Abstract 抽象
Osteoarthritis (OA) is a common joint disease characterized by cartilage degeneration. It can cause severe pain, deformity and even amputation risk. However, existing clinical treatment methods for cartilage repair present certain deficiencies. Meanwhile, the repair effect of cartilage tissue engineering is also unsatisfactory. Cartilage organoids are multicellular aggregates with cartilage-like three-dimensional structure and function. On the one hand, cartilage organoids can be used to explore the pathogenesis of OA by constructing disease models. On the other hand, it can be used as filler for rapid cartilage repair. Extracellular matrix (ECM)-like three-dimensional environment is the key to construct cartilage organoids. Silk fibroin (SF)-based hydrogels not only have ECM-like structure, but also have unique mechanical properties and remarkable biocompatibility. Therefore, SF-based hydrogels are considered as ideal biomaterials for constructing cartilage organoids. In this review, we reviewed the studies of cartilage organoids and SF-based hydrogels. The advantages of SF-based hydrogels in constructing cartilage organoids and the iterative optimization of cartilage organoids through designing hydrogels by using artificial intelligence (AI) calculation are also discussed. This review aims to provide a theoretical basis for the treatment of OA using SF-based biomaterials and cartilage organoids.
骨关节炎 （OA） 是一种以软骨退化为特征的常见关节疾病。它会导致严重的疼痛、畸形，甚至有截肢的风险。然而，现有的临床软骨修复治疗方法存在一定的不足。同时，软骨组织工程的修复效果也不尽如人意。软骨类器官是具有软骨样三维结构和功能的多细胞聚集体。一方面，软骨类器官可用于通过构建疾病模型来探索 OA 的发病机制。另一方面，它可以用作快速软骨修复的填充剂。细胞外基质 （ECM） 样三维环境是构建软骨类器官的关键。基于丝素蛋白 （SF） 的水凝胶不仅具有类似 ECM 的结构，而且还具有独特的机械性能和显著的生物相容性。因此，基于 SF 的水凝胶被认为是构建软骨类器官的理想生物材料。在本综述中，我们回顾了软骨类器官和基于 SF 的水凝胶的研究。还讨论了基于 SF 的水凝胶在构建软骨类器官方面的优势，以及通过使用人工智能 （AI） 计算设计水凝胶来迭代优化软骨类器官。本文旨在为使用 SF 基生物材料和软骨类器官治疗 OA 提供理论依据。
Keywords: Osteoarthritis, Silk fibroin, Hydrogels, Cartilage organoids, Cartilage regeneration.
关键字：骨关节炎、丝素蛋白、水凝胶、软骨类器官、软骨再生。
1. Introduction 1. 引言
Osteoarthritis (OA) is a joint disease characterized by cartilage degeneration 1-5. Pathogenic factors of OA are mainly associated with obesity and age 6. As the global aging and obese population rises, the number of OA patients will increase sharply in the next decade 7, 8. Statistically, approximately 595 million people worldwide are affected by OA. And by 2050, the prevalence is projected to increase by over 200%, resulting in substantial economic burdens on both individuals and society 9. Cartilage degeneration plays a significant role in the progression of OA, as it contributes not only to the disease outcome but also to its worsening 10, 11. Therefore, repairing cartilage is a valuable strategy for the treatment and prevention of OA.
骨关节炎 （OA） 是一种以软骨退化 1 为特征的关节疾病 - 5 。OA 的致病因素主要与肥胖和年龄 6 有关。随着全球老龄化和肥胖人口的增加，未来十年 7 OA 患者的数量将急剧增加。 8 据统计，全球约有 5.95 亿人受到 OA 的影响。到 2050 年，患病率预计将增加 200% 以上，给个人和社会带来沉重的经济负担 9 。软骨变性在 OA 的进展中起着重要作用，因为它不仅会导致疾病结果，还会导致其恶化 10 。 11 因此，修复软骨是治疗和预防 OA 的宝贵策略。
Cartilage has limited self-repair capacity 12-14. As an immune-privileged tissue, it makes engineered cartilage analogs attractive candidates for off-the-shelf grafts in allogeneic transplantation. Organoids are simplified multicellular structures that develop from stem cells or organ progenitors through in vitro 3D culture combined with targeted induction technology, enabling the formation of organ-specific architectures and functions 15-17. Specifically, cartilage organoids are tissues with cartilage structure, function, physiological and pathological characteristics through cultivating and assembling stem cells or chondrocytes 18, 19. This offers a novel approach for cartilage regeneration. Compared to autologous chondrocyte implantation (ACI), cartilage organoid transplantation eliminates the need for a secondary surgery to harvest cells, streamlining the procedure and reducing postoperative complications. Techniques like ACI and matrix-induced autologous chondrocyte implantation (MACI), which rely on 2D chondrocyte expansion, are time-consuming and increase the risk of dedifferentiation into fibrocartilage 20-22. In contrast, cartilage organoids have distinct advantages: they replicate the natural cartilage structure and can be directly implanted to repair defects, ensuring that the regenerated tissue closely resembles native cartilage. Furthermore, as they already mimic cartilage properties, cartilage organoids can seamlessly integrate with native tissue, minimizing the need for further regeneration post-implantation and thus enhancing the overall repair efficiency 23.
软骨的自我修复能力 12 有限 - 14 。作为一种免疫特权组织，它使工程软骨类似物成为同种异体移植中现成移植物的有吸引力的候选者。类器官是从干细胞或器官祖细胞通过体外 3D 培养结合靶向诱导技术发育而来的简化多细胞结构，能够形成器官特异性结构和功能 17 15 。具体来说，软骨类器官是通过培养和组装干细胞或软骨细胞 18 而具有软骨结构、功能、生理和病理特征的组织。 19 这为软骨再生提供了一种新的方法。与自体软骨细胞植入 （ACI） 相比，软骨类器官移植无需二次手术来采集细胞，简化了手术并减少了术后并发症。像 ACI 和基质诱导的自体软骨细胞植入 （MACI） 这样的技术依赖于 2D 软骨细胞扩增，非常耗时，并且增加了去分化为纤维软骨 20 的风险 - 22 。相比之下，软骨类器官具有明显的优势：它们复制天然软骨结构，可以直接植入以修复缺损，确保再生组织与天然软骨非常相似。此外，由于它们已经模拟了软骨的特性，软骨类器官可以与天然组织无缝集成，最大限度地减少植入后进一步再生的需要，从而提高整体修复效率 23 。
Organoid construction methods are broadly divided into scaffold-free self-organization and biomaterial-based co-cultivation. Scaffold-free self-organization allows mesenchymal stem cells (MSCs) to naturally form complex tissue structures. This method is straightforward and has a lower risk of contamination, but it struggles with achieving consistent organoid size and uniformity. On the other hand, biomaterial-assisted co-cultivation uses scaffolds like hydrogels to provide structural support, allowing precise control over the organoid architecture and customization of the environment. This makes it ideal for developing organoids with consistent and predictable properties. Currently, Matrigel is the most commonly used material for organoid construction. However, its undefined composition and batch-to-batch variability result in inconsistent mechanical strength, making it less reliable for precise experimental needs. Additionally, Matrigel lacks the flexibility for customization in specific organoid culture contexts 18, 24, 25. Silk fibroin (SF) hydrogels, as a natural macromolecular material, offer several advantages over Matrigel, including a well-defined structure, controllable mechanical properties, and high customizability. These features make SF hydrogels more suitable for precise tissue engineering applications 26-30. Moreover, SF's excellent processability allows it to be adapted to various processing methods and functional modifications, making it versatile for preparing different types of cartilage tissue engineering materials 31-33. Furthermore, SF hydrogels have excellent printability, enabling their use in 3D bioprinting for advanced biofabrication techniques 34-36. Therefore, SF-based hydrogel has a great prospect for cartilage regeneration and cartilage organoids construction 37.
