Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (34): 5540-5547.doi: 10.3969/j.issn.2095-4344.2014.34.023
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Zhao Wen1,2, Liu Yu-ying3, Liu Zi-hao4, Wang Mei4
Revised:
2014-07-02
Online:
2014-08-20
Published:
2014-08-20
Contact:
Zhao Wen, Department of Orthopedic Surgery, the General Hospital of Chinese PLA (301 Hospital), Beijing 100853, China; Department of Orthopedic Surgery, Beijing Aerospace General Hospital, Beijing 100076, China
About author:
Zhao Wen, Master, Professor, Chief physician, Department of Orthopedic Surgery, the General Hospital of Chinese PLA (301 Hospital), Beijing 100853, China; Department of Orthopedic Surgery, Beijing Aerospace General Hospital, Beijing 100076, China
Supported by:
the Science and Technology Fund of Beijing Aerospace General Hospital
CLC Number:
Zhao Wen, Liu Yu-ying, Liu Zi-hao, Wang Mei. Degradable natural polymer hydrogels in articular cartilage repair: safety and effectiveness assessment [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(34): 5540-5547.
2.1 天然水凝胶 天然生物水凝胶材料的生物相容性好,免疫原性低,在原位组织中产生免疫应答率低。研究调查显示,天然水凝胶是组织工程和修复医学中具有较高应用价值的生物材料。以天然水凝胶为材料制成的软骨细胞+水凝胶合成体,可为机体提供促进软骨细胞增殖分化的细胞及生长因子等。另一方面,如果植入物具备良好的机械性能对手术操作及机体应力环境具有至关重要的作用。然而,许多天然水凝胶缺乏强度及韧性,故为了提高水凝胶材料的生物学活性、强度及韧性,常需要对其进行理化性质的功能化修饰,即水凝胶材料的物理、化学改性(如氢键、静电作用),使其获得特定功能。 2.2 蛋白质类 2.2.1 胶原蛋白 人体胶原蛋白是人体细胞外基质内含量最高的结构蛋白,其中关节软骨内Ⅱ型胶原的质量占固体质量的90%。然而其余几种胶原蛋白也参与构成胶原蛋白网或软骨细胞间生物大分子间的反应过程。胶原蛋白网中尽管含有70%的水,但足以提供关节的机械强度,塑造软骨形态。