CN102272289A - 用于活化干细胞的方法和装置 - Google Patents
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Abstract
本文描述的本发明实施方案包括用于刺激干细胞源中的间充质干细胞分化为能够形成骨的成骨细胞的方法和装置。所述装置和方法包括以有效形成活化干细胞的方式将干细胞源(诸如骨髓抽吸物,脂肪组织和/或纯化的异体干细胞)暴露于活化剂。
Description
相关申请
本专利申请要求享有下列申请的申请日:2008年10月31日提交的美国临时专利申请系列号61/110,096,标题为″利用生长因子的体外刺激活化骨髓抽吸物以改善骨形成的装置(DEVICE FOR ACTIVATING BONEMARROW ASPIRATE USING EX VIVO STIMULATION BY GROWTHFACTORS FOR IMPROVED BONE FORMATION)″,和2009年2月13日提交的美国临时专利申请系列号61/152,335,标题为″用于形成骨髓抽吸产物的方法和装置(METHOD AND DEVICE FOR FORMING A BONE MARROWASPIRATE PRODUCT)″,其内容在此完整引入作为参考。
技术领域
本文描述的发明主题涉及用于活化干细胞的装置和方法,包括使用体外刺激活化骨髓抽吸物中的干细胞。发明主题还涉及包含这类活化干细胞的植入物。
发明背景
为了提供最多的骨形成,希望移植已经显示成骨细胞表型的细胞,因为这类细胞可能显示骨形成活性。但是,骨髓干细胞在体外分化为成骨细胞涉及在成骨培养基中的培养(Jaiswal et al.1997.J Cell Biochem 64:295-312),可能导致这类细胞体外增殖的降低。此外,成骨培养基的使用包括向细胞中添加成分(例如,生长因子),如果这些成分与细胞一起给患者施用可能具有非故意的副作用。
因此,本领域需要从干细胞(例如,人骨髓干细胞)体外产生骨祖细胞,成骨细胞或成骨细胞表型细胞的简单和可靠的方法,其中可以方便的将将所需细胞与用于产生骨祖细胞,成骨细胞或成骨细胞表型细胞的因子分离。
发明概述
本发明的一个方面是制备用于促进哺乳动物骨生长的植入组合物的方法,包括(a)将干细胞与一种或更多种活化剂接触24小时或更短时间以制备活化干细胞,(b)从活化干细胞分离活化剂以形成基本上不含活化剂的活化干细胞群,和(c)将基本上不含活化剂的活化干细胞群与骨移植替代物混合从而制备用于促进哺乳动物骨生长的植入组合物,其中至少一种活化剂促进干细胞分化为成骨细胞或成骨前体细胞。成骨细胞和/或成骨祖细胞可以是骨祖细胞,成骨细胞或成骨细胞表型细胞。在一些实施方案中,将干细胞与一种或更多种活化剂接触5分钟到1小时。在其他实施方案中,将干细胞与一种或更多种活化剂接触5分钟到0.5小时。干细胞可以,例如,获得自或分离自骨髓,脂肪组织,肌肉组织,脐血,胚胎卵黄囊,胎盘,脐带,骨膜,胎儿皮肤,青少年皮肤,或血液。干细胞可以是胚胎干细胞,出生后干细胞或成人干细胞。在一些实施方案中,干细胞是自体的(autologous),异体的(allogeneic)或异种的(xenogeneic)。干细胞可以包括间充质干细胞。这类间充质干细胞可以包括自体骨髓抽吸物。骨髓抽吸物可以在手术中吸出。获得干细胞后,可以将它们浓缩以去除不必要的液体。可选的,干细胞可以从最初获得它们的组织或液体分离。
活化剂可以,例如,调节细胞生长和/或分化,并且可以选自小分子,肽,生长因子,细胞因子,配体,激素及其组合。活化剂的实例包括活化剂诸如转化生长因子beta(TGF-β),成纤维细胞生长因子(FGF),血小板衍生生长因子(PDGF),骨形态发生蛋白(BMP),胰岛素生长因子(IGF),白介素-1(IL-1),白介素-11(IL-11),斯伐他汀(simvastatsin),地塞米松,氧甾酮,音猬因子(sonic hedgehog),干扰素,肿瘤坏死因子,神经生长因子(NGF),纤连蛋白,RGD肽,整合素,表皮生长因子(EGF),肝细胞生长因子(HGF),角质形成细胞生长因子,成骨蛋白,及其组合。在一些实施方案中,活化剂选自BMP-2,TGF-beta3,PSGF-AB,PDGF-BB,FGF-2,TGF-beta1,BMP-4,BMP-7,BMP-6,FGF-8,IL-11,斯伐他汀,地塞米松,氧甾酮,音猬因子及其组合。在其他实施方案中,活化剂包括TGF-β和FGFb,还可以包括PDGF。活化剂可以来自于自体来源。活化剂可用于采用溶液的方法。当活化剂用于采用溶液的方法时,通过包括过滤,凝胶过滤,切向流动过滤,免疫沉淀,免疫吸附,柱层析或其组合的方法从活化剂按照步骤(b)分离活化干细胞。在其他实施方案中,将活化剂连接到固相支持物。例如,通过共价粘附,吸附,非共价相互作用和/或其组合,将至少一些活化剂直接或间接连接到固相支持物。在一些实施方案中,至少一些活化剂通过肽,抗体,化学交联剂,烷撑链或其组合连接到固相支持物。
骨移植替代物可以包括材料诸如钙盐。这类钙盐可以,例如,包括一水合磷酸一钙,α-磷酸三钙,β-磷酸三钙,碳酸钙,或其组合。骨移植替代物还可以包括脱矿质骨(demineralized bone),磷酸钠盐,多聚体或其组合。这类多聚体可以是胶原,明胶,透明质酸,透明质酸盐,羟丙基纤维素(HPC),羧甲基纤维素(CMC),羟丙基甲基纤维素(HPMC),羟乙基纤维素(HEC),黄原胶(xantham gum),瓜耳豆胶(guar gum),藻酸盐或其组合。在一些实施方案中,所述方法增加干细胞中碱性磷酸酶和/或骨形态发生蛋白(BMP)受体亚单位的表达。这类方法还可以包括将植入组合物植入患者。
本发明的另一个方面是通过本文所述方法制备的植入组合物。
本发明的另一个方面是治疗受试者骨损伤、病症或疾病的方法,包括给受试者的骨损伤、病症或疾病部位施用本文所述的植入组合物。这类骨损伤、病症或疾病可以是断裂的骨,骨缺陷,骨移植物(bone transplant),骨移植(bonegraft),骨癌,关节替换(joint replacement),关节修复(joint repair),骨融合,骨小平面修复(bone facet repair),骨退化,牙齿植入,牙齿修复,关节炎,骨重建,或其组合。
本发明的另一个方面是用于活化干细胞的装置,包括固相支持物和至少一种促进干细胞分化为成骨细胞或成骨前体细胞的活化剂,其中所述装置适于:(i)将干细胞与至少一种活化剂温育,和(ii)在温育步骤(i)后将至少一种活化剂与干细胞分离。例如,至少一种活化剂选自转化生长因子beta(TGF),成纤维细胞生长因子(FGF),血小板衍生生长因子(PDGF),骨形态发生蛋白(BMP),胰岛素生长因子(IGF),白介素-1(IL-1),白介素-11(IL-11),斯伐他汀,地塞米松,氧甾酮,音猬因子,干扰素,肿瘤坏死因子,神经生长因子(NGF),纤连蛋白,RGD肽,整合素,表皮生长因子(EGF),肝细胞生长因子(HGF),角质形成细胞生长因子,成骨蛋白,及其组合。
在一些实施方案中,至少一种活化剂选自BMP-2,TGF-beta3,PSGF-AB,PDGF-BB,FGF-2,TGF-beta1,BMP-4,BMP-7,BMP-6,FGF-8,IL-11,斯伐他汀,地塞米松,氧甾酮,音猬因子及其组合。在其他实施方案中,至少一种活化剂包括TGF-β和FGFb,还可包括PDGF。至少一种活化剂可以,例如,来自于自体来源。活化剂可以位于固相支持物的溶液内或连接到固相支持物。活化剂可以,例如,通过结合至少一种活化剂的抗体或肽连接到固相支持物。
固相支持物可以包括柱基质材料,滤器,培养板,管或平皿,微量滴定板,珠子,盘,或其组合。固相支持物可以是容器。固相支持物可以包括塑料,纤维素,纤维素衍生物,磁性颗粒,硝酸纤维素,玻璃,玻璃纤维,胶乳,或其组合。固相支持物还可以包括亲和基质以去除至少一种活化剂。当滤器存在于固相支持物中时,滤器可以保留细胞和骨移植替代材料但允许至少一种活化剂穿过。可选的,滤器可以保留至少一种活化剂但允许干细胞穿过。在一些实施方案中,固相支持物不结合干细胞或不与干细胞不利地相互作用。在其他实施方案中,固相支持物可以结合干细胞但没有与干细胞不利地相互作用。