类器官构建方法大致分为无支架自组织和基于生物材料的共培养。无支架的自组织使间充质干细胞 （MSC） 能够自然形成复杂的组织结构。这种方法简单明了，污染风险较低，但难以实现一致的类器官大小和均匀性。另一方面，生物材料辅助共培养使用水凝胶等支架提供结构支撑，从而可以精确控制类器官结构和环境定制。这使其成为开发具有一致和可预测特性的类器官的理想选择。目前，Matrigel 是类器官构建最常用的材料。然而，其不确定的成分和批次间的可变性导致机械强度不一致，使其在满足精确实验需求方面不太可靠。此外，Matrigel 缺乏在特定类器官培养环境中 18 进行定制的灵活性， 24 、 25 。丝素蛋白 （SF） 水凝胶作为一种天然大分子材料，与 Matrigel 相比具有多项优势，包括结构明确、机械性能可控和高度可定制性。这些特性使 SF 水凝胶更适合于精确的组织工程应用 26 - 30 .此外，SF 出色的加工性能使其能够适应各种加工方法和功能修饰，使其可用于制备不同类型的软骨组织工程材料 31 - 33 。 此外，SF 水凝胶具有出色的可打印性，使其能够用于 3D 生物打印，用于先进的生物制造技术 34 - 36 。因此，SF 基水凝胶在软骨再生和软骨类器官构建 37 方面具有很大的前景。
Herein, we summarize recent research on cartilage organoids and SF-based hydrogels, highlighting the advantages of SF-based hydrogels for cartilage organoid construction. The iterative optimization of cartilage organoids through designing hydrogels by using artificial intelligence (AI) calculation is also discussed (Figure 1). We hope that this review can provide a reference for cartilage organoids construction and a promising therapeutic strategy for OA.
在此，我们总结了最近关于软骨类器官和 SF 基水凝胶的研究，强调了 SF 基水凝胶在软骨类器官构建中的优势。还讨论了通过使用人工智能 （AI） 计算设计水凝胶来迭代优化软骨类器官（图 1 ）。我们希望本文能为软骨类器官的构建提供参考，并为 OA 的治疗策略提供有前途的治疗策略。
Figure 1. 图 1.
Schematic diagram of SF-based hydrogels for construction of cartilage organoids. Created with BioRender.com.
用于构建软骨类器官的基于 SF 的水凝胶示意图。使用 BioRender.com 创建。
2. Research progress in cartilage organoids
2. 软骨类器官的研究进展
Cartilage is a critical component of the human body, providing essential support for mechanical reinforcement, cushioning, and protection (Figure 2). Cartilage development commences with a cartilaginous template constituted by embryonic mesenchymal stem cells. During this stage, primitive embryonic mesenchymal cells initiate differentiation into chondroblasts 38-40. These chondroblasts proliferate and synthesize collagen fibers and glycosaminoglycans, establishing the extracellular matrix of cartilage. The distribution and orientation of collagen fibers and glycosaminoglycans, along with the degree of chondrocyte calcification, contribute to the multi-layered structure of cartilage. For example, articular cartilage can be broadly categorized into hyaline cartilage and calcified cartilage layers. Cartilage serves various physiological roles, with the most crucial being to provide cushioning and support within joints. It reduces friction between bones and protects them from wear and tear. Moreover, cartilage effectively disperses pressure generated during joint movement, thereby protecting joint tissues. In structures like the nose and ears, the elasticity and flexibility of cartilage allow it to maintain specific shapes 41, 42. Currently, artificially engineered cartilage organoids structures are predominantly composed of uniformly spherical cell clusters or uniformly layered tissues. Research in cartilage organoids mainly focuses on understanding cartilage-related mechanisms, advancing plastic surgery, and promoting cartilage repair (Figure 3 and Figure 4).
软骨是人体的关键组成部分，为机械加固、缓冲和保护提供必要的支撑（图 2 ）。软骨发育始于由胚胎间充质干细胞构成的软骨模板。在此阶段，原始胚胎间充质细胞开始分化为成软骨母细胞 38 - 40 。这些软骨母细胞增殖并合成胶原纤维和糖胺聚糖，建立软骨的细胞外基质。胶原纤维和糖胺聚糖的分布和取向，以及软骨细胞钙化的程度，有助于软骨的多层结构。例如，关节软骨大致可分为透明软骨和钙化软骨层。软骨具有多种生理作用，其中最重要的是在关节内提供缓冲和支撑。它减少了骨骼之间的摩擦并保护它们免受磨损。此外，软骨可有效分散关节运动过程中产生的压力，从而保护关节组织。在鼻子和耳朵等结构中，软骨的弹性和柔韧性使其能够保持特定的形状 41 。 42 目前，人工工程软骨类器官结构主要由均匀的球形细胞簇或均匀分层的组织组成。软骨类器官的研究主要集中在了解软骨相关机制、推进整形手术和促进软骨修复（图 3 和图 4 ）。
Figure 2. 图 2.
Differences between articular cartilage and cartilage organoids in origin, location, structure, and function. Created with BioRender.com.
关节软骨和软骨类器官在起源、位置、结构和功能上的差异。使用 BioRender.com 创建。
Figure 3. 图 3.
Cartilage organoids in mechanism exploration and plastic surgery: (A) Reproduced with permission from ref 43; Copyright 1990, Elsevier. (B) Reproduced with permission from ref 44; Copyright 2016, Elsevier. (C) Reproduced with permission from ref 45; Copyright 2018, MDPI. (D) Reproduced with permission from ref 46; Copyright 2021, Royal Society of Chemistry. (E) Reproduced with permission from ref 48; Copyright 1997, Wolters Kluwer Health, Inc. (F) Reproduced with permission from ref 49; Copyright 2014, Elsevier. (G) Reproduced with permission from ref 50; Copyright 2019, Royal Society of Chemistry. (H) Reproduced with permission from ref 51; Copyright 2023, Springer Nature.
机制探索和整形手术中的软骨类器官：（A） 经参考文献许可转载 43 ;版权所有 1990，爱思唯尔。（B） 经 ref 许可转载 44 ;版权所有 2016，爱思唯尔。（C） 经 ref 许可转载 45 ;版权所有 2018，MDPI。（D） 经 ref 许可转载 46 ;版权所有 2021，英国皇家化学学会。（E） 经 ref 许可转载 48 ;版权所有 1997， Wolters Kluwer Health， Inc. （F） 经参考文献许可转载 49 ;版权所有 2014，爱思唯尔。（G） 经 ref 许可转载 50 ;版权所有 2019，英国皇家化学学会。（H） 经 ref 许可转载 51 ;版权所有 2023，施普林格自然。
Figure 4. 图 4.
Cartilage organoids in tissue engineering: (A) Reproduced with permission from ref 52; Copyright 2020, Elsevier. (B) Reproduced with permission from ref 53; Copyright 2020, American Chemical Society. (C) Reproduced with permission from ref 54; Copyright 2021, Elsevier. (D) Reproduced with permission from ref 55; Copyright 2023, Springer Nature. (E) Reproduced with permission from ref 56; Copyright 2023, Elsevier. (F) Reproduced with permission from ref 57; Copyright 2023, Wiley-VCH GmbH.
组织工程中的软骨类器官：（A） 经参考文献许可转载 52 ;版权所有 2020，爱思唯尔。（B） 经 ref 许可转载 53 ;版权所有 2020，美国化学会。（C） 经 ref 许可转载 54 ;版权所有 2021，爱思唯尔。（D） 经 ref 许可转载 55 ;版权所有 2023，施普林格自然。（E） 经 ref 许可转载 56 ;版权所有 2023，爱思唯尔。（F） 经 ref 许可转载 57 ;版权所有 2023，Wiley-VCH GmbH。
Cartilage organoids were initially applied to explore cartilage-related mechanisms. In 1990, Somogyi et al. pioneered the in vitro construction of cartilage organoids. By examining their morphology and ECM, they found that osteoblasts promoted mineralization within cartilage, whereas fibroblasts had inhibitory effects (Figure 3A) 43. With the advancement of technology, cartilage organoids have also been utilized to investigate the impact of growth factors (TGF-β) and osmotic pressure on cartilage development (Figure 3B-C) 44, 45. Furthermore, cartilage organoids have also been employed to construct organ-on-a-chip models to study inter-tissue interactions. For example, Ertl et al. constructed the chondro-synovial organoid chip to simulate cross-talk between individual synovial and cartilage organoids. Co-culturing with synovial organoids, it was demonstrated that cartilage organoids induced a heightened degree of cartilage physiology and structure, along with distinct cellular cytokine responses compared to their respective monocultures, underscoring the significance of inter-tissue cross-talk at the organ level in models of arthritic diseases (Figure 3D) 46. In addition to exploring cartilage-related mechanisms, cartilage organoids have also demonstrated remarkable potential in plastic surgery. Notably, one of the most matured applications involves the construction of auricular-shaped cartilage organoids, particularly for reconstructing human ears 47. In 1997, Cao et al. constructed the first human-ear mouse with a polyglycolic acid fiber scaffold (Figure 3E) 48. Subsequently, Zhou et al. advanced the field by constructing human auricular cartilage organoids through co-culturing microtia chondrocytes and bone mesenchymal stem cells (BMSCs) (Figure 3F) 49. This approach not only enhanced the shape stability of human auricular cartilage organoids but also effectively reduced construction costs. In 2019, Alsberg et al. further advanced the field by enhancing the shape resolution of human auricular cartilage organoids using 3D bioprinting technology (Figure 3G) 50. Building on these advances, Lei et al. constructed homogeneous and mature human auricular cartilage organoids using synthetically engineered fiber-reinforced SF super elastic absorbent sponges (Figure 3H) 51.
