壳聚糖-明胶-果胶仿生网络膜对间充质干细胞增殖和矿化的影响
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摘要
目的:探讨采用壳聚糖、明胶和果胶制备的仿生网络膜对间充质干细胞(MSCs)增殖和矿化的影响,评价其用于构建组织工程骨的可行性。方法:采用仿生学方法,将壳聚糖、明胶和果胶按照一定比例制作成新型仿生网络膜。实验分为对照组(MSCs+常规培养基)、材料组(MSCs+网络膜+常规培养基)和材料+成骨诱导培养基(OS)组(MSCs+网络膜+OS培养基)。倒置相差显微镜下观察细胞形态表现,扫描电镜(SEM)观察细胞生长及细胞外基质分泌情况,MTT法检测细胞增殖情况(MSCs分为阴性对照组和材料组,分别以空白培养液和含材料的培养液培养),茜素红染色检测细胞中钙无机物表达情况,实时聚合酶链反应(Real time-PCR)测定细胞中骨钙素(OC)mRNA和骨桥蛋白(OPN)mRNA表达水平。结果:网络膜材料呈半透明薄膜状,倒置相差显微镜下MSCs为短梭形,细胞成簇状聚集生长。SEM下观察,细胞培养第7天可见梭形细胞;培养第14天时细胞数量增多,以伪足状突起锚定于材料表面;培养第21天时,细胞聚集并分泌大量细胞外基质。材料组细胞增殖水平与阴性对照组比较差异无统计学意义(P>0.05)。茜素红染色,网络膜上细胞被染成橘红色。Real time-PCR法,材料组和材料+OS组MSCs中OC mRNA在接种后第7和14天时表达水平较低,第21天时其表达水平明显升高,达到峰值;OPN mRNA在第7天明显表达,第14天时表达水平达峰值,第21天略有下降;与对照组比较,不同时间点材料组和材料+OS组细胞中OC mRNA和OPN mRNA表达水平明显升高(P<0.01),而材料组与材料+OS组比较差异无统计学意义(P>0.05)。结论:壳聚糖-明胶-果胶仿生网络膜具有良好的生物相容性,MSC在其表面可正常生长和增殖,在不加诱导剂的情况下可以诱导MSCs表达矿化相关的基因和蛋白。
Objective::To explore the effects of the biomimetic network membrane prepared by chitosan/gelatin/pectin on the proliferation and mineralization of mesenchymal stem cells(MSCs),and to evaluate its feasibility of constructing tissue engineering bone.Methods:Chitosan,gelatin and pectin were made into a new biomimetic network membrane in a certain ratio by biomimetics.The experiment was divided into control group(MSCs+conventional medium),material group(MSCs+network membrane+conventional medium)and material+OS group(MSCs+ network membrane+OS medium).The cell morphology was observed by inverted phase contrast microscope;the growth and secretion of extracellular matrix of the MSCs were observed under scanning electron microscope(SEM).The proliferation of cells was determined by MTT assay(The MSCs were divided into negative control group and material group,and they were cultivated with blank medium and medium including materials).The expression of calcium in MSCs was detected by Alizarin Red staining.Real-time polymerase chain reaction(RT-PCR)was used to determine the expression levels of osteocalcin(OC)mRNA and osteopontin(OPN)mRNA in the MSCs.Results:The network membrane was semitransparent thin film.The MSCs were short shuttle and clustered under inverted phase contrast microscope.After cultured for 7 d,the MSCs were shuttle;after cultured for 14 d,the number of MSCs was increased,with pseudo feet on the membrane;after cultured for21 d,the MSCs clustered with a lot of neo-formed extracellular matrix.The MTT results showed that there was no significant difference in the proliferation level of MSCs between material group and negative control group(P>0.05).The Alizarin Red staining results showed that the MSCs in the network membrane were dyed orange red.The RT-PCR results showed that the expression levels of OC mRNA in the MSCs in material group and material+OS group were lower on the 7 th and 14 th days,but on the 21 th day,the expression levels were significantly increased and reached the peak;the expression level of OC mRNA in the MSCs in material group was significantly increased on the 7 th day,and the expression level reached the peak on the 14 th day,then fell slightly on the 21 th day;compared with control group,the expression levels of OC mRNA and OPN mRNA in the cells in material group and material+OS group at different time points were significantly increased(P<0.01),but there were no significant differences between material group and material+OS group(P>0.05).Conclusion:Chitosan/gelatin/pectin biomimetic network membrane has good biocompatibility,and MSCs can grow and proliferate well on the membrane.The membrane can induce the MSCs to express mineralization-related genes and proteins without inducers.
