琼脂糖/明胶/透明质酸/细胞外基质水凝胶的制备与性能表征
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摘要
背景:由于软骨组织无血管结构,无神经组织,缺乏自我修复与再生的内在能力,因此临床上软骨损伤的修复仍然是一个重大挑战。目的:旨在制备一种温敏性可注射生物活性水凝胶,用于软骨组织工程。方法:将0.4%琼脂糖、10%明胶和10 g/L透明质酸溶液混合,制备琼脂糖/明胶/透明质酸水凝胶,然后向其内分别加入0,10,20,30 g/L的软骨细胞外基质,制备4种温敏性可注射生物活性水凝胶,进行扫描电镜、透射电镜、平衡溶胀率、生物力学强度和体外降解率检测。将含20 g/L软骨细胞外基质的温敏性可注射生物活性水凝胶与软骨细胞-骨髓间充质干细胞共培养,培养1,3,7 d,通过活/死细胞荧光双染法评价该水凝胶的细胞相容性。结果与结论:①扫描电镜显示,4种水凝胶均具有良好的三维网格结构,孔径较大;透射电镜显示,细胞外基质颗粒均匀分布于水凝胶中,颗粒粒径主要在50-200 nm;②随着加入细胞外基质颗粒质量浓度的增加,水凝胶的平衡溶胀率降低;③浸泡于PBS溶液4周时,含0,10,20,30 g/L细胞外基质水凝胶的体外降解率分别为42.32%,67.36%,89.05%和99.47%;④当添加细胞外基质颗粒质量浓度在0-20 g/L时,水凝胶的生物力学强度逐渐增加;⑤培养7 d内,软骨细胞-骨髓间充质干细胞在水凝胶内均匀地分布与生长,并不断增殖,细胞存活率均在90%以上;⑥结果表明细胞外基质颗粒修饰的琼脂糖/明胶/透明质酸水凝胶,是一种可用于软骨组织工程的良好医用生物材料。
BACKGROUND: Due to no vascular structure or nerve tissue, cartilage tissue lacks the intrinsic ability of self-repair and regeneration.Therefore, the repair of cartilage defect in clinic remains a challenge.OBJECTIVE: To prepare a thermo-sensitive injectable bioactive hydrogel used for cartilage tissue engineering.METHODS: Agarose/gelatin/hyaluronic acid hydrogel was prepared by mixing 0.4% agarose, 10% gelatin and 10 g/L hyaluronic acid solution.Then 0, 10, 20, and 30 g/L cartilage extracellular matrix particles were added into the hydrogel respectively to prepare four kinds of thermo-sensitive injectable bioactive hydrogels, for further scanning electron microscope, transmission electron microscope, equilibrium swelling ratio, biomechanical strength and degradation rate. The thermo-sensitive injectable bioactive hydrogel containing 20 g/L cartilage extracellular matrix was co-cultured with chondrocyte-bone marrow mesenchymal stem cells for 1, 3, and 7 days, and the cytocompatibility was assessed by live/dead fluorescence double staining.RESULTS AND CONCLUSION:(1) Scanning electron microscope showed that all four hydrogels had good three-dimensional grid structure and large pore size. Transmission electron microscope showed that extracellular matrix particles were evenly distributed in the hydrogels, and the particle size was mainly in the range of 50-200 nm.(2) With the extracellular matrix particle concentration increasing, the equilibrium swelling rate of the hydrogel was decreased.(3) The degradation rate in vitro in the four groups of hydrogels containing 0, 10, 20, and 30 g/L extracellular matrix particles immersed in PBS for 4 weeks was 42.32%, 67.36%, 89.05% and 99.47%, respectively.(4) When the concentration of extracellular matrix particles was added at 0-20 g/L, the biomechanical strength of the hydrogel was gradually increased.(5)Within 7 days after culture, chondrocytes-bone marrow mesenchymal stem cells were uniformly distributed and grew in the hydrogel and proliferated continuously, with the cell survival rate of above 90%.(6) These results showed that the agarose/gelatin/hyaluronic acid hydrogel modified with extracellular matrix particles is a good medical biomaterial that can be used for cartilage tissue engineering.