现有的人造异体胶原蛋白组织网或海绵主要通过酶促反应、盐/酸提纯等方法从动物组织中分离提取而来,所以此种类型的胶原蛋白结构必须经过纯化以降低其免疫原性后才能植入人体。研究证实Ⅰ、Ⅱ型胶原纤维蛋白均可以促进软骨细胞增殖、分化,诱导软骨组织的生成,但Ⅰ型胶原纤维同时可诱导软骨细胞的去分化[15-17]。Ⅰ型胶原凝胶的三维网状基质结构是其产生、分泌及储存Ⅱ型胶原蛋白,以及新生软骨细胞外基质内矿物沉积的关 键[18]。Yamaoka等[19]还报道了将软骨细胞在胎牛血清中嵌入到Ⅰ型胶原凝胶中可迅速增殖,并产生出大量的Ⅱ型胶原及黏多糖。在研究骨形态发生蛋白2对Ⅰ型胶原凝胶调节控制作用过程的兔模型中,证实了软骨细胞的分化起源于骨髓间充质干细胞,并得出结论:合适的Ⅰ型胶原凝胶三维环境及与软骨细胞之间良好的相互作用,能够促进软骨细胞的增殖、软骨基质的储存[20-21]。然而随着时间的推移,新生组织形成,人工合成的Ⅰ型胶原凝胶会逐渐失去作用,被机械稳定性更好的新生组织替代[22]。 影响胶原蛋白凝胶在软骨再生组织工程应用中的最大障碍是其缺乏机械强度,难以应用于原位关节软骨的修复,有报道表明Ⅰ型胶原凝胶的杨氏模量为65.5 kPa[19],远低于人体关节软骨的杨氏模量。为解决该问题,有学者将胶原蛋白与强度较高的可降解聚合支架材料相结合。在组织工程中理想的具体做法是:在多聚材料的小孔中植入胶原蛋白微海绵,再将软骨细胞接种到这些胶原蛋白微海绵中,由多聚材料充当组织的外形骨架,胶原蛋白微海绵则模拟细胞生长的微环境,促进软骨细胞增殖、分化及新组织再生。其中一项动物对照实验显示,分别在裸鼠皮下直接植入乳酸乙醇酸共聚物胶原蛋白海绵或乳酸乙醇酸共聚物海绵后,发现乳酸乙醇酸共聚物胶原蛋白海绵中比乳酸乙醇酸共聚物海绵中有更多的均质性物质生成,并且新生成的物质还保持着乳酸乙醇酸共聚物胶原蛋白海绵原有的形态。但在最初10周的新生组织形成过程中,由于降解作用,乳酸乙醇酸共聚物混合胶原蛋白海绵和乳酸乙醇酸共聚物海绵的质量均减少了约36.9%[23]。合成的乳酸乙醇酸共聚物海绵可以充当支架,并且该支架容易改造成任何想要的形状,提供相应的机械强度以此来限定最终制备的工程组织形态。掺入的胶原微海绵则有助于细胞的种植、均质性细胞的分布,创造一个适合细胞分化、增殖的环境。因此,混合应用生物材料和合成材料制备三维支架对于软骨组织工程是未来发展的趋向。在另外一项研究中,使用合成材料聚乳酸和天然生物材料胶原制备一种新型的三维支架用于治疗关节软骨损伤修复[24]。在此项研究中,三维聚乳酸网络提供机械强度,而天然聚合物胶原则模拟软骨组织内软骨细胞生长的微环境。结果显示,聚乳酸胶原混合支架显示出高度孔性结构,支架的硬度明显增强,细胞的活力、黏附性增强。研究者还发现,使用PLCL和胶原制备一种膜式支架并将软骨细胞种植其上,体外培养一段时间后将其植入体内软骨缺损的地方[25]。术后8周植入物出现软骨类组织,并且随时间推移,类软骨组织越来越多。除此之外,还可将胶原与其他材料混合制备各种支架,如透明质酸和硫酸软骨素[26]。另外,研究人员还发现支架内孔的大小虽然不影响细胞的增殖但却影响体内组织再生的效率[27]。在所观察的4种具有不同大小孔的支架中,孔径为150-250 μm的支架能促进软骨形成,使其具有一定的机械特性。这些研究表明胶原确实是一种合适的软骨组织工程生物材料,但为了提高其机械强度及便于改变其形状,最好与其他材料混合应用。 