软骨类器官最初用于探索软骨相关机制。1990 年，Somogyi 等人率先在体外构建软骨类器官。通过检查它们的形态和 ECM，他们发现成骨细胞促进软骨内的矿化，而成纤维细胞具有抑制作用（图 3 A）。 43 随着技术的进步，软骨类器官也被用于研究生长因子 （TGF-β） 和渗透压对软骨发育的影响（图 3 BC）， 44 45 。此外，软骨类器官也被用于构建器官芯片模型以研究组织间相互作用。例如，Ertl 等人构建了软骨滑膜类器官芯片，以模拟单个滑膜和软骨类器官之间的串扰。与滑膜类器官共培养，证明软骨类器官诱导了更高程度的软骨生理学和结构，以及与它们各自的单一培养相比，具有不同的细胞因子反应，强调了器官水平组织间串扰在关节炎疾病模型中的重要性（图 3 D） 46 .除了探索软骨相关机制外，软骨类器官在整形手术中也显示出显着的潜力。值得注意的是，最成熟的应用之一涉及耳廓形状软骨类器官的构建，特别是用于重建人耳 47 。1997 年，Cao 等人构建了第一只具有聚乙醇酸纤维支架的人耳小鼠（图 3 E）。 48 随后，周 et al.通过共培养小耳畸形软骨细胞和骨间充质干细胞 （BMSC） 构建人耳软骨类器官，推动了该领域的发展（图 3 F）。 49 这种方法不仅增强了人类耳软骨类器官的形状稳定性，而且有效地降低了施工成本。2019 年，Alsberg 等人通过使用 3D 生物打印技术提高人类耳软骨类器官的形状分辨率，进一步推动了该领域的发展（图 3 G）。 50 在这些进展的基础上，Lei 等人使用合成工程纤维增强 SF 超弹性吸收海绵构建了均质和成熟的人耳软骨类器官（图 3 H）。 51
In recent years, researchers have increasingly directed their focus towards applying cartilage organoids in cartilage repair. For instance, Lin et al. and Yin et al. achieved the stacking of cartilage microsphere organoids using materials with self-assembling properties, enabling the construction of larger-volume cartilage organoids (Figure 4A-B) 52, 53. Papantoniou et al. assembled cartilage microtissues derived from iPSC-derived chondrocytes with callus organoids (COs) sourced from human plasmacytoid dendritic cells to constructed layered osteochondral organoids (Figure 4C) 54. It is worth noting that cartilage organoids constructed by Tsumaki et al. and Xing et al. successfully achieved cartilage repair in primates and canids (Figure 4D-E) 55, 56. In addition, Ouyang et al. constructed macromass cartilage organoids up to 3 mm in diameter by culturing human polydactyly chondrocytes in customized culture, which can be used as implants to facilitate cartilage defect repair (Figure 4F) 57. Based on these studies, it is evident that constructing cartilage organoids requires providing material support for seed cells and directing their chondrogenic differentiation. Consequently, to construct cartilage organoids that closely mimic the natural cartilage structure and function, novel smart biomaterials need to be designed to furnish the chondrogenic microenvironment necessary for seed cells.
近年来，研究人员越来越多地将注意力转向将软骨类器官应用于软骨修复。例如，Lin 等人和 Yin 等人使用具有自组装特性的材料实现了软骨微球类器官的堆叠，从而能够构建更大容量的软骨类器官（图 4 A-B）。 52 53 Papantoniou 等人将源自 iPSC 衍生的软骨细胞的软骨微组织与源自人浆细胞样树突状细胞的愈伤组织类器官 （CO） 组装成构建的分层骨软骨类器官（图 4 C）。 54 值得注意的是，Tsumaki 等人和 Xing 等人构建的软骨类器官成功地实现了灵长类动物和犬科动物的软骨修复（图 4 D-E）、 55 56 .此外，Ouyang 等人通过在定制培养物中培养人多指软骨细胞构建了直径达 3 mm 的大质量软骨类器官，其可用作植入物以促进软骨缺损修复（图 4 F）。 57 基于这些研究，很明显，构建软骨类器官需要为种子细胞提供物质支持并指导其软骨形成分化。因此，为了构建与天然软骨结构和功能紧密相似的软骨类器官，需要设计新型智能生物材料来提供种子细胞所需的软骨形成微环境。
3. Preparation of silk fibroin-based hydrogel
3. 丝素蛋白基水凝胶的制备
3.1. Characteristics of silk fibroin
3.1. 丝素蛋白的特点
SF is a naturally occurring macromolecular material produced by a range of animals, including silkworms, spiders, scorpions, mites, and flies 58. It is worth noting that SF of different origins has obvious differences in structure and properties. Among them, silkworms-derived SF has been widely studied and applied in the clinic because of its unique mechanical properties and abundant yield 59, 60. Hence, this review only discusses the silkworms derived SF. A single silk filament is composed of two strands of SF, enveloped in sericin (Figure 5A) 61.
SF 是一种天然存在的大分子材料，由一系列动物产生，包括蚕、蜘蛛、蝎子、螨虫和苍蝇 58 。值得注意的是，不同产地的 SF 在结构和性质上存在明显差异。其中，家蚕来源的SF因其独特的力学性能和丰富的产量 59 ，在临床上得到了广泛的研究和应用。 60 因此，本文仅讨论家蚕来源的 SF。单丝由两股 SF 组成，包裹在丝胶蛋白中（图 5 A）。 61
Figure 5. 图 5.
Source and structure of SF: (A) SF can be extracted from cocoon of silkworm. (B) SF molecule is composed of heavy chain and light chain connected by disulfide bond. (C) Crystal structure consists of GAGAGS, GAGAGY and GAGAGSGAAS repetitive sequences. Created with BioRender.com.
SF的来源和结构： （A） SF可以从蚕茧中提取。（B） SF 分子由二硫键连接的重链和轻链组成。（C） 晶体结构由 GAGAGS、GAGAGY 和 GAGAGSGAAS 重复序列组成。使用 BioRender.com 创建。
The molecular structure of SF is very complex, which is composed of disulfide-linked heavy chain and light chain (Figure 5B). The heavy chain includes non-repetitive C-terminal and N-terminal, along with 11 hydrophilic segments composed of 31 amino acid residues and 12 hydrophobic segments. The hydrophobic segments mainly contain Gly-X repeats, where X can be Ala (65%), Ser (23%), or Tyr (9%). Repeated sequences include Gly-Ala-Gly-Ala-Gly-Ser (GAGAGS), Gly-Ala-Gly-Ala-Gly-Tyr (GAGAGY), and Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Ser (GAGAGSGAAS). These repetitive sequences can form crystalline β-sheet structures through hydrophobic interactions (Figure 5C)
26. Contrary to the heavy chain, the amino acid sequence of the light chain is disordered and tends to form an amorphous structure 62. Recent studies have revealed that SF achieves interfacial self-assembly due to its amphiphilic molecular structure, which promotes the formation of β-sheets at interfaces. This property is particularly useful in hydrogel formation, as the conformation of SF can be modulated by adjusting the water-to-organic phase ratio. These adjustments enable the creation of hydrogels specifically optimized for cartilage regeneration, offering enhanced mechanical properties and bioactivity for improved tissue repair 63.
SF 的分子结构非常复杂，由二硫键连接的重链和轻链组成（图 5 B）。重链包括非重复的 C 端和 N 端，以及由 31 个氨基酸残基和 12 个疏水片段组成的 11 个亲水片段。疏水片段主要包含 Gly-X 重复序列，其中 X 可以是 Ala （65%）、Ser （23%） 或 Tyr （9%）。重复序列包括 Gly-Ala-Gly-Ala-Gly-Ser （GAGAGS）、Gly-Ala-Gly-Ala-Gly-Tyr （GAGAGY） 和 Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Ser （GAGAGSGAAS）。这些重复序列可以通过疏水相互作用形成结晶β片结构（图 5 C）。 26 与重链相反，轻链的氨基酸序列是无序的，倾向于形成无定形结构 62 。最近的研究表明，SF 由于其两亲性分子结构实现了界面自组装，这促进了界面处β片的形成。这种特性在水凝胶形成中特别有用，因为可以通过调整水与有机相比来调节 SF 的构象。这些调整能够产生专门针对软骨再生优化的水凝胶，从而提供增强的机械性能和生物活性，以改善组织修复 63 。
SF exhibits exceptional physical and chemical properties due to its unique structure and composition. It can adopt four distinct conformations—silk I, silk II, silk III, and an amorphous structure—through intra- and intermolecular interactions. Among these, the amorphous structure and β-sheet-rich silk II conformation endow SF with high mechanical strength and toughness. Furthermore, silk I, silk II, silk III and amorphous structures can be transformed into each other by external effects (temperature, ultrasound, electric field, shear force and pH value) 62. This adaptability makes SF highly suitable for diverse tissue regeneration applications, as its mechanical properties can be finely tuned. As a macromolecular protein material, SF also offers excellent cytocompatibility and biodegradability. It degrades in response to multiple proteases, with its degradation rate primarily controlled by the content of silk II. The degradation products—amino acids and peptides—are non-toxic and can be absorbed by cells, providing essential building blocks for tissue regeneration 76. In addition to these favorable properties, SF's processability allows it to be adapted into various processing methods to meet the complex demands of tissue repair 77. To provide a clearer understanding of SF's properties relative to other biomaterials commonly used in cartilage regeneration, Table 1 presents a quantitative comparison of SF with collagen, alginate, hyaluronic acid (HA), and Matrigel across key parameters, including mechanical properties, degradation rates, and biological performance.
