SDF-1α缓释水凝胶包被去细胞基质特性研究
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摘要
目的将牛心包去细胞基质(AM)包被基质细胞衍生因子-1α(SDF-1α)并进行相关研究,对其作为功能支架材料应用于组织工程心脏瓣膜(TEHV)作以评价。方法将新鲜牛心包经胰酶+TritonX-100处理后得到AM;将含SDF-1α的缓释水凝胶包被AM。取大鼠(SD)5只,抽取骨髓,分离间充质干细胞(MSCs),作为种子细胞。在体外条件下,将MSCs与含SDF-1α的缓释水凝胶包被AM共培养(实验组),诱导其黏附、增殖,并与未包被基质进行对照(对照组)。结果胰酶+Triton X-100可彻底去除基质中细胞和相关成分;包被后的AM可持续释放SDF-1α,7 d内SDF-1α累计释放率为74.36%;体外实验表明MSCs黏附、增殖数量[(6.93±0.39)×104/cm2,7 d]较对照组[(2.25±0.25)×104/cm2,7 d]显著增加(P <0.05)。光学显微镜观察提示,实验组材料孔隙内黏附大量细胞,细胞生长和增殖情况良好;电子显微镜扫描提示,MSCs在支架材料表面贴附生长,相互连接成片,对照组中,细胞仅在材料表面散在生长,材料内部无细胞生长。结论含SDF-1α缓释水凝胶包被AM可通过持续释放SDF-1α显著诱导MSCs归巢、增殖,具备进一步体内构建TEHV的特性。
Objective To evaluate the property of acellular matrix(AM) coated with controlled-release stromal cell-derived factor 1 alpha(SDF-1α) hydrogel, and its potential usage in construction of tissue engineering heart valve(TEHV). Methods Bovine pericardium was treated with the method of trypsinase + TritonX-100 to get AM. The sustained release of hydrogel containing SDF-1α was coated with AM. Five rats(SD) were sacrificed, bone marrow was taken, and mesenchymal stem cells(MSCs) were isolated as seed cells. Under in vitro conditions, MSCs were co-cultured with AM-containing slow-release hydrogel containing SDF-1α(experimental group) to induce adhesion and proliferation, and compared with uncoated matrix(control group). Results Trypsinase + TritonX-100 method were removed cellular components in the matrix completely. The coated AM could release SDF-1α, and the cumulative release rate of SDF-1α in 7 days was 74.36 %; in vitro experiment results showed that the adhesion and proliferation of MSCs[(6.93 ± 0.39) × 104/cm2, 7-day] was significantly increased compared to that of control group[(2.25 ± 0.25) × 104/cm2, 7-day](P < 0.05). The optical microscopy observation results showed that a large number of cells adhered to the pores in experimental group, and the cell growth and proliferation were good. The electron microscopy results showed that MSCs adhered to the surface of scaffold material and connected to each other. In control group, the cells were scattered only on the surface of the material, while no cell growth was observed inside the material. Conclusion It is demonstrated that the AM coated SDF-1α hydrogel could induce MSCs homing and proliferation significantly, which is the property for construction of tissue engineering heart valve.
Objective To evaluate the property of acellular matrix(AM) coated with controlled-release stromal cell-derived factor 1 alpha(SDF-1α) hydrogel, and its potential usage in construction of tissue engineering heart valve(TEHV). Methods Bovine pericardium was treated with the method of trypsinase + TritonX-100 to get AM. The sustained release of hydrogel containing SDF-1α was coated with AM. Five rats(SD) were sacrificed, bone marrow was taken, and mesenchymal stem cells(MSCs) were isolated as seed cells. Under in vitro conditions, MSCs were co-cultured with AM-containing slow-release hydrogel containing SDF-1α(experimental group) to induce adhesion and proliferation, and compared with uncoated matrix(control group). Results Trypsinase + TritonX-100 method were removed cellular components in the matrix completely. The coated AM could release SDF-1α, and the cumulative release rate of SDF-1α in 7 days was 74.36 %; in vitro experiment results showed that the adhesion and proliferation of MSCs[(6.93 ± 0.39) × 104/cm2, 7-day] was significantly increased compared to that of control group[(2.25 ± 0.25) × 104/cm2, 7-day](P < 0.05). The optical microscopy observation results showed that a large number of cells adhered to the pores in experimental group, and the cell growth and proliferation were good. The electron microscopy results showed that MSCs adhered to the surface of scaffold material and connected to each other. In control group, the cells were scattered only on the surface of the material, while no cell growth was observed inside the material. Conclusion It is demonstrated that the AM coated SDF-1α hydrogel could induce MSCs homing and proliferation significantly, which is the property for construction of tissue engineering heart valve.