2.2.2 明胶 明胶是胶原蛋白部分水解变性的衍生物,它保留了胶原蛋白的一些氨基酸序列,如精氨酸-甘氨酸-天冬氨酸(RGD)序列,能促进软骨细胞黏附、增生及分化。相比其前体胶原蛋白,明胶的抗原性更低。但由于其机械强度过低,故很少单独用作关节软骨组织支架材料。文章前述的适于胶原蛋白的化学交联剂同样适用于明胶。通过在明胶胶体上接种及培养大鼠软骨细胞,发现与京尼平交联形成的水凝胶在促进软骨细胞黏附及生长方面,比戊二醛交联的水凝胶更好,而且在细胞接种后9 d可以观察到胶原蛋白和黏多糖的生成[28]。另外通过化学功能化也可以得到新的二硫化明胶及光交联型明胶[29-30]。其中光交联明胶的双网络结构,使此种交联后的明胶胶体获得了满意的机械强度,使其有可能成为一种具有研究前景的软骨细胞胶原蛋白支架。 由于已知的凝胶法对于明胶水凝胶胶体的强度及其多孔结构的形成起着关键作用,而多孔结构及孔径大小直接影响软骨细胞的黏附、分化,所以为了提高明胶基复合结构与功能,纳米纤维-明胶复合体形成的三维多孔支架结构也进入实验研究阶段[31]。初步实验表明此种水凝胶复合体可为人骨髓间充质干细胞来源软骨细胞的增殖、分化及细胞外基质的形成提供更好的机械支撑和生长环境[32]。目前以明胶为基础的多种复合仿生多相支架已研制成功,不同支架主要包含有三磷酸钙[33]、羟磷灰石和多磷酸钙等成分[34-36],这类仿生多相支架对于骨软骨组织工程研究具有较高价值,其中部分研究者认为双相支架可用于模拟机体软骨及软骨下成分,明胶层及碳酸钙/磷酸复合层有助于软骨细胞的黏附与增殖,促进软骨组织的再生和软骨板的形成,同时加强再生软骨与软骨下骨的紧密结合[33]。另外研究者为了增强明胶水凝胶的机械强度及各种材料间的协同作用,通过光聚合制备了pMHMGCL/PCL/明胶甲基丙烯酰胺混合水凝胶,该水凝胶机械强度相比明胶水凝胶至少增强了5倍,此支架内两种材料间通过共价键结合,更耐反复轴向和旋转力[37]。并且体内外实验证实,种植于此支架内的软骨细胞能够形成软骨特异性基质。因此,用明胶制备水凝胶支架为了达到需要的机械强度应该与其他材料混合应用,并且应用时需要考虑材料间的协同作用。 介于明胶的理化特性,明胶微粒也被广泛地用作载体来转运细胞或生长因子,以促进局部特殊细胞高浓度及同时释放相关生长因子,增强软骨细胞的附着、增殖、分化,同时增强间充质干细胞的软骨分化及新生软骨组织的再生能力[38]。研究证实以明胶微粒为载体运输及释放的转化生长因子β和胰岛素样生长因子1,能明显促进软骨细胞的形成与分化[39]。兔模型体内实验表明,在软骨再生不良的情况下,载有转化生长因子β1的明胶微粒寡聚乙二醇富马酸盐水凝胶复合材料比单纯寡聚乙二醇富马酸盐水凝胶复合材料能更好地促进软骨下骨的形成[40]。另一实验证实,由一层软骨层及一层成骨层构成的双层水凝胶复合材料,两层均载有转化生长因子β3明胶微粒的寡聚乙二醇富马酸盐水凝胶,在软骨层,转化生长因子β3主要刺激骨髓基质细胞向软骨细胞分化,而另一层骨髓基质细胞则可向成骨分化[41]。 2.3 多糖 多糖是一类可降解天然高分子,在组织工程中应用广泛。这些天然聚合物具有非常丰富的资源,广泛存在于微生物、植物、动物中。多糖不是一种纯粹的化学物质,而是聚合程度不同物质的混合物,是一类分子机构复杂且庞大的糖类物质。尽管来源不同,但所有多糖都是由多个单糖分子缩合、失水而成,糖单位由糖苷键、共价键结合而成。各种多糖因糖单元的取代基、结合位点、类型及分子量不同可导致多糖特性的差异[42-43]。