SF 由于其独特的结构和成分而表现出卓越的物理和化学性能。它可以通过分子内和分子间相互作用采用四种不同的构象——丝 I、丝 II、丝 III 和无定形结构。其中，无定形结构和富含 β 片的 silk II 构象赋予 SF 高机械强度和韧性。此外，蚕丝 I、蚕丝 II、蚕丝 III 和非晶结构可以通过外部作用（温度、超声波、电场、剪切力和 pH 值） 62 相互转化。这种适应性使 SF 非常适合各种组织再生应用，因为它的机械性能可以微调。作为一种大分子蛋白质材料，SF 还具有出色的细胞相容性和生物降解性。它响应多种蛋白酶而降解，其降解速率主要受丝 II 的含量控制。降解产物（氨基酸和肽）无毒，可被细胞吸收，为组织再生 76 提供必需的组成部分。除了这些有利的特性外，SF 的可加工性使其能够适应各种加工方法，以满足组织修复 77 的复杂需求。为了更清楚地了解 SF 相对于软骨再生中常用的其他生物材料的特性，下表 1 列出了 SF 与胶原蛋白、藻酸盐、透明质酸 （HA） 和基质胶在关键参数（包括机械性能、降解速率和生物性能）上的定量比较。
Table 1. 表 1.
Comparison of the mechanical properties, degradation, cell viability, and chondrocyte differentiation of SF and other biomaterials used in cartilage tissue engineering.
SF 和其他用于软骨组织工程的生物材料的机械性能、降解、细胞活力和软骨细胞分化的比较。
| Property 财产 | SF | Col 山坳 | Alginate 海藻 酸 | HA | Matrigel 基质胶 |
|---|---|---|---|---|---|
| Young's Modulus (MPa) 杨氏模量 （MPa） | 300-700 MPa | 0.1-10 MPa | 0.01-1.5 MPa | 0.01-0.1 MPa | 0.00004-0.00045 MPa |
| Breaking Elongation (%) 断裂伸长率 （%） | 4%-26% | 10-30% | 10-20% | 5-20% | Not reported 未报告 |
| Toughness (MJ/m3) 韧性 （MJ/m3） |
70-78 MJ/m3 70-78 兆焦耳/立方米 | 1-5 MJ/m³ 1-5 MJ/立方米 | 1-5 MJ/m³ 1-5 MJ/立方米 | 1-2 MJ/m³ 1-2 MJ/立方米 | Not reported 未报告 |
| Degradation Time 退化时间 | Tunable (weeks to months) 可调（数周至数月） |
Days to weeks 天到周 | Days to weeks 天到周 | Days to weeks 天到周 | Within a few days 几天内 |
| Cell Viability (%) 细胞活力 （%） | >90% | >80% | >80% | >85% | >90% |
| Chondrocyte Differentiation 软骨细胞分化 |
Supports viability and promotes collagen type II synthesis 支持活力并促进 II 型胶原蛋白合成 |
Promotes moderate chondrocyte differentiation but limited stable phenotype 促进中等软骨细胞分化，但稳定表型有限 |
Supports chondrocyte viability with moderate differentiation capacity 支持具有中等分化能力的软骨细胞活力 |
Supports viability, requires additional cues for stable phenotype 支持活力，需要额外的线索才能获得稳定的表型 |
Supports growth but does not inherently promote chondrocyte differentiation 支持生长，但本身并不促进软骨细胞分化 |
| References 引用 | 64, 65 | 66, 67 | 68-70 | 71, 72 | 73-75 |
3.2. Cross-linking methods for silk fibroin-based hydrogels preparation
3.2. 丝素蛋白基水凝胶制备的交联方法
As previously mentioned, SF can be processed using various methods, including hydrogel preparation through cross-linking. Cross-linking methods are generally categorized into chemical and physical approaches 78. Chemical cross-linking promotes the formation of covalent bonds by adding enzymes, cross-linking agents and photo-initiators to accelerate SF gelation. In contrast, physical cross-linking involves the self-assembly of SF into hydrogels by regulating physical parameters such as temperature, pH, shear force, ultrasound, and electric fields, each method offering distinct advantages and limitations (Figure 6, Table 2). The schematic in Figure 7 provides an overview of the mechanisms and preparation techniques employed in these cross-linking strategies for SF hydrogels.
如前所述，SF 可以使用各种方法加工，包括通过交联制备水凝胶。交联方法通常分为化学方法和物理方法 78 。化学交联通过添加酶、交联剂和光引发剂来加速 SF 凝胶化，从而促进共价键的形成。相比之下，物理交联涉及通过调节温度、pH 值、剪切力、超声波和电场等物理参数将 SF 自组装成水凝胶，每种方法都有明显的优点和局限性（图 6 ， 表 2 ）。图 7 中的示意图概述了 SF 水凝胶的这些交联策略中采用的机制和制备技术。
Figure 6. 图 6.
Cross-linking methods of SF-based hydrogels. Chemical cross-linking: enzymatic cross-linking, photo-polymerization and cross-linking agent. Physical cross-linking: temperature, pH, shear force ultrasonication and electric field. Created with BioRender.com.
SF 基水凝胶的交联方法。化学交联：酶交联、光聚合和交联剂。物理交联：温度、pH、剪切力、超声波和电场。使用 BioRender.com 创建。
Table 2. 表 2.
Cross-linking methods for SF-based hydrogels: advantages and limitations.
SF 基水凝胶的交联方法：优点和局限性。
| Type 类型 | Cross-linking Method 交联方法 | Advantages 优势 | Limitations 局限性 |
|---|---|---|---|
| Chemical cross-linking 化学交联 | Enzymatic cross-linking 酶交联 | High selectivity; biocompatibility; tunable properties 选择性高;生物相容性;可调属性 |
High cost; low reaction velocity; limited scalability 成本高;反应速度低;可扩展性有限 |
| Photo-polymerization 光聚合 | Rapid cross-linking; precise control 快速交联;精确控制 |
Potential cytotoxicity; limited tissue penetration 潜在的细胞毒性;组织渗透受限 |
|
| Cross-linking agents 交联剂 | Cost-effective; enhances mechanical properties 成本效益高;增强机械性能 |
Non-specific reactions; cytotoxicity (for some agents) 非特异性反应;细胞毒性（对于某些药物） |
|
| Physical cross-linking 物理交联 | Temperature 温度 | Non-toxic; simple process 无毒;简单的流程 |
Lacks precision; risk of denaturation at high temperatures 缺乏精确性;高温下变性的风险 |
| pH 酸碱度 | Effective control over gelation 有效控制凝胶化 |
Requires careful pH control; potential impact on cell viability 需要仔细控制 pH 值;对细胞活力的潜在影响 |
|
| Shear force 剪切力 | Creates directional structures; anisotropic properties 创建定向结构;各向异性特性 |
Requires specialized equipment; limited scalability 需要专门的设备;可扩展性有限 |
|
| Ultrasonication 超声检查 | Non-toxic; controllable process; tailored porosity 无毒;过程可控;定制孔隙率 |
Weaker mechanical properties; limited load-bearing capacity 机械性能较弱;承重能力有限 |
|
| Electric field 电场 | Enables gradient structures; useful for tissue engineering 启用渐变结构;适用于组织工程 |
Requires specialized equipment; potential for uneven cross-linking 需要专门的设备;交联不均匀的可能性 |
Figure 7. 图 7.
Schematic representation of the mechanisms and preparation methods of different cross-linking approaches for SF-based hydrogels. Created with BioRender.com.