装置还可以包括用于控制干细胞与至少一种活化剂温育时间的计时器。例如,在温育步骤(i)后计时器可以触发至少一种活化剂与干细胞的分离。在一些实施方案中,具有计时器的装置控制干细胞与至少一种活化剂的温育时间为24小时或更短。在其他实施方案中,具有计时器的装置控制干细胞与至少一种活化剂的温育时间为5分钟到1小时。
本发明的另一个方面是用于骨形成的装置,包括用于处理干细胞源的第一部件;和用于以有效刺激干细胞源中间充质干细胞分化为成骨细胞的方式将干细胞源暴露于活化剂的第二部件,其中可以将成骨细胞掺入可用于骨修复和/或生成的植入组合物干细胞源可以是骨髓抽吸物,包括自体骨髓抽吸物在内,脂肪组织和/或纯化的异体干细胞。活化剂可以包括,但不限于BMP-2,TGF-beta3,PSGF-AB,PDGF-BB,FGF-2,TGF-beta1,BMP-4,BMP-7,BMP-6,FGF-8,IL-11,斯伐他汀,地塞米松,氧甾酮和/或音猬因子。活化剂可以例如通过接头直接连接到装置的固相支持物或栓系到固相支持物。在一个实施方案中,栓系选自烷撑链,肽,抗体,化学交联剂,及其组合。
在一个实施方案中,活化剂位于溶液中,并且第二部件还包括用于从间充质干细胞去除活化剂的滤器部件或亲和基质(例如,含有肽或抗体)。
一种实施方案包括用于活化骨髓抽吸物的装置。该装置包括用于将骨移植替代物和从患者抽取的骨髓抽吸物混合以形成混合物的部件。该装置还包括以有效触发间充质干细胞以增强成骨细胞表型的方式将混合物短暂暴露于固定或栓系的活化剂的部件。
另一个实施方案包括用于活化骨髓抽吸物的装置,其包括用于处理从患者抽取的骨髓抽吸物的部件。该装置还包括以有效触发间充质干细胞以增强成骨细胞表型的方式将骨髓抽吸物暴露于活化剂的部件。
本文还描述了另一活化骨髓抽吸物的装置,包括用于将骨移植替代物和从患者抽取的骨髓抽吸物混合以形成混合物的部件。该装置还包括以有效触发间充质干细胞以增强成骨细胞表型的方式将混合物暴露于固定或栓系的活化剂的部件。暴露后,从栓系或固定的活化剂分离BMA,并将其与骨移植替代物混合。
在一个实施方案中,一种方法包括将干细胞源(例如,骨髓抽吸物,包括自体骨髓抽吸物)暴露于活化剂,其中干细胞中的间充质干细胞被刺激分化为成骨细胞。在一个实施方案中,骨髓抽吸物在手术中抽取和/或使用从患者抽取的形式。可选的,在从患者抽取后可将骨髓抽吸物进一步浓缩。该方法还包括将刺激或活化的干细胞(例如,间充质干细胞)与合成的骨移植替代物混合以形成混合物,其中暴露包括以有效触发间充质干细胞增强成骨细胞表型的方式将混合物短暂暴露于固定或栓系的活化剂。
在一个实施方案中,活化剂与基质键合形成键合基质。在该实施方案中,将干细胞源与键合活化剂温育一段时间,诸如5分钟到24小时,或5分钟到1小时,或15分钟到1小时。在一个实施方案中,温育引起骨中骨形态发生蛋白(BMP)受体亚单位的上调。
在一个实施方案中,干细胞源暴露于活化剂发生在干细胞溶液中,该方法还包括从干细胞溶液去除活化剂,诸如利用亲和基质的形成和/或过滤。
在另一个实施方案中,提供方法以形成能够刺激骨形成的祖细胞。方法操作包括将骨髓抽吸物(BMA)与活化剂混合,并使用活化剂以触发间充质干细胞(MSCs)来增强成骨细胞表型。这类方法可以触发间充质干细胞来增强或发展成骨细胞表型。该方法还包括将骨移植替代物于从患者抽取的骨髓抽吸物混合以形成混合物;并以有效触发间充质干细胞增强或发展成骨细胞表型的方式将混合物短暂暴露于固定或栓系的活化剂。
在一些实施方案中,所述方法还包括给患者植入刺激的间充质干细胞,包括植入活化剂以及干细胞源。
附图说明
图1图示说明本发明实施方案中原代人MSCs表达的碱性磷酸酶水平。在用成纤维细胞生长因子(下文中称为″FGF-2″)(100ng/ml)或TGF beta3(250ng/ml)处理1小时后将细胞在分化培养基中培养。然后使用实施例1描述的测定法测量细胞表达的碱性磷酸酶水平。
图2图示说明在将细胞暴露于活化剂24小时后,原代人间充质细胞(下文中称为″MSCs″)中3类骨形态发生蛋白(下文中称为″BMP″)受体单位的相对mRNA拷贝数,即BMPR-1A(″IA″),BMPR-1B(″IB″)和BMPR-II(″II″)。使用的活化剂是浓度为1ng/ml,10ng/ml和100ng/ml的血小板衍生生长因子(下文中称为″PDGF BB″),成纤维细胞生长因子(下文中称为″FGF b″),和转化生长因子(下文中称为″TGF beta3″),暴露24小时。收集细胞,并针对实施例1描述的全部3种BMP受体单位(即-1A,-1B和II)进行定量聚合酶链式反应(下文中称为″PCR″)。
图3图示说明在将原代MSCs暴露于PDGF BB,FGF b和TGF beta3(使用1ng/ml,10ng/ml和100ng/ml的浓度)一(1)小时后,3种BMP受体单位(即-1A,-1B和II)的相对mRNA拷贝数。收集细胞,并针对实施例1描述的全部3种BMP受体单位(即-1A,-1B和II)进行定量PCR。
详细说明
在本领域的当前状态,骨髓抽吸物(下文中称为″BMA″)在手术中从患者抽取,与骨移植替代物混合并重新植入手术部位以促进骨治愈。但是,尽管骨髓抽吸物可能包含一些能够产生成骨细胞的结缔组织祖细胞,但是在实际能够促进骨形成的成骨细胞数目和类型方面患者与患者之间存在巨大差异。
本文描述了改善干细胞(例如,BMA)性能的方法,装置和植入物,通过增加细胞数目或通过增加BMA或干细胞混合物中祖细胞的成骨潜能。本文描述的实施方案包括方法和装置,简单来说,手术中在体外将干细胞暴露于一种或更多种外源活化剂,从而产生能够刺激骨生长的祖细胞,然后从干细胞(即,通过活化剂活化的祖细胞)去除活化剂。活化剂可以是,例如,小分子,肽,生长因子,细胞因子,配体或其他因子。活化剂可以从自体来源捕获,获自商品化来源或制备得到(例如,通过重组方法)。
在一些实施方案中,活化剂能够增加干细胞混合物的祖细胞中骨形态发生蛋白(下文中称为BMP)受体亚单位的表达。活化剂的这种处理有助于,例如,在骨损伤或骨手术部位增强祖细胞对内源性BMPs产生应答的能力。活化剂的这种处理还可以驱使干细胞沿着成骨细胞途径分化。如本文所述,虽然通过用细胞因子处理较长时段(例如,几天)已经活化干细胞,但是将干细胞暴露于活化剂所述较长时段不是必需的:在只暴露于活化剂约15分钟到约3小时后干细胞就可以被活化并显示成骨潜能。
此外,一些研究表明存在骨前体细胞体外生长必需的辅助细胞群。因此,即使在存在成骨细胞因子混合物的条件下,高度纯化的骨前体细胞也可能不能扩增。因此,干细胞的延长培养不一定是有益的。本文描述的方法和装置不涉及培养细胞的延长时段,它们是更快的,从而避免了与延长的细胞培养有关的污染和花费的可能。
因为本文描述的干细胞具有成骨潜能的增强、活化和/或分化可以通过将活化剂栓系到装置上或装置内实现,以便干细胞混合物内包含的细胞可以被活化,并方便的与活化剂分离,例如,通过从装置去除细胞。在细胞移植前从细胞分离活化剂减轻了活化剂本身不需要的副作用的可能(例如,刺激不想要的应答或诱导有害的免疫应答)。
干细胞源
本文描述的方法,装置和植入物可以使用来自任何方便来源的干细胞。但是,优选的是具有成骨潜能或可以被处理(例如,分化)以产生具有成骨潜能的细胞的干细胞。
可用于本文所述方法,装置和植入物的干细胞源包括骨髓,脂肪组织,肌肉组织,体外培养的自体间充质干细胞,异体的制成(off-the-shelf)间充质干细胞,脐血,胚胎卵黄囊,胎盘,脐带,骨膜,胎儿和青少年皮肤,和血液。在一些实施方案中,干细胞是间充质干细胞或包括间充质干细胞的细胞混合物(例如,骨髓抽吸物)。干细胞可以是自体,异体或异种来源。干细胞可以是胚胎来源,出生后来源或成人来源。
骨髓抽吸物是可用于本文所述方法,装置和植入物的一种干细胞源。这类骨髓抽吸物可以是自体,异体或异种来源,在一些实施方案中,骨髓抽吸物是自体的。
