《Advanced Healthcare Materials》：一种基于天然聚合物的高强度、可注射、组织粘合的复合水凝胶

来源：生物医用高分子材料

近年来，研究者采用人类重组蛋白(MeTro)开发出了一类可降解的密封胶。与商用密封胶(如Evicel、Coseal和Progel)相比，具有更好的组织黏附性能和力学强度，但是较高的生产成本限制了它的临床应用。因此，来自加州大学Ali Khademhosseini教授团队和Amir Sheikhi教授团队开发了一种韧性良好、低成本、可降解、具有良好生物相容性的可注射水凝胶。研究者设计了一种以明胶和海藻酸盐为基础的复合水凝胶，并评估其密封伤口的能力。实验证明改性的海藻酸盐(AlgMA)在与改性的明胶(GelMA)共价键合的过程中可以形成离子网络，从而赋予了这种复合水凝胶独特的力学性能。

这种新型复合水凝胶由改性的GelMA和AlgMA组成。水凝胶中的甲基丙烯酸(MA)基团通过光引发剂形成具有亲水的复合聚合物网络。并且，MA基团与胺基和巯基形成的共价键以及GelMA与AlgMA之间形成的氢键可以提高水凝胶的组织黏附能力。最后，由于Ca2+的加入进一步促进AlgMA的交联，从而制备出一种具有良好力学性能与组织黏附性能的新型水凝胶（图1）。

Figure 1. Schematic of a GelMA-AlgMA hybrid hydrogel undergoing photo/ionic crosslinking and tissue adhesion. Both AlgMA and GelMA undergo covalent crosslinking through the photo-initiated polymerization of methacrylate/methacryloyl (MA) groups. In AlgMA, the G blocks on the polymer chains form ionic bonds with Ca2+, providing a reversibly crosslinked network. Crosslinking hybrid GelMA/AlgMA hydrogels yields two types of polymer networks intertwined and connected by covalent bonds (via MA groups) supplemented by the Ca2+-mediated physical bonds of AlgMA. GelMA may interact with amine-rich biological tissues through the formation of hydrogen bonds as well as covalent bonding of amine-MA and thiol-MA groups. AlgMA can interact with the tissue via hydrogen bonding, covalent bonding, and/or electrostatic interactions between the carboxylate and amino groups.

研究者研究了AlgMA浓度对复合水凝胶性能的影响。结果显示单纯GelMA水凝胶的断裂应变只有40%，在加入2-3% w/v的AlgMA时后，复合水凝胶的断裂应变可以增加到100%，然而进一步增加AlgMA的浓度，水凝胶的断裂应变则会降低（图 2a-c）。随着AlgMA浓度的增加，水凝胶的拉伸模量、拉伸强度、韧性不断增加，然而当AlgMA浓度过高时复合水凝胶的各项性能会显著下降（图2d-f）。水凝胶压缩后的图像显示，GelMA水凝胶在50%的应变下会有严重的损伤，而含有2% AlgMA的复合水凝胶可以承受3倍的压力，并在超过80%的应变下保持其结构完整性（图 2g）。从复合水凝胶的应力应变曲线图中可以看出，复合水凝胶的抗压强度和压缩模量也会随着AlgMA浓度的增加而显著增加(图 2h-j)。复合水凝胶的循环压缩应力应变曲线显示增加AlgMA浓度后，水凝胶的滞后作用会更加明显，这是由于AlgMA与Ca2+之间动态离子键的作用。当AlgMA浓度为2-3%时，从GelMA水凝胶的15%到45%的压缩滞后曲线内的能量损耗有明显增加（图 2k-l）。复合水凝胶的储存模量(G′)和损耗模量(G″)随着AlgMA浓度的增加而不断增加。AlgMA对损耗模量的影响更明显，可能是因为钙离子的动态交联减弱了能量耗散。损失系数或阻尼系数定义为G′与G″的比值，随着AlgMA浓度的增加，能量耗散和损失系数也随之增加（图 2m-o）。

Figure 2. Mechanical and rheological properties of hybrid hydrogels composed of GelMA (20% w/v) and varying concentrations of AlgMA. a) Images of the hybrid hydrogels containing 0% and 2% w/v AlgMA undergoing stretching. b) Representative tensile stress–strain curves, c) tensile strain at break, d) Young's modulus, e) tensile strength, and f) toughness for the hybrid hydrogels containing varying concentrations of AlgMA. g) Images of the hybrid hydrogels undergoing compression, and h) representative compressive stress–strain curves, i) compressive strength, j) compressive modulus, k) cyclic compressive stress–strain curves, and l) energy loss for the hybrid hydrogels. The m) storage modulus, n) loss modulus, and o) loss factor at angular frequency = 1 rad s−1 and strain = 0.1% for the hybrid hydrogels. Data are reported as the mean values of at least 5 experiments ± their standard deviation. The statistical analysis was done according to the methods explained in “Statistical Analysis” section. Asterisks show the results that are statically significant with p-values < 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****).