Objective::To explore the effects of the biomimetic network membrane prepared by chitosan/gelatin/pectin on the proliferation and mineralization of mesenchymal stem cells(MSCs),and to evaluate its feasibility of constructing tissue engineering bone.Methods:Chitosan,gelatin and pectin were made into a new biomimetic network membrane in a certain ratio by biomimetics.The experiment was divided into control group(MSCs+conventional medium),material group(MSCs+network membrane+conventional medium)and material+OS group(MSCs+ network membrane+OS medium).The cell morphology was observed by inverted phase contrast microscope;the growth and secretion of extracellular matrix of the MSCs were observed under scanning electron microscope(SEM).The proliferation of cells was determined by MTT assay(The MSCs were divided into negative control group and material group,and they were cultivated with blank medium and medium including materials).The expression of calcium in MSCs was detected by Alizarin Red staining.Real-time polymerase chain reaction(RT-PCR)was used to determine the expression levels of osteocalcin(OC)mRNA and osteopontin(OPN)mRNA in the MSCs.Results:The network membrane was semitransparent thin film.The MSCs were short shuttle and clustered under inverted phase contrast microscope.After cultured for 7 d,the MSCs were shuttle;after cultured for 14 d,the number of MSCs was increased,with pseudo feet on the membrane;after cultured for21 d,the MSCs clustered with a lot of neo-formed extracellular matrix.The MTT results showed that there was no significant difference in the proliferation level of MSCs between material group and negative control group(P>0.05).The Alizarin Red staining results showed that the MSCs in the network membrane were dyed orange red.The RT-PCR results showed that the expression levels of OC mRNA in the MSCs in material group and material+OS group were lower on the 7 th and 14 th days,but on the 21 th day,the expression levels were significantly increased and reached the peak;the expression level of OC mRNA in the MSCs in material group was significantly increased on the 7 th day,and the expression level reached the peak on the 14 th day,then fell slightly on the 21 th day;compared with control group,the expression levels of OC mRNA and OPN mRNA in the cells in material group and material+OS group at different time points were significantly increased(P<0.01),but there were no significant differences between material group and material+OS group(P>0.05).Conclusion:Chitosan/gelatin/pectin biomimetic network membrane has good biocompatibility,and MSCs can grow and proliferate well on the membrane.The membrane can induce the MSCs to express mineralization-related genes and proteins without inducers.
引文
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[17]TAGHIYAR L,HOSSEINI S,HESARAKI M,et al.Isolation,characterization and osteogenic potential of mouse digit tip blastema cells in comparison with bone marrowderived mesenchymal stem cells In vitro[J].Cell J,2018,19(4):585-598.
[18]SON H E,KIM E J,JANG W G.Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2on osteoblast cells[J].Life Sci,2018,15(193):34-39.
[19]WU G,FENG C,QUAN J,et al.In situ controlled release of stromal cell-derived factor-1αand antimiR-138 for ondemand cranial bone regeneration[J].Carbohydr Polym,2018,15(182):215-224.
[20]ZHANG Y D,ZHAO S C,ZHU Z S,et al.Cx43-and Smad-mediated TGF-β/BMP signaling pathway promotes cartilage differentiation of bone marrow mesenchymal stem cells and inhibits osteoblast differentiation[J].Cell Physiol Biochem,2017,42(4):1277-1293.
[2]LI S,CHOW T,CHU J.Engineering microdent structures of bone implant surfaces to enhance osteogenic activity in MSCs[J].Biochem Biophys Rep,2016,10(9):100-105.
[3]CHEN G,KAWAZOE N.Porous scaffolds for regeneration of cartilage,bone and osteochondral tissue[J].Adv Exp Med Biol,2018,1058:171-191.
