可注射性乙二醇壳聚糖/双醛功能化聚乙二醇水凝胶的细胞相容性
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摘要
背景:课题组前期研究自主研发了可注射性乙二醇壳聚糖/双醛功能化聚乙二醇水凝胶(glycolchitosan/dibenzaldehyde-terminated poly-ethyleneglycol,GCS/DF-PEG),研究显示其具有较好的可注射性和自愈性。目的:进一步检测可注射性GCS/DF-PEG的物理学性质及细胞相容性。方法:将质量分数1.5%的乙二醇壳聚糖溶液与质量分数分别为2%,4%,8%的双醛功能化聚乙二醇溶液等体积混合,制备3组可注射性GCS/DF-PEG水凝胶,检测其弹性模量。将3组可注射性水凝胶分别浸入PBS中4周,检测凝胶的体外降解。提取第2代SD乳鼠脂肪间充质干细胞,实验组加入可注射性GCS/DF-PEG水凝胶浸提液,对照组常规培养,MTT法检测细胞增殖。将质量分数3%的乙二醇壳聚糖溶液与第2代SD乳鼠脂肪间充质干细胞混合,再与质量分数为4%的双醛功能化聚乙二醇溶液混合,培养第1,5天,采用死活染色法检测细胞在水凝胶内死活状态。将质量分数3%的乙二醇壳聚糖溶液与第2代SD乳鼠脂肪间充质干细胞混合,再与质量分数分别为2%,4%,8%的双醛功能化聚乙二醇溶液混合,MTT法检测细胞增殖。结果与结论:(1)质量分数分别为2%,4%,8%双醛功能化聚乙二醇组水凝胶的弹性模量分别为13.48,22.21,33.19kPa;(2)随时间的延长,3组水凝胶的降解率均逐渐增加,质量分数2%双醛功能化聚乙二醇组水凝胶的降解速率明显快于其他两组;(3)培养7d内,实验组细胞增殖与对照组比较无差异;(4)死活染色显示,脂肪间充质干细胞在水凝胶内呈球形,存活率在90%以上,且随时间延长细胞数量明显增多;(5)随着时间的延长,脂肪间充质干细胞在含不同质量分数双醛功能化聚乙二醇GCS/DF-PEG水凝胶内的数量逐渐增多,且含质量分数2%双醛功能化聚乙二醇组水凝胶内的细胞增殖快于含质量分数4%,8%双醛功能化聚乙二醇组(P <0.05);(6)结果表明,可注射性GCS/DF-PEG水凝胶的细胞相容性良好,力学强度及降解具有可调性,有望成为软骨组织工程中干细胞移植的良好载体。
BACKGROUND: Our research group independently developed an injectable glycol chitosan/dibenzaldehyde-terminated poly-ethyleneglycol(GCS/DF-PEG) hydrogel, which has good injectability and self-healing properties. OBJECTIVE: To test the physical properties and cytocompatibility of the GCS/DF-PEG hydrogel. METHODS: The injectable GCS/DF-PEG hydrogel was prepared by mixing GCS solution at a mass fraction of 1.5% with an equal volume of DF-PEG solution at a mass fraction of 2%, 4%, and 8%, respectively. Their moduli of elasticity were measured. Three groups of injectable hydrogels were immersed in PBS for 4 weeks to detect the in vitro degradation of the hydrogels. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were cultured in injectable GCS/DF-PEG hydrogel leaching solution as experimental group or cultured routinely as control group. MTT assay was used to detect the cell proliferation. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 4% DF-PEG solution. On the 1 st and 5 th days of culture, the cell survival and death in the hydrogel were tested by live/dead staining. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 2%, 4% and 8% DF-PEG solution, respectively. MTT method was used for testing the cell proliferation. RESULTS AND CONCLUSION:(1) The moduli of elasticity of GCS/DF-PEG hydrogel with 2%, 4%, 8% DF-PEG were 13.48, 22.21 and 33.19 kPa, respectively.(2) In vitro degradation experiments showed that GCS/DF-PEG hydrogels gradually degraded in PBS over time. And the degradation rate of the 2% DF-PEG hydrogel was significantly faster than the other two groups.(3) Within 7 days of culture, there was no difference in the cell proliferation between the experimental and control groups.(4) Live/dead staining results showed that adipose-derived mesenchymal stem cells were spherical in the hydrogel, the survival rate was over 90%, and the number of cells increased significantly with time.(5) Over time, the number of adipose-derived mesenchymal stem cells in the GCS/DF-PEG hydrogel with different mass fractions gradually increased. And the cell proliferation in the hydrogel containing 2% DF-PEG was faster than 4% and 8% groups(P < 0.05). In conclusion, the injectable GCS/DF-PEG hydrogel has good cytocompatibility, mechanical strength and degradation, and it is expected to be a good carrier for stem cell transplantation in cartilage tissue engineering.
