CN101883593A - 制造负载生长因子的颗粒的方法以及由此制得的颗粒 - Google Patents
制造负载生长因子的颗粒的方法以及由此制得的颗粒 Download PDFInfo
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Abstract
本发明涉及制造负载有生长因子的颗粒材料(颗粒)的方法、由此制得的颗粒以及用于改善植入材料向骨质中生长的应用。
Description
本发明涉及制造负载生长因子的颗粒材料(颗粒)的方法、由此制得的颗粒及其用于提高植入材料(尤其是金属或陶瓷材料)生长入骨质中的应用，这些植入材料用于诸如人造骨骼、关节、牙齿植入物以及微植入物的植入物中。
最近几年，植入人造关节或骨骼对于例如治疗关节发育异常或者脱臼以及因关节错位导致的关节磨损所产生的疾病越来越重要。植入物的功能以及用于制造它们的材料(包括金属，如钛或者金属合金；以及陶瓷或塑料，如特氟隆(Teflon)或者聚交酯)得到不断改进，使得植入物在90-95％的病例中能在成功的康复过程之后具有高达10年的使用寿命。
尽管有这些进步以及改进的手术方法，植入仍旧是一种艰难和有压力的介入措施，尤其是当这种植入关系到针对植入物的长期康复过程，包括长时间呆在具有康复检测的诊所和康复中心。除了疼痛之外，长时间的治疗以及与熟悉环境的分离也会给所涉及的患者带来沉重的压力。而且，由于需要精心的护理，长期康复过程要付出高昂的人力成本和治疗成本。
近年来，为满足植入物成功长入所需，对该方法在分子水平的理解越来越迫切。结构相容性和表面相容性对植入物的组织相容性来说是致关重要的。狭义上说，生物相容性仅取决于表面。蛋白质对所有水平的融合来说很重要。如下文所述，在植入手术过程中，由于起始吸附蛋白质层的形成(如首批生长在这一层的细胞)，它们已决定了植入物生长的进一步方法。
就植入物(也称为生物材料)与组织之间的分子相互作用而言，它们之间发生许多显示为极具分级结构的反应。作为第一生物反应，在生物材料表面上发生了蛋白质的吸附。在由此形成的蛋白质层中，单一蛋白质分子随后通过例如构象改变而转化为位于表面上的信号传导物质，或者蛋白质片段通过催化(蛋白质水解)反应作为信号传导物质(分子信号(cue))传递。
在分子信号的激发下，在下一阶段发生细胞沉积，该沉积包括许多类细胞，例如白细胞、巨噬细胞、免疫细胞和最终的组织细胞(成纤维细胞、纤维囊肿(fibrocyst)、成骨细胞和骨细胞)。在这一阶段，其它信号传导物质(也称为介质，如细胞因子、趋化因子、形态发生因子(morphogene)、组织激素和真激素(realhormone))发挥重要的作用。在生物相容的情况下，发生植入物与整个器官的整合，理想的是形成永久的植入物。
从近年来对骨生成分子水平的研究来看，化学信号传导物质(对骨骼生长有影响的所谓的“骨形态发生蛋白质”(BMP-1-BMP-15))变得越来越重要。BMP(尤其是BMP-2和BMP-4、BMP-5、BMP-6、BMP-7)是骨诱导性蛋白，通过影响前体细胞到成骨细胞的增殖和分化来刺激骨骼形成和骨骼康复。而且，它们有助于形成碱性磷酸酶、激素受体、骨骼特异性物质(如I型胶原质)、成骨素、骨桥蛋白(osteopontine)和最终的矿化作用。
BMP分子调节各前体细胞的三种关键的反应：趋化性、有丝分裂和分化。而且，BMP在胚胎形成、骨骼和其它组织的器官形成中起到重要作用，这样，已知成骨细胞、成软骨细胞、成肌细胞和血管平滑肌细胞为靶细胞(通过BMP-2阻断增殖)。