复合水凝胶的溶胀性能和降解性能对其封闭伤口和促进组织再生的能力很重要。高溶胀比的凝胶可能会在组织和水凝胶的界面产生应力而不利于凝胶与组织间的粘附。在37 ℃时复合水凝胶的溶胀能力随DPBS培养时间的变化而变化。无论AlgMA浓度如何，混合水凝胶在交联后约4 h达到溶胀平衡（图 3a）。在DPBS中孵育4 h后水凝胶的平衡膨胀率显示：当AlgMA浓度为3%左右时，增加AlgMA浓度可使溶胀率从5%提高到20%。随后研究者研究了胶原酶对混合水凝胶的降解作用，胶原酶是体内降解胶原蛋白的主要酶之一（图 3b）。在胶原酶的作用下，所有的水凝胶都会随着时间的推移而降解。在低AlgMA浓度(2%)时，在1周内超过40%的水凝胶会降解，到第5周时超过80%的凝胶会降解（图 3c）。胶原酶溶液中浸泡的水凝胶的降解情况显示随着AlgMA浓度的增加，水凝胶的降解率会显著降低（图 3d）。

Figure 3. Physical properties of hybrid hydrogels composed of GelMA (20% w/v) and varying concentrations of AlgMA. a) Time-dependent swelling ratio of hydrogels immersed in DPBS at 37 °C. b) The swelling ratio of the hydrogels after 4 h incubation in DPBS. c) Time-dependent degradation of the hydrogels immersed in DPBS containing collagenase (1.25 U mL−1) at 37 °C. d) The degradation of hydrogels after 5 weeks of incubation in DPBS containing collagenase (1.25 U mL−1) at 37 °C. Data are reported as the mean values of at least 5 experiments ± their standard deviation. The statistical analysis was done according to the methods explained in “Statistical Analysis” section. Asterisks show the results that are statically significant with p-values < 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****).

研究者采用组织黏附法对复合水凝胶的黏附性能进行了评价。研究者设计了体外模拟压力装置，在胶原蛋白片上造孔以模拟伤口，然后将预凝胶滴在穿孔上，接着暴露在可见光下形成凝胶以密封孔洞。水凝胶对孔洞的粘附性能是通过测量压力来评估的。图 4b显示了从实验中获得的压力随时间的分布曲线，可见随着空气被引入密封孔下，压力呈线性增加直到水凝胶破坏。结果显示存在一个使孔洞密封强度最大化的最佳AlgMA浓度，在此浓度下孔洞密封强度可以提高250%以上。拉伸应变-应力曲线如图所示，在没有AlgMA时水凝胶胶通常会破裂，在2-3% AlgMA时密封胶也会由于水凝胶从猪皮肤上脱落而破裂（图 4e）。研究者发现伤口闭合强度，即水凝胶在脱落前所能承受的最大应力，存在一个使穿口密封强度最大的AlgMA浓度，该浓度一般在2-3%范围内，可以使创面闭合强度达到最高值(图 4f,g)。

Figure 4. In vitro sealing properties of hybrid hydrogels composed of GelMA (20% w/v) and varying concentrations of AlgMA. a) Images showing the burst pressure assessment of double-network hybrid hydrogels prepared via successive photochemical and ion-mediate crosslinking, b) representative pressure–time curves obtained from the burst pressure tests, and c) the burst pressure values of hybrid hydrogels containing varying AlgMA concentrations. d) Wound closure assessment setup, e) representative stress-strain curves from wound closure experiments, f) wound closure strength, g) and adhesion energy of hybrid hydrogels. Data are reported as the mean values of at least 5 experiments ± their standard deviation. The statistical analysis was done according to the methods explained in “Statistical Analysis” section. Asterisks show the results that are statically significant with p-values less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****).

