一种具有结构支撑性、自愈性和高渗透性的水凝胶生物墨水用于建立多种均质类组织结构|BAM
目前即用型生物墨水难以同时满足结构支撑和高渗透性需求，不利于构建均质化类组织体。本研究基于醛基化透明质酸/N-羧甲基壳聚糖体系和明胶/聚乙二醇琥珀酰亚胺戊二酸酯体系的凝胶化时间差，提出分时(TSH)凝胶化策略，制备了一种分时结构支撑(TSHSP)水凝胶生物墨水，该墨水便于室温打印且具备高渗透性，能构建多种均质化类组织结构。
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研究内容简介
生物3D打印能便捷、有效地整合细胞和生物材料在体外制造类组织、类器官及其功能单元以模拟各种临床研究。已经有诸多具有生物安全性、生物相容性、利于外形和微结构维持的水凝胶生物墨水被用于微挤出式生物3D打印这一最常见、最经济的生物打印方式。常见的即用型溶胶相生物墨水除了会因为细胞沉降导致打印结构中的细胞分布不均匀外，通常还需要低温、辅助交联或牺牲材料等额外辅助手段来实现打印过程中的结构稳定。凝胶相生物墨水能较好地确保细胞分散，如纳米工程化κ-卡拉胶、蛋白质工程化藻酸盐和β-环糊精/金刚烷功能化透明质酸主客体等体系，这类墨水的凝胶化基于动态交联，因此具有剪切变稀和/或自愈的特性，但仍不能完全脱离低温或其它辅助手段来独立实现结构支撑。当上述打印过程中的辅助手段控制不佳时，则不利于细胞存活和形状保持。而另一些具有结构支撑性的水凝胶生物墨水则存在耗时、打印效果不理想等不足。因此，开发一种新的即用型、结构支撑性生物墨水具有重要意义。
基于席夫碱键的可注射水凝胶强度通常跨越三个数量级，这足以满足各种结构支撑选择。最简便的希夫碱水凝胶由醛基化透明质酸（AHA）和水溶性壳聚糖（如N-羧甲基壳聚糖，CMC）组成，其不仅具有良好的生物相容性，而且可在室温下几分钟内实现快速凝胶化。对于生物3D打印构建体而言，水凝胶的结构支撑性不仅要实现一般的形状维持，还应满足微结构保持的要求。因此，如何平衡AHA/CMC水凝胶基于动态酰亚胺键的自愈行为以避免微结构融合仍具有一定的挑战性，另外该水凝胶体系的易水解性也制约了其在生物3D打印中的应用。
无细胞毒性的琥珀酰亚胺活性酯功能化聚乙二醇（如四臂聚乙二醇琥珀酰亚胺戊二酸酯，PEG-SG）可与常见蛋白质基的材料（如明胶，GEL）的氨基发生反应，形成基于稳定酰胺键的水凝胶，该特性可以减缓席夫碱水凝胶构建体的微结构过度融合及快速水解。GEL/PEG-SG水凝胶通常需要数小时才能达到满足打印的凝胶强度，这无疑会降低打印效率，而快速凝胶化的AHA/CMC体系则反过来可以弥补这一缺陷。此外，由于物质交换效率低下，低渗透性水凝胶可导致封装细胞凋亡或非均质化增殖，而AHA/CMC水凝胶优异的吸水性和保水性则使水凝胶具备了高渗透性，这能确保深层细胞的生长并促进组织均质化修复。
根据AHA/CMC和GEL/PEG-SG水凝胶体系之间的凝胶时间差异，我们提出了分时（Time-sharing，TSH）凝胶化策略，通过简单混合的方式制备了一种分时结构支撑性（Time-sharing structure-supporting, TSHSP）生物墨水，并验证了其在室温下打印多种均质类组织构建体的能力(Fig. 1)。我们优化了配方并验证了该复合水凝胶中每种凝胶体系的交联效果，该复合水凝胶具有平衡的粘弹性、自愈性和高渗透性。然后我们测试了水凝胶的可打印性和打印构建体的水环境耐久性，并在21天的培养过程中评估了打印构建体中的细胞活力和增殖。类神经、类肌肉和类软骨构建体也被成功打印，每种构建体中的细胞都能均匀生长，并表现出优异的生物学特性。
Fig.1. Schematic illustration of TSHSP formulation and the uniform proliferation pattern of cells in tissue-like constructs.