同时，已知包含15BMP的许多同种型。除了BMP-1外，所有BMP都是“转化生长因子β(TGF-β)超家族”的一部分，在各细胞的表面上已经找到它们的特异性受体。由于在鼠、狗、兔和猴子的缺损康复实验中已成功使用重组BMP-2和/或BMP-7，似乎没有任何物种出现特异性。
在现有技术中，已知许多关于用来促进骨质生长的负载材料和颗粒领域的实验。有关将BMP-2结合到羟基磷灰石(hydroxyl apatite，HAP)的报道又回到BMP研究的起始阶段，即Urist在1984年发现BMP可以通过在羟基磷灰石柱上色谱分离纯化。在那一年，Urist提到引起软骨在小鼠中形成的BMP和TCP聚集(US4,596,574)。在之后的20年中，发表了大量有关磷酸钙(羟基磷灰石、磷酸三钙)和BMP-2组合的应用的报道。此外，也提到将BMP-2与指定量的胶原或羟基磷灰石混合，然后立刻将混合物冻干，并在冻干之后使用。在另一报道中还研究了在变性剂(如脲)存在下，将变性的rh-BMP-2吸附到羟基磷灰石。即便在这种激烈的条件下，仅有少量的BMP-2结合到羟基磷灰石上。
目前，BMP-2作为“可吸收胶原海绵”上的Induct
(Wyeth，惠氏)或者
(Stryker，斯特瑞克)用于治疗用途上。对那些材料来说相同的是每体积单位使用的BMP的浓度相对较低，即～2ml颗粒或海绵的所需体积对应于1mgBMP-2。仅仅在非生理条件(如在碱性或酸性的极端pH值下或者在中性范围时在去污剂存在下)，较大量的BMP-2才是可溶的。在许多情况下，这些量对于根据伤口大小施用BMP-2以及最佳地刺激骨骼生长(尤其是存在其它骨替换物质时)是不够的。通过这些施用形式(由于BMP-2与胶原的不充分结合)以及在单独的早期递送阶段(“突释期”，burst phase)将BMP-2提供给器官会存在干扰。
下文中，本发明基于对于BMP-2在颗粒材料，尤其是无机骨替换材料如羟基磷灰石、磷酸三钙、碳酸钙、氧化铝或者它们混合物(尤其是它们两相或三相混合物)上吸附的观察，如果吸附步骤在受控pH值下进行足够长的时间，与上文所述材料相比，在固相上每体积份可以获得更多量的BMP，尤其是BMP-2。在吸附步骤之后，第二步较好用至少10倍液体体积(相比所用的固相体积)进行大量的洗涤。这样，可确保除去可溶于液相中的BMP-2。由此，将所谓的突释期显著降低到1-2％的吸附的BMP-2量。因此，可实现在小隔室中施加高剂量的BMP-2的选择。可以在pH 4-5(尤其是pH 4.5)的酸性范围或者在pH 9-11(尤其是pH 10)的弱碱性范围内在缓冲水溶液中涂覆所述表面。而且，当颗粒材料是可生物再吸收的(bioresorbable)材料时很有利。
通过本发明的方法，不能从颗粒上洗去的大量骨生长因子通过化学键合的形式被吸附到表面上，这不同于：
·与HAP或TCP的混合/组合(即混合物)
·例如包含/捕获在孔隙中
·通过例如液体的冻干和材料中的沉积进行结合
·涂覆金属或陶瓷，其中，例如，将颗粒或模塑体浸没在BMP溶液中，并立即进行干燥步骤以除去溶液[BMP-2干燥成表面上的一层](即不是吸附(adsorption)，而是附着(adhesion))
在BMP-2(表1)对各种羟基磷灰石的结合研究中，发明人发现BMP，尤其是BMP-2大量地在大范围内线性结合到磷酸钙上。
在表1中，显示了在pH 4.5下进行的吸附实验的结果(20mM乙酸钠pH 4.5)，其中，吸附/孵育已经进行了至少30分钟(t1/2＝16d)，较好至少1-2小时(t1/2＝19d)，尤其优选至少4-6小时(t1/2＝20d)。最长的半寿期值为孵育15-17小时(t1/2＝23d)。较好在去污剂如SDS存在下，在碱性范围内进行涂覆(缓冲液：125mM硼酸盐/0.066％SDS，pH 10.0)。在吸附步骤之后，通过用10倍于材料体积的PBS缓冲液(pH 7.4，137mM NaCl，8.1mM Na2HPO4，2.7mM KCl，1.5mM KH2PO4)洗涤5次来除去去污剂。
