荨麻提取物诱导骨膜细胞增殖分化:构建组织工程骨及超微结构的变化
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摘要
背景:荨麻作为一种传统的中草药,目前认为其主要的药理作用是抗炎、抗风湿等作用,其对骨膜细胞的效果的相关研究较少。目的:探讨荨麻诱导人骨膜细胞增殖及分化特性,并诱导其体外构骨,为荨麻运用于骨组织工程提供理论依据。方法:实验取材于成人锁骨。体外组织块培养法获得骨膜原代细胞,恒温培养扩增后,取第3代骨膜细胞种植于细胞培养板中。实验分为3组,实验组分别加入10-6,10-5及10-4 g/L荨麻提取物,阳性对照组加入50μg/L骨形态发生蛋白7,对照组仅加入纯净水。分别在第1,4,7和10天MTT法检测骨膜细胞增殖情况,碱性磷酸酶染色和Von Kossa染色检测碱性磷酸酶和钙结节的表达。分别将10-4 g/L荨麻提取物及骨形态发生蛋白7加入到含有聚己内酯三维支架中和骨膜细胞复合培养构建组织工程骨,培养24 d,电镜下观察不同组细胞形态学情况。结果与结论:(1)MTT结果显示各时间点荨麻提取物不同浓度组和阳性对照组的吸光度值均显著高于对照组(P<0.05),荨麻提取物不同浓度组的各时间点吸光度值相比,10-4 g/L组>10-5 g/L组>10-6 g/L组(P<0.05),表明10-4 g/L为最佳浓度;(2)实验组各时间点碱性磷酸酶和钙结节的染色阳性率与阳性对照组相比,差异无显著性意义(P>0.05),但2组各时间点表达量均显著高于对照组(P<0.05);(3)电镜下观察实验组细胞膜表面有大量微绒毛状突起,线粒体基质致密有少量嵴,细胞增殖和分化活跃。且阳性对照组和荨麻组细胞内线粒体数量显著多于对照组(P<0.05);(4)结果提示,荨麻能促进骨膜细胞增殖,同时能诱导骨膜细胞分化为成骨细胞,可作为组织工程中的诱导剂。另外,荨麻可诱导骨膜细胞在聚己内酯三维支架中扩增并构建组织工程骨。
BACKGROUND: Urtica, a traditional Chinese herbal, holds anti-inflammatory and anti-rheumatic effects, but its influence on periosteal cells is little reported. OBJECTIVE: To investigate the proliferation and differentiation of human periosteal cells induced by urtica and to construct tissue-engineered bone by periosteal cells in vitro, thereby providing theoretical basis for the application of urtica in bone tissue engineering. METHODS: Human clavicle periosteum was obtained. Primary periosteal cells were cultured by tissue culture method in vitro, passaged, and the 3 generations of periosteal cells were seeded into cell culture plate, and treated with urtica extracts(10-6, 10-5 and 10-4 g/L, experimental group), 50 μg/L bone morphogenetic protein-7(positive control group) or purified water(control group). The absorbance values of periosteal cells were measured by MTT assay, and the expression levels of alkaline phosphatase and calcium nodules were detected by alkaline phosphatase staining and Von Kossa staining at 1, 4, 7 and 9 days after culture. 10-4 g/L urtica extracts and bone morphogenetic protein-7 were added into the three-dimensional polyclonal lactone scaffold and periosteal cells to construct tissue-engineered bone. After 24 days of culture, the morphological changes of periosteal cells were observed under electron microscope. RESULTS AND CONCLUSION: MTT results revealed that the absorbance values of periosteal cells in the experimental and positive control groups were significantly higher than those in the control group at different time points(P < 0.05); the order of absorbance values was as follows: 10-4 group > 10-5 group > 10-6 g/L group(P < 0.05), suggesting 10-4 g/L was the optimal concentration. The positive rates of alkaline phosphatase and calcium nodules in the experimental and positive control groups were significantly higher than those in the control group(P < 0.05), but the experimental and positive control groups did not differ significantly(P > 0.05). In the experimental group, there were abundant microvilli on the cell membrane, the mitochondria matrix was dense and appeared with crista, and active proliferation and differentiation of cells were detected. The number of mitochondria in the experimental and positive control groups was significantly more than that in the control group(P < 0.05). These findings indicate that periosteal cells can be induced by urtica to amplify and differentiate into osteoblasts, and construct tissue-engineered bone in the three-dimensional polyclonal lactone scaffold.