仅仅化学结构上的差异,足以使不同的多糖具有不同的理化特性,比如机械强度、溶解度、静电特性、黏度、胶凝作用及在表层、界面的特性。多糖可以与生物大分子、蛋白质或其他多糖通过氢键和静电引力进行络合,化学修饰亦可以引入功能化结构团来与这些多糖结合,以赋予其更多功能,如生物相容性、胶凝潜能、交联能力、形成多糖凝胶支架或进行结构降解动力学的调控[44-46]。 2.3.1 壳聚糖 壳聚糖是甲壳素的脱乙酰化产物,由是β1,4连接的二氨基二脱氧基葡萄糖(GlcNH2)残基和随机分布的N-乙酰葡糖胺(GlcNAc)基团组成的线性多糖。它分子质量介于(50-1 000) ku之间。在弱酸性溶液中,胺基去乙酰后质子化,使得壳聚糖成为聚阳离子,此时壳聚糖的结晶度和电荷密度,以及许多其他重要的物理化学特性都决定于脱乙酰度。市售壳聚糖产品有度脱乙酰范围从50%到90%的,目前市面上壳聚糖产品的脱乙酰度在50%-90%。由于壳聚糖具有良好的生物相容性、生物可降解性,病理性炎症反应率和诱发感染及内毒素率低,同时固有的抗菌能力好,使其成为最重要的生物材料之一,胺基化学修饰为其获得更优的生物活性和生物功能[47],例如通过N-乙酰化来调控细胞黏附功能和壳聚糖膜降 解[48]。 作为氨基葡萄糖类似物,壳聚糖是一种在软骨组织工程及软骨修复中格外受关注的生物材料。壳聚糖及其降解产物参与了关节软骨中多种物质(包括硫酸软骨素、透明质酸、Ⅱ型胶原蛋白)的合成。尽管壳聚糖的主要缺点是不溶于中性溶剂,但酸性溶液能使聚合物链中的-NH2基被部分质子化,故壳聚糖能溶于酸性溶液中,所以通常用醋酸来制造壳聚糖溶液,所需要的酸浓度取决于壳聚糖的量、氨基的数目和离子的量[49]。另外,通过化学修饰方法可以制成许多水溶性壳聚糖衍生物,并且这些衍生物能被加工成理想的支架材料应用于软骨组织工程,代表性的例子是通过乙酸将氨基嵌合入壳聚糖中形成水溶性的脱乙酰壳多糖。目前,市面上出售的可注射、可交联二硫 键[50-51],具有光交联性的壳聚糖水凝胶,都是利用壳聚糖的化学改性形成的水溶性壳聚糖。 壳聚糖水凝胶已被广泛应用于骨组织工程中,作为支架材料,壳聚糖水凝胶能够模拟原位关节软骨水合胶原网,持软骨细胞的增殖、生长及新软骨组织的再生[52]。Gupta等[53]制备了壳聚糖-琼脂糖-明胶支架,将其植入兔膝关节软骨缺损处,发现植入后4周即出现显著的软骨再生,并且持续到术后8周,而未植入支架的对照组则没有出现软骨再生。另有研究者利用壳聚糖和聚乙烯醇制备一种具有热敏感性、可注射的壳聚糖-聚乙烯醇水凝胶,该水凝胶能够携带经转化生长因子β1转化的骨髓基质细胞,将该水凝胶注射到兔受损伤部位能够显著修复关节软骨缺损[54]。通常可将聚阳离子壳聚糖与阴离子聚电解质或硫酸氨基葡萄糖混合而生成离子水凝胶,形成不溶于水的复合物[55],壳聚糖也能在带多价阴离子小分子存在的条件下形成物理凝胶,如甘油磷酸酯[56]。Naderi-Meshkin等[57]利用甘油磷酸酯制备了壳聚糖-甘油磷酸钠羟乙基纤维水凝胶(CH-GP-HEC),该水凝胶具有生物相容性以及可降解性,在37 ℃条件下呈固态,并且此凝胶能够携带软骨生长因子或者骨髓基质细胞。将携带胰岛素样生长因子1 的壳聚糖-甘油磷酸钠羟乙基纤维水凝胶水凝胶注射到软骨损伤部位,发现其能持续释放胰岛素样生长因子1至少 8 d,并且适合骨髓基质细胞的分化和生长。除了物理交联外,壳聚糖还可通过化学交联形成凝胶。化学交联剂包括醛、京尼平,可以通过酶促反应与壳聚糖的胺基团作 用[58]。