BACKGROUND: Our research group independently developed an injectable glycol chitosan/dibenzaldehyde-terminated poly-ethyleneglycol(GCS/DF-PEG) hydrogel, which has good injectability and self-healing properties. OBJECTIVE: To test the physical properties and cytocompatibility of the GCS/DF-PEG hydrogel. METHODS: The injectable GCS/DF-PEG hydrogel was prepared by mixing GCS solution at a mass fraction of 1.5% with an equal volume of DF-PEG solution at a mass fraction of 2%, 4%, and 8%, respectively. Their moduli of elasticity were measured. Three groups of injectable hydrogels were immersed in PBS for 4 weeks to detect the in vitro degradation of the hydrogels. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were cultured in injectable GCS/DF-PEG hydrogel leaching solution as experimental group or cultured routinely as control group. MTT assay was used to detect the cell proliferation. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 4% DF-PEG solution. On the 1 st and 5 th days of culture, the cell survival and death in the hydrogel were tested by live/dead staining. Passage 2 adipose-derived mesenchymal stem cells from Sprague-Dawley neonatal rats were mixed with 3% GCS solution, and then mixed with 2%, 4% and 8% DF-PEG solution, respectively. MTT method was used for testing the cell proliferation. RESULTS AND CONCLUSION:(1) The moduli of elasticity of GCS/DF-PEG hydrogel with 2%, 4%, 8% DF-PEG were 13.48, 22.21 and 33.19 kPa, respectively.(2) In vitro degradation experiments showed that GCS/DF-PEG hydrogels gradually degraded in PBS over time. And the degradation rate of the 2% DF-PEG hydrogel was significantly faster than the other two groups.(3) Within 7 days of culture, there was no difference in the cell proliferation between the experimental and control groups.(4) Live/dead staining results showed that adipose-derived mesenchymal stem cells were spherical in the hydrogel, the survival rate was over 90%, and the number of cells increased significantly with time.(5) Over time, the number of adipose-derived mesenchymal stem cells in the GCS/DF-PEG hydrogel with different mass fractions gradually increased. And the cell proliferation in the hydrogel containing 2% DF-PEG was faster than 4% and 8% groups(P < 0.05). In conclusion, the injectable GCS/DF-PEG hydrogel has good cytocompatibility, mechanical strength and degradation, and it is expected to be a good carrier for stem cell transplantation in cartilage tissue engineering.
引文
[1]Rai V,Dilisio MF,Dietz NE,et al.Recent strategies in cartilage repair:A systemic review of the scaffold development and tissue engineering.J Biomed Mater Res A.2017;105(8):2343-2354.
[2]Laudy AB,Bakker EW,Rekers M,et al.Efficacy of platelet-rich plasma injections in osteoarthritis of the knee:a systematic review and meta-analysis.Br J Sports Med.2015;49(10):657-672.
[3]Lynch TS,Patel RM,Benedick A,et al.Systematic review of autogenous osteochondral transplant outcomes.Arthroscopy.2015;31(4):746-754.