骨髓抽吸物包含以下细胞的复杂混合物:造血干细胞,红细胞和白细胞及它们的前体,间充质干细胞和祖细胞,间质细胞及它们的前体,以及形成称为“基质”的结缔组织网络的包括成纤维细胞,网织红细胞,脂肪细胞,和内皮细胞的一组细胞。基质细胞通过细胞表面蛋白的直接相互作用和生长因子的分泌从形态上调节造血细胞分化,并涉及骨结构的建立和支持。研究显示骨髓包含“前间质(pre-stromal)”细胞,其具有分化为软骨,骨和其他结缔组织细胞的能力。Beresford″Osteogenic Stem Cells and the StromalSystem of Bone and Marrow″,Clin.Orthop.,240:270,1989。新近的证据显示这些细胞(称为多能性间质干细胞或间充质干细胞)在活化时具有产生数种不同类型的细胞系(即,骨细胞,软骨细胞,脂肪细胞,等)的能力。但是,间充质干细胞经常以极少量与多种其他细胞(即,红细胞,血小板,中性粒细胞,淋巴细胞,单核细胞,嗜酸性粒细胞,嗜碱性粒细胞,脂肪细胞,等)存在于骨髓抽吸物中。此外,它们分化为结缔组织组合的能力不仅取决于抽吸物中生物活性因子的存在(其可以不同),但是在某种程度上还取决于供体的年龄。本文描述的方法和装置通过提高干细胞样品中细胞的数目和干细胞分化为成骨细胞的潜能来解决这些问题。
在一些实施方案中,干细胞包括间充质干细胞。间充质干细胞可以通过本领域技术人员能够得到的方法进行鉴定。例如,可以通过集落形成单位测定(CFU-F)或利用间充质干细胞通常表达的标记物通过流式细胞术鉴定间充质干细胞。间充质干细胞通常表达诸如CD271+,CD105+,CD73+的标记物,但显示CD34-和CD45-表型。
当使用骨髓细胞时,这些细胞可以获自髂嵴,股骨,胫骨,脊柱,肋骨或其他骨髓空隙。在一些实施方案中,干细胞来自于自体液体(例如,骨髓抽吸物)。内腔骨髓抽吸物是间充质干细胞的良好来源。
在一些实施方案中,将干细胞用于分离步骤诸如离心,大小过滤,免疫磁性挑选等,以便筛除“无关”细胞,并提高活化步骤的效率,或预选间充质干细胞以有利于植入材料中的骨形成。尽管不是必需分离细胞类型和/或纯化间充质干细胞,在一些实施方案中这是所希望的。
当需要分离细胞类型时,可以将例如包括骨髓的生物样品进行离心以将样品成分基于密度分离为各种组分,包括富含结缔组织生长促进成分的组分诸如间充质干细胞。从而分离富含结缔组织生长促进成分的组分。此外,被离心的生物样品可以不含细胞培养基材料。在一些实施方案,被离心的生物样品基本上由来自患者的组织材料(例如骨髓材料任选的以及血液或其他组织材料)组成,随后将给所述患者植入所获分离和活化的干细胞。
活化剂
在本文描述的方法和装置中使用活化剂以促进干细胞形成和/或分化为成骨细胞。这类活化剂可以是,例如,小分子,肽,生长因子,细胞因子,配体,激素,以及调节生长和分化的其他分子。活化剂可以从自体来源捕获,获自商品化来源或制备得到(例如,通过重组方法)。
可以使用的活化剂实例包括TGF,FGF,PDGF,BMP,IGF,白介素,IL-1,IL-11,TGF,NGF,EGF,HGF,斯伐他汀,地塞米松,氧甾酮,音猬因子,干扰素,纤连蛋白,“RGD”或整合素肽和/或蛋白,角质形成细胞生长因子,成骨蛋白,MSX1,NFKB1,RUNX2,SMAD1,SMAD2,SMAD3,SMAD4,SOX9,TNF,TWIST1,VDR,AHSG,AMBN,AMELY,BGLAP,ENAM,MINPP1,STATH,TUFT1,BMP1,COL11A1,SOX9,ALPL,AMBN,AMELY,BGLAP,CALCR,CDH11,DMP1,DSPP,ENAM,MINPP1,PHEX,RUNX2,STATH,TFIP11,TUFT1,BGLAP,BMP3,BMP5,BMP6,COL10A1,COL12A1,COL1A1,COL1A2,COL2A1,COMP,FGFR1,GDF10,IGF1,IGF2,MSX1,ANXA5,CALCR,CDH11,COMP,DMP1,EGF,MMP2,MMP8,COL10A1,COL14A1,COL15A1,COL3A1,COL4A3,COL5A1,EGFR,FGF1,FGF3,IGF1R,TGFB2,VEGFA,VEGFB,COL4A3,CSF3,FLT1,IGF1,IGF1R,IGF2,PDGFA,SMAD3,TGFB1,TGFB2,TGFB3,TGFBR2,VEGFA,VEGFB,BMP1,CSF2,CSF3,FGFR1,FGFR2,FLT1,GDF10,IGF1,IGF1R,IGF2,PDGFA,TGFB1,TGFB2,TGFB3,TGFBR1,TGFBR2,VEGFA,VEGFB,AHSG,SERPINH1,CTSK,MMP10,MMP9,PHEX,AMBN,AMELY,ENAM,STATH,TUFT1,BGN,COMP,DSPP,GDF10,CDH11,ICAM1,ITGB1,VCAM1,ITGA1,ITGA2,ITGA3,ITGAM,ITGB1,CD36,COMP,SCARB1,AMH,GDF2(BMP9),GDF3(Vgr-2),GDF5(CDMP-I),GDF6,GDF7,IGFBP3,IL6,INHA(抑制素a),INHBA(抑制素BA),LEFTY 1,LTBP 1,LTBP2,LTBP4,NODAL,ACVR1(ALK2),ACVR2 A,ACVRL1(ALK1),AMHR2,BMPR1A(ALK3),BMPR1B(ALK6),BMPR2,ITGB5(整合素B5),ITGB7(整合素B7),LTBP1,NROB1,STAT1,TGFB1I1,TGFBR1,(ALK5)TGFBR2,TGFBR3,TGFBRAP1,CDC25A,CDKN1A(p2 IWAF1/p2 ICIP1),CDKN2B(p15LNK2B),FOS,GSC(goosecoid),IGFBP3,ITGB5(整合素B5),ITGB7(整合素B7),JUN,JUNB,MYC,SERPINE 1(PAI-I),TGFB1I1,TSC22D1(TGFB 1I4),TGIF1,DLX2,ID1,ID2,JUNB,SOX4,STAT1,BAMBI,BMPER,CDKN2B(p15LNK2B),CER1(cerberus),CHRD(chordin),CST3,ENG(Evi-1),EVI1,FKBP1B,HIPK2,NBL1(DAN),NOG,PLAU(uPA),RUNX1(AML1),SMURF1和调节生长和分化的其他分子,以及这些因子的组合。
在一些实施方案中,活化剂包括转化生长因子beta(TGF-β),成纤维细胞生长因子(FGF,包括酸性或碱性成纤维细胞生长因子(FBFa或FBFb),和/或成纤维细胞生长因子-8(FGF-8)),血小板衍生生长因子(PDGF),骨形态发生蛋白(BMP)家族(诸如BMP-1,BMP-2,BMP-3,BMP-4,BMP-5,BMP-6和/或BMP-7),胰岛素生长因子(IGF)家族的成员(例如,胰岛素样生长因子-I和/或II),白介素-1(IL-1),IL-11,斯伐他汀,地塞米松,氧甾酮,音猬因子,干扰素,肿瘤坏死因子,神经生长因子(NGF),纤连蛋白,″RGD″或整合素序列,表皮生长因子(EGF),肝细胞生长因子(HGF),角质形成细胞生长因子,成骨蛋白(Ops;诸如OP-1,OP-2,OP-3),和调节生长和分化的其他分子,以及这些因子的组合。
还可以使用与完整蛋白一样引发相同活化应答的肽活化剂。例如,可以使用与TGF-β,FGFb和/或PDGF具有类似作用的蛋白的氨基酸片段或肽。可以使用来自本文所述任意活化剂的或已知可用于活化干细胞的肽活化剂。在其他实施方案中,可以使用小分子活化剂来活化干细胞。
在许多实施方案中,在本文描述的方法和装置中包括TGF-β家族作为活化剂。TGF-β家族包括一组结构相关蛋白,其在胚胎发育期间影响各种各样的分化过程。包括在TGF β家族中的是基于包括7个半胱氨酸残基的保守性的初级氨基酸序列同源性。该家族包括,例如,Mullerian抑制物(MIS),其是正常男性发育需要的(Behringer,et al.,Nature,345:167,1990),果蝇decapentaplegic(DPP)基因产物,其是成虫盘的背-腹轴形成和形态发生需要的(Padgett,et al.,Nature,325:81-84,1987),非洲爪蟾Vg-基因产物,其位于卵的植物极(Weeks,et al.,Cell,51:861-867,1987),活化素(Mason,et al.,Biochem,Biophys.Res.Commun.,135:957-964,1986),其能够诱导非洲爪蟾胚胎的中胚层和前部结构的形成(Thomsen,et al.,Cell,63:485,1990),和骨形态发生蛋白(BMP′s,诸如BMP-2到BMP-15),其能够诱导重新的软骨和骨形成(Sampath,et al.,J.Biol.Chem.,265:13198,1990)。TGF-β基因产物可以影响各种分化过程,包括脂肪形成,肌形成,软骨形成,造血作用,和上皮细胞分化(综述参见Massague,Cell 49:437,1987),其在此完整引入作为参考。