引文
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[2]HU Xing-jian,SHI Jia-wei,DONG Nian-guo,et al.Biological evaluation of novel tissue engineering heart valve material in vitro[J].Journal of Clinical Cardiology(China),2014,30(1):71-74.[胡行健,史嘉玮,董念国,等.新型组织工程化心脏瓣膜材料的体外生物学性能评价[J].临床心血管病杂志,2014,30(1):71-74.]
[3]Dong X,Wei X,Yi W,et al.RGD-modified acellular bovine pericardium as a bioprosthetic scaffold for tissue engineering[J].J Mater Sci Mater Med,2009,20(11):2327-2336.
[4]Shen L,Zhang P,Zhang S,et al.C-X-C motif chemokine ligand 8 promotes endothelial cell homing via the Akt-signal transducer and activator of transcription pathway to accelerate healing of ischemic and hypoxic skin ulcers[J].Exp Ther Med,2017,13(6):3021-3031.
[5]LIU Yang,LIU Wei-yong.Acellular tissue engineering heart valve[J].Chinese Journal of Clinical Thoracic and Cardiovascular Surgery,2005,12(1):38-41.[刘洋,刘维永.去细胞组织工程心脏瓣膜[J].中国胸心血管外科临床杂志,2005,12(1):38-41.]
[6]Straka F,Schornik D,Masin J,et al.A human pericardium biopolymeric scaffold for autologous heart valve tissue engineering:cellular and extracellular matrix structure and biomechanical properties in comparison with a normal aortic heart valve[J].J Biomater Sci Polym Ed,2018,29(6):599-634.
[7]Kaku M,Kitami M,Rosales Rocabado JM,et al.Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis[J].J Periodontal Res,2017,52(4):686-694.
[8]Patry C,Stamm D,Betzen C,et al.CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients[J].J Inflamm(Lond),2018,15:10-10.
[9]Park S,Jang H,Kim BS,et al.Directional migration of mesenchymal stem cells under an SDF-1αgradient on a microfluidic device[J].PLoS One,2017,12(9):e0184595-e0184595.
[10]Purcell BP,Elser JA,Mu A,et al.Synergistic effects of SDF-1αchemokine and hyaluronic acid release from degradable hydrogels on directing bone marrow derived cell homing to the myocardium[J].Biomaterials,2012,33(31):7849-7857.
[11]Stuermer EK,Lipenksy A,Thamm O,et al.The role of SDF-1 in homing of human adipose-derived stem cells[J].Wound Repair Regen,2015,23(1):82-89.
[12]Hong W,Tatsuo S,Shou-Dong W,et al.Resveratrol upregulates cardiac SDF-1 in mice with acute myocardial infarction through the deacetylation of cardiac p53[J].PLoS One,2015,10(6):e0128978-e0128978.
[13]Sharma S,Kumar A,Deepak,et al.Development of a novel chitosan based biocompatible and self-healing hydrogel for controlled release of hydrophilic drug[J].Int J Biol Macromol,2018,116:37-44.
[14]Ianchis R,Ninciuleanu C,Gifu IC,et al.Novel hydrogel-advanced modified clay nanocom-posites as possible vehicles for drug delivery and controlled release[J].Nanomaterials(Basel),2017,7(12):pii:443-pii:443.
[15]Hoerstrup SP,Kadner A,Melnitchouk S,et al.Tissue engineering of functional trileaflet heart valves from human marrow stromal cells[J].Circulation,2002,106(12 Suppl 1):I143-I150.
[16]Perry TE,Kaushal S,Sutherland FW,et al.Thoracic surgery directors association award.Bone marrow as a cell source for tissue engineering heart valves[J].Ann Thorac Surg,2003,75(3):761-767,discussion 767-767.