摘要
开发具有成骨诱导的骨填充材料是促进骨再生的重要研究方向。自组装多肽水凝胶凭借其高度的仿生人工细胞外基质结构、低免疫原性、易于合成及修饰、载药灵活等优势为骨组织修复提供了一个高效治疗手段。本文讨论了自组装多肽水凝胶的设计原则,报道了自组装多肽结构特征及组装机制,重点介绍了自组装多肽骨修复水凝胶在递送干细胞、血管内皮细胞、骨形成蛋白、成骨因子以及小分子化合物等方面的最新研究进展,总结了限制自组装多肽水凝胶发展的瓶颈和未来发展方向,为构建高成骨性能凝胶递送系统提供理论参考。
随着高强度体育活动的流行和人口老龄化加剧,我国每年因各种原因导致的骨伤患者达数百万,尤其是60岁以上的老人,而骨质疏松直接导致骨折率上升。另外,由于年龄的增加,骨折部位血管新生、淋巴循环以及祖细胞等机能降低,受损骨组织的自我修复功能受限,导致病程延
目前研究的可注射凝胶递送系统基质多由明
自组装是自然界中普遍存在的现象,例如细胞膜的形成、蛋白质折叠和DNA双螺旋结构,这是生命科学中最重要的组成部分之一。通过模仿具有自组装功能的蛋白结构可以设计出多种具有组装功能的仿生多肽。除了仿生设计,从头设计也是自组装多肽设计的主要途径。从头设计自组装肽的关键是理解各种分子间相互作用,包括氢键、静电相互作用、π-π堆积、范德华力和色散力、金属-配体配合物和熵力,如疏水性和排除体积相互作
自组装肽的结构包括:多肽氨基酸序列即一级结构,以及相邻氨基酸之间的分子内相互作用形成的二级结构,如α-螺旋、β-折叠和无规则卷曲,以及多肽分子间形成的具有特定折叠结构的分子组
亲疏水作用、静电作用、氢键以及π-π堆积等分子间相互作用是驱动多肽自组装实现凝胶化的主要作用力。亮氨酸拉链(leucine zipper,LZ)是典型的基于疏水作用形成的α-螺旋结构,这种卷曲的螺旋结构能够在疏水作用下进一步互相聚集,形成物理交联凝胶。Huang
胶原蛋白是细胞外基质(ECM)的主要结构成分,占人体蛋白质含量的25% ~ 35%。目前,已至少发现了30余种胶原蛋白链的编码基因,可以形成16种以上的胶原蛋白分子。根据其结构和超分子组织,它们可分为原纤维形成胶原蛋白(Ⅰ、Ⅱ、Ⅲ、Ⅴ和XI型)、基膜胶原蛋白(Ⅳ型)、微纤维胶原蛋白(Ⅵ型)、六边网状胶原蛋白(Ⅷ、Ⅹ型)

Figure 1 Self-assembly and potential characterization of peptide amphiphiles
A: Mechanism of self-assembly of peptide amphiphiles; B: Self-assembly of peptide amphiphiles in their respective environment, leading to hydrogel formation at different pHs through counter ion stabilization, and upon mixing them together, they showed gelation at physiological pH because of electrostatic interaction; C: Zeta potential graph showing the overall surface charge at different mixing ratios of the peptide
通常,离子自互补型肽的结构是由疏水氨基酸残基(丙氨酸、缬氨酸、异亮氨酸或苯丙氨酸)和带有正电荷(赖氨酸或精氨酸)或负电荷(天冬氨酸或谷氨酸)的亲水氨基酸残基交替排

Figure 2 Charge distribution pattern and assembly mechanism of ionic self-complementary peptides
A: Self-assembly of a peptide consisting of alanine, glutamic acid and lysine (dEAK) into nanofibers, which were then entangled into 3D scaffolds. The self-assembling process was a consequence of complementary ionic interactions between charged residues (red for negatively charged glutamic acid and blue for the positively charged lysine residues), which could have different arrangements: modulus I, ----++++; modulus II, --++--++; modulus III, -+-+-+-
两亲性多肽(PA)是另一种典型的自组装多肽,PA结构中主要包含N端疏水端、诱导形成β折叠的中间肽序列和一个亲水的C端肽序

Figure 3 Construction and characterization of amphiphilic tripeptides
A: Schematic illustration of the amphiphilic tripeptides, designed with an N-terminal phenylalanine residue and a C-terminal aspartic acid residue flanking a variable residue (X). This variable residue was modified to alter R-group volume and hydrogen bonding propensity by including a glycine (G), alanine (A), valine (V), leucine (L), or isoleucine (I) residue at this position; B: (Top row) Representative images of 20 mmol/L tripeptide solutions in water demonstrating those that form sol (FGD and FAD) and others that form hydrogels (FVD, FLD, and FID). (Second and third row) Representative TEM images for each tripeptide sequence, acquired from films cast from the 20 mmol/L solutions and negatively stained with uranyl acetat
自组装多肽水凝胶能够仿生模拟细胞外基质,有助于细胞的黏附、迁移,具有低免疫原性、易于负载多种细胞及生物活性物质等优势,一直以来,被广泛用于递送干细胞、成骨细胞、软骨细胞、内皮细胞以及多种成骨蛋白或因子等,此外,水凝胶还可以通过掺入不同的纳米填料,增强水凝胶的机械性能,在体内外表现出良好的成骨性能。
将功能性细胞如干细胞直接递送至在骨伤处能够通过成骨分化以及分泌相关生长因子促进骨再生。自组装多肽水凝胶因其良好的生物相容性以及能够为细胞黏附、迁移和培养提供良好条件已成为多种干细胞递送和培养的重要基
为了促进骨组织再生,多种成骨活性剂,包括骨形成蛋白(BMPs

Figure 4 Schematic illustration of the construction of NapFFY-OGP hydrogel and its application in promoting bone regeneratio
近年来,自组装多肽作为新兴生物材料,其良好的生物相容性、可注射性、低免疫原性以及灵活负载细胞、蛋白、小分子药物的能力使之在骨再生领域大放异彩。然而,骨再生是一个复杂的过程,受到多种因素的影响,基于凝胶灵活多样的负载性能和仿生性能,可探索利用多肽水凝胶同时负载多种干细胞及活性因子,发挥多策略协同增效的作用。此外,通过对自组装多肽进行功能化修饰,结合3D打印等技术进一步模拟骨组织再生微环境,除了负载干细胞及活性因子,还可以尝试在凝胶中进行骨组织的类器官培养再植入体内,以提高大段骨缺损的再生能力。本课题组在多肽长效凝胶递释系统研究中发现,在多肽凝胶中引入MMPs的降解序列,可调控多肽凝胶在体内的降解速率。同时,天然存在的L型氨基酸具有快速水解的特点,非天然D-氨基酸残基组成的D型多肽具有抗蛋白水解性,使得D型多肽具有比L型多肽具有更高的稳定性。此外,多肽组装序列的交联度决定了凝胶的硬度和孔隙大小,对凝胶降解及其负载药物的释放速率都产生影响。为了调控凝胶的降解速率,本课题组研究了氨基酸构型和交联度这两大影响凝胶降解的因素,调控凝胶的降解动力学,能够满足长期降解释放药物的治疗需求。总之,在未来的发展中,自组装多肽骨修复水凝胶的开发应集中于:构建多策略联用的仿生凝胶、提高水凝胶力学强度和控制凝胶降解速率。随着骨组织工程的不断进步以及多技术的交叉融合,自组装多肽水凝胶有望为骨缺损提供更多的临床治疗方案。
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