[4]Oussedik S,Tsitskaris K,Parker D.Treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation:a systematic review.Arthroscopy.2015;31(4):732-744.
[5]Johnstone B,Alini M,Cucchiarini M,et al.Tissue engineering for articular cartilage repair--the state of the art.Eur Cell Mater.2013;25:248-267.
[6]刘雪婷,支晓亮,白春雨,等.脂肪间充质干细胞生物学特性及分泌功能研究[J].农业科学与技术,2016,17(6):1331-1335.
[7]Kasir R,Vernekar VN,Laurencin CT.Regenerative Engineering of Cartilage Using Adipose-Derived Stem Cells.Regen Eng Transl Med.2015;1(1):42-49.
[8]Pleumeekers MM,Nimeskern L,Koevoet JLM,et al.Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.PloS One.2018;13(2):1-23.
[9]Zhang J,Du C,Guo W,et al.Adipose Tissue-Derived Pericytes for Cartilage Tissue Engineering.Curr Stem Cell Res Ther.2017;12(6):513-521.
[10]Yang J,Zhang YS,Yue K,et al.Cell-laden hydrogels for osteochondral and cartilage tissue engineering.Acta Biomater.2017;57:1-25.
[11]Liu M,Zeng X,Ma C,et al.Injectable hydrogels for cartilage and bone tissue engineering.Bone Res.2017;5(2):75-94.
[12]Zhang Y,Tao L,Li S,et al.Synthesis of multiresponsive and dynamic chitosan-based hydrogels for controlled release of bioactive molecules.Biomacromolecules.2011;12(8):2894-2901.
[13]Li Y,Zhang Y,Shi F,et al.Modulus-regulated 3D-cell proliferation in an injectable self-healing hydrogel.Colloids Surf B Biointerfaces.2017;149:168-173.
[14]Qi C,Liu J,Jin Y,et al.Photo-crosslinkable,injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage.Biomaterials.2018;163:89-104.
[15]Sun AX,Lin H,Fritch MR,et al.Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional,photocrosslinked hydrogel constructs:Effect of cell seeding density and material stiffness.Acta Biomaterialia.2017;58:302-311.
[16]徐丽娟.小鼠成体干细胞体外诱导成肝能力比较及与丝素蛋白纤维相容性的实验研究[D].北京:解放军医学院,2016.
[17]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107.
[18]Tsukamoto J,Naruse K,Nagai Y,et al.Efficacy of a Self-assembling Peptide Hydrogel,SPG-178-Gel,for Bone Regeneration and Three-dimensional Osteogenic Induction of Dental Pulp Stem Cells.Tissue Eng Part A.2017;23(23-24):1394-1402.
[19]Stevens M,George JH.Exploring and engineering the cell surface interface.Science.2011;100(3):1135-1138.
[20]Lim SM,Oh SH,Lee HH,et al.Dual growth factor-releasing nanoparticle/hydrogel system for cartilage tissue engineering.J Mater Sci Mater Med.2010;21(9):2593-2600.
[21]Gonzalez-Fernandez T,Tierney EG,Cunniffe GM,et al.Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.Tissue Eng Part A.2016;22(9-10):776-787.
[22]Simson JA,Strehin IA,Lu Q,et al.An adhesive bone marrow scaffold and bone morphogenetic-2 protein carrier for cartilage tissue engineering.Biomacromolecules.2013;14(3):637-643.
[23]王刚,李晓萍,王进.植入壳聚糖体内降解的机理研究[J].当代医学,2015,21(34):5-8.
[24]Sheehy EJ,Mesallati T,Vinardell T,et al.Engineering cartilage or endochondral bone:A comparison of different naturally derived hydrogels.Acta Biomaterialia.2015;13:245-253.
[25]Ansari S,Diniz IM,Chen C,et al.Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage.J Mater Sci Mater Med.2017;28(10):162-173.