BACKGROUND: Urtica, a traditional Chinese herbal, holds anti-inflammatory and anti-rheumatic effects, but its influence on periosteal cells is little reported. OBJECTIVE: To investigate the proliferation and differentiation of human periosteal cells induced by urtica and to construct tissue-engineered bone by periosteal cells in vitro, thereby providing theoretical basis for the application of urtica in bone tissue engineering. METHODS: Human clavicle periosteum was obtained. Primary periosteal cells were cultured by tissue culture method in vitro, passaged, and the 3 generations of periosteal cells were seeded into cell culture plate, and treated with urtica extracts(10-6, 10-5 and 10-4 g/L, experimental group), 50 μg/L bone morphogenetic protein-7(positive control group) or purified water(control group). The absorbance values of periosteal cells were measured by MTT assay, and the expression levels of alkaline phosphatase and calcium nodules were detected by alkaline phosphatase staining and Von Kossa staining at 1, 4, 7 and 9 days after culture. 10-4 g/L urtica extracts and bone morphogenetic protein-7 were added into the three-dimensional polyclonal lactone scaffold and periosteal cells to construct tissue-engineered bone. After 24 days of culture, the morphological changes of periosteal cells were observed under electron microscope. RESULTS AND CONCLUSION: MTT results revealed that the absorbance values of periosteal cells in the experimental and positive control groups were significantly higher than those in the control group at different time points(P < 0.05); the order of absorbance values was as follows: 10-4 group > 10-5 group > 10-6 g/L group(P < 0.05), suggesting 10-4 g/L was the optimal concentration. The positive rates of alkaline phosphatase and calcium nodules in the experimental and positive control groups were significantly higher than those in the control group(P < 0.05), but the experimental and positive control groups did not differ significantly(P > 0.05). In the experimental group, there were abundant microvilli on the cell membrane, the mitochondria matrix was dense and appeared with crista, and active proliferation and differentiation of cells were detected. The number of mitochondria in the experimental and positive control groups was significantly more than that in the control group(P < 0.05). These findings indicate that periosteal cells can be induced by urtica to amplify and differentiate into osteoblasts, and construct tissue-engineered bone in the three-dimensional polyclonal lactone scaffold.
引文
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[18]张晓,王啸,张鹏,等.丹参对小鼠前成骨细胞的增殖分化的影响其与细胞外信号调节激酶1/2信号通路的关系[J].中华实验外科杂志,2017,34(1):86-89.
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[25]Lee J,Noh AL,Zheng T,et al.Eriodicyol inhibits osteoclast differentiation and ovariectomy-induced bone loss in vivo.Exp Cell Res.2015;339:380-388.
[26]Ming LG,Chen KM,Xian CJ.Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling.J Cell Physiol.2013;228:513-521.
[27]Lappe J,Kunz I,Bendik I,et al.Effect of a combination of genistein,polyunsaturated fatty acids and vitamins D3 and K1on bone mineral density in postmenopausal women:a randomized,placebo-controlled,double-blind pilot study.Eur J Nutr.2013;52:203-215
[28]Behloul N,Wu G.Genistein:a promising therapeutic agent for obesity and diabetes treatment.Eur J Pharmacol.2013;698:31-38.
[29]Yang L,Yu Z,Qu H,et al.Comparative effects of hispidulin,genistein,and icariin with estrogen on bone tissue in ovariectomized rats.Cell Biochem Biophys.2014;70:485-490.