在颗粒材料上吸附BMP-2之后，测量脱附。为此，将样品各自转移到2ml缓冲液(50mM Tris/HCl，150mMNaCl，pH 7.4)中。在预定间隔之后，将样品取出，进行洗涤(3×2ml缓冲液(50mM Tris/HCl，150mM NaCl，pH 7.4))，并在γ-计数器中计数。然后，将它们转移到2ml新鲜缓冲液中，进行下一次释放间隔。被固定的BMP-2的量使用125碘辐射标记蛋白质并在γ计数器中计数来确定。
使所有骨替换材料在室温下，在溶有指定浓度的BMP-2的20mM乙酸钠-缓冲液(pH 4.5)中孵育15小时。在50mM Tris/HCl，150mM NaCl(pH 7.4)中测定脱附。在所谓的突释期中释放的半寿期时间(仅仅涉及1-2％吸附量的BMP-2)为0.4-1.1天(未显示)。APS：氨丙基三乙氧基硅烷；
(密度～0.63g/cm3；～1.3×104个颗粒/克)，
(Dichte～0.63g/cm3；～1.3×104个颗粒/克)，Co.Algoss GmbH，(阿高斯公司，维也纳)；
Company DOT GmbH(DOT股份有限公司，罗斯托克)；Collagen Matrix ACE Surgical Supply Co.(胶原基质ACE外科用品公司，布鲁克敦(Brockton)，MA，USA).
本发明通过附图进一步举例说明：
图1显示了从石灰石藻类获得的
-颗粒的超微结构(单位对应10μm)。
具有相同的结构(摘自“Bone Augmentation in Oral Implantology”《口腔移植学中的骨质增大》，Khoury，F.等人，第349页，2007，QuintessenzVerlags-GmbH，国际精萃出版集团，柏林)。
图2显示了在含有MC3T3-E1细胞的细胞培养中，吸附在Algisorb(C和D)上的rhBMP-2的生物活性的证据，其中：
A：Algisorb，阴性对照-在培养基中没有可溶rhBMP-2)；
B.Algisorb，阳性对照-将50nM可溶rhBMP-2加入到培养基中)；
C.rhBMP-2吸附到Algisorb(～0.5mg rhBMP-2每克Algisorb)(保持潮湿)；
D.rhBMP-2吸附到Algisorb(～0.5mg rhBMP-2每克Algisorb)(干燥的)；
图3A显示rhBMP-2的亲水和疏水吸附；和
图3B显示rhBMP-2的释放(第一顺序的反应)。
图1显示了从石灰石藻类获得的微结构
(阿高斯公司，维也纳)的电子显微镜图，相比其它多孔羟基磷灰石，其具有显著提高的康复和再吸收性能。红色石灰石辣根菜(Cochlearia officinalis)的原始CaCO3被羟基磷灰石(HAP)
或者磷酸三钙(TCP)替换，保持原微结构[1]。
如图2所示，吸附在Algisorb上的rhBMP-2的生物活性证据在含有MC3T3-E1细胞的细胞培养基中是成功的。因此，5×105个新鲜用胰蛋白酶消化的MC3T3-E1细胞在无菌条件下被接种在Algisorb-颗粒上(这些颗粒用纤维蛋白粘合剂固定在48孔微滴定板的孔底部)，并在含有10％FCS的α-MEM培养基(Gibco，吉必可)中孵育。在6-12小时之后，用新鲜的含有1％FCS的α-MEM培养基替换融合生长在板中的细胞的培养基，所述细胞进一步在对照-Algisorb或者吸附有BMP-2的Algisorb(没有用APS功能化)上生长6天。6天之后，用杜尔贝科(Dulbecco)磷酸缓冲液洗涤长有细胞的Algisorb-颗粒，并用2％的多聚甲醛固定。用磷酸酶检测试剂盒ELF-97(Molecular Probes，Inc.，分子探针公司，奥勒冈，USA)，使用荧光显微镜(Nikon Eclipse E400，10兆像素照相机，尼康公司，杜塞尔多夫，德国，激发波长345nm，发射波长530nm)，对碱性磷酸酶(绿色荧光染料)进行成像和测定。