[26]Huang SJ,Fu RH,Shyu WC,et al.Adipose-derived stem cells:isolation,characterization,and differentiation potential.Cell Transplant.2013;22(4):701-709.
[27]Panadero JA,Lanceros-Mendez S,Ribelles JL.Differentiation of mesenchymal stem cells for cartilage tissue engineering:Individual and synergetic effects of three-dimensional environment and mechanical loading.Acta Biomaterialia.2016;33:1-12.
[28]Yousefi AM,James PF,Akbarzadeh R,et al.Prospect of Stem Cells in Bone Tissue Engineering:A Review.Stem Cells Int.2016;2016(10):1-13.
[29]Quintiliano K,Crestani T,Silveira D,et al.Neural Differentiation of Mesenchymal Stem Cells on Scaffolds for Nerve Tissue Engineering Applications.Cell Reprogram.2016;18(6):369-381.
[30]Alhadlaq A,Mao JJ.Mesenchymal stem cells:isolation and therapeutics.Stem Cells Dev.2004;13(4):436-448.
[31]Fellows CR,Matta C,Zakany R,et al.Adipose,Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair.Front Genet.2016;7(Pt 6):213-232.
[32]Kang H,Peng J,Lu S,et al.In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds.J Tissue Eng Regen Med.2014;8(6):442-453.
[33]Zhao MC,Liu C.Research Progress on Adipose-Derived Stem Cells in Cartilage Tissue Engineering.Chin J Biomed Eng.2014;33(4):475-481.
[2]Laudy AB,Bakker EW,Rekers M,et al.Efficacy of platelet-rich plasma injections in osteoarthritis of the knee:a systematic review and meta-analysis.Br J Sports Med.2015;49(10):657-672.
[3]Lynch TS,Patel RM,Benedick A,et al.Systematic review of autogenous osteochondral transplant outcomes.Arthroscopy.2015;31(4):746-754.
[4]Oussedik S,Tsitskaris K,Parker D.Treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation:a systematic review.Arthroscopy.2015;31(4):732-744.
[5]Johnstone B,Alini M,Cucchiarini M,et al.Tissue engineering for articular cartilage repair--the state of the art.Eur Cell Mater.2013;25:248-267.
[6]刘雪婷,支晓亮,白春雨,等.脂肪间充质干细胞生物学特性及分泌功能研究[J].农业科学与技术,2016,17(6):1331-1335.
[7]Kasir R,Vernekar VN,Laurencin CT.Regenerative Engineering of Cartilage Using Adipose-Derived Stem Cells.Regen Eng Transl Med.2015;1(1):42-49.
[8]Pleumeekers MM,Nimeskern L,Koevoet JLM,et al.Trophic effects of adipose-tissue-derived and bone-marrow-derived mesenchymal stem cells enhance cartilage generation by chondrocytes in co-culture.PloS One.2018;13(2):1-23.
[9]Zhang J,Du C,Guo W,et al.Adipose Tissue-Derived Pericytes for Cartilage Tissue Engineering.Curr Stem Cell Res Ther.2017;12(6):513-521.
[10]Yang J,Zhang YS,Yue K,et al.Cell-laden hydrogels for osteochondral and cartilage tissue engineering.Acta Biomater.2017;57:1-25.
[11]Liu M,Zeng X,Ma C,et al.Injectable hydrogels for cartilage and bone tissue engineering.Bone Res.2017;5(2):75-94.
[12]Zhang Y,Tao L,Li S,et al.Synthesis of multiresponsive and dynamic chitosan-based hydrogels for controlled release of bioactive molecules.Biomacromolecules.2011;12(8):2894-2901.
[13]Li Y,Zhang Y,Shi F,et al.Modulus-regulated 3D-cell proliferation in an injectable self-healing hydrogel.Colloids Surf B Biointerfaces.2017;149:168-173.
[14]Qi C,Liu J,Jin Y,et al.Photo-crosslinkable,injectable sericin hydrogel as 3D biomimetic extracellular matrix for minimally invasive repairing cartilage.Biomaterials.2018;163:89-104.