[4]CAPLAN A I.MSCs:The sentinel and safe-guards of injury[J].J Cell Physiol,2016,231(7):1413-1416.
[5]于祥茹,韩晓谦,袁浩天,等.PLGA/CPC支架材料复合骨髓基质干细胞构建组织工程骨的体外效果评价[J].吉林大学学报:医学版,2014,40(2):294-299.
[6]KUMAR S,STOKES LII J A,DEAN D,et al.Biphasic organo-bioceramic fibrous composite as a biomimetic extracellular matrix for bone tissue regeneration[J].Front Biosci(Elite Ed),2017,9:192-203.
[7]HU Y,CHEN J,FAN T,et al.Biomimetic mineralized hierarchical hybrid scaffolds based on in situ synthesis of nanohydroxyapatite/chitosan/chondroitin sulfate/hyaluronic acid for bone tissue engineering[J].Colloids Surf BBiointerfaces,2017,157:93-100.
[8]AZEVEDO H S,PASHKULEVA I.Biomimetic supramolecular designs for the controlled release of growth factors in bone regeneration[J].Adv Drug Deliv Rev,2015,94:63-76.
[9]TOPOLUK N,HAWKINS R,TOKISH J,et al.Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells[J].Am J Sports Med,2017,45(11):2637-2646.
[10]李硕峰,崔菁,刘国良,等.ECM-多糖/蛋白质多孔复合材料对BMSCs定向软骨分化的影响[J].广东医学,2018,39(8):1137-1141.
[11]ALEXANDRE N,RIBEIRO J,GRTNER A,et al.Biocompatibility and hemocompatibility of polyvinyl alcohol hydrogel used for vascular grafting-In vitro and in vivo studies[J].J Biomed Mater Res A,2014,102(12):4262-4275.
[12]CHEN X N,GU Y X,LEE J H,et al.Multifunctional surfaces with biomimetic nanofibres and drug-eluting micropatterns for infection control and bone tissue formation[J].Eur Cell Mater,2012,24:237-248.
[13]ZHANG S,PRABHAKARAN M P,QIN X,et al.Biocomposite scaffolds for bone regeneration:Role of chitosan and hydroxyapatite within poly-3-hydroxybutyrate-co-3-hydroxyvalerate on mechanical properties and in vitro evaluation[J].J Mech Behav Biomed Mater,2015,51(1):88-98.
[14]MLLER W E,NEUFURTH M,WANG S,et al.Morphogenetically active scaffold for osteochondral repair(polyphosphate/alginate/N,O-carboxymethyl chitosan)[J].Eur Cell Mater,2016,31:174-190.
[15]PANSERI S,RUSSO A,CUNHA C,et al.Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration[J].Knee Surg Sports Traumatol Arthrosc,2012,20(6):1182-1191.
[16]QI Y,DU Y,LI W,et al.Cartilage repair using mesenchymal stem cell(MSC)sheet and MSCs-loaded bilayer PLGA scaffold in a rabbit model[J].Knee Surg Sports Traumatol Arthrosc,2014,22(6):1424-1433.
[17]TAGHIYAR L,HOSSEINI S,HESARAKI M,et al.Isolation,characterization and osteogenic potential of mouse digit tip blastema cells in comparison with bone marrowderived mesenchymal stem cells In vitro[J].Cell J,2018,19(4):585-598.
[18]SON H E,KIM E J,JANG W G.Curcumin induces osteoblast differentiation through mild-endoplasmic reticulum stress-mediated such as BMP2on osteoblast cells[J].Life Sci,2018,15(193):34-39.
[19]WU G,FENG C,QUAN J,et al.In situ controlled release of stromal cell-derived factor-1αand antimiR-138 for ondemand cranial bone regeneration[J].Carbohydr Polym,2018,15(182):215-224.
[20]ZHANG Y D,ZHAO S C,ZHU Z S,et al.Cx43-and Smad-mediated TGF-β/BMP signaling pathway promotes cartilage differentiation of bone marrow mesenchymal stem cells and inhibits osteoblast differentiation[J].Cell Physiol Biochem,2017,42(4):1277-1293.