如图3所示，以下可以看到本发明高密度固相BMP-2的性能。从～1.3×104个颗粒/克Algisorb(6.7mg/g的rhBMP-2负载量)的颗粒数来计算，每个颗粒结合0.5微克rhBMP-2。这意味着2个颗粒(1微克)足够在绵羊实验中提供显著的骨诱导[2]。
按照发明人的发现，Algipore的改进性能一方面基于互连孔隙系统以及各向同性(无定形)羟基磷灰石颗粒的存在，这与Bio-Oss(Geistlich，盖思特利公司)的高结晶羟基磷灰石相反。在本研究中，与Algipore相比，包含磷酸三钙形式的石灰石藻类，
因此具有进一步提高的再吸收性能。
结合行为不仅在Algipore和Algisorb中显示，类似行为也在其它羟基磷灰石中出现。Algipore和Algisorb的特别之处在于结合的量在1-2mg/g或以上。这种量迄今在本领域中也是未知的。使用本发明的方法，可以获得超过7mgBMP-2/克颗粒(2.8-4.4mg/cm3)(高密度固相BMP-2)。
用在本发明中的材料Algipore和Algisorb的信息可以在文献[1]中找到。因此，
98％羟基磷灰石HA-单相，和
80％磷酸三钙β-TCP，19.3％HA，0.7％方解石CaCO3-在本发明中可以使用两相/三相。对于后者，β-TCP和HA的所有两相/三相形式均可以使用，它们具有相同的电子显微镜结构。方解石为0.3-0.7％。
发明人有关高密度固相BMP-2性质的研究进一步揭示了从～1.3×104个颗粒/克Algisorb(6.7mg/g的rhBMP-2负载量，图1)的颗粒数来计算，每个颗粒结合0.5微克rhBMP-2。这意味着2个颗粒(1微克)足够在绵羊实验中提供显著的骨诱导[2]。因此，在本发明的方法中，可以在体内和临床中合理且无扩散损失地使用rhBMP-2。
对于制造本发明的高密度固相BMP-2的方法，该方法在BMP-2含量/每体积单位高于在水溶液中获得的浓度的范围内有效。较好的是，在本发明中，使用BMP(优选BMP-2)的缓冲溶液，其浓度为0.1-1.5mg/ml缓冲溶液，较好是0.5-1.5mg/ml缓冲溶液。将这样浓度的包含BMP的缓冲溶液以一定量加入到颗粒中，加入量为能获得以毫克BMP/克颗粒为单位的所需的负载量。例如，如果想要2.5毫克BMP/克颗粒的负载量，则将包含1mg/ml BMP的5ml缓冲溶液加入到2g颗粒中。如果试验体积相比净重增大4倍，则得到7mg/g颗粒，而不是结合10mg/g颗粒。
而且，发明人未完成的研究表明可以获得更高量(4-5毫克BMP～8-10毫克BMP/克颗粒)。因此，可以使用一定量(单位为ml)的根据浓度来预定的包含BMP的缓冲溶液。由此，如果在使用过程中仅仅使用一些BMP-HAP组合物颗粒(例如，和植入物一起或者在上颌窦的骨质增大过程中)来再产生骨诱导，则使用本发明负载有骨生长因子的颗粒是足够的。发明人的体外研究显示，结合到HAP的BMP-2是具有生物活性的。发明人在绵羊上的进一步研究目前正在进行。
而且，发明人惊奇的发现和羟基磷灰石组合用作骨替换材料的磷酸三钙(在上述
中占约80％)可以和活化剂(如氨丙基三乙氧基硅烷)发生活化反应，从而使BMP-2对颗粒表面的吸附提高至少2倍。
