血管网形成,随后添加3张鼠共培养心肌细胞片至已完成血管化的细胞片上,添加的细胞片也被血管化,然后将体外的股部肌肉瓣连同其上的心肌细胞片组织游离移植至鼠心肌缺损区,心肌细胞片组织完全成活,并能功能性搏动。2001年,Terheyden 等[2]用小型猪的背阔肌包裹重组人骨形态发生蛋白(rhBMP-7)及异体骨基质材料(BioOss),6周后,植入物在背阔肌内形成血管化骨块;2004年,他们使用同样的方法,在患者背阔肌内构建出血管化骨块,并以背阔肌的天然供养血管-胸背动静脉为血管蒂,将背阔肌连同其内的血管化骨瓣游离移植至患者下颌角骨缺损区,成功修复下颌骨缺损[3]。
以轴型骨膜瓣为“组织培育器”
2015年,Alexander M 等[4]用聚甲基丙烯酸甲酯制成一面敞开、五面关闭的长方体盒子,敞开面对着绵羊肋骨膜,盒壁和肋骨膜围成一个密室(isolation chamber),室内充填磷酸三钙-羟基磷灰石支架(β-TCP/HA),骨膜内骨祖细胞和成骨细胞在β-TCP/HA 的骨诱导作用下,不断增殖分化形成新骨,同时骨膜内丰富的血管丛通过出芽生长,长入新生骨内,9周后,密室内的β-TCP/HA 转变成血管化骨组织,然后以肋骨膜的天然供养血管-肋间动静脉为血管带,将肋骨膜连同其内的血管化骨块游离移植至绵阳下颌角骨缺损部位,成功修复下颌骨缺损。
以轴型肉膜瓣为“组织培育器”
2008年,Oliver Scheufler等[5]用兔的背部肉膜瓣包裹已接种骨髓间充质干细胞(BMSC)的羟基磷灰石支架,也成功获得了可带蒂移植的血管化骨瓣。
参考文献:
1.Sekine H, Shimizu T, Sakaguchi K, et al. In vitro fabrication offunctional three-dimensional tissues with perfusable blood vessels. NatCommun. 2013; 1399(4):1-10.
2.Hendrik Terheyden, Patrick Warnke, Anton Dunsche. Mandibularreconstruction with prefabricated vascularized bone grafts using recombinanthuman osteogenic protein-1: an experimental study in miniature pigs. Part II:Transplantation. Int. J. Oral Maxillofac. Surg. 2001; 30: 373-379.
3.Warnke PH, Springer IN, Wiltfang J, Growth and transplantation of acustom vascularised bone graft in a man. Lancet. 2004; 364(9436):766-70.
4.Alexander M. Tatara, James D. Kretlow ,Patrick P. Spicer. et al.Autologously Generated Tissue-Engineered Bone Flaps for Reconstruction ofLarge Mandibular Defects in an Ovine Model. Tissue Eng. Part A. 2015;21(10):1520-1528.
5.Oliver Scheufler a, Dirk J. Schaefer a, Claude Jaquiery, et al. Spatialand temporal patterns of bone formation in ectopically pre-fabricated,autologous cell-based engineered bone flaps in rabbits. J. Cell. Mol. Med.2008; 12( 4): 1238-1249.