[30]陆海涛,袁峰,张峻玮,等.低氧环境下共培养的骨膜细胞和髓核细胞骨向分化能力的研究[J].中国矫形外科杂志,2016,24:839-844.
[31]张国营,方加虎,李翔,等.周期性流体静压对骨膜细胞血管内皮生长因子表达的影响及其机制[J].江苏医药,2015,41:999-1001.
[32]张峻玮,陆海涛,杨宇明,等.低氧培养对骨膜细胞成软骨分化的影响[J].中华实验外科杂志,2016,33:1281-1283.
[33]Kessler S,Koepp HE,Mayr-Wohlfart U,et al.Bone morphogeneric protein2 accelerates osteointegration and remodelling of sovent-dehydrated bone substitutes.Arch Orthop Trauma Surg.2004;2124(2006):2410-2414.
[34]Zhang X,Aubin JE,Inman RD.Molecular and cellular biology of new bone formation:insights into the ankylosis of ankylosing spondylitis.Curr Opin Rheumatol.2003;2015(2004):2387-2393.
[2]Chrubasik JE,Roufogalis BD,Wagner H,et al.A comprehensive review on the stinging nettle effect and efficacy profiles.Part II:urticae radix.Phytomedicine.2007;14:568-579
[3]Chrubasik JE,Roufogalis BD,Wagner H,et al.A comprehensive review on nettle effect and efficacy profiles,Part I:herba urticae.Phytomedicine.2007;14:423-435.
[4]Koch E.Extracts from fruits of saw palmetto(Sabal serrulata)and roots of stinging nettle(Urtica dioica):viable alternatives in the medical treatment of benign prostatic hyperplasia and associated lower urinary tracts symptoms.Planta Med.2001;67:489-500.
[5]Wang M,Zhang Y,Zhang H,et al.The active glycosides from Urtica fissa rhizome decoction.J Nat Med.2018.doi:10.1007/s11418-018-1172-3.
[6]Abu-Darwish MS,Efferth T.Medicinal Plants from Near East for Cancer Therapy.Front Pharmacol.2018;9:56.
[7]Yatoo MI,Gopalakrishnan A,Saxena A,et al.Anti-inflammatory drugs and herbs with special emphasis on herbal medicines for countering inflammatory diseases and disorders-a Review.Recent Pat Inflamm Allergy Drug Discov.2018.doi:10.2174/1872213X12666180115153635.8.
[8]Zouari BK,Bardaa S,Khimiri M,et al.Exploring theUrtica dioicaLeaves Hemostatic and Wound-Healing Potential.Biomed Res Int.2017;2017:1047523.
[9]Akkouh O,Ng TB,Singh SS,et al.Lectins with anti-HIV activity:a review.Molecules.2015;20(1):648-68.
[10]Cui D,Li H,Xu X,et al.mesenchymal stem cells for cartilage regeneration of TMJ Osteoarthritis.Stem Cells Int.2017;2017:5979741.
[11]Park HC,Son YB,Lee SL,et al.Effects of osteogenic-conditioned medium from human periosteum-derived cells on osteoclast differentiation.Int J Med Sci.2017 Nov 2;14(13):1389-1401.
[12]Park JH,Park BW,Kang YH,et al.Lin28a enhances in vitro osteoblastic differentiation of human periosteum-derived cells.Cell Biochem Funct.2017;35(8):497-509.
[13]Luo H,Zhang Y,Li G,et al.Sacrificial template method for t synthesis of three-dimensional nanofibrous 58S bioglass scaffold and its in vitro bioactivity and cell responses.J Biomater Appl.2017;32(2):265-275.
[14]Almeida HV,Sathy BN,Dudurych I,et al.Anisotropic shape-memory alginate scaffolds functionalized with either type I or type II collagen for cartilage tissue Engineering.Tissue Eng Part A.2017;23(1-2):55-68.
[15]Cecen B,Kozaci LD,Yuksel M,et al.Biocompatibility and biomechanical characteristics of loofah based scaffolds combined with hydroxyapatite,cellulose,poly-l-lactic acid w chondrocyte-like cells.Mater Sci Eng C Mater Biol Appl.20169:437-446.