为了模拟对可拉伸器官的封闭作用，研究者对猪膀胱和输尿管进行了体外封闭实验。图5a显示了一个离体猪膀胱进行人工穿孔的模型，然后加入预凝胶溶液，采用可见光进行交联，然后加入CaCl2和水（图 5a）。从密封体系承受的压力可以看出，复合水凝胶的组成对破裂压力有直接的影响。不含AlgMA的凝胶的破裂压力只有2 KPa，当AlgMA浓度增加到2%时，破裂压力增加到5 KPa。进一步增加AlgMA浓度至5%，破裂压力会降低。AlgMA对破裂压力的影响与对拉伸强度、韧性和创口闭合强度的影响相似。研究者还评估了复合水凝胶在连接撕裂组织(如输尿管)的能力。结果显示复合水凝胶可以很容易地将两块切开的输尿管连接在一起，并通过光交联和Ca2+介导的物理结合将它们粘接在一起（图 5c）。通过拉伸试验得到含有不同浓度AlgMA的复合水凝胶的拉伸应力与应变曲线(图 5e)。在无AlgMA或AlgMA浓度低于2%时，复合凝胶的力学性能较差，在凝胶中加入了2%的AlgMA后抗拉强度会提高6倍。实验证明了在凝胶中加入少量的AlgMA后，凝胶的抗拉强度和韧性有了显著的改善。黏附应力与AlgMA浓度的关系显示无AlgMA密封剂的失效应力为20 kPa，而复合凝胶（2% AlgMA）在90 kPa时破裂，在凝胶中包含5% AlgMA时失效应力降低到60  kPa（图 5f）。

Figure 5. Ex vivo sealing capability of hybrid hydrogels composed of GelMA (20% w/v) and varying concentrations of AlgMA. a) Porcine bladder incision model: images show a,i) a healthy porcine bladder, a,ii) a superficial wound created in the bladder before sealing, a,iii) the wound covered with the hydrogel, a,iv) the subsequent crosslinking of hydrogel with visible light and a,v) with a CaCl2 solution, and a,vi) the sealed bladder filled with water at pressure ≈6 kPa. b) Burst pressure of bioadhesive hybrid sealants at varying AlgMA concentrations. c) Porcine ureter anastomosis model: c,i–iii) images illustrating the method used for sealing a fully torn porcine ureter, followed by d) stretching the tissue to test the wound closure capability of the bioadhesive. e) Representative tensile stress–strain curves and some examples of bioadhesive failure modes during the anastomosis tensile tests. f) Anastomosis strength of hybrid sealants at varying AlgMA concentrations. Data are reported as the mean values of at least 5 (b) and 4 (f) experiments ± their standard deviation. The statistical analysis was done according to the methods explained in “Statistical Analysis” section. Asterisks show the results that are statically significant with p-values < 0.05 (*) or 0.01 (**).

研究者利用NIH/3T3成纤维细胞对新型复合水凝胶的细胞毒性进行了评估。在0%、2%和5% AlgMA的复合水凝胶上培养3天和7天的活死染色荧光图像显示在凝胶中添加AlgMA对细胞活性没有显著影响。成纤维细胞成功地粘附在复合水凝胶上并在整个培养期间保持良好状态（图 6a-f）。如图所示含有5% AlgMA的复合水凝胶的细胞存活率为90%，这与对照水凝胶的细胞存活率相似（图 6g）。在第1,3,7天接种于复合水凝胶上的细胞的代谢活动如图所示。黏附在含有0-5% AlgMA的水凝胶上的细胞的代谢活动没有显著差异。在第3天和第5天，细胞活性较第1天分别增加了2和4倍，因此，复合水凝胶不仅没有细胞毒性，而且还支持细胞粘附和生长（图 6h）。

Figure 6. In vitro cytotoxicity assessment of hybrid hydrogels composed of GelMA (20% w/v) and varying concentrations of AlgMA. a–f) Fluorescence images of live and dead cells stained in green and red colors, respectively, after culturing them on the composite hydrogels, g) viability of fibroblast cells cultured on composite hydrogels, and h) metabolic activity of fibroblast cells represented by the fluorescence intensity of resazurin converted to fluorescent resorufin. Data are reported as the mean values of at least n = 5 (g) and 4 (h) replicates ± their standard deviation. The statistical analysis was done according to the methods explained in “Statistical Analysis” section. Asterisks show the results that are statically significant with p-values less than 0.05 (*), 0.01 (**), 0.001 (***), or 0.0001 (****).

本研究由来自于加州大学生物工程系的Ali Khademhosseini教授团队和来自于加州大学的加州纳米研究所的Amir Sheikhi教授团队合作完成，并于2020年4月发表于Advanced Healthcare Materials。

论文信息：Maryam Tavafoghi, Amir Sheikhi*, Rumeysa Tutar, Jamileh Jahangiry, Avijit Baidya,Reihaneh Haghniaz, and Ali Khademhosseini*. Engineering Tough, Injectable, Naturally Derived, Bioadhesive Composite Hydrogels. Adv Healthcare Mater 2020, 9: 1901722.

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