[15]Sun AX,Lin H,Fritch MR,et al.Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional,photocrosslinked hydrogel constructs:Effect of cell seeding density and material stiffness.Acta Biomaterialia.2017;58:302-311.
[16]徐丽娟.小鼠成体干细胞体外诱导成肝能力比较及与丝素蛋白纤维相容性的实验研究[D].北京:解放军医学院,2016.
[17]Tabatabaei Qomi R,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107.
[18]Tsukamoto J,Naruse K,Nagai Y,et al.Efficacy of a Self-assembling Peptide Hydrogel,SPG-178-Gel,for Bone Regeneration and Three-dimensional Osteogenic Induction of Dental Pulp Stem Cells.Tissue Eng Part A.2017;23(23-24):1394-1402.
[19]Stevens M,George JH.Exploring and engineering the cell surface interface.Science.2011;100(3):1135-1138.
[20]Lim SM,Oh SH,Lee HH,et al.Dual growth factor-releasing nanoparticle/hydrogel system for cartilage tissue engineering.J Mater Sci Mater Med.2010;21(9):2593-2600.
[21]Gonzalez-Fernandez T,Tierney EG,Cunniffe GM,et al.Gene Delivery of TGF-β3 and BMP2 in an MSC-Laden Alginate Hydrogel for Articular Cartilage and Endochondral Bone Tissue Engineering.Tissue Eng Part A.2016;22(9-10):776-787.
[22]Simson JA,Strehin IA,Lu Q,et al.An adhesive bone marrow scaffold and bone morphogenetic-2 protein carrier for cartilage tissue engineering.Biomacromolecules.2013;14(3):637-643.
[23]王刚,李晓萍,王进.植入壳聚糖体内降解的机理研究[J].当代医学,2015,21(34):5-8.
[24]Sheehy EJ,Mesallati T,Vinardell T,et al.Engineering cartilage or endochondral bone:A comparison of different naturally derived hydrogels.Acta Biomaterialia.2015;13:245-253.
[25]Ansari S,Diniz IM,Chen C,et al.Alginate/hyaluronic acid hydrogel delivery system characteristics regulate the differentiation of periodontal ligament stem cells toward chondrogenic lineage.J Mater Sci Mater Med.2017;28(10):162-173.
[26]Huang SJ,Fu RH,Shyu WC,et al.Adipose-derived stem cells:isolation,characterization,and differentiation potential.Cell Transplant.2013;22(4):701-709.
[27]Panadero JA,Lanceros-Mendez S,Ribelles JL.Differentiation of mesenchymal stem cells for cartilage tissue engineering:Individual and synergetic effects of three-dimensional environment and mechanical loading.Acta Biomaterialia.2016;33:1-12.
[28]Yousefi AM,James PF,Akbarzadeh R,et al.Prospect of Stem Cells in Bone Tissue Engineering:A Review.Stem Cells Int.2016;2016(10):1-13.
[29]Quintiliano K,Crestani T,Silveira D,et al.Neural Differentiation of Mesenchymal Stem Cells on Scaffolds for Nerve Tissue Engineering Applications.Cell Reprogram.2016;18(6):369-381.
[30]Alhadlaq A,Mao JJ.Mesenchymal stem cells:isolation and therapeutics.Stem Cells Dev.2004;13(4):436-448.
[31]Fellows CR,Matta C,Zakany R,et al.Adipose,Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair.Front Genet.2016;7(Pt 6):213-232.
[32]Kang H,Peng J,Lu S,et al.In vivo cartilage repair using adipose-derived stem cell-loaded decellularized cartilage ECM scaffolds.J Tissue Eng Regen Med.2014;8(6):442-453.
[33]Zhao MC,Liu C.Research Progress on Adipose-Derived Stem Cells in Cartilage Tissue Engineering.Chin J Biomed Eng.2014;33(4):475-481.