该新型生物墨水配方中，快速凝胶化的AHA/CMC可以实现墨水的即用性(室温下达到凝胶化时间为99.83 ± 14.28秒，达到可维持结构强度时间为500.36 ± 29.79秒)，缓慢持续凝胶化的GEL/PEG-SG可以在打印中保持外形及微结构稳定(室温下30分钟后仍有交联行为)。通过优化各组分配比，我们最终获得了含有1%AHA、0.75%CMC、1%GEL和0.5%PEG-SG的TSHSP水凝胶生物墨水(Fig. 2)。
Fig. 2. Optimization of TSHSP hydrogel formulation. (A) Schematic diagram of HA oxidation. (B) Actual oxidation degree and oxidation efficiency profile of AHA under different sodium periodate equivalents (n = 3, error bars, mean ± SD). (C) G'max distribution of AHA/CMC (i-iv) and GEL/PEG-SG (v) hydrogels under different formulations, macroscopic appearance of AHA/CMC hydrogels with different G'max (vi). (D)-(F) Gelation profile of AHA/CMC (D), GEL/PEG-SG (E), and TSHSP (AHA/CMC-GEL/PEG-SG, F) formulation. (G)-(I) Macroscopic appearance of AHA/CMC, GEL/PEG-SG, TSHSP formulations (G), AHA-GEL mixture (H), and CMC-PEG-SG mixture (I). *P < 0.05, over previous oxidation degree.
TSHSP生物墨水在室温下具有均衡的粘弹性和自愈性，这为墨水的室温可打印性提供了保证。此外，配方中的AHA/CMC和GEL/PEG-SG体系分别确保了打印构建体的高渗透性和水环境耐久性(Fig. 3-5)。
Fig. 3. Frequency dependence of shear moduli (A), |η*| (B), and tan δ (C) of AHA/CMC, GEL/PEG-SG, and TSHSP hydrogels.
Fig. 4. Self-healing behavior and permeability of TSHSP hydrogel. (A)-(C) Response of AHA/CMC (A), GEL/PEG-SG (B), and TSHSP(C) hydrogels to increasing strains. (D)-(F) Recovery performance of AHA/CMC (D), GEL/PEG-SG (E), and TSHSP(F) hydrogels after repeated deformations. (G) Macroscopic self-healing behavior of TSHSP hydrogels (i), healed TSHSP hydrogels were lifted against their own weights (ii-iii). Hydrogels were stained with blue and red food dyes. (H) The dyes in the healed TSHSP hydrogel (fused after 4 h at room temperature) almost entirely escaped into PBS after 2 h at 37°C (i), the new added yellow food dyes infiltrated into the hydrogel after another 2 h (ii).
Fig. 5. Printability of TSHSP hydrogels and subaqueous durability of cell-free constructs. (A) Continuous extrusion of TSHSP hydrogel ink. (B) Printing of 6 grid structures at once with TSHSP ink (i) and schematic view of the gelation mechanism of TSHSP ink (ii). (C) Photographs of grid structure printed with TSHSP ink and its top view (i), laboriously printed non-integrated GEL/PEG-SG structure that shattered after being immersed in PBS for 10 min at room temperature (ii), and printed bulk AHA/CMC constructs (iii). (D)-(F) Subaqueous images of the integrity of cell-free constructs printed with different proportions of TSHSP formulations during incubation. (G) Printing fidelity and dimensional change profile of cell-free constructs compared to design (n = 3, error bars, mean ± SD).