近期1篇综述集中讲述了如何将氨基酸通过化学反应交联到壳聚糖中[59]。 为了使壳聚糖水凝胶用于软骨组织工程,需要解决的关键问题是提高细胞黏附力,实验证实,含有硫酸软骨素质酸凝胶,通过与促软骨细胞黏附、增殖和软骨分化的生物活性物质偶联,可更好地支持细胞生命活动和软骨分化。另一实验,将牛关节软骨细胞植入一层薄硫酸软骨 素-壳聚糖水凝胶复合支架中,可观察到细胞呈圆形外观、限制性有丝分裂,Ⅱ型胶原蛋白及蛋白多糖的生成[55]。此外尚有其他天然来源的生物材料也可制作成复合水凝胶支架,如海藻酸钠[60]、明胶[61]、半乳糖和乳糖等[62-63]。有研究者将生长因子通过辛二酸及活化的N-羟基琥珀酰亚胺嵌入到冻干壳聚糖支架中,并将ATDC5小鼠软骨细胞接种于该支架上,再根据MTT测定法及产生出GAG和DNA评估细胞增殖情况,发现表皮生长因子较精氨酸-甘氨酸-天冬氨酸有更好的促细胞增殖效果[64]。对此近期有篇综述介绍了有关生长因子共价固定在组织工程的应用进展[65]。 2.3.2 透明质酸 透明质酸是由250-25 000个由N-乙酰基-D-葡糖胺和D-葡糖醛酸的二糖单元组合成的线性多糖结构,广泛存在于机体软骨、滑液和眼的玻璃体液中,它通过组装、聚集蛋白聚糖、大软骨蛋白多糖使关节软骨细胞外基质成为弹性结构,故透明质酸是糖胺聚糖的重要成分。该蛋白聚糖/透明质酸可为细胞外基质蓄积大量的水分,对软骨的黏弹性能和润滑起着重要作用。关节腔内注射透明质酸是目前骨关节炎早期治疗中一种行之有效的治疗方法,但透明质酸分子量的高低对治疗效果具有决定性作用,低分子量使其具有致炎作用,而高分子量使其具有抗炎保护作用[66]。 透明质酸对蛋白质的黏附及提供软骨细胞和细胞外基质之间附着位点都具有关键作用。已有研究者将透明质酸复合材料作为支架用于软骨再生。Solchaga等[67]把透明质酸制成海绵支架,用于治疗骨软骨缺损,达到促进受损细胞的软骨分化及透明软骨细胞再生的目的。另外透明质酸可通过羧酸基团反应而进行化学改性,透明质酸的羧基基团与不同的醇类进行酯化反应可以产生出不同降解度具备生物相容性的衍生物。如交联二硫化物[68]、二胺和酪胺等[69],可使透明质酸化学改性成为许多不同功能的可交联透明质酸衍生物,这些衍生物则可以用来生产不同的透明质酸水凝胶。目前光聚合或光交联已成为在可注射透明质酸内整合入软骨细胞最重要的方法,其中透明质酸分子改性的部分称光交联功能团,例如甲基丙烯酸甲 酯[70-72]、甲基丙烯酸缩水甘油酯[73-74],光交联反应可通过可见光或低能量的紫外线照射激活,多种光交联系统具有低度的黏性,因而方便注射和填充成不规则软骨缺损。 尽管透明质酸水凝胶具有颇多促进软骨再生的优势,但因透明质酸材料中的交联密度不够导致其在体内不能提供足够的机械性能,使透明质酸水凝胶作为软骨修复支架材料受到一定限制[72-75]。如何控制透明质酸材料中的交联密度,以便能够提高其机械性能和可降解性,Nettles等[72]利用含伯醇甲基丙烯酸对透明质酸进行改性,光交联后,透明质酸-含甲基丙烯酸复合体表现出0.63 kPa的压缩模量和0.3 kPa的剪切模量,合成的透明质酸网络结构交联密度可以通过控制甲基丙烯酸脂的程度、大分子单体的分子量和大分子单体浓度来调整。不同条件控制下,透明质酸-含甲基丙烯酸复合体表现出大体情况是:根据大单体浓度(2%-20%)的不同,凝胶网的抗压模量可从2-100 kPa。在100 U/mL透明质酸酶存在的条件下,水凝胶网的降解时间可以从少于1 h改变为超过38 d,但随着大单体浓度增加,可植入细胞数减少。