TGF-β家族的蛋白最初合成为大的前体蛋白,其随后在C端的大约110-140个氨基酸的一簇碱性残基处经历蛋白水解切割。蛋白的C端区域都是结构相关的,不同的家族成员基于它们的同源性程度被划分到不同的亚组。尽管特定亚组的同源性范围是70%到90%氨基酸序列同一性,但亚组之间的同源性是显著更低的,通常只有20%到50%。每种情况下,活性物看起来是C端片段的二硫键连接的二聚体。对于已经被研究的大多数家族成员来说,发现同二聚体物是有生物活性的,而对其他家族成员来说,例如抑制素(Ung,et al.,Nature,321:779,1986)和TGF-βs(Cheifetz,et al.,Cell,48:409,1987),,还检测到异二聚体,这些看起来比相应的同二聚体具有不同的生物学特性。
TGF-β基因超家族的成员包括TGF-β3,TGF-β2,TGF-β4(鸡),TGF-β1,TGF-β5(非洲爪蟾),BMP-2,BMP-4,果蝇DPP,BMP-5,BMP-6,Vgrl,OP-1/BMP-7,果蝇60A,GDF-1,非洲爪蟾Vgf,BMP-3,抑制素-βA,抑制素-βB,抑制素-α,和MIS。这些基因描述于Massague,Ann.Rev.Biochem.67:753-791,1998,其在此完整引入作为参考。
在一些实施方案中,本文所述装置和方法中使用的TGF-β家族成员是TGF-β3。
成纤维细胞生长因子和它们的受体
成纤维细胞生长因子(FGFS)包括进化上保守的多肽家族,涉及各种生物过程包括形态发生,血管生成,和组织重建以及涉及许多疾病的发病机理(综述于Ornitz,Bioessays 22:108,2000,在此特别完整引入作为参考)。该家族的各种成员在体外和体内刺激较大范围细胞的增殖,范围从间充质到上皮和神经外胚层来源。FGFs在发育期间以严格的时间和空间模式表达,在模式形成(patterning)和肢形成中具有重要作用(Omitz,Bioessays 22:108,2000)。
FGF家族的所有成员都具有约120个氨基酸的同源性核心结构域,其中28个氨基酸残基是高度保守的,6个是相同的。对数种FGFs的结构研究鉴定出12个反平行β链,每个均邻近包括核心区(在整个家族中都是保守的)的β-环。核心结构域包括主要的FGFR和肝素结合位点。受体结合区与肝素结合区不同(综述于Ornitz and Itoh,Gen.Biol.2,3005.1,2001)。
在一些实施方案中,本文描述的装置和方法中使用的FGF家族成员是FGF-2。
来自人血小板的血小板衍生生长因子(PDGF)包含两条多肽序列-PDGF-B和PDGF-A多肽(Antoniades,H.N.and Hunkapiller,M.,Science220:963-965,1983)。PDGF-B由位于染色体7上的基因编码(Betsholtz,C.et al.,Nature 320:695-699),PDGF-A由位于染色体22上(Dalla-Favera,R.,Science218:686-688,1982)的sis癌基因编码(Doolittle,R.et al.,Science 221:275-277,1983)。sis基因编码猿肉瘤病毒(SSV)的转化蛋白,其与PDGF-2多肽密切相关。人细胞c-sis也编码PDGF-A链(Rao,C.D.et al.,Proc.Natl.Acad.Sci.USA83:2392-2396,1986)。因为PDGF的两条多肽链由位于分离染色体中的两个不同基因编码,人PDGF可以由二硫键连接的PDGF-B和PDGF-A异二聚体组成,或由两种同二聚体(PDGF-BB同二聚体和PDGF-AA同二聚体)的混合物组成,或由异二聚体和两种同二聚体的混合物组成。
PDGF可以商购获得,或获自人组织或细胞,例如,血小板,通过固相肽合成,或通过重组DNA技术。培养的感染猿肉瘤病毒(其包含编码PDGF-A链的基因)的哺乳动物细胞可以合成PDGF-A多肽,并将其加工为二硫键连接的同二聚体(Robbins et al.,Nature 305:605-608,1983)。此外,PDGF-A同二聚体与经激发抗人PDGF的抗血清反应,分泌的PDGF-A同二聚体的功能特性与血小板来源的PDGF类似。可以利用表达载体将c-sis/PDGF-B基因的cDNA克隆导入小鼠细胞,获得重组PDGF-B同二聚体。用于所述表达的c-sis/PDGF-B克隆获自培养的正常人内皮细胞(Collins,T.,et al.,Nature216:748-750,1985)。
尽管许多活化剂已经用于活化成骨干细胞,本发明人产生的数据表明,在一些实施方案中,TGF-β3,FGF-2和各种形式的PDGF是有用的。在其他实施方案中,用于活化干细胞的装置和方法包括至少TGF-β3和FGF-2作为活化剂。
在一些实施方案中,活化剂能够增加干细胞混合物的祖细胞中骨形态发生蛋白受体亚单位的表达。利用这类活化剂的这种处理有助于,例如,在骨损伤或骨手术部位增强祖细胞对内源性骨形态发生蛋白产生应答的能力。活化剂的这种处理还可以驱使干细胞沿着成骨细胞途径分化。
骨形态发生蛋白(BMPs)不仅诱导骨和软骨形成,而且是多功能性细胞因子,对许多细胞类型具有各种各样的作用(Hogan et al.,Genes Dev.10:1580-1594(1996);Reddi et al.,Cytokine Growth Factor Rev.8:11-20(1997))。BMPs是TGFβ超家族的成员。在人类中存在15-20种BMPs基因,3种BMP受体,和大量的起BMP拮抗剂作用的BMP相关蛋白(Yamashita etal.Bone 19:569-574(1996))。BMP通过3种BMP受体(BMPR-IA,-IB和II)的Smad信号转导途径起作用。当BMP二聚体结合II型受体时,它形成复合体并磷酸化I型受体,其激活Smad途径。
将原代成骨细胞暴露于外源生长因子可以调节这些受体的表达。Singhatanadgit等(J.Cell Physiol.209(3):912-22(2006))检测了原代成骨细胞的TGF-beta1,FGF-2,PDGF-AB和BMP-2处理,以及这些生长因子对细胞表面的细胞内受体的作用。Yeh等(J.Cell.Physiol.190(3):322-31(2002);J.Cell Physiol.191(3):298-309(2002))观察到在暴露于OP-1后,胎儿大鼠颅盖细胞中受体亚单位mRNA的差异调节模式。Xu等(Growth Factors 24(4):268-78(2006))检测了TGFbeta3对BMPR-IB的作用。尽管已经泛泛了解了涉及BMPs结合它们的相应受体的信号转导途径的因子,它们受体亚单位的调节和表达模式还没有被完全阐明。此外,研究人员还未意识到,用生长因子处理干细胞(例如,骨髓)较短时间,然后去除生长因子,可以刺激成骨细胞和/或成骨前体的形成。
活化干细胞
如本文所述,可以通过短暂暴露于活化剂将干细胞成骨活化。这类活化的干细胞分化为骨祖细胞,成骨细胞和/或成骨细胞表型细胞。此外,从活化的干细胞去除活化剂产生不包括生长因子和细胞因子(当移植入受试者时它们可能具有非故意的副作用)的细胞混合物。因此,不需要在生物活性分子混合物中的数天干细胞培养,这导致等待治疗患者的进行中的疼痛和不能活动,骨损伤初次修复后用于插入植入物的额外手术,培养细胞的污染,干细胞群或骨抽吸物中不想要的细胞类型的生长,以及维持培养和照顾受伤患者的额外时间和花费。
相反,可以通过与活化剂温育较短时间将干细胞活化用于移植和骨生长的刺激。因此,例如,当患者被允许接受骨损伤或疾病的治疗时,可以活化自体或异体的干细胞(例如,骨髓抽吸物),当当患者进行手术时,立刻植入活化的干细胞(以及骨移植替代物,如果所需的话)。