将完成打印的负载细胞构建体与4%PEG-SG交联仅3分钟，即可获得稳定的内部和外部 GEL/PEG-SG网络。在NIH/3T3的TSHSP生物打印构建体中，细胞表现出持久的活力和伸展的形态。在培养至21天细胞处于生长平台期时，TSHSP构建体中的细胞仍有12.35 ± 1.65%增殖率，细胞活力高达89.97 ± 1.48% (Fig. 6)。
Fig. 6. Cell proliferation and viability performance of NIH/3T3 in bioprinted constructs. (A) Comparison of cell proliferation profiles in TSHSP hydrogel constructs and GEL-ALG constructs (Alamar Blue assay) (n = 3, error bars, mean ± SD). (B) Comparison of DNA replication in TSHSP hydrogel constructs and GEL-ALG constructs (i) (EdU incorporation assay) (n = 3, error bars, mean ± SD), cytometry plots of DNA replication in the above two constructs on day 9 and 21 (ii-v). (C)-(D) Micrographs and cytometry plots of live/dead stained cells in TSHSP hydrogel constructs (C) and GEL-ALG constructs (D). (E) Comparison of the changing trend of the proportion of cells in different states in the two constructs (n = 3, error bars, mean ± SD). **P < 0.01, between different constructs at each time point.
在使用NE-4C、C2C12和软骨细胞的TSHSP生物墨水打印的类神经、类肌肉和类软骨体外构建体中，不同种类的细胞均表现出均匀生长的特点和显著的特异性标志物表达(Fig. 7和Fig. 8)。
Fig. 7. Properties of in vitro tissue-like constructs after 21 days of culture. (A)-(C) Photographs of NE-4C (A), C2C12 (B), and Chondrocyte (C) constructs printed with TSHSP formulation after 21 days of culture. (D)-(F) Images of DAPI stained NE-4C (D), C2C12 (E), and chondrocyte (F) constructs. (G) Cell density and distribution plots of tissue-like constructs evaluated by DAPI staining (n = 10). (H) Cells/cell clusters dispersion degree plots of tissue-like constructs evaluated by CoV (SD/mean) of % area of DAPI. Dotted lines, microfilament range.
Fig. 8. Immunohistological images and semi-quantitative analyses of specific markers of in vitro tissue-like constructs after 21 days of culture. (A)-(C) Nestin and tublin staining in NE-4C constructs and semi-quantitative analyses of their immunoreactivity (n = 5, error bars, mean ± SD). (D)-(F) Desmin and fast myosin skeletal heavy chain staining in C2C12 constructs and semi-quantitative analyses of their immunoreactivity (n = 5, error bars, mean ± SD). (G)-(I) Collagen II and alcian blue staining in chondrocyte constructs and their semi-quantitative analyses (n = 5, error bars, mean ± SD). Yellow and orange squares: the position of the high-magnification images below. Dotted lines, microfilament range. *P < 0.05, **P < 0.01, ***P < 0.001, between different constructs.
本研究中，我们通过简单混合具有动态共价键的快速 AHA/CMC 凝胶体系和具有稳定共价键的慢速 GEL/PEG-SG凝胶体系，成功制备了TSHSP 生物墨水。该生物墨水凝胶化的TSH特性可实现室温即时打印，避免打印纤维丝断裂，实现完整结构支撑性构建体的均匀打印，确保打印过程中形状和微观结构的稳定性以及孵育过程中构建体的耐久性。这种生物墨水的低刚度、高保水性、高渗透性、生物相容性和均匀分布的细胞使类组织构建体中的细胞活力、细胞形态和生物特异性表现良好。我们认为这种生物墨水未来可用于各种软组织工程，以细胞载体的形式与高强度材料结合用于硬组织工程，或用于靶向细胞治疗。此外，当利用其它凝胶体系开发具有类似要求的生物墨水或细胞载体时，该生物墨水的TSH策略也可以为之提供一种思路。