已见报道的“组织培育器”为构建轴型骨瓣或心肌瓣提出了很好的方案,但缺点也很明显:首先,作为“组织培育器”的股部肌肉、背阔肌、骨膜或肉膜均为机体固有重要结构,牺牲这些结构同样会对供区造成严重损伤;其次,肌肉、骨膜等随所培育组织移植至受区后,给受区带来额外的不必要的组织成分,影响治疗效果。
发明内容
本发明目的在于:提供一种轴型血管化组织瓣培育器,在不牺牲机体重要结构的前提下,构建新型“组织培育器”。
本发明的再一目的在于:提供所述轴型血管化组织瓣培育器的应用,用以培育可带血管蒂移植的血管化组织瓣或目标器官。
本发明目的通过下述方案实现:一种轴型血管化组织瓣培育器,利用移出体外的在患者体内构建的含完整血管系统的血管化包膜、或者含轴心血管的血管化轴型包膜瓣,该培育器由血管床和体外人工灌流系统构成,由体外人工灌流系统供养含完整血管系统的血管化包膜或轴型包膜瓣,该血管化包膜或轴型包膜瓣平铺在血管床上,将血管化包膜或轴型包膜瓣轴心血管的动静脉断端分别与人工灌流系统的管道连接,灌流系统将人造血液灌注至血管化包膜或包膜瓣,并将包膜瓣的代谢产物及时运走,成为组织培育器。
本发明中含完整血管系统的血管化包膜、或者含轴心血管的血管化轴型包膜瓣为扩张刺激形成的扩张包膜。
在上述方案基础上,所述的体外人工灌流系统包括:由管道连接的置换液容器、血浆泵、血液分离器和废液收集容器,轴型包膜瓣平铺在包膜血管床上,轴型包膜瓣的轴心血管与体外人工灌流系统的管路连通,置换液经血浆泵泵入血液分离器的血浆槽内,血浆槽内的合格血浆进入轴型包膜瓣,循环出的血浆泵入血液分离器进行分离,将包膜瓣的代谢产物排出至废液收集容器内,分离出的合格血浆调节压力、pH值后与置换液在血浆槽内混合后重新进入轴型包膜瓣,形成血浆的体外人工灌流系统。该在血管床上的包膜瓣经体外血液循环得到可带蒂移植的轴型组织瓣。
所述轴心血管可以是人工血管或组织工程血管等。
进一步的,所述的血管床置于组织培养箱内,组织培养箱上有置换液入口和代谢产物排出口,在置换液入口处设有与压力控制器连接的压力传感器,由压力控制器控制空气与CO2混合气体与置换液的混合比和pH值,并控制置换液输送压力和流量,数据被传送到显示器上。
本发明提供所述轴型血管化组织瓣培育器在培育目标组织或器官方面的应用。
具体的,活检患者拟修复组织,原代培养及扩增目标细胞,抽取外周血,分离培养内皮祖细胞,在温敏培养皿内形成共培养各种肌细胞片、粘膜细胞片等,将共培养细胞片平铺于包膜血管床上,进行组织器官的培育,获得可带蒂移植的轴型组织瓣。
将所述的肌细胞片或粘膜细胞片反复多次平铺于轴型包膜血管床上,形成带血管蒂的扩张包膜。
或者,所述的肌细胞片或粘膜细胞片平铺于轴型包膜血管床上,将人工假体或接种成骨细胞、成软骨细胞的可降解支架包裹于包膜瓣内,培育带血管蒂的目标器官。
其中,所述的接种成骨细胞、成软骨细胞的可降解支架是将培养的成骨细胞或成软骨细胞接种至可降解多孔支架材料上,单纯假体或接种细胞的支架包裹于包膜内,进行组织器官的培育,获得可带蒂移植的目标器官。
本发明的优越性在于:
(1)包膜内轴心血管不是机体的主干血管,它可以是人工血管或组织工程血管,避免造成血管供区的血供障碍;
(2)包膜的主要组织成分为胶原纤维和毛细血管,不含肌肉、骨、软骨等功能组织,包膜培育的组织器官转移至修复区后,不会为修复区带来额外的功能性组织。而且,失去扩张刺激一年左右,包膜会自发降解消失,最终修复区仅含所培育的目标组织或器官;
(3)扩张刺激形成的扩张包膜为机体新生物,而非机体固有组织结构,利用扩张包膜不会对机体造成损伤;