本文使用的短语“活化的干细胞”表示干细胞被诱导分化为成骨前体细胞,能够增殖并随后分化为骨形成细胞。这类骨形成细胞包括成骨细胞和成骨组细胞。骨形成细胞可以通过它们的骨特异性标记物的表达来识别,诸如碱性磷酸酶,骨钙素,骨桥蛋白和BMP受体。如本文所示,干细胞的活化可以只进行较短时间,例如,5分钟到24小时的时间段。将干细胞暴露于活化剂的其他最佳时段是10分钟到2小时,或15分钟到1小时。在一些实施方案中,将干细胞与一种或更多种活化剂接触5分钟到1小时,或将干细胞与一种或更多种活化剂接触5分钟到0.5小时。如本文所述,将骨髓抽吸物暴露于活化剂仅仅1小时就引起碱性磷酸酶和BMP受体亚单位的表达。
因此,本发明的一个方面是制备用于促进哺乳动物骨生长的植入物的方法。该方法包括将干细胞暴露于一种或更多种活化剂24小时或更短(例如,约5分钟到24小时,或约10分钟到2小时,或约15分钟到1小时)以形成活化的干细胞,将活化的干细胞与一种或更多种活化剂分离以形成基本上不含活化剂的活化干细胞群,并将基本上不含活化剂的活化干细胞群与骨移植替代物混合,从而制备用于促进哺乳动物骨生长的植入物。
将干细胞暴露于足以活化干细胞为成骨或成骨前体表型的浓度的一种或多种活化剂。本领域技术人员可以容易的确定这种浓度是什么样的,例如,通过观察何种浓度导致碱性磷酸酶和BMP受体亚单位表达的增加或上调。合适活化剂浓度的实例包括使用浓度为约0.01ng/ml到约1μg/ml的活化剂。在一些实施方案中,活化剂使用的浓度为约0.1ng/ml到约500ng/ml,或约1ng/ml到约100ng/ml。
如本文所述,干细胞可以来自于任何方便的来源。但是,优选的是具有成骨潜能或可以被处理(例如,分化)以产生具有成骨潜能的细胞的干细胞。可用于本文所述方法,装置和植入物的干细胞源包括骨髓,脂肪组织,肌肉组织,脐血,胚胎卵黄囊,胎盘,脐带,骨膜,胎儿和青少年皮肤,和血液。在一些实施方案中,干细胞是间充质干细胞或包括间充质干细胞的细胞混合物。干细胞可以是自体,异体或异种来源。干细胞可以是自体,异体或异种来源。干细胞可以是胚胎来源,出生后来源或成人来源。在一些实施方案中,干细胞是自体或异体的骨髓抽吸物。
通常,不需要将干细胞与非干细胞分离,或从其他细胞类型纯化活化的(成骨的)干细胞。但是,如果本领域技术人员希望从非干细胞纯化干细胞,或从未活化的干细胞和其他细胞类型纯化活化的、成骨的干细胞,本领域技术人员可以通过任何方便的鹅方法这么做。例如,可以离心骨髓以基于密度将抽吸物成分分离为各种组分,从而获得富含间充质干细胞的组分。还可以使用识别并结合活化的成骨干细胞的细胞表面表达的因子(例如,BMP受体)的抗体,将细胞进行免疫纯化。
如本文所示,本文所述活化干细胞的方法和装置中使用的活化剂可以是能够活化干细胞成骨潜能的任何活化剂。实例将在本文叙述和说明。
当干细胞被活化时,它们开始表达具有成骨祖细胞特征的因子。例如,如本文所述,碱性磷酸酶(成骨细胞分化的早期标记物,由原代人间充质干细胞表达)的水平增加。参见,例如,图1,其中与未处理的对照细胞相比,用TGFβ3(三角形)或FGF-2(正方形)处理间充质干细胞仅仅1小时就观察到碱性磷酸酶表达随时间的增加,表明接受处理的细胞显示更有效的分化应答。
在一些实施方案中,使用本文所述方法和装置的干细胞活化还可以增加BMP受体亚单位的水平。如本文所述,在暴露于活化剂(1ng/ml,10ng/ml和100ng/ml的PDGF BB,FGF b和TGF beta3)仅仅一(1)小时(图3)或24小时(图2)后,原代间充质干细胞中3种BMP受体单位(即BMPR-IA,BMPR-IB和BMPR-II)的mRNA拷贝数增加。因此,在植入手术部位前用活化剂处理干细胞(例如,间充质干细胞)仅仅较短时间就可以增强细胞在植入后对内源BMP的更有效应答。
通过任何方便的方法将活化干细胞与它们已经接触的一种或多种活化剂分离,从而形成基本上不含活化剂的活化干细胞群。当包含干细胞的组合物(例如,植入组合物)不显示活化剂的副作用(这将阻止干细胞组合物(或植入组合物)的施用)时,干细胞群是基本上不含活化剂的。因此,少量的活化剂仍可以存在于活化干细胞群(或植入组合物)中,只要活化剂的量是,例如,小于10ng/ml,或小于1ng/ml,或小于0.1ng/ml或小于0.01ng/ml。
将细胞与小分子和大分子分离的方法是本领域技术人员可以获得的。例如,通过将细胞悬浮于培养基或盐水并通过离心收集细胞来清洗干细胞。数种这类清洗产生基本上不含活化剂的活化干细胞群。在另一个实例中,将细胞通过保留活化剂但允许细胞穿过的柱来将细胞与活化剂分离。这类柱可以具有结合或保留活化剂的基质;例如,所述柱可以是凝胶过滤柱,亲和纯化柱,或离子交换柱。
因此,可以将活化剂引入溶液中的干细胞源(即,骨髓抽吸物)。在指定温育时间后,通过包括过滤,凝胶过滤,免疫沉淀,免疫吸附,柱层析或其组合的方法将活化干细胞与活化剂分离。例如,利用固定活化剂并允许活化剂从干细胞去除或分离的抗体或结合蛋白或肽来去除活化剂。在一些实施方案中,可以通过手术中的过滤法(诸如切向流动过滤,利用合适的分子量筛截以允许活化剂从溶液的手术中去除)从细胞去除溶液中的活化剂。
在另一个实施方案中,将分离的干细胞掺入骨移植替代物,然后暴露于本文描述的活化剂。可以利用任何方便的方法将从骨髓干细胞/骨移植替代物的复合物中去除活化剂,例如,通过数轮骨髓干细胞/骨移植替代物的复合物的沉淀以及去除包含活化剂的液体上清洗涤液。
在一些实施方案中,通过使用本文描述的装置将活化的干细胞与它们已经接触的一种或更多种活化剂分离。
用于活化干细胞的装置
本发明的另一个方面是活化干细胞例如分化为可以刺激骨生长的细胞(例如,成骨祖细胞,成骨细胞等等)的装置。该装置在本文所述方法中的使用允许将干细胞,干细胞混合物和干细胞组合物(例如,植入组合物)与至少一种活化剂温育足以活化干细胞的时间,然后将活化干细胞与至少一种活化剂分离,以产生基本上不含活化剂的活化干细胞和/或活化的干细胞组合物。
因此,例如,本发明的一个方面是包括固相支持物和连接到该固相支持物的一种或更多种活化剂的装置。活化剂可以直接连接到固相支持物(例如,通过吸附或通过共价键)或活化剂可以间接连接到固相支持物(例如,通过接头,抗体,肽,适体,烷撑链,生物素-链霉亲和素,等)。
固相支持物可以是任何材料,活化剂可与其直接或间接连接,其中所述材料不结合干细胞或不与干细胞不利地相互作用。因此,固相支持物可以是柱基质材料,滤器,培养板,管或平皿,微量滴定板(或微量滴定板的孔),珠子(例如,磁珠),盘,以及与干细胞相容的其他材料。固相支持物可以从各种材料制备,诸如塑料,纤维素,纤维素衍生物,磁性颗粒,硝酸纤维素,玻璃,玻璃纤维,胶乳,和其他基质材料。如果需要,固相支持物可以用抑制干细胞结合或降低固相支持物中材料反应性的物质进行包被。
可以使用本领域技术人员已知的各种技术(这在专利和科学文献中进行了充分描述)将活化剂连接到固相支持物。在本发明的背景下,术语“连接的”或“连接”表示非共价结合,诸如吸附,共价连接(其可以通过活化剂和支持物上功能团之间的直接连接或可以是通过间接连接)。
可以通过将溶于合适缓冲液的活化剂与固相支持物接触合适量的时间来实现一些固相支持物材料(例如,塑料)上的吸附。接触时间随温度而变,但通常在约1小时到约1天。通常,例如,将塑料微量滴定板(诸如聚苯乙烯或聚氯乙烯)的孔与约10ng到约10μg(和优选的约100ng到约1μg)的活化剂量接触足以固定足够量的活化剂。
活化剂与固相支持物的共价连接通常通过首先将支持物与双功能试剂反应来实现,所述双功能试剂将与支持物与活化剂上的功能团(诸如羟基或氨基)两者均发生反应。例如,利用苯醌或者通过支持物上的醛基与结合伴侣的胺以及活性氢的缩合,活化剂可以被共价连接到具有合适聚合物涂层的支持物(参见,例如,Pierce Immunotechnology Catalog and Handbook,1991,A12-A13)。双功能试剂可以是交联剂,具有双功能团的接头,肽,烷撑链,适体,或其他双功能分子。
活化剂可以通过结合剂诸如抗体非共价连接到固相支持物,其中抗体连接或吸附到固相支持物并非共价结合活化剂。可选的,活化剂可以通过生物素-链霉亲和素非共价连接到固相支持物,其中生物素或链霉亲和素连接到固相支持物。当链霉亲和素连接到固相支持物时,生物素连接到活化剂。连接到活化剂的生物素将结合链霉亲和素,从而将活化剂固定到固相支持物上。