BACKGROUND: Due to no vascular structure or nerve tissue, cartilage tissue lacks the intrinsic ability of self-repair and regeneration.Therefore, the repair of cartilage defect in clinic remains a challenge.OBJECTIVE: To prepare a thermo-sensitive injectable bioactive hydrogel used for cartilage tissue engineering.METHODS: Agarose/gelatin/hyaluronic acid hydrogel was prepared by mixing 0.4% agarose, 10% gelatin and 10 g/L hyaluronic acid solution.Then 0, 10, 20, and 30 g/L cartilage extracellular matrix particles were added into the hydrogel respectively to prepare four kinds of thermo-sensitive injectable bioactive hydrogels, for further scanning electron microscope, transmission electron microscope, equilibrium swelling ratio, biomechanical strength and degradation rate. The thermo-sensitive injectable bioactive hydrogel containing 20 g/L cartilage extracellular matrix was co-cultured with chondrocyte-bone marrow mesenchymal stem cells for 1, 3, and 7 days, and the cytocompatibility was assessed by live/dead fluorescence double staining.RESULTS AND CONCLUSION:(1) Scanning electron microscope showed that all four hydrogels had good three-dimensional grid structure and large pore size. Transmission electron microscope showed that extracellular matrix particles were evenly distributed in the hydrogels, and the particle size was mainly in the range of 50-200 nm.(2) With the extracellular matrix particle concentration increasing, the equilibrium swelling rate of the hydrogel was decreased.(3) The degradation rate in vitro in the four groups of hydrogels containing 0, 10, 20, and 30 g/L extracellular matrix particles immersed in PBS for 4 weeks was 42.32%, 67.36%, 89.05% and 99.47%, respectively.(4) When the concentration of extracellular matrix particles was added at 0-20 g/L, the biomechanical strength of the hydrogel was gradually increased.(5)Within 7 days after culture, chondrocytes-bone marrow mesenchymal stem cells were uniformly distributed and grew in the hydrogel and proliferated continuously, with the cell survival rate of above 90%.(6) These results showed that the agarose/gelatin/hyaluronic acid hydrogel modified with extracellular matrix particles is a good medical biomaterial that can be used for cartilage tissue engineering.
引文
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[2]安勇,王丹亭,岳宏.基质诱导的自体软骨细胞移植治疗膝关节软骨损伤的研究进展[J].医学研究杂志,2017,46(7):177-180.
[3]Min BH,Choi WH,Lee YS,et al.Effect of different bone marrow stimulation techniques(BSTs)on MSCs mobilization.J Orthop Res.2013;31(11):1814-1819.
[4]Bedi A,Feeley BT,Williams RJ 3rd.Management of articular cartilage defects of the knee.J Bone Joint Surg Am.2010;92(4):994-1009.
[5]Chiang H,Liao CJ,Wang YH,et al.Comparison of articular cartilage repair by autologous chondrocytes with and without in vitro cultivation.Tissue Eng Part C Methods.2010;16(2):291-300.
[6]Langer R,Vacanti JP.Tissue Engineering.Science.2000;260(5110):920-926.
[7]神艳.骺板软骨组织工程支架的研究进展[J].中华小儿外科杂志,2015,36(5):397-400.
[8]张柏青,张仲文,孙磊.Ⅱ型胶原修饰脱细胞真皮基质胶原膜修复关节软骨缺损[J].中国组织工程研究,2017,21(6):871-876.
[9]陈维明.明胶/聚乳酸纳米纤维三维多孔支架的制备及应用于关节软骨组织再生[D].上海:东华大学,2017.
[10]李奇龙.一种光聚合PEGDA-HA水凝胶递送纳米粒药物用于非小细胞肺癌的治疗[D].上海:华东师范大学,2017.
[11]刘环宇,叶静仪,梁佩莹.水凝胶的制备[J].化工时刊,2014,28(1):11-14.
[12]朱文,段世锋,丁建东.组织工程用水凝胶材料[J].功能高分子学报,2004,17(4):689-697.
[13]Ninan N,Forget A,Shastri VP,et al.Antibacterial and Anti-Inflammatory p H-Responsive Tannic Acid-Carboxylated Agarose Composite Hydrogels for Wound Healing.ACS Appl Mater Interfaces.2016;8(42):28511-28521.