SF 基水凝胶不同交联方法的机理和制备方法的示意图。使用 BioRender.com 创建。
3.2.1. Chemical cross-linking
3.2.1. 化学交联
Enzymatic cross-linking 酶交联
In recent years, enzyme cross-linked hydrogels have attracted wide attention in the biomedicine field. For SF-based hydrogels, enzymes facilitate the formation of intermolecular covalent bonds by activating functional groups within SF. Additionally, enzymatic cross-linking induces the formation of an ECM-like elastic structure by controlling β-sheet formation, resulting in hydrogels with stable structures, controllable mechanical properties, and non-toxic effects on cells 79. This method also supports cell encapsulation due to its cross-linking process being conducted at physiological pH and temperature 80.
近年来，酶交联水凝胶在生物医学领域引起了广泛关注。对于基于 SF 的水凝胶，酶通过激活 SF 内的官能团来促进分子间共价键的形成。此外，酶促交联通过控制 β 片的形成诱导形成类似 ECM 的弹性结构，从而产生结构稳定、机械性能可控且对细胞 79 无毒作用的水凝胶.该方法还支持细胞封装，因为它的交联过程是在生理 pH 值和温度下 80 进行的。
Among the numerous enzymatic cross-linking reactions, horseradish peroxidase (HRP) mediated enzymatic cross-linking reaction is the most commonly used 81, 82. It has the advantages of high selectivity, mild reaction conditions and no toxic components 83, 84. HRP is typically combined with H2O2 to induce SF cross-linking by oxidizing tyrosine residues into o-quinone residues. These o-quinone residues then react with phenol or aniline to form covalent bonds, leading to intermolecular or intramolecular cross-linking 85. For example, Hasturk et al. prepared SF/tyramine-substituted SF (SF-TA) composite hydrogel by using HRP and H2O2
86. The composite hydrogel exhibited adjustable mechanical properties, degradability, and excellent cytocompatibility, making it promising for cartilage defect repair due to its cell encapsulation capability. Li et al. further developed an SF-gelatin (SF-GT) hydrogel with a macroporous structure using HRP/H2O2 in combination with 3D bioprinting 87. SF-GT hydrogel had structural stability, mechanical properties and adjustable degradation rate for cartilage reconstruction. Additionally, SF-GT hydrogel could induce stem cells to synthesize Col II at a higher level and show hyaline cartilage phenotype.
在众多的酶促交联反应中，辣根过氧化物酶 （HRP） 介导的酶促交联反应是最常用的 81 82 。它具有选择性高、反应条件温和、无毒成分 83 等优点。 84 HRP 通常与 H 2 O 2 结合，通过将酪氨酸残基氧化成邻醌残基来诱导 SF 交联。然后，这些邻醌残基与苯酚或苯胺反应形成共价键，导致分子间或分子内交联 85 。例如，Hasturk 等人使用 HRP 和 H2O2 86 制备了 SF/酪胺取代的 SF （SF-TA） 复合水凝胶。复合水凝胶表现出可调节的机械性能、可降解性和出色的细胞相容性，由于其细胞封装能力，使其有望用于软骨缺损修复。Li 等人使用 HRP/H2O2 结合 3D 生物打印 87 进一步开发了一种具有大孔结构的 SF-明胶 （SF-GT） 水凝胶。SF-GT 水凝胶具有结构稳定性、力学性能和可调节的软骨重建降解速率。此外，SF-GT 水凝胶可以诱导干细胞在更高水平上合成 Col II，并显示透明软骨表型。
Photo-polymerization 光聚合
Photo-polymerization is a widely used chemical cross-linking method that utilizes a photo-initiator and light (ultraviolet, visible, or gamma rays) to control the cross-linking process 88. During photo-polymerization, the photo-initiator absorbs light energy and cleaves to produce free radicals, which subsequently react with unsaturated bonds in SF to induce cross-linking. The primary advantage of photo-polymerization is its extremely rapid cross-linking rate 89, 90. For instance, Cui et al. successfully cross-linked SF within 1 minute using tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate and sodium persulfate as photo-initiators 91. The SF-based hydrogels had stable mechanical properties and supported the long-term culture of human articular chondrocytes and cartilage tissue regeneration. In addition, there is a special photo-polymerization method using high-intensity gamma-ray without adding photo-initiators. This method can completely remove the toxic effects caused by photo-initiators residues 92. For example, Kim et al. prepared chemically cross-linked SF hydrogels by using Co-60 derived gamma-ray (SF C-gel) 93. They found that SF C-gel was biocompatible and could promote the attachment and proliferation of hMSCs.
光聚合是一种广泛使用的化学交联方法，它利用光引发剂和光（紫外线、可见光或伽马射线）来控制交联过程 88 。在光聚合过程中，光引发剂吸收光能并裂解产生自由基，自由基随后与 SF 中的不饱和键反应以诱导交联。光聚合的主要优点是其极快的交联速率 89 。 90 例如，Cui 等人使用三（2,2-联吡啶基）二氯钌 （II） 六水合物和过硫酸钠作为光引发剂，在 1 分钟内成功交联了 SF 91 。基于 SF 的水凝胶具有稳定的机械性能，并支持人关节软骨细胞的长期培养和软骨组织再生。此外，还有一种特殊的光聚合方法，使用高强度伽马射线，无需添加光引发剂。这种方法可以完全去除光引发剂残留物引起的毒性作用 92 。例如，Kim 等人使用 Co-60 衍生的伽马射线 （SF C 凝胶） 93 制备了化学交联的 SF 水凝胶。他们发现 SF C-gel 具有生物相容性，可以促进 hMSC 的附着和增殖。
Cross-linking agents 交联剂
Cross-linking agent molecules can accelerate SF cross-linking by reacting with reactive groups such as -OH, -NH2 and -COOH in SF 94. Compared to enzymes and photo-initiators, cross-linking agents are more cost-effective and can improve the mechanical properties of hydrogels 59. Glutaraldehyde (GTA) is the most widely used cross-linking agent, which can promote SF cross-linking by reacting with the phenolic group of tyrosine. For instance, Srisawasdi et al. prepared polycarbazole/SF (SF/PCZ) hydrogels with glutaraldehyde as cross-linking agent 95. They found that SF/PCZ hydrogel had good dielectric properties and excellent toughness. However, the biotoxicity of GTA limits its applications in tissue engineering and medicine. In contrast, genipin is a promising natural small molecule cross-linking agent due to its excellent biocompatibility. Considering this, Min et al. designed chitosan/SF hydrogels loaded with kartogenin (KGN) and platelet-derived growth factor BB (PDGF-BB) by using genipin as cross-linking agent 96. The hydrogels allowed for the sustained release of KGN and PDGF-BB, supporting the growth of seed chondrocytes and maintaining their phenotype, demonstrating potential in cartilage tissue engineering.
交联剂分子通过与 SF 中的反应性基团如 -OH、-NH2 和 -COOH 反应来加速 SF 交联 94 。与酶和光引发剂相比，交联剂更具成本效益，并且可以改善水凝胶的机械性能 59 。戊二醛 （GTA） 是使用最广泛的交联剂，它可以通过与酪氨酸的酚基反应来促进 SF 交联。例如，Srisawasdi 等人用戊二醛作为交联剂 95 制备了聚咔唑/SF （SF/PCZ） 水凝胶。他们发现 SF/PCZ 水凝胶具有良好的介电性能和优异的韧性。然而，GTA 的生物毒性限制了其在组织工程和医学中的应用。相比之下，genipin 由于其优异的生物相容性，是一种很有前途的天然小分子交联剂。考虑到这一点，Min 等人使用京尼平作为交联剂 96 ，设计了载有 kartogenin （KGN） 和血小板衍生生长因子 BB （PDGF-BB） 的壳聚糖/SF 水凝胶。水凝胶允许 KGN 和 PDGF-BB 的持续释放，支持种子软骨细胞的生长并维持其表型，在软骨组织工程中显示出潜力。
3.2.2. Physical cross-linking
3.2.2. 物理交联
Temperature 温度
Temperature significantly affects the cross-linking of proteins, including SF 97. Increasing the temperature can promote SF cross-linking by enhancing the Brownian motion of SF molecules and increasing the effective collision rate between them. Additionally, elevated temperatures can disrupt the free energy state of SF molecules, exposing internal hydrophobic regions and facilitating the transition from random coil to β-sheet structures, thereby enhancing hydrophobic interactions and accelerating cross-linking 98. For instance, Kim et al. researched the effect of cross-linking temperature for SF hydrogels 99. They found that the cross-linking rate and compressive modulus of SF hydrogels increased with increasing cross-linking temperature within a certain range.