在一个实例中,包括柱基质或过滤材料,活化剂与其共价结合。然后将干细胞(例如,骨髓抽吸物)通过该基质一段时间或在该基质中温育一段时间,例如5分钟到24小时的时间段,最佳时间段是15分钟到1小时。
对活化剂的这种暴露已经显示增加BMP受体亚单位和碱性磷酸酶表达。例如,图2和3说明3种活化剂(PDGF BB,FGF b和TGF-β3)上调BMPR-IB亚单位,并且在特定浓度,BMPR-IA和BMPR-II亚单位相对于背景增加。对这类活化剂的暴露用于活化或触发间充质干细胞以增强成骨细胞表型。
因此活化剂可以是FGF b,TGF-β3和/或PDGF BB。在其他实施方案中,固相支持物上的活化剂包括BMP多肽。其他活化剂可以同样连接到固相支持物上,例如,本文所列活化剂的任意一种。活化剂可以来自自体来源,和异体来源,或通过制备得到(例如通过重组技术)。
数种或许多活化剂可以连接到相同固相支持物上。例如,普遍接受的是BMPs在异二聚体(即BMP-2/BMP-7组合)中比在同二聚体制剂(它们目前是商品化供应的)中更有效。因此,对于本文描述的装置和方法实施方案来说,活化剂可以连接到固相支持物,随后在再植入术之前分离。因此,在本文描述的现有设备和方法的使用中,对于使用多种活化剂的可能性具有增加的灵活性。
在一些实施方案中,装置适于将植入组合物暴露于一种或更多种活化剂选定的时间(例如,24小时或更少,和/或本文描述的其他时间),以便活化植入组合物中的干细胞。这类装置适于将植入组合物与本文描述的至少一种活化剂温育,然后允许活化剂与植入组合物的分离。因此,植入材料(例如,骨移植替代物)和植入组合物内的干细胞保留在装置中,而活化剂被去除。装置的使用产生基本上不含活化剂的植入组合物。
该装置因此包括将干细胞和/或骨移植替代物与活化剂分离的工具。例如,该装置可以包括滤器,其排除较大的材料诸如干细胞和/或骨移植替代物,但允许溶液中的活化剂穿过过滤材料,从而将干细胞和/或骨移植替代物与活化剂分离。在将植入组合物与活化剂温育后,将容纳植入组合物和活化剂的温育室引流并用合适的介质(例如,缓冲液,盐水,缓冲盐水,培养基)清洗。因此,该装置可以产生包含活化干细胞并且基本上不含活化剂的干细胞组合物(例如,植入组合物)。
本文描述的装置还可以包括用于控制干细胞与至少一种活化剂温育时间的计时器。例如,在温育步骤(i)后计时器可以触发至少一种活化剂与干细胞的分离。因此,例如,计时器可以起始包含至少一种活化剂的溶液的引流或去除。此外,或可选的,计时器可以起始溶液的添加以清洗干细胞和/或骨移植替代材料。在一些实施方案中,具有计时器的装置控制干细胞与至少一种活化剂的温育时间为24小时或更短。在其他实施方案中,具有计时器的装置控制干细胞与至少一种活化剂的温育时间为5分钟到1小时。
植入物
在一个实施方案中,将如本文所述制备的活化干细胞(例如,骨髓抽吸物)与合成骨移植替代物(诸如beta磷酸三钙(beta-TCP))以形成植入组合物。在一个实例中,在组织移植处使用活化干细胞(例如,活化的骨髓抽吸物)和合成骨移植替代物的混合物。可以在通过暴露于活化剂将干细胞活化之前或之后,将干细胞与合成骨移植替代物组合。但是,在移植前,将干细胞暴露于活化剂或接触活化剂以便植入组合物中的干细胞是活化干细胞。
骨移植替代物可以是固体材料,当在合适条件下将其放置于活性骨中或其附近时,固体材料可以作为通过骨形成活化干细胞形成新骨的支架。可以使用的骨移植替代物的实例描述于美国专利号5,383,931;6,461,632;7,044,972;7,494,950;和美国申请公开号20060008504,其中每篇均在此完整具体引入作为参考。
骨移植替代物可以包括含钙盐的成分。例如,骨移植替代物可以包括一水合磷酸一钙,α-磷酸三钙,碳酸钙,脱矿质骨,磷酸钠盐和,任选的,多聚体。多聚体可以是可吸收的多聚体。在一些实施方案中,多聚体包括同聚体或共聚纤维,它们具有不超过约15mm的纤维长度,约50∶1到约1000∶1的长宽比,或两者(和任选的还包括连续的强化纤维)。
多聚体可以是胶原,明胶,透明质酸,透明质酸盐,羟丙基纤维素(HPC),羧甲基纤维素(CMC),羟丙基甲基纤维素(HPMC),羟乙基纤维素(HEC),黄原胶,瓜耳豆胶,和/或藻酸盐。
可以使用的骨移植替代物的实例包括,但不限于,beta-TCP(例如,Synthes制备的chronOS),胶原,生物玻璃(例如,45S5生物玻璃),BioOss(基于磷酸钙的骨移植替代物),Pepgen P-15(结合天然形式的羟磷灰石的合成P-15肽)和AlloGraft(脱矿质骨基质,基于异源移植的骨移植替代物)。
通常,将活化干细胞与骨移植替代物温育或混合以形成植入组合物。在一些实施方案中,植入组合物是浆(putty);在其他实施方案中,植入组合物是足以流过注射器针头的液体。例如,植入组合物可以具有约0.3到约0.0或约0.41到约0.55的液体成分与固体成分的比例。
另一个实施方案包括在体外使用活化剂以及干细胞源(BMA)较短时间(即,5分钟到60分钟)以便刺激干细胞进入成骨细胞途径。在体外温育后,将干细胞和活化剂的组合一起植入。
治疗方法
包含活化干细胞和骨移植替代物的植入组合物可用于修复和治疗骨损伤,病症或疾病。这类骨损伤,病症或疾病的特征在于骨丢失(骨量减少或骨质溶解)或骨破坏或损伤。这类骨损伤、病症或疾病包括但不限于断裂的骨,骨缺陷,骨移植物,骨移植,骨癌,关节替换,关节修复,融合,小平面修复,骨退化,牙齿植入和修复,源于疾病的骨缺陷(例如,关节炎),源于重建手术的骨缺陷,和与骨和骨组织有关的其他疾病。骨缺陷的实例包括但不限于骨的间隙,变形或不结合的骨折。骨退化的实例包括但不限于骨量减少或骨质疏松症。在一个实施方案中,骨缺陷源于侏儒症。组合物还可用于关节替换或修复,其中所述关节是脊椎,膝,臀部,跗骨,指骨,肘,踝,骶髂的关节或其他关节联接/非关节联接的关节。
可以通过将组合物压入或掺入骨部位,或注射植入组合物,来施用所述植入组合物。当通过注射施用时,注射器可以具有约12到约18 gauge的针头,其中使用的最大注射压力不超过约40磅。在一个实施方案中,所述组合物还包括连续的强化纤维。
下列非限制性的实施例进一步说明本发明的一些方面。
实施例1:材料和方法
使用下列材料和方法实施本发明的一些方面。
细胞的分化
将传代3次的人间充质干细胞(Lonza,Walkersville,MD)按照6×104细胞/35mm孔的密度种于基础培养基(Stem Cell Technologies,Vancouver,Canada),并在37℃温育2天。将细胞用PBS清洗,并用溶于新鲜基础培养基的100ng/ml生长因子(FGF-2或TGFβ3,R&D Systems,Minneapolis,MN)活化1小时,用PBS清洗,然后在新鲜基础培养基或成骨分化培养基(StemCell Technologies,Vancouver,Canada)中37℃温育7-14天。
实时PCR
利用RNeasy Plus Mini Kit和QIA shredder Mini Spin柱(Qiagen)从用各种活化剂处理的细胞中制备总RNA。还从未处理的细胞制备总RNA作为对照。通过TaqMan Reverse Transcription Kit(Applied Biosystems)使用随机六聚物和Oligo dT产生cDNA。用于实时的引物和探针组如下:
huBMPRlA_2 fwd (SEQ ID NO:1):5′-TAACCAGTATTTGCAACCCACACT-3′.
huBMPRlA_2 rev(SEQ ID NO:2):5′-GAGC AAAACC AGCCATCGAA-3′.
huBMPRlA_2探针(SEQ ID NO:3):5′-CCC CCT GTT GTC ATA GGTCCG TTT TTT GAT-3′(FAM/TAMRA).
huBMPRlB_1 fwd(SEQ ID NO:4):5′-CCA AAG GTC TTG CGT TGTAAATG-3′.
huBMPRlB_1 rev(SEQ ID NO:5):5′-CAT CGT GAA ACAATA TCCGTCTGT-3′.