[14]Singh YP,Bhardwaj N,Mandal BB.Potential of Agarose/Silk Fibroin Blended Hydrogel for in Vitro Cartilage Tissue Engineering.ACSAppl Mater Interfaces.2016;8(33):21236-21249.
[15]Zheng L,Sun J,Chen X,et al.In vivo cartilage engineering with collagen hydrogel and allogenous chondrocytes after diffusion chamber implantation in immunocompetent host.Tissue Eng Part A.2009;15(8):2145-2153.
[16]Chao PH,Yodmuang S,Wang X,et al.Silk hydrogel for cartliage tissue enginee-ring.J Biomed Mater Res.2010;95(1):84-90.
[17]Buwalda SJ,Boere KW,Dijkstra PJ,et al.Hydrogels in a historical perspective:from simple networks to smart materials.J Control Release.2014;190:254-273.
[18]Smeets N,Bakaic E,Patenaude M,et al.Injectable poly(oligoethylene glycol methacrylate)-based hydrogels with tunable phase transition behaviours:physicochemical and biological responses.Acta Biomater.2014;10(10):4143-4155.
[19]Elias PZ,Liu GW,Wei H,et al.A functionalized,injectable hydrogel for localized drug delivery with tunable thermosensitivity:synthesis and characterization of physical and toxicological properties.JControl Release.2015;208:76-84.
[20]Lin C,Zhao P,Li F,et al.Thermosensitive in situ-forming dextranpluronic hydrogels through Michael addition.Mater Sci Eng C.2010;30(8):1236-1244.
[21]Yu L,Ding J.Injectable hydrogels as unique biomedical materials.Chem Soc Rev.2008;37(8):1473-1481.
[22]Ni P,Ding Q,Fan M,et al.Injectable thermosensitive PEG-PCL-PEG hydrogel/acellular bone matrix composite for bone regeneration in cranial defects.Biomaterials.2014;35(1):236-248.
[23]Hudson SP,Langer R,Fink GR,et al.Injectable in situ cross-linking hydrogels for local antifungal therapy.Biomaterials.2010;31(6):1444-1452.
[24]Li L,Wang N,Jin X,et al.Biodegradable and injectable in situ crosslinking chitosan-hyaluronic acid based hydrogels for postoperative adhesion prevention.Biomaterials.2014;35(12):3903-3917.
[25]Xie B,Jin L,Luo Z,et al.An injectable thermosensitive polymeric hydrogel for sustained release of Avastin to treat posterior segment disease.Int J Pharm.2015;490(1-2):375-383.
[26]倪培艳,罗静聪,钱志勇.可注射的温度敏感型水凝胶复合材料在骨组织工程中的应用研究[EB/OL].北京:中国科技论文在线[2012-02-22].http://www.paper.edu.cn/releasepaper/content/201202-823.
[27]孙斌.可注射温敏壳聚糖/胶原/甘油磷酸钠水凝胶应用于骨组织工程的初步研究[D].北京:解放军军医进修学院,2011.
[28]赵守军,熊文化,许柯.细胞因子基因转染的骨骺干细胞复合明胶-硫酸软骨素-透明质酸钠支架修复大鼠骨骺生长板缺损的实验研究[J].中医正骨,2018,30(2):10-15.
[29]Tibbitt MW,Anseth KS.Hydrogels as extracellular matrix mimics for 3D cell culture.Biotechnol Bioeng.2009;103:655-663.
[30]Yin HY,Wang Y,Sun Z,et al.Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles.Acta Biomater.2016;33:96-109.
[31]Buckley CT,Kelly DJ.Expansion in the presence of FGF-2enhances the functional development of cartilaginous tissues engineered using infrapatellar fat pad derived MSCs.J Mech Behav Biomed Mater.2012;11:102-111.
[32]Pia M,Alexander T,Hagen Schmal,et al.Nanomechanics of Human Adipose-Derived Stem Cells:Small GTPases Impact Chondrogenic Differentiation.Tissue Eng Part A.2012;18(9-10):1035-1044.
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