实验中还发现,含有少量烷基(如十二烷基)侧链的透明质酸,可形成具有可逆性分子间疏水作用的可注射水凝胶,并且这种水凝胶的化学交联得到进一步增强[76]。 综上所述,虽然透明质酸水凝胶在动物模型研究中表现出很高的促细胞增值及软骨再生作用,但由于其无法提供足够要求的机械力学性能使其在应用中仍面临许多问题。 2.4 天然高聚物混合水凝胶 大多数天然高聚物是聚合高分子电解质,含有丰富的氨基、羟基、羧基、硫酸根或磷酸基团,故可根据静电作用、氢键或酯化作用将几种天然高聚物结合起来,这样可以避免一些有毒物质的引入。许多多糖能在适当pH值环境下形成聚电解质,例如脱乙酰壳多糖在pH<6的环境下可携带正电荷,而透明质酸是聚阴离子,由于聚阴离子存在,使其具有更佳的机械性能及软骨细胞依附能力,且这类支架材料对pH、温度、离子键强度敏感,故适合用于药物、基因及细胞的传导。 将明胶与壳聚糖[77-79]、透明质酸等混合也能促进其机械性能的提升及降解性的控制[79]。冻干技术制造的壳聚 糖-明胶复合支架材料有利于软骨细胞的黏附、分化和增殖。将种植软骨细胞的复合支架植入皮下以支持透明软骨细胞生长,9周后观察到新生软骨组织的结构与强度类似于16周的原生软骨组织。另一项研究显示,壳聚糖基明胶混合溶液经过冷冻、交联戊二醛、解冻、冻干等步骤得到大孔冷冻凝胶,这种凝胶具有良好的促细胞黏附、增殖及产生分泌细胞外基质的功能[78]。另外,可将壳聚糖与丝蛋白混合产生丝蛋白基生物材料。Bhardwaj等[80]开发了一种由丝蛋白与壳聚糖混合组成的三维多孔聚电解质复合支架,这种复合支架较单一高聚物生成的支架具有更高的抗压强度和弹性模量。此外,该支架材料与壳聚糖支架相比酶降解性更低,且壳聚糖的存在又使其抗菌能力得到提升。丝蛋白壳聚糖共混支架有利于软骨细胞的黏附和生 长[81],以及大鼠间充质干细胞向软骨分化[82]。Garcia-Fuentes等[83]用透明质酸作为制孔剂,将丝蛋白与透明质酸混合水溶液冻干处理后制备出多孔支架,并植入间充质干细胞,发现该复合支架兼备丝蛋白的强度及透明质酸的生物性能,其促进细胞及软组织生成的作用较单纯丝蛋白支架更佳。 2.5 水凝胶复合支架在软骨组织工程研究中的设计原则 合成聚合物水凝胶旨在促进及支持软骨细胞表型、生长、增殖和新软骨组织的再生、水凝胶的物质营养传输功能。多孔结构有助于支架材料和周围环境之间的物质传递,能使植入其中的软骨细胞获得类似于原生软骨的内环境。此外,周围组织能长入支架的多孔结构,使支架与原位组织融合一体。冷冻-解冻是最重要的制备多孔水凝胶方法之一,在冷冻过程中凝胶形成实体材料与冰分离的结构,解冻后,由于冰的融化而制备出多孔结构。另一种制备方法是将水凝胶与大小不同的微粒相混合,在选择性地除去这些粒子后可以获得多孔材料。同时这些微粒能作为药物、基因或细胞的载体,随着培养过程中降解而释放出生物学活性物质。 目前,多孔水凝胶用于软骨组织工程最主要的问题是其较低的机械力学性能,混合糖胺聚糖后其机械力学性能得以改善,但要在其结构与机械性能之间求得平衡尚需进一布研究。因为随着深度的变化,关节软骨的基质结构、软骨细胞类型及密度具有差异[84-85],所以良好的水凝胶材料应模拟这种基于深度的差异性,由此在软骨组织工程中应制备出以分层水凝胶为基础的复合支架结构。"
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