HuBMPRIB_1探针(SEQ ID NO:6):5′-CCA CCA TTG TCC AGA AGACTC AGT CAA CAA-3′(F AM/TAMRA).
huBMPR2_2fwd(SEQ ID N0:7):5′-TGC CCT GGC TAC CAT GGA-3′
huBMPR2_2rev(SEQ ID NO:8):5′-CGC ACA TAG CCGTTCTTGATT-3′
huBMPR2_2探针(SEQ ID NO:9):5′-TCA GCA CTG CGG CTG CTTCOGS′(F AM/TAMRA)
在Applied Biosystems 7500Fast Real-Time PCR System中进行样品的多重反应,利用beta 2小球蛋白内源对照(VIC/T AMRA)(Applied Biosystems)。
碱性磷酸酶测定
将传代3次的人间充质干细胞(Lonza,Walkersville,MD)按照6×104细胞/35mm孔的密度种于基础培养基(Stem Cell Technologies,Vancouver,Canada),并在37℃温育2天。将细胞用PBS清洗,并用溶于新鲜基础培养基的100ng/ml生长因子(FGF-2或TGFβ3,R&D Systems,Minneapolis,MN)活化1小时,用PBS清洗,然后在新鲜基础培养基或成骨分化培养基(StemCell Technologies,Vancouver,Canada)中37℃温育7-14天。测定时,将细胞用PBS清洗两次,收集于100l/35mm孔裂解缓冲液,并在液氮中冻融两次。通过将20μl裂解物与20μl 1mg/ml/7-硝基苯基磷酸盐温育3分钟并测量405nm产生的发光来确定碱性磷酸酶活性。根据CyQuant(Invitrogen,Carlsbad,CA)DNA定量确定的细胞浓度将碱性磷酸酶活性归一化。
固定
对于初步研究,如下将活化剂(例如生长因子)固定到微量滴定孔上。
将生物素化的抗生长因子抗体(500ng)(R&D Systems,Minneapolis,MN)在包被链霉亲和素的96孔板的200μl PBS/孔中温育30分钟,用PBS清洗3次,然后与溶于200μlPBS的量不断增加的生长因子结合30分钟。用PBS清洗3次后,通过与溶于200μl PBS(含0.1%BSA)1μg未标记的抗生长因子二抗温育30分钟来检测生长因子的存在。孔用PBS清洗3次,与溶于200μl PBS(含0.1%BSA)的1μg偶联辣根过氧化物酶的二抗温育30分钟,再用PBS清洗3次。然后将固定的抗体-生长因子-二抗与200μl增强的化学发光底物温育1分钟。通过测量可见光波长的化学发光发射来定量固定的偶联辣根过氧化物酶的二抗的量。
活性测定
为了在干细胞活化步骤前确定栓系活化剂的生物活性,进行下列测定。对于包括FGF-2的研究,将Swiss Albino 3T3细胞(ATCC,Manassas,VA)按照4×1O5/35mm孔的密度种于10%血清基础培养基,37℃温育1天,用PBS清洗3次,然后在37℃0.5%血清基础培养基中同步化1天。将FGF2(R&DSystems,Minneapolis,MN)和50倍过量的生物素化抗FGF2抗体(R&DSystems,Minneapolis,MN)在0.5%血清基础培养基中复合15分钟。将同步化细胞用PBS清洗3次,然后用FGF2/生物素化的抗FGF2抗体复合物在37℃处理30分钟。在测定的终点,将细胞用PBS清洗3次,收集到SDS样品缓冲液,并在90℃加热10分钟。通过SDS-PAGE分离所获裂解物,转移到PVDF膜,并用抗磷酸ERK抗体(Cell Signaling Technology,Beverly,MA)的1∶10000稀释物,继之以偶联辣根过氧化物酶的二抗(Santa CruzBiotechnology,Santa Cruz,CA)的1∶10000稀释物顺序检测。通过暴露于增强的化学发光底物1分钟,CCD照相机显像和利用ImageJ分析程序进行密度测量定量来确定辣根过氧化物酶活性。
对于TGFβ3活性测定,将Mv1Lu貂肺细胞(ATCC,Manassas,VA)按照96孔板4×103细胞/孔的密度种于基础培养基,并在37℃温育1天。将TGFβ3(R&D Systems,Minneapolis,MN)和50倍过量的生物素化抗TGFβ3抗体(R&D Systems,Minneapolis,MN)在0.5%血清基础培养基中复合15分钟。用PBS清洗3次后,将细胞用TGFβ3/生物素化的抗TGFβ3抗体复合物在37℃处理3天。在测定时,向TGFβ3处理培养基中添加CellTiter-Glo ATP检测试剂(Promega,Madison,WI),温育5分钟,通过测量可见光波长的化学发光发射来定量细胞数目。
实施例2:结果
原代人间充质干细胞能够沿着成骨细胞系分化。这通过在成骨混合物(包含,但不限于,地塞米松,抗坏血酸和β-甘油磷酸盐)中培养细胞在体外得到证明。在这种培养条件下细胞显示成骨细胞分化标记物(其最常见的是早期标记物碱性磷酸酶)的上调。
图1显示从第10到12天,未处理的间充质干细胞(圆)中碱性磷酸酶活性的轻微上调,与预期一致。但是,当间充质干细胞用TGFβ3(三角形)或FGF-2(正方形)预处理1小时,然后除去分化培养基中的试剂和培养基,碱性磷酸酶活性的水平显著增加2-3倍。
这些数据表明在第0天用TGFβ3或FGF-2处理间充质干细胞仅仅1小时就能够影响成骨细胞分化,这样的话成骨细胞形成的标记物在处理后10+天被上调。
间充质干细胞在体外和体内均对骨形态发生蛋白产生应答以诱导成骨细胞表型。BMPs通过由3个亚单位(BMPR-IA,BMPR-IB和BMPR-II)组成的受体复合物起作用。当用挑选的活化剂(PDGF-BB,TGFbeta3和FGF-2)处理MSCs 24小时时,BMPR-EB基因的相对拷贝数相对于未处理的细胞显著增加。但是,图3显示在处理后仅仅1小时观察到类似的应答。
这些数据表明仅仅1小时的手术间隙足以用活化剂处理MSCs,这样的话BMP配体的受体水平相对未处理的细胞将增加。间充质干细胞上BMP受体增加水平的存在增强了这些细胞的体内BMP-2成骨应答,从而导致更有效的骨治愈。
本文参考或提及的所有专利和出版物显示本发明相关领域技术人员的技术水平,因此将各种这类参考专利或出版物特别引入按照相同程度进行参考,就像它是分别单独完整引入作为参考或在本文完整阐述一样。申请人保留将来自任何引用的这类专利或出版物的任意和全部材料和信息实际整合入本说明书的权利。
本文描述的具体方法和组合物代表优选的实施方案,只是示例性的,而非旨在限制本发明的范围。在考虑本说明书后本领域技术人员将清楚其他目的,方面和实施方案,这也包括在权利要求范围限定的本发明精神之内。本领域技术人员显而易见的是,在不脱离本发明范围和精神的情况下可以对本文公开的发明进行各种不同的取代和改变。本文说明性描述的发明可以在没有任何元件或限制(其没有被本文具体公开是重要的)的条件下合适的实施。本文说明性描述的方法和过程可以按照不同的步骤顺序实施,而且它们无需局限于本文或权利要求所述的步骤顺序。当在本文和所附权利要求中使用时,单数形式“一个(a)”、“一个(an)”和“the”包括复数含义,除非上下文明确另外规定。因此,例如,“一种抗体”的含义包括多种(例如,抗体溶液或一系列抗体制品)这类抗体,等等。在任何情况下该专利都不能被解释为限于本文具体公开的特定实施例或实施方案或方法。在任何情况下该专利都不能被解释为受到专利与商标局(Patent and Trademark Office)的任何审查员或任何其他公务员或雇员的任何声明的限制,除非这类声明是具体和无条件的或在申请人的回复文字中明确采用的保留意见。
使用的术语和表述被用做说明书的术语而非限制,使用这类术语和表述没有意图来排除显示和描述的特征的任何等同物或其部分,但是应承认各种改变也可能在要求的本发明范围内。因此,应理解尽管本发明已经被优选的实施方案和可选的特征具体公开,但是本领域技术人员可以使用本文公开概念的改变和变化,而且这类改变和变化被认为属于所附权利要求和本发明声明所限定的本发明范围。
本文已经大致和一般性的描述了发明。属于一般性内容内的更小的各个种类和亚属群组也形成本发明的一部分。这包括从所述种类排除任何主题的限制(proviso)或负限制的本发明种类描述,不管排除的材料是否在本文具体叙述。
其他实施方案在下列权利要求范围内。此外,当按照Markush组描述本发明的特征或方面时,本领域技术人员应意识到还按照Markush组成员的任何单独成员或亚组来描述本发明。
Claims (52)
1.一种制备用于促进哺乳动物骨生长的植入组合物的方法,包括(a)将干细胞与一种或更多种活化剂接触24小时或更短时间以制备活化干细胞,(b)从活化干细胞分离活化剂以形成基本上不含活化剂的活化干细胞群,和(c)将基本上不含活化剂的活化干细胞群与骨移植替代物混合从而制备用于促进哺乳动物骨生长的植入组合物,其中至少一种活化剂促进干细胞分化为成骨细胞或成骨前体细胞。
2.权利要求1的方法,其中将干细胞与一种或更多种活化剂接触5分钟到1小时。
3.权利要求1或2的方法,其中将干细胞与一种或更多种活化剂接触5分钟到0.5小时。