因此，发明人揭示了每克由～80％的TCP和～20％的HAP组成的颗粒材料(Algisorb)，可以结合与98％的HAP(Algipore)相同的结合量。更令人惊奇的是，本领域技术人员所预期的是，如果仅是HAP在反应，则相比具有98％HAP的Algipore而言，仅可以结合额外的20％BMP(即Algisorb中的HAP份额)。发明人假设额外的60-70％的结合的BMP-2被改性的TCP结合。因此，本发明也公开了在骨生长因子吸附之前，通过用活化方式处理来活化颗粒材料。所述活化剂可选自硅烷，其中优选使用氨基烷基烷氧基硅烷，如氨丙基三乙氧基硅烷。这种活化处理通常如下所述进行：在3-氨丙基三乙氧基硅烷(APS，Sigma-Aldrich，西格马-奥觉齐公司，Taufkirchen，塔吉城)在无水甲苯的5体积％溶液混合物(50ml)中，于惰性气体气氛(氮气5.0)中，在加热的玻璃设备中加热骨替换材料(参见表1)至沸腾，回流3.5小时。在反应结束后，冷却样品，并各自在10ml的氯仿中洗涤3次，在丙酮中洗涤3次，并在甲醇中洗涤3次。
之后，将如此活化的颗粒材料(特别是由TCP、HAP或它们混合物组成(见表1))在pH 4-5的酸性范围，在pH 4.5(20mM乙酸钠-缓冲液，pH 4.5)或者在pH9-11，优选在pH 10(125mM硼酸盐，0.066％SDS pH 10.0)的弱碱性范围内，在骨生长因子(优选BMP-2或BMP-7)的缓冲溶液中处理至少30分钟(t1/2＝26d)，优选至少4小时(t1/2＝36d)，尤其优选至少15小时(t1/2＝43d)。为此，在用氨丙基三乙氧基硅烷(APS)化学改性之后，用水洗涤颗粒材料，随后转移到小的反应容器(2ml)中，其中各自装有在20mM乙酸钠-缓冲液(pH 4.5)中或者在125mM硼酸盐/0.066％SDS-缓冲液(pH 10.0)中的1.0ml BMP-2溶液。对于将rhBMP-2吸附到材料，使用了三种不同的蛋白质浓度：0.1、0.2和0.3mg/ml。使用用125碘辐射标记的蛋白质确定被固定的BMP-2的量。
为了防止突释期(例如，过量释放没有吸附到颗粒表面上而仅仅是保留在颗粒上的骨生长因子)，在吸附步骤之后优选洗涤该颗粒，较好用10倍于颗粒材料体积的不含骨生长因子的缓冲溶液(20mM乙酸钠-缓冲液，pH 4.5或者125mM硼酸盐，0.066％SDS pH 10.0)分别洗涤三次。之后，在pH 7.4的PBS-缓冲液(137mM NaCl，8.1mM Na2HPO4，2.7mM KCl，1.5mM KH2PO4，pH 7.4)中再洗涤5次。
通过提供本发明的高密度固相BMP，可以用新的方式在体内和临床上合理且无任何扩散损失地使用rhBMP-2。已经显示所述高密度固相BMP在冻干之后可以存储数星期，而活性不发生任何损失任何(见图2)。本发明最早的研究显示本发明的高密度固相BMP的储存期可延长至超过1-2年。由此，发明人认为BMP生物活性的保持可归功于优选在无菌条件下使用的所述材料。
文献
[1]Spassova，E.，Gintenreiter，S.，Halwax，E.，Moser，D.，Schopper，C.，& Ewers，R.(2007)Chemistry，Ultrastructure and Porosity of Monophasicand Biphasic Bone Forming Materials Derived from Marine Algae.(“源自海藻的单相和双相骨形成材料的化学、超微结构和多孔性”)Materialwiss.Werkstofftech.，38，1BMP-27-1034.
