近年來，研究者採用人類重組蛋白(MeTro)開發出了一類可降解的密封膠。與商用密封膠(如Evicel、Coseal和Progel)相比，具有更好的組織黏附性能和力學強度，但是較高的生產成本限制了它的臨床應用。因此，來自加州大學Ali Khademhosseini教授團隊和Amir Sheikhi教授團隊開發了一種韌性良好、低成本、可降解、具有良好生物相容性的可注射水凝膠。研究者設計了一種以明膠和海藻酸鹽為基礎的複合水凝膠，並評估其密封傷口的能力。實驗證明改性的海藻酸鹽(AlgMA)在與改性的明膠(GelMA)共價鍵合的過程中可以形成離子網絡，從而賦予了這種複合水凝膠獨特的力學性能。
這種新型複合水凝膠由改性的GelMA和AlgMA組成。水凝膠中的甲基丙烯酸(MA)基團通過光引發劑形成具有親水的複合聚合物網絡。並且，MA基團與胺基和巰基形成的共價鍵以及GelMA與AlgMA之間形成的氫鍵可以提高水凝膠的組織黏附能力。最後，由於Ca2+的加入進一步促進AlgMA的交聯，從而製備出一種具有良好力學性能與組織黏附性能的新型水凝膠（圖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濃度對複合水凝膠性能的影響。結果顯示單純GelMA水凝膠的斷裂應變只有40%，在加入2-3% w/v的AlgMA時後，複合水凝膠的斷裂應變可以增加到100%，然而進一步增加AlgMA的濃度，水凝膠的斷裂應變則會降低（圖 2a-c）。隨著AlgMA濃度的增加，水凝膠的拉伸模量、拉伸強度、韌性不斷增加，然而當AlgMA濃度過高時複合水凝膠的各項性能會顯著下降（圖2d-f）。水凝膠壓縮後的圖像顯示，GelMA水凝膠在50%的應變下會有嚴重的損傷，而含有2% AlgMA的複合水凝膠可以承受3倍的壓力，並在超過80%的應變下保持其結構完整性（圖 2g）。從複合水凝膠的應力應變曲線圖中可以看出，複合水凝膠的抗壓強度和壓縮模量也會隨著AlgMA濃度的增加而顯著增加(圖 2h-j)。複合水凝膠的循環壓縮應力應變曲線顯示增加AlgMA濃度後，水凝膠的滯後作用會更加明顯，這是由於AlgMA與Ca2+之間動態離子鍵的作用。當AlgMA濃度為2-3%時，從GelMA水凝膠的15%到45%的壓縮滯後曲線內的能量損耗有明顯增加（圖 2k-l）。複合水凝膠的儲存模量(G′)和損耗模量(G″)隨著AlgMA濃度的增加而不斷增加。AlgMA對損耗模量的影響更明顯，可能是因為鈣離子的動態交聯減弱了能量耗散。損失係數或阻尼係數定義為G′與G″的比值，隨著AlgMA濃度的增加，能量耗散和損失係數也隨之增加（圖 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 (****).
複合水凝膠的溶脹性能和降解性能對其封閉傷口和促進組織再生的能力很重要。高溶脹比的凝膠可能會在組織和水凝膠的界面產生應力而不利於凝膠與組織間的粘附。在37 ℃時複合水凝膠的溶脹能力隨DPBS培養時間的變化而變化。無論AlgMA濃度如何，混合水凝膠在交聯後約4 h達到溶脹平衡（圖 3a）。在DPBS中孵育4 h後水凝膠的平衡膨脹率顯示：當AlgMA濃度為3%左右時，增加AlgMA濃度可使溶脹率從5%提高到20%。隨後研究者研究了膠原酶對混合水凝膠的降解作用，膠原酶是體內降解膠原蛋白的主要酶之一（圖 3b）。在膠原酶的作用下，所有的水凝膠都會隨著時間的推移而降解。在低AlgMA濃度(2%)時，在1周內超過40%的水凝膠會降解，到第5周時超過80%的凝膠會降解（圖 3c）。膠原酶溶液中浸泡的水凝膠的降解情況顯示隨著AlgMA濃度的增加，水凝膠的降解率會顯著降低（圖 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 (****).