温度显着影响蛋白质的交联，包括 SF 97 。提高温度可以通过增强 SF 分子的布朗运动并提高它们之间的有效碰撞率来促进 SF 交联。此外，高温会破坏 SF 分子的自由能状态，暴露内部疏水区域并促进从无规卷曲过渡到β片结构，从而增强疏水相互作用并加速交联 98 。例如，Kim 等人研究了交联温度对 SF 水凝胶的影响 99 。他们发现，在一定范围内，SF 水凝胶的交联速率和压缩模量随着交联温度的升高而增加。
pH 酸碱度
In addition to temperature, the pH of the SF solution is also a critical factor in SF cross-linking. When the pH of the solution approaches the isoelectric point of SF (pH = 3.8-4.0), the electrostatic repulsion between SF molecules is minimized, making the molecules more prone to aggregation and cross-linking 94. In this condition, the SF molecules are unstable and prone to aggregation and cross-linking. Therefore, adjusting the pH value of the solution is an effective method to induce SF cross-linking. For example, Nagarkar et al. investigated SF cross-linking by changing pH via adding HCl 100. They found that adjusting the solution pH from 8 to 2 could prepare weak SF hydrogels. Additionally, Fini et al. designed and developed SF-based hydrogel though regulating pH via adding citric acid to SF solution 101. They found that the hydrogel had good mechanical properties and excellent cytocompatibility. Additionally, in vivo experiments demonstrated that the hydrogel showed non-inflammatory response after implantation and stimulated cells to produce TGF-β1 to induce tissue regeneration.
除了温度，SF 溶液的 pH 值也是 SF 交联的关键因素。当溶液的 pH 值接近 SF 的等电点（pH = 3.8-4.0）时，SF 分子之间的静电排斥最小，使分子更容易聚集和交联 94 。在这种情况下，SF 分子不稳定，容易聚集和交联。因此，调节溶液的 pH 值是诱导 SF 交联的有效方法。例如，Nagarkar 等人通过添加 HCl 100 来改变 pH 值，从而研究了 SF 交联。他们发现，将溶液 pH 值从 8 调整到 2 可以制备弱 SF 水凝胶。此外，Fini 等人设计并开发了基于 SF 的水凝胶，通过向 SF 溶液 101 中添加柠檬酸来调节 pH 值。他们发现水凝胶具有良好的机械性能和出色的细胞相容性。此外，体内实验表明，水凝胶在植入后表现出非炎症反应，并刺激细胞产生 TGF-β1 以诱导组织再生。
Shear force 剪切力
The shear force cross-linking method typically involves high-speed vortex treatment on SF solution 102. High-speed vortex treatment accelerates β-sheet generation by stretching SF molecules and changing their orientation to promote SF cross-linking 103. Moreover, this method can also be used to prepare SF-based hydrogels with directional structures. For example, Chen et al. fabricated SF/sodium surfactin hydrogels with directional three-dimensional structure by vortex treatment 104. The hydrogel could accelerate 3D directed tissue regeneration due to its excellent anisotropy. Moreover, Kasoju et al. fabricated SF hydrogel with good mechanical properties by combining methanol treatment and vortex treatment 105. These hydrogels demonstrated effective cell encapsulation and controlled drug release capabilities.
剪切力交联法通常涉及对 SF 溶液 102 进行高速涡流处理。高速涡旋处理通过拉伸 SF 分子并改变其方向以促进 SF 交联 103 来加速β片的生成。此外，该方法还可用于制备具有定向结构的 SF 基水凝胶。例如，Chen 等人通过涡旋处理 104 制备了具有定向三维结构的 SF/钠表面活性素水凝胶。由于其优异的各向异性，水凝胶可以加速 3D 定向组织再生。此外，Kasoju 等人通过结合甲醇处理和涡旋处理 105 制备了具有良好机械性能的 SF 水凝胶。这些水凝胶表现出有效的细胞封装和受控的药物释放能力。
Ultrasonication 超声检查
Ultrasonication is a physical cross-linking method commonly used to prepare SF hydrogels. The effect of this method is similar to the anterior silk gland of silkworm, which promotes SF cross-linking through local temperature increase and shear force 106. This method is highly stable and controllable, as it allows for adjustments in output energy and duration. Importantly, it effectively avoids the toxicity issues associated with additives like photo-initiators and cross-linking agents 107. For example, Byram et al. designed and developed SF/xanthan gum hydrogels by ultrasonication 108. Additionally, due to their cartilage ECM-like microstructure, these hydrogels showed potential for applications in cartilage tissue engineering.
超声处理是常用于制备 SF 水凝胶的一种物理交联方法。这种方法的效果类似于蚕的前丝腺，通过局部温度升高和剪切力 106 促进SF交联。这种方法非常稳定和可控，因为它允许调整输出能量和持续时间。重要的是，它有效地避免了与光引发剂和交联剂 107 等添加剂相关的毒性问题。例如，Byram 等人通过超声设计并开发了 SF/黄原胶水凝胶 108 。此外，由于其软骨 ECM 样微观结构，这些水凝胶在软骨组织工程中显示出应用潜力。
Electric field 电场
As we mentioned before, SF is negatively charged in neutral solutions due to its isoelectric point (pH=3.8-4.0). Under an electric field, SF molecules aggregate near the anode, forming micelles that subsequently assemble into hydrogels through physical entanglement of molecular chains 109. For instance, Liu et al. constructed SF electrogels with excellent mechanical properties via a low-voltage electric field 110. They found that the hydrogel had excellent biocompatibility and drug loading capacity. In addition, gradient structure hydrogels which are more suitable for cartilage repair can be prepared by the electric field. Consider this, Xu et al. developed multi-functional beta-sheet rich silk nanofibers (BSNF) hydrogels with adjustable gradient mechanical strength and structure in electric field 111. BSNF hydrogel could regulate BMSCs to differentiate into chondrocytes to promote cartilage repair due to its gradients structure.
正如我们之前提到的，SF 由于其等电点 （pH=3.8-4.0） 而在中性溶液中带负电。在电场下，SF 分子在阳极附近聚集，形成胶束，随后通过分子链 109 的物理纠缠组装成水凝胶。例如，Liu 等人通过低压电场 110 构建了具有优异机械性能的 SF 电凝胶。他们发现水凝胶具有出色的生物相容性和载药能力。此外，可以通过电场制备更适合软骨修复的梯度结构水凝胶。考虑到这一点，Xu 等人开发了多功能富含 β 折叠的蚕丝纳米纤维 （BSNF） 水凝胶，在电场 111 中具有可调节的梯度机械强度和结构。BSNF 水凝胶由于其梯度结构，可以调节 BMSCs 分化为软骨细胞以促进软骨修复。
3.3. Functional modifications
3.3. 功能修改
As we mentioned in 3.1. Characteristics of silk fibroin, SF has excellent biocompatibility, biodegradability and mechanical properties. However, after various physical and chemical treatments, the molecular structure of SF is destroyed, which leads to the unsatisfactory mechanical strength of pure SF hydrogel. In addition, pure SF hydrogel has certain disadvantages, such as insufficient water retention, poor antibacterial properties and unsatisfactory cartilage repair properties 138. In order to improve SF hydrogel, it is an effective strategy to prepare composite SF hydrogel by mixing some functional materials into SF solution 139. These functional materials can be divided into synthetic materials and natural materials (Table 3).
正如我们在 3.1 中提到的。丝素蛋白的特点，SF 具有优异的生物相容性、生物降解性和机械性能。然而，经过各种物理和化学处理后，SF 的分子结构被破坏，导致纯 SF 水凝胶的机械强度不理想。此外，纯SF水凝胶存在一定的缺点，如保水性不足、抗菌性能差、软骨修复性能 138 不理想等。为了改善SF水凝胶，将一些功能材料混合到SF溶液 139 中制备复合SF水凝胶是一种有效的策略。这些功能材料可分为合成材料和天然材料（表 3 ）。
Table 3. 表 3.
The functional materials that have been used in combination with SF to prepare composite hydrogels for cartilage repair.