(4)扩张包膜厚度菲薄,不会造成修复区外观臃肿,而且包膜质地柔软,容易塑形,可培育特定形状的血管化骨或软骨结构,省去术中塑形骨块或软骨块的操作,故简化手术。
附图说明
图1,轴型血管化组织瓣培育器示意图,包括体外人工灌流系统和组织培养箱;
图2,在患者体内构建轴型血管化包膜瓣 A:将皮下的轴心血管从周围组织中分离出来;B:贴着轴心血管埋置皮肤扩张器,并注水扩张数周;C:掀起含轴心血管的包膜瓣,并移出体外;
图3,免疫缺陷动物心血管系统供养轴型包膜瓣; 轴型血管化组织瓣
图4,体外人工灌流系统供养轴型包膜瓣;
图5:肌细胞片或粘膜细胞片等反复多次平铺于轴型包膜血管床上,人工假体或接种成骨细胞、成软骨细胞的可降解支架包裹于包膜瓣内,培育带血管蒂的组织瓣;
图中标号说明:
实施例1中:
1——血管床;
21——置换液容器;22——废液收集容器;
3——轴型包膜瓣;
4——组织培养箱;
5——压力控制器;
51——压力传感器;52——pH值传感器;53——流量传感器;
6——CO2源
7——空气源
8——泵;
9——显示器;
实施例2中:
1’——血管床;
2’——人工灌流系统;
21’——置换液容器;22’——废液收集容器;
23——血液分离器;
24、25、26——血浆泵一、二、三;
图5中:
1’’——扩张器包膜的血管床;
31——包膜的轴心血管;
32——粘膜细胞片;
33——肌细胞片;
34——人工材料。
具体实施方式
实施例1
如图1所示,本发明一种轴型血管化组织瓣培育器,利用移出体外的在患者体内构建的含完整血管系统的血管化包膜、或者含轴心血管的血管化轴型包膜瓣,由血管床1和体外人工灌流系统构成,由体外人工灌流系统2供养含完整血管系统的血管化包膜或轴型包膜瓣3,该血管化包膜或轴型包膜瓣3平铺在血管床1上,将血管化包膜或轴型包膜瓣3轴心血管的动静脉断端分别与人工灌流系统的管道连接,人工灌流系统将人造血液灌注至血管化包膜或包膜瓣,并将包膜瓣的代谢产物及时运走,成为组织培育器。人工灌流系统系统可选用如图1所示的现有设备。
如图1所示,所述的血管床1置于组织培养箱4内,组织培养箱4上有置换液入口和代谢产物排出口,在置换液入口处设有与压力控制器5连接的压力传感器51,压力控制器5还设有与置换液容器21连接的pH值传感器52、与泵连接的流量传感器53,由压力控制器5控制CO2源6和7空气源中空气与CO2混合气体与置换液的混合比和pH值,并控制置换液输送压力和流量,用泵8输送置换液时,数据被传送到显示器9上。
实施例2
如图4体外人工灌流系统供养轴型包膜瓣所示,体外人工灌流系统包括:由管道连接的置换液容器21’、废液收集容器22’、血浆泵和血液分离器23,轴型包膜瓣3’平铺在包膜血管床1’上,轴型包膜瓣3’的轴心血管的动静脉断端分别与人工灌流系统的管道连通,置换液经血浆泵一24泵入血液分离器23的血浆槽231内,血浆槽231内的合格血浆进入轴型包膜瓣3’,循环出的血浆被血浆泵二25泵入血液分离器23进行分离,将轴型包膜瓣3’的代谢产物经血浆泵三26泵入至废液收集容器21’内,分离出的合格血浆调节压力PV、pH值后与置换液容器21’内的置换液在血浆槽221内混合后重新灌入轴型包膜瓣3’,形成血浆的体外人工灌流系统。
含完整血管系统的血管化包膜还可以通过其他方法获得。
本发明所述的轴型血管化组织瓣培育器在培育目标组织或器官方面的应用。
应用例
本发明在培养目标组织中的应用
(1)体内构建含完整血管系统的血管化包膜