[2]Lichtinger，T.K.，Muller，R.T.，Schurmann，N.，Wiemann，M.，Chatzinikoleidou，M.，Rumpf，H.M.，& Jennissen，H.P.(2001)Osseointegration of Titanium Implants by Addition of Recombinant BoneMorphogenetic Protein 2(rhBMP-2).(“通过添加重组骨形态形成蛋白2(rhBMP-2)进行的钛移植物的骨整合”)Materialwiss.Werkstofftech.，32，937-941.
Claims (12)
1.一种制造负载有生长因子的无机颗粒材料的方法，其中，选自陶瓷材料的所述颗粒材料在pH 4-5的酸性范围或者在pH 9-11的弱碱性范围的缓冲水溶液中用生长因子处理至少30分钟。
2.如权利要求1所述的方法，其特征在于，所述缓冲水溶液的pH值为4.3-4.7，尤其是4.5。
3.如权利要求1所述的方法，其特征在于，所述缓冲水溶液的pH值为9.5-10.5，尤其是10。
4.如权利要求1-3中任一项所述的方法，其特征在于，所述颗粒材料的粒度为10-500微米，并且具有互连孔隙结构。
5.如权利要求1-4中任一项所述的方法，其特征在于，所述颗粒材料由羟基磷灰石、磷酸三钙、碳酸钙、氧化铝或者它们的混合物组成。
6.如权利要求1-5中任一项所述的方法，其特征在于，使用具有化学活化的表面的颗粒材料。
7.如权利要求6所述的方法，其特征在于，具有化学活化的表面的所述颗粒材料通过用活化剂处理颗粒材料来获得，优选所述活化剂选择硅烷类。
8.如以上任一项权利要求所述的方法，其特征在于，在负载步骤之后，将所述无机颗粒材料与缓冲水溶液分离，然后用不含生长因子的至少相同体积的缓冲溶液洗涤至少一次，并优选进行干燥。
9.如权利要求8所述的方法，其特征在于，所述干燥步骤包括冻干。
10.如上述任一项权利要求所述的方法，其特征在于，使用BMP-2或BMP-7作为生长因子。
11.按照上述任一项权利要求所述方法制得的颗粒材料。
12.权利要求11所述颗粒材料在制造促进骨生长的药物组合物中的应用。
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| CN114404651A (zh) * | 2022-01-25 | 2022-04-29 | 中科南京绿色制造产业创新研究院 | 一种碳酸钙复合的二肽水凝胶支架及其制备方法和应用 |
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| US8998925B2 (en) | 2011-06-20 | 2015-04-07 | Rdc Holdings, Llc | Fixation system for orthopedic devices |
| WO2012177759A1 (en) | 2011-06-20 | 2012-12-27 | Rdc Holdings, Llc | System and method for repairing joints |
| KR102147554B1 (ko) | 2012-07-27 | 2020-08-25 | 레스메드 피티와이 엘티디 | 환자 인터페이스 및 그 제조 방법 |
| KR101461159B1 (ko) * | 2013-01-29 | 2014-11-24 | 단국대학교 산학협력단 | 약물 전달층을 포함하는 임플란트의 제조방법 및 이를 포함하는 생체이식용 임플란트 조성물 |
| KR101405859B1 (ko) | 2013-04-24 | 2014-06-12 | 오스템임플란트 주식회사 | pH 완충 물질과 술폰기를 갖는 유기 양친성 물질의 혼합 용액으로 코팅된 치과용 임플란트 및 그 제조방법 |
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| CA2704123C (en) | 2015-12-22 |
| AU2008320842B2 (en) | 2011-09-22 |
| KR20100085982A (ko) | 2010-07-29 |
| AU2008320842A1 (en) | 2009-05-07 |
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| DE102007051914A1 (de) | 2009-05-07 |
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