与 SF 联合用于制备用于软骨修复的复合水凝胶的功能材料。
| Type 类型 | Functional materials 功能性材料 | Preparation method 制备方法 | Advantages of composite hydrogels in cartilage repair 复合水凝胶在软骨修复中的优势 |
References 引用 |
|---|---|---|---|---|
| Synthetic 合成 | GO | Photo-polymerization with RuBPY as photo-initiators 以 RuBPY 作为光引发剂的光聚合 |
Excellent mechanical properties 优异的机械性能 |
112, 113 |
| PEG | Ultrasonication 超声检查 | Injectable; rapid gelation; suitable microenvironment 注射;快速凝胶化;适宜的微环境 |
114 | |
| PEGDMA | UV photo-polymerization UV 光聚合 | Adjustable mechanical properties 可调节的机械性能 |
115 | |
| PEGDA | Photo-polymerization with LAP as photo-initiators 以 LAP 为光引发剂的光聚合反应 |
Excellent mechanical properties; bioprinting-compatible 优良的机械性能;兼容 BioPrinting |
116 | |
| poly(N-vinylcaprolactam) 聚（N-乙烯基己内酰胺） | Photo-polymerization with tris(2,2-bipyridyl)dichlororuthenium(II) and ammonium peroxodisulfate as photo-initiators 以三（2,2-联吡啶基）二氯钌 （II） 和过氧化二硫酸铵为光引发剂进行光聚合 |
Enhanced water uptake capacity, elasticity and toughness 增强的吸水能力、弹性和韧性 |
117 | |
| PVA | pH adjustment pH 值调节 | Excellent mechanical properties; high porosity; adjustable swelling ratio 优良的机械性能;高孔隙率;可调节的溶胀率 |
118 | |
| POL | Enzyme cross-linking catalyzed by HRP and H2O2 HRP 和 H2O2 催化的酶交联 |
Thermosensitive; injectable 热 敏;注射 |
119, 120 | |
| MXene | Enzyme cross-linking catalyzed by HRP and H2O2; HRP 和 H2O 2 催化的酶交联; Ultrasound technique 超声技术 |
Metallic conductivity; piezoelectricity; excellent hydrophilicity; diverse surface chemical properties; injectable 金属导电性;压电;优良的亲水性;多种表面化学性质;注射 |
121, 122 | |
| Natural 自然的 | Collagen 胶原 | Ultrasonication 超声检查 | Promotes MSC proliferation; chondrogenic differentiation 促进 MSC 增殖;软骨形成分化 |
123, 124 |
| Gelatin 明胶 | Enzyme cross-linking catalyzed by transglutaminase 转谷氨酰胺酶催化的酶交联 |
Cell attachment, proliferation; excellent mechanical integrity 细胞附着、增殖;出色的机械完整性 |
125, 126 | |
| HPMC | Heating 加热 | Excellent mechanical properties 优异的机械性能 |
127 | |
| CS | Enzyme cross-linking catalyzed by HRP and H2O2 HRP 和 H2O2 催化的酶交联 |
Environmentally sensitive; controlled release of drugs and growth factors 对环境敏感;药物和生长因子的控释 |
128-131 | |
| HA | Enzyme cross-linking catalyzed by HRP and H2O2 HRP 和 H2O2 催化的酶交联 |
MSC recruitment; cell adhesion; cartilage-like mechanical properties MSC 招聘;细胞粘附;类似软骨的机械性能 |
132-135 | |
| GA | Photo-polymerization 光聚合 | Reduced mechanical stress; lower in vivo friction coefficients 减少机械应力;更低的体内摩擦系数 |
136 | |
| DNA | Enzyme cross-linking catalyzed by HRP and H2O2 and complementary base pairing HRP 和 H2O2 催化的酶交联以及互补碱基配对 |
Regulate chondrogenic differentiation of BMSCs 调节 BMSCs 的软骨生成分化 |
137 |
3.3.1. Synthetic material modification
3.3.1. 合成材料改性
Graphene oxide is a kind of functional carbon allotrope. Recently, it has attracted increasing attention in the field of materials due to its excellent mechanical strength, attractive surface volume ratio, high water solubility, easy solution processing and chemical functionality 140. The incorporation of GO into SF solutions can significantly enhance the mechanical properties of SF-based hydrogels. For example, Wang et al. developed nano-hydroxyapatite-GO/SF hydrogels using click chemistry, resulting in enhanced mechanical strength compared to pure SF hydrogels, with a compressive modulus of 95.4 ± 2.0 kPa 112. Furthermore, the addition of GO can improve the toughness of SF hydrogels. Balu et al. fabricated regenerated SF (RSF)/GO nanocomposite hydrogels using RuBPY as a photo-initiator, achieving mechanical properties superior to natural cartilage, with a Young's modulus of 8 MPa and tensile toughness of 2.4 MJ/m3
113.
氧化石墨烯是一种功能性碳同素异形体。近年来，由于其优异的机械强度、有吸引力的表面体积比、高水溶性、易于固溶处理和化学功能 140 ，它在材料领域引起了越来越多的关注。将 GO 掺入 SF 溶液中可以显著提高 SF 基水凝胶的机械性能。例如，Wang 等人使用点击化学开发了纳米羟基磷灰石-GO/SF 水凝胶，与纯 SF 水凝胶相比，机械强度更高，压缩模量为 95.4 ± 2.0 kPa 112 。此外，GO 的添加可以提高 SF 水凝胶的韧性。Balu 等人使用 RuBPY 作为光引发剂制造了再生 SF （RSF）/GO 纳米复合水凝胶，实现了优于天然软骨的机械性能，杨氏模量为 8 MPa，拉伸韧性为 2.4 MJ/m3 113 。
Synthetic polymers are also commonly used to prepare composite hydrogels. Among them, polyethylene glycol (PEG) has received wide attention due to its versatility in molecular weight, topology (linear, branched, star-shaped) 139, 141. Interestingly, the incorporation of PEG can endow the SF composite hydrogel with the injectable property. For example, Zhang et al. prepared injectable BMSC-encapsulated dual-network SF-PEG composite hydrogels via ultrasonication, resulting in hydrogels with a high cross-linking rate, excellent biocompatibility, and strong mechanical strength. Additionally, these dual-network SF-PEG hydrogels promoted cartilage repair by enhancing BMSC chondrogenic differentiation 114. Furthermore, derivatives of PEG can also strengthen the cartilage repair ability of SF hydrogels. For instance, Achachelouei et al. fabricated SF/poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogels with adjustable mechanical properties by photo-polymerization 115. They found that the compression modulus of SF/PEGDMA hydrogel was related to the ratio of SF to PEGDMA. Meanwhile, Bandyopadhyay et al. constructed silk methacrylate (SilMA)-PEG diacrylate hydrogel with excellent mechanical properties and adjustable degradability via photo-polymerization 116. Importantly, this hydrogel was suitable for chondrocytes-laden 3D biological printing to accelerate cartilage repair.
合成聚合物也常用于制备复合水凝胶。其中，聚乙二醇 （PEG） 因其在分子量、拓扑结构（线性、支链、星形） 139 等方面的多功能性而受到广泛关注。 141 有趣的是，PEG 的掺入可以赋予 SF 复合水凝胶可注射的特性。例如，Zhang 等人通过超声处理制备了可注射的 BMSC 封装的双网络 SF-PEG 复合水凝胶，得到的水凝胶具有较高的交联速率、优异的生物相容性和较强的机械强度。此外，这些双网络 SF-PEG 水凝胶通过增强 BMSC 软骨形成分 114 化来促进软骨修复。此外，PEG 的衍生物还可以增强 SF 水凝胶的软骨修复能力。例如，Achachelouei 等人通过光聚合制备了具有可调节机械性能的 SF/聚乙二醇二甲基丙烯酸酯 （PEGDMA） 水凝胶 115 。他们发现 SF/PEGDMA 水凝胶的压缩模量与 SF 与 PEDMA 的比率有关。同时，Bandyopadhyay 等人通过光聚合构建了具有优异机械性能和可调节降解性的甲基丙烯酸丝 （SilMA）-PEG 二丙烯酸酯水凝胶 116 。重要的是，这种水凝胶适用于载有软骨细胞的 3D 生物打印，以加速软骨修复。
Apart from PEG, the performance of SF hydrogels can be enhanced by mixing with poly(N-vinylcaprolactam), poly vinyl alcohol, poloxamer 142. For instance, Whittaker et al. constructed RSF-poly(N-vinylcaprolactam) double network (DN) hydrogel by a rapid one-pot method 117. Compared with SF hydrogels, poly(N-vinylcaprolactam) enhanced elasticity and toughness of hybrid hydrogels. Furthermore, Subramanian et al. developed Mo3Se3-PVA-SF nanowire hydrogel with remarkable mechanical properties by using glutaraldehyde as cross-linking agent 118. They found the composite could stimulate the expression of collagen to accelerate tissue repair. It is worth noting that POL can impart injectability to SF hydrogel 120. For example, Min et al. constructed an injectable alginate-poloxamer (ALG-POL)/SF hydrogel though using HRP and H2O2
119. The injectability of this hydrogel was attributed to its solution-gel transition properties at physiological temperature. In addition, ALG-POL/SF hydrogel accelerated cartilage regeneration by promoting chondrocyte proliferation.