贴着体内可进行显微吻合的血管(非主干血管,可以是天然知名血管或组织工程血管)埋置皮肤扩张器,间歇性注水扩张一段时间,扩张囊表面形成含轴心血管的扩张包膜,轴心血管两旁的包膜内富含新生小血管(该区域包膜称之为血管化包膜),本申请人的前期研究已证实,血管化包膜内新生小血管与轴心血管相连通,即轴心血管与其两旁的血管化包膜构成完整的轴型血管载体。
于患者皮下埋置皮肤扩张器,构建血管化包膜:
需要进行组织修复或器官再造的患者,可将其旋髂浅动静脉束(SCI)的起始段从腹股沟脂肪垫中分离出来,贴着该血管束埋置皮肤软组织扩张器,缝合皮肤。术后每周1-2次向扩张囊内注入生理盐水,扩张4-6周,扩张囊表面自发形成一薄层包膜,SCI的起始段走行于包膜内,且该段血管束的两旁包膜内富含新生小血管, SCI起始段与其两旁的血管化包膜构成有完整血供系统的新型血管载体。
如图2,在患者体内构建轴型血管化包膜瓣,如图2A将皮下的轴心血管从周围组织中分离出来;图2B贴着轴心血管埋置皮肤扩张器,并注水扩张数周;图2C:掀起含轴心血管的包膜瓣,并移出体外。
(2)制备轴型血管化包膜瓣
于血管化包膜边缘作两个平行于包膜内轴心血管的切口,经钝性分离,将血管化包膜与周围组织分开,包膜内轴心血管的近、远端分别予结扎及切断,于轴心血管的两断端分别作垂直于轴心血管的包膜切口,形成游离的矩形包膜瓣,轴心血管位于包膜瓣的中轴线。将游离的包膜瓣连同其轴心血管移出体外备用。
掀起SCI两旁的包膜,制备游离的轴型包膜瓣:
注水扩张4-6周后,沿原切口切开皮肤,取出扩张器,于血管化包膜边缘作两个平行于包膜内SCI的切口,经钝性分离,将血管化包膜与周围组织分开,包膜内SCI位于包膜的中轴线,作两个垂直于包膜内SCI的包膜切口并切断SCI,形成游离的矩形包膜瓣。将游离的含轴心血管的包膜瓣移出体外,皮肤切口间断缝合。
(3)为轴型包膜瓣提供血液灌流,形成新型“组织培育器”
方案:i.由免疫缺陷动物心血管系统提供血液灌流;
麻醉状态下,于免疫缺陷动物的腹股沟皮下分离一腔隙,显露股动静脉,将已制备的轴型包膜瓣移入该腔隙内,包膜瓣轴心血管的动静脉分别与免疫缺陷动物的股动静脉显微吻合,免疫缺陷动物的动脉血经股动脉输送给血管化包膜瓣,血管化包膜瓣代谢产生的静脉血由股静脉运走,包膜瓣与周围组织之间用半透膜隔开,以免周围组织长入包膜内。完成血管吻合的包膜瓣也可置入固定于动物体表的特制培养室内。位于免疫缺陷动物皮下或体表的包膜瓣得以供养而成为新型“组织培育器”。
如图3所示免疫缺陷动物心血管系统供养轴型包膜瓣。由裸鼠心血管系统为轴型包膜瓣提供血液灌流,构建新型“组织培育器”:
麻醉状态下,于裸鼠的腹股沟区皮下分离一腔隙,显露股动静脉,将已制备的轴型包膜瓣移入该腔隙内,包膜瓣内的SCI断端分别与裸鼠的股动静脉显微吻合,包膜瓣与周围组织之间用半透膜隔开,包膜瓣得以血液供养而成为新型“组织培育器”。
ii.由体外人工灌流系统提供血液灌流
如图1,可参照国外现有技术构建体外人工灌流系统[1](如图1所示),已制备的血管化包膜瓣移至体外培养室内,其轴心血管的动静脉断端分别与人工灌流系统的管道连接,灌流系统将人造血液灌注至包膜瓣,并将包膜瓣的代谢产物及时运走。由人造血液灌注的轴型包膜瓣也可成为新型“组织培育器”。
图4所示,由体外人工灌流系统为轴型包膜瓣提供血液灌流,构建新型“组织培育器”:
构建体外人工灌流系统,患者体内的血管化包膜瓣移至体外培养室内,其轴心血管SCI的动静脉分别与人工灌流系统的管道连接,灌流系统将人造血液灌注至包膜瓣,并将包膜瓣的代谢产物及时运走。由人造血液灌注的轴型包膜瓣也可作为新型“组织培育器”。