4.权利要求1-3中任一项的方法,其中所述干细胞来自于骨髓,脂肪组织,肌肉组织,脐血,胚胎卵黄囊,胎盘,脐带,骨膜,胎儿皮肤,青少年皮肤,或血液。
5.权利要求1-4中任意一项的方法,其中所述干细胞是自体的,异体的或异种的。
6.权利要求1-5中任意一项的方法,其中所述干细胞是胚胎干细胞,出生后干细胞或成人干细胞。
7.权利要求1-6中任意一项的方法,其中所述干细胞包括间充质干细胞。
8.权利要求1-4中任意一项的方法,其中所述干细胞包括自体骨髓抽吸物。
9.权利要求8的方法,其中所述骨髓抽吸物是在手术中抽取的。
10.权利要求8的方法,其中分离或浓缩骨髓抽吸物中的细胞。
11.权利要求1-10中任意一项的方法,其中所述活化剂可以调节细胞生长和/或分化,并选自小分子,肽,生长因子,细胞因子,配体,激素及其组合。
12.权利要求1-11中任意一项的方法,其中所述活化剂选自转化生长因子beta(TGF-β),成纤维细胞生长因子(FGF),血小板衍生生长因子(PDGF),骨形态发生蛋白(BMP),胰岛素生长因子(IGF),白介素-1(IL-1),白介素-11(IL-11),斯伐他汀,地塞米松,氧甾酮,音猬因子,干扰素,肿瘤坏死因子,神经生长因子(NGF),纤连蛋白,RGD肽,整合素,表皮生长因子(EGF),肝细胞生长因子(HGF),角质形成细胞生长因子,成骨蛋白,及其组合。
13.权利要求1-12中任意一项的方法,其中所述活化剂选自BMP-2,TGF-beta3,PSGF-AB,PDGF-BB,FGF-2,TGF-beta1,BMP-4,BMP-7,BMP-6,FGF-8,IL-11,斯伐他汀,地塞米松,氧甾酮,音猬因子及其组合。
14.权利要求1-13中任意一项的方法,其中所述活化剂包括TGF-β和FGFb。
15.权利要求14的方法,其中所述活化剂包括PDGF。
16.权利要求1-15中任意一项的方法,其中所述活化剂位于溶液中。
17.权利要求1-15中任意一项的方法,其中将所述活化剂连接到固相支持物。
18.权利要求17的方法,其中通过肽,抗体,化学交联剂或其组合将至少一些活化剂连接到固相支持物。
19.权利要求1-18中任意一项的方法,其中通过包括过滤,凝胶过滤,切向流动过滤,免疫沉淀,免疫吸附,亲和层析,柱层析或其组合的方法将活化干细胞与活化剂分离。
20.权利要求1-19中任意一项的方法,其中所述骨移植替代物包括钙盐。
21.权利要求20的方法,其中所述钙盐包括一水合磷酸一钙,α-磷酸三钙,β-磷酸三钙,碳酸钙,或其组合。
22.权利要求20或21的方法,其中所述骨移植替代物还包括脱矿质骨,磷酸钠盐,多聚体或其组合。
23.权利要求22的方法,其中所述多聚体是胶原,明胶,透明质酸,透明质酸盐,羟丙基纤维素(HPC),羧甲基纤维素(CMC),羟丙基甲基纤维素(HPMC),羟乙基纤维素(HEC),黄原胶,瓜耳豆胶,藻酸盐或其组合。
24.权利要求1-23中任意一项的方法,其中所述方法增加干细胞中碱性磷酸酶和/或骨形态发生蛋白(BMP)受体亚单位的表达。
25.权利要求1-24中任意一项的方法,还包括将植入组合物植入患者。
26.通过权利要求1-25中任意一项的方法制备的植入组合物。
27.一种治疗受试者骨损伤、病症或疾病的方法,包括给受试者的骨损伤、病症或疾病部位施用权利要求26的植入组合物。
28.权利要求27的方法,其中所述骨损伤、病症或疾病包括断裂的骨,骨缺陷,骨移植物,骨移植,骨癌,关节替换,关节修复,骨融合,骨小平面修复,骨退化,牙齿植入,牙齿修复,关节炎,骨重建,或其组合。
29.一种用于活化干细胞的装置,包括固相支持物和至少一种促进干细胞分化为成骨细胞或成骨前体细胞的活化剂,其中所述装置适于:(i)将干细胞与所述至少一种活化剂温育,和(ii)在温育步骤(i)后将所述至少一种活化剂与干细胞分离。
30.权利要求29的方法,其中至少一种活化剂选自转化生长因子beta(TGF-β),成纤维细胞生长因子(FGF),血小板衍生生长因子(PDGF),骨形态发生蛋白(BMP),胰岛素生长因子(IGF),白介素-1(IL-1),白介素-11(IL-11),斯伐他汀,地塞米松,氧甾酮,音猬因子,干扰素,肿瘤坏死因子,神经生长因子(NGF),纤连蛋白,RGD肽,整合素,表皮生长因子(EGF),肝细胞生长因子(HGF),角质形成细胞生长因子,成骨蛋白,及其组合。
31.权利要求29或30的装置,其中至少一种活化剂选自BMP-2,TGF-beta3,PSGF-AB,PDGF-BB,FGF-2,TGF-beta1,BMP-4,BMP-7,BMP-6,FGF-8,IL-11,斯伐他汀,地塞米松,氧甾酮,音猬因子及其组合。
32.权利要求29,30或3 1的装置,其中至少一种活化剂包括TGF-β和FGFb。
33.权利要求32的装置,其中至少一种活化剂还包括PDGF。
34.权利要求29-33中任意一项的装置,其中至少一种活化剂位于溶液中。
35.权利要求29-33中任意一项的装置,其中将至少一种活化剂连接到固相支持物。
36.权利要求29-35中任意一项的装置,其中所述固相支持物包括柱基质材料,滤器,培养板,管或平皿,长颈瓶,微量滴定板,珠子,盘,或其组合。
37.权利要求29-36中任意一项的装置,其中所述固相支持物包括塑料,纤维素,纤维素衍生物,磁性颗粒,硝酸纤维素,玻璃,玻璃纤维,胶乳,或其组合。
38.权利要求29-37中任意一项的装置,其中所述固相支持物包括亲和基质以去除至少一种活化剂。
39.权利要求29-38中任意一项的装置,其中所述固相支持物包括,与至少一种活化剂结合的,链霉亲和素,生物素,交联剂,抗体或肽。
40.权利要求29-39中任意一项的装置,其中所述固相支持物包括容器。
41.权利要求29-40中任意一项的装置,其中所述固相支持物包括滤器。
42.权利要求41的装置,其中所述滤器保留细胞和骨移植替代材料但允许至少一种活化剂穿过。
43.权利要求41的装置,其中所述滤器保留至少一种活化剂但允许干细胞穿过。
44.权利要求29-43中任意一项的装置,其中所述固相支持物材料不结合干细胞或不会与干细胞发生不利地相互作用。
45.权利要求29-44中任一项的装置,其中所述干细胞来自于骨髓,脂肪组织,肌肉组织,脐血,胚胎卵黄囊,胎盘,脐带,骨膜,胎儿皮肤,青少年皮肤,或血液。
46.权利要求29-45中任意一项的装置,其中所述干细胞包括间充质干细胞。
47.权利要求29-46中任意一项的装置,其中所述干细胞包括自体骨髓抽吸物。
48.权利要求29-47中任意一项的装置,还包括用于控制干细胞与至少一种活化剂温育时间的计时器。
49.权利要求48的装置,其中在温育步骤(i)后计时器触发至少一种活化剂与干细胞的分离。
50.权利要求48的装置,其中计时器控制干细胞与至少一种活化剂的温育时间为24小时或更短。
51.权利要求48的装置,其中计时器控制干细胞与至少一种活化剂的温育时间为5分钟到1小时。
52.权利要求48的装置,其中计时器控制干细胞与至少一种活化剂的温育时间为5分钟到0.5小时。
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CN107849539A (zh) * | 2015-08-13 | 2018-03-27 | 富士胶片株式会社 | 多能干细胞的培养方法、培养容器的制造方法、培养容器以及细胞培养用支架材料 |
TWI745027B (zh) * | 2019-08-14 | 2021-11-01 | 南韓商美迪帕克醫療器械有限公司 | 骨移植組合物、其製備方法及骨移植組合物試劑盒 |
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EP2344631A1 (en) | 2011-07-20 |
CA2741426C (en) | 2020-04-21 |
EP3613845A1 (en) | 2020-02-26 |
CN104357381A (zh) | 2015-02-18 |
US10494604B2 (en) | 2019-12-03 |
KR20160132133A (ko) | 2016-11-16 |
JP5932000B2 (ja) | 2016-06-08 |
EP2344631B1 (en) | 2019-10-16 |
US20100119492A1 (en) | 2010-05-13 |
KR20110097785A (ko) | 2011-08-31 |
US20160222351A1 (en) | 2016-08-04 |
JP2015037433A (ja) | 2015-02-26 |
BRPI0921726B1 (pt) | 2021-01-19 |
CA2741426A1 (en) | 2010-05-06 |
BRPI0921726B8 (pt) | 2021-07-27 |
BRPI0921726A2 (pt) | 2016-12-06 |
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JP5701765B2 (ja) | 2015-04-15 |
JP2012507285A (ja) | 2012-03-29 |
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