除 PEG 外，SF 水凝胶的性能还可以通过与聚（N-乙烯基己内酰胺）、聚乙烯醇、泊洛沙姆 142 混合来提高。例如，Whittaker 等人通过快速一罐法 117 构建了 RSF-聚（N-乙烯基己内酰胺）双网络 （DN） 水凝胶。与 SF 水凝胶相比，聚 （N-乙烯基己内酰胺） 增强了杂化水凝胶的弹性和韧性。此外，Subramanian 等人通过使用戊二醛作为交联剂 118 开发了具有显着机械性能的 Mo3Se 3-PVA-SF 纳米线水凝胶。他们发现这种复合物可以刺激胶原蛋白的表达以加速组织修复。值得注意的是，POL 可以赋予 SF 水凝胶 120 注射性。例如，Min 等人使用 HRP 和 H2O2 119 构建了一种可注射的藻酸盐-泊洛沙姆 （ALG-POL）/SF 水凝胶。这种水凝胶的可注射性归因于其在生理温度下的溶液-凝胶过渡特性。此外，ALG-POL/SF 水凝胶通过促进软骨细胞增殖来加速软骨再生。
MXenes, as novel 2D nanomaterials composed of transition metal complexes, had been applied in biomedicine due to their metallic conductivity, piezoelectricity, excellent hydrophilicity, and diverse surface chemical properties. Jiang et al. utilized enzyme crosslinking to combine MXene nanosheets with SF, forming a piezoresistive nanocomposite hydrogel that facilitated bone tissue regeneration by restoring the electrical microenvironment 121. Additionally, Yang et al. developed an injectable SF/MXene conductive hydrogel developed through ultrasound techniques, which acted as a stem cell carrier and enabled in vivo electrical stimulation for repairing brain tissue damage 122.
MXenes 作为由过渡金属配合物组成的新型二维纳米材料，由于其金属导电性、压电性、优异的亲水性和多样化的表面化学性质而已应用于生物医学。江等人利用酶交联将MXene纳米片与SF结合，形成压阻纳米复合水凝胶，通过恢复电微环境 121 促进骨组织再生。此外，Yang 等人开发了一种通过超声技术开发的可注射 SF/MXene 导电水凝胶，它充当干细胞载体，能够在体内电刺激以修复脑组织损伤 122 。
3.3.2. Natural material modification
3.3.2. 天然材料改性
The mixing of synthetic materials in SF hydrogels may introduce cytotoxicity. Conversely, the combination of SF with natural materials can avoid the toxicity problems associated with synthetic materials 143. Currently, composite hydrogels have been prepared by combining with GT, collagen, cellulose or HA 144, 145.
合成材料在 SF 水凝胶中的混合可能会引入细胞毒性。相反，SF 与天然材料的结合可以避免与合成材料 143 相关的毒性问题。目前，已经通过与 GT、胶原蛋白、纤维素或 HA 144 、 结合制备了复合水凝胶。 145
Collagen (Col) is the most abundant natural polymer in ECM with remarkable biocompatibility, negligible immunogenicity and strong biological activity. Notably, collagen can enhance cartilage regeneration effects as it promotes the attachment and chondrogenic differentiation of MSCs 123. For example, Zhang et al. developed an injectable BMSC-laden collagen-PEG/SF DN hydrogel via ultrasonication, which exhibited enhanced mechanical strength and cytocompatibility, ultimately accelerating cartilage regeneration 124.
胶原蛋白 （Col） 是 ECM 中最丰富的天然聚合物，具有显著的生物相容性、可忽略不计的免疫原性和强大的生物活性。值得注意的是，胶原蛋白可以增强软骨再生作用，因为它促进 MSC 的附着和软骨形成分化 123 。例如，Zhang 等人通过超声开发了一种可注射的载有 BMSC 的胶原蛋白-PEG/SF DN 水凝胶，它表现出增强的机械强度和细胞相容性，最终加速软骨再生 124 。
GT is a mixture of large polypeptides, denatured from collagen. GT is a promising material in tissue engineering attributed to its high swelling and thermal inversion properties 125. In addition, GT can form an interpenetrating network (IPN) with SF to enhance the compressive moduli of SF-based hydrogel. Park et al. fabricated GT-SF IPN hydrogels through microbial transglutaminase-induced cross-linking, resulting in biodegradable, non-cytotoxic hydrogels with superior mechanical properties compared to individual GT or SF hydrogels. The resulting composite hydrogel also promoted cell adhesion and proliferation 126.
GT 是由胶原蛋白变性的大多肽混合物。GT 是一种很有前途的组织工程材料，因为它具有高溶胀和热反转特性 125 。此外，GT 可以与 SF 形成互穿网络 （IPN），以增强 SF 基水凝胶的压缩模量。Park 等人通过微生物转谷氨酰胺酶诱导的交联制备了 GT-SF IPN 水凝胶，与单个 GT 或 SF 水凝胶相比，产生了可生物降解、无细胞毒性的水凝胶，具有优异的机械性能。所得的复合水凝胶还促进了细胞粘附和增殖 126 。
Cellulose is a polysaccharide biomolecular material available in various sources 146-148. Noticeably, cellulose can be modified in several ways to generate cellulose derivatives 149. These cellulose derivatives strengthen the compressive modulus of SF-based hydrogels by inducing the generation of β-sheets. For instance, Luo et al. fabricated RSF/hydroxypropyl methyl cellulose (HPMC) hydrogel with excellent mechanical properties 127. The maximum compressive and tensile modulus of this hydrogel exceeded 1.0 MPa.
纤维素是一种多糖生物分子材料，有多种来源 146 - 148 。值得注意的是，纤维素可以通过多种方式进行改性以生成纤维素衍生物 149 。这些纤维素衍生物通过诱导 β 片的产生来增强 SF 基水凝胶的压缩模量。例如，Luo 等人制造了具有优异机械性能 127 的 RSF/羟丙基甲基纤维素 （HPMC） 水凝胶。这种水凝胶的最大压缩模量和拉伸模量超过 1.0 MPa。
Chitosan (CS) is a kind of polysaccharide biopolymer, which has an inherent linear structure, excellent biocompatibility and environmental responsiveness. It has been proved that SF hydrogels can be endowed with environmental responsiveness by combining with CS 128. For example, Xu et al. developed pH responsiveness SF/CS hydrogel by chemical cross-linking 129. Meanwhile, Yu et al. prepared CS/SF/amino-functionalized mesoporous silica hydrogel through using genipin as cross-linking agent 130. They found that the excellent injectability of the composite hydrogel was due to its sensitive thermal responsiveness at physiological pH. Additionally, CS-SF composite hydrogels can achieve controlled drug release. Dong et al. prepared a p-hydroxybenzene propanoic acid-modified chitosan (PC)/SF hydrogel using an HRP-mediated enzymatic cross-linking reaction, which sequentially released bioactive molecules to induce MSC homing and chondrogenic differentiation 131.
壳聚糖 （CS） 是一种多糖生物聚合物，具有固有的线性结构、优异的生物相容性和环境响应性。已经证明，SF 水凝胶可以通过与 CS 结合来赋予环境响应性 128 。例如，Xu 等人通过化学交联 129 开发了 pH 响应性 SF/CS 水凝胶。同时，Yu 等人以 genipin 为交联剂 130 制备了 CS/SF/氨基功能化介孔二氧化硅水凝胶。他们发现，复合水凝胶出色的注射性能是由于它在生理 pH 值下敏感的热响应性。此外，CS-SF 复合水凝胶可以实现药物的控释。Dong 等人使用 HRP 介导的酶促交联反应制备了对羟基苯丙酸改性壳聚糖 （PC）/SF 水凝胶，该反应依次释放生物活性分子以诱导 MSC 归巢和软骨形成分 131 化。
HA is a common biodegradable, non-immunogenic and non-inflammatory polysaccharide in the human body. It has been reported that mixing HA can enhance the storage modulus and compressive modulus of SF hydrogels 132, 150. For example, Ziadlou et al. designed and developed HA-TA /SF composite hydrogels via enzymatic cross-linking 133. Compared with pure SF hydrogel, they found this hybrid hydrogel had enhanced storage modulus and drug releasing ability to stimulate cartilage regeneration, remarkable mechanical properties and drug release ability. Moreover, Wang et al. also fabricated an SF-HA composite hydrogel scaffold by combining SF/HA-Tyr hydrogel with SF sponge, finding that this biomaterial accelerated cartilage repair due to its excellent mechanical properties and MSC recruitment ability 134. Fan et al. compounded SF with aldehyde-HA to create a dynamic network that delayed or interrupted the β-sheet-induced hardening of SF chains, producing a hydrogel matrix with mechanical properties si