[16]Hernandez I,Kumar A,Joddar B.A Bioactive hydrogel and3D printed polycaprolactone system for bone tissue engineering.Gels.2017;3(3):26.
[17]Wozney JM.The bone morphogenetic protein family:multifunctional cellular regulators in the embryo and adult.Eur J Oral Sci.1998;106(Suppl1):160-166.
[18]张晓,王啸,张鹏,等.丹参对小鼠前成骨细胞的增殖分化的影响其与细胞外信号调节激酶1/2信号通路的关系[J].中华实验外科杂志,2017,34(1):86-89.
[19]Antunes-Ricardo M,Gutierrez-Uribe J,Serna-Saldivar SO.Anti-inflammatory glycosylated flavonoids as therapeutic agents for treatment of diabetes-impaired wounds.Curr Top Med Chem.2015;15:2456-2463.
[20]Nguyen TY,To DC,Tran MH,et al.Anti-inflammatory Flavonoids Isolated from Passiflora foetida.Nat Prod Commun.2015;10:929-931.
[21]Al Mamun MA,Hosen MJ,Islam K,et al.Tridax procumben flavonoids promote osteoblast differentiation and bone formation.Biol Res.2015;48:65
[22]Leotoing L,Davicco MJ,Lebecque P,et al.The flavonoid fisetin promotes osteoblasts differentiation through Runx2transcriptional activity.Mol Nutr Food Res.2014;58:1239-1248.
[23]Nash LA,Sullivan PJ,Peters SJ,et al.Rooibos flavonoids,orientin and luteolin,stimulate mineralization in human osteoblasts through the Wnt pathway.Mol Nutr Food Res.2015;59:443-453.
[24]Al Mamun MA,Islam K,Alam MJ,et al.Flavonoids isolated from Tridax procumbens(TPF)inhibit osteoclasts differentiation and bone resorption.Biol Res.2015;48:51.
[25]Lee J,Noh AL,Zheng T,et al.Eriodicyol inhibits osteoclast differentiation and ovariectomy-induced bone loss in vivo.Exp Cell Res.2015;339:380-388.
[26]Ming LG,Chen KM,Xian CJ.Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling.J Cell Physiol.2013;228:513-521.
[27]Lappe J,Kunz I,Bendik I,et al.Effect of a combination of genistein,polyunsaturated fatty acids and vitamins D3 and K1on bone mineral density in postmenopausal women:a randomized,placebo-controlled,double-blind pilot study.Eur J Nutr.2013;52:203-215
[28]Behloul N,Wu G.Genistein:a promising therapeutic agent for obesity and diabetes treatment.Eur J Pharmacol.2013;698:31-38.
[29]Yang L,Yu Z,Qu H,et al.Comparative effects of hispidulin,genistein,and icariin with estrogen on bone tissue in ovariectomized rats.Cell Biochem Biophys.2014;70:485-490.
[30]陆海涛,袁峰,张峻玮,等.低氧环境下共培养的骨膜细胞和髓核细胞骨向分化能力的研究[J].中国矫形外科杂志,2016,24:839-844.
[31]张国营,方加虎,李翔,等.周期性流体静压对骨膜细胞血管内皮生长因子表达的影响及其机制[J].江苏医药,2015,41:999-1001.
[32]张峻玮,陆海涛,杨宇明,等.低氧培养对骨膜细胞成软骨分化的影响[J].中华实验外科杂志,2016,33:1281-1283.
[33]Kessler S,Koepp HE,Mayr-Wohlfart U,et al.Bone morphogeneric protein2 accelerates osteointegration and remodelling of sovent-dehydrated bone substitutes.Arch Orthop Trauma Surg.2004;2124(2006):2410-2414.
[34]Zhang X,Aubin JE,Inman RD.Molecular and cellular biology of new bone formation:insights into the ankylosis of ankylosing spondylitis.Curr Opin Rheumatol.2003;2015(2004):2387-2393.