研究者採用組織黏附法對複合水凝膠的黏附性能進行了評價。研究者設計了體外模擬壓力裝置，在膠原蛋白片上造孔以模擬傷口，然後將預凝膠滴在穿孔上，接著暴露在可見光下形成凝膠以密封孔洞。水凝膠對孔洞的粘附性能是通過測量壓力來評估的。圖 4b顯示了從實驗中獲得的壓力隨時間的分布曲線，可見隨著空氣被引入密封孔下，壓力呈線性增加直到水凝膠破壞。結果顯示存在一個使孔洞密封強度最大化的最佳AlgMA濃度，在此濃度下孔洞密封強度可以提高250%以上。拉伸應變-應力曲線如圖所示，在沒有AlgMA時水凝膠膠通常會破裂，在2-3% AlgMA時密封膠也會由於水凝膠從豬皮膚上脫落而破裂（圖 4e）。研究者發現傷口閉合強度，即水凝膠在脫落前所能承受的最大應力，存在一個使穿口密封強度最大的AlgMA濃度，該濃度一般在2-3%範圍內，可以使創面閉合強度達到最高值(圖 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 (****).
為了模擬對可拉伸器官的封閉作用，研究者對豬膀胱和輸尿管進行了體外封閉實驗。圖5a顯示了一個離體豬膀胱進行人工穿孔的模型，然後加入預凝膠溶液，採用可見光進行交聯，然後加入CaCl2和水（圖 5a）。從密封體系承受的壓力可以看出，複合水凝膠的組成對破裂壓力有直接的影響。不含AlgMA的凝膠的破裂壓力只有2 KPa，當AlgMA濃度增加到2%時，破裂壓力增加到5 KPa。進一步增加AlgMA濃度至5%，破裂壓力會降低。AlgMA對破裂壓力的影響與對拉伸強度、韌性和創口閉合強度的影響相似。研究者還評估了複合水凝膠在連接撕裂組織(如輸尿管)的能力。結果顯示複合水凝膠可以很容易地將兩塊切開的輸尿管連接在一起，並通過光交聯和Ca2+介導的物理結合將它們粘接在一起（圖 5c）。通過拉伸試驗得到含有不同濃度AlgMA的複合水凝膠的拉伸應力與應變曲線(圖 5e)。在無AlgMA或AlgMA濃度低於2%時，複合凝膠的力學性能較差，在凝膠中加入了2%的AlgMA後抗拉強度會提高6倍。實驗證明了在凝膠中加入少量的AlgMA後，凝膠的抗拉強度和韌性有了顯著的改善。黏附應力與AlgMA濃度的關係顯示無AlgMA密封劑的失效應力為20 kPa，而複合凝膠（2% AlgMA）在90 kPa時破裂，在凝膠中包含5% AlgMA時失效應力降低到60 kPa（圖 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成纖維細胞對新型複合水凝膠的細胞毒性進行了評估。在0%、2%和5% AlgMA的複合水凝膠上培養3天和7天的活死染色螢光圖像顯示在凝膠中添加AlgMA對細胞活性沒有顯著影響。成纖維細胞成功地粘附在複合水凝膠上並在整個培養期間保持良好狀態（圖 6a-f）。如圖所示含有5% AlgMA的複合水凝膠的細胞存活率為90%，這與對照水凝膠的細胞存活率相似（圖 6g）。在第1,3,7天接種於複合水凝膠上的細胞的代謝活動如圖所示。黏附在含有0-5% AlgMA的水凝膠上的細胞的代謝活動沒有顯著差異。在第3天和第5天，細胞活性較第1天分別增加了2和4倍，因此，複合水凝膠不僅沒有細胞毒性，而且還支持細胞粘附和生長（圖 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 (****).
本研究由來自於加州大學生物工程系的Ali Khademhosseini教授團隊和來自於加州大學的加州納米研究所的Amir Sheikhi教授團隊合作完成，並於2020年4月發表於Advanced Healthcare Materials。
論文信息：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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