(4)“组织培育器”培育目标组织或器官
活检患者拟修复组织,原代培养及扩增目标细胞,抽取外周血,分离培养内皮祖细胞,在温敏培养皿内形成共培养肌细胞片、粘膜细胞片等,将培养的成骨细胞或成软骨细胞接种至多孔可降解支架材料上。将共培养细胞片反复多次平铺于包膜血管床上,每次间隔1-2天;将预制的单纯假体或接种成骨细胞或成软骨细胞的支架包裹于包膜内,进行组织器官的培育,收获可带蒂移植的轴型组织瓣或器官。
如图5所示,粘膜细胞片32、肌细胞片33、各种人工材料多次平铺于扩张器包膜的血管床1’’上,自包膜的轴心血管31提供血液灌流,人工假体或接种成骨细胞、成软骨细胞的可降解支架包裹于包膜瓣内,培育带血管蒂的组织瓣。
Claims (9)
1.一种轴型血管化组织瓣培育器,利用移出体外的在患者体内构建的含完整血管系统的血管化包膜、或者含轴心血管的血管化轴型包膜瓣,其特征在于:由血管床和体外人工灌流系统构成,由体外人工灌流系统供养含完整血管系统的血管化包膜或轴型包膜瓣,该血管化包膜或轴型包膜瓣平铺在血管床上,将血管化包膜或轴型包膜瓣轴心血管的动静脉断端分别与人工灌流系统的管道连接,灌流系统将人造血液灌注至血管化包膜或包膜瓣,并将包膜瓣的代谢产物及时运走,成为组织培育器。
2.根据要求3所述的轴型血管化组织瓣培育器,其特征在于,所述的体外人工灌流系统包括:由管道连接的置换液容器、血浆泵、血液分离器和废液收集容器,轴型包膜瓣平铺在包膜血管床上,轴型包膜瓣的轴心血管的动静脉断端分别与人工灌流系统的管道连通,置换液经血浆泵泵入血液分离器的血浆槽内,血浆槽内的合格血浆进入轴型包膜瓣,循环出的血浆泵入血液分离器进行分离,将包膜瓣的代谢产物排出至废液收集容器内,分离出的合格血浆调节压力、pH值后与置换液在血浆槽内混合后重新进入轴型包膜瓣,形成血浆的体外人工灌流系统。
3.根据要求1或2所述的轴型血管化组织瓣培育器,其特征在于:所述轴心血管是人工血管或组织工程血管。
4.根据要求1或2所述的轴型血管化组织瓣培育器,其特征在于,所述的血管床置于组织培养箱内,组织培养箱上有置换液入口和代谢产物排出口,在置换液入口处设有与压力控制器连接的压力传感器,由压力控制器控制空气与CO2混合气体与置换液的混合比和pH值,并控制置换液输送压力和流量,数据被传送到显示器上。
5.一种根据权利要求1至4之任一项所述的轴型血管化组织瓣培育器在培育目标组织或器官方面的应用。
6.一种根据要求1至4之任一项所述的轴型血管化组织瓣培育器的应用,特征在于:活检患者拟修复组织,原代培养及扩增目标细胞,抽取外周血,分离培养内皮祖细胞,在温敏培养皿内形成共培养各种肌细胞片、粘膜细胞片,将共培养细胞片平铺于包膜血管床上,进行组织器官的培育,获得可带蒂移植的轴型组织瓣。
7.根据要求6所述的轴型血管化组织瓣培育器的应用,其特征在于:将所述的肌细胞片或粘膜细胞片反复多次平铺于轴型包膜血管床上,形成带血管蒂的扩张包膜。
8.根据要求6所述的轴型血管化组织瓣培育器的应用,其特征在于:所述的肌细胞片或粘膜细胞片平铺于轴型包膜血管床上,将人工假体或接种成骨细胞、成软骨细胞的可降解支架包裹于包膜瓣内,培育带血管蒂的目标器官。
9.根据要求7所述的轴型血管化组织瓣培育器的应用,其特征在于:所述的接种成骨细胞、成软骨细胞的可降解支架是将培养的成骨细胞或成软骨细胞接种至可降解多孔支架材料上,单纯假体或接种细胞的支架包裹于包膜内,进行组织器官的培育,获得可带蒂移植的目标器官。
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