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首页> 外文会议>World biomaterials congress >Poly(L-lactide) nanofibrous meshes loaded with detonation diamond particles as substrates for the adhesion, growth and osteogenic differentiation of human osteoblast-like cells
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Poly(L-lactide) nanofibrous meshes loaded with detonation diamond particles as substrates for the adhesion, growth and osteogenic differentiation of human osteoblast-like cells

机译：聚（L-丙交酯）纳米纤维网，其上装有引爆金刚石颗粒，作为人类成骨样细胞粘附，生长和成骨分化的基质

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Introduction: Diamond nanoparticles (DNPs) are considered as a highly biocompatible material for biomedical applications. In our earlier studies, nanocrystalline diamond films proved as excellent substrates for the adhesion, growth and osteogenic differentiation of human-bone derived cells, particularly after their doping with boron or termination with oxygen. In addition, diamond nanoparticles could be added into nanofibrous polymeric scaffolds in order to improve their mechanical properties and bioactivity for bone tissue engineering. Thus, composite poly(L-lactide) (PLLA) nanofibrous membranes with six different concentrations of DNPs were prepared, and the adhesion, growth and osteogenic differentiation of human osteoblast-like MG-63 and Saos-2 cells were studied on these matrices. Materials and Methods: Five grams of PLLA were dissolved in 100 ml of chloroform, and DNPs (NanoAmando, Nanocarbon Research Institute Co., Ltd., Japan) were added in 6 concentrations ranging from 0.021875 to 0.7 g/100 ml of PLLA solution, i.e. from 0.38 to 12.28 wt.% in dry PLLA. Nanofibrous membranes were then prepared by needle-less electrospinning. The cell growth was estimated using XTT test, measuring the activity of mitochondrial enzymes. The concentrations of focal adhesion proteins talin and vinculin, and also of osteogenic proteins collagen Ⅰ, alkaline phosphatase, osteopontin and osteocalcin, were measured in cell homogenates by an enzyme linked-immunosorbent assay (ELISA) on day 7 after seeding. The expression of mRNA for these proteins was measured by real-time PCR on day 14 after seeding. Results and Discussion: The activity of mitochondrial enzymes decreased with increasing nanoparticle concentration in both MG-63 and Saos-2 cells. The concentration of focal adhesion and osteogenic proteins, measured on day 7 at the protein level, was similar in cells on pure PLLA scaffolds and scaffolds with all concentrations with DNPs, except of osteocalcin in MG-63 cells, which decreased with increasing nanoparticle concentration. On day 14 after seeding, the mRNA expression of talin and vinculin, as well as of collagen Ⅰ, alkaline phosphatase and osteopontin showed a general tendency to decrease with increasing DNP concentration. On the other hand, the addition of DNPs to PLGA nanofibers in our earlier studies supported the growth of MG-63 cells and human bone marrow mesenchymal stem cells, even in a higher concentration (23 wt.%). This disproportion could be explained by different origin and physicochemical properties of DNPs used for PLLA (detonation diamond) and for PLGA (DNPs prepared by a RF-PACVD method). The DNPs used for PLLA scaffolds were hydrogen-terminated and hydrophobic, which markedly increased the hydrophobicity of the PLLA-DNP scaffolds, making them less appropriate for the colonization with cells. A direct cytotoxicity of DNPs cannot also be excluded. DNPs can damage cells by oxidative mechanisms and by an increased influx of Na+ ions into cells (for a review, see). Conclusion: Addition of diamond nanoparticles produced by detonation and terminated with H into nanofibrous scaffolds had negative effects on the adhesion, growth and osteogenic differentiation of human bone-derived cells, while the effects of diamond nanoparticles produced by RF-PACVD method were beneficial. Thus, the mode of preparation and surface properties of diamond nanoparticles could be important for their biocompatibility and for their use as components of biomaterials.
机译：简介：金刚石纳米粒子（DNP）被认为是生物医学应用中具有高度生物相容性的材料。在我们较早的研究中，纳米晶金刚石膜被证明是人骨衍生细胞粘附，生长和成骨分化的极好基质，尤其是在掺杂硼或终止于氧之后。另外，可以将金刚石纳米颗粒添加到纳米纤维聚合物支架中，以改善其机械性能和对骨组织工程的生物活性。因此，制备了具有六种不同浓度DNP的复合聚（L-丙交酯）（PLLA）纳米纤维膜，并在这些基质上研究了人类成骨样MG-63和Saos-2细胞的粘附，生长和成骨分化。材料和方法：将5克PLLA溶于100毫升氯仿中，然后以0.021875至0.7克/ 100毫升PLLA溶液的6种浓度添加DNP（NanoAmando，日本纳米碳研究所有限公司）。即在干燥PLLA中为0.38至12.28重量％。然后通过无针电纺丝制备纳米纤维膜。使用XTT测试评估细胞生长，测量线粒体酶的活性。在接种后第7天，通过酶联免疫吸附测定法（ELISA）测量粘着斑蛋白塔林和纽蛋白的浓度，以及成骨蛋白胶原Ⅰ，碱性磷酸酶，骨桥蛋白和骨钙素的浓度。在接种后第14天通过实时PCR测量这些蛋白质的mRNA表达。结果与讨论：MG-63和Saos-2细胞中的线粒体酶活性均随着纳米颗粒浓度的增加而降低。在第7天在蛋白水平上测定的粘着斑和成骨蛋白的浓度在纯PLLA支架上的细胞中以及在所有具有DNP浓度的支架中的细胞中都相似，除了MG-63细胞中的骨钙素随着纳米颗粒浓度的增加而降低。接种后第14天，随着DNP浓度的升高，塔林和纽扣蛋白的mRNA表达以及Ⅰ型胶原，碱性磷酸酶和骨桥蛋白的mRNA表达总体呈下降趋势。另一方面，在我们较早的研究中，向PLGA纳米纤维中添加DNP甚至可以以更高的浓度（23 wt。％）支持MG-63细胞和人骨髓间充质干细胞的生长。可以通过PLLA（爆破钻石）和PLGA（通过RF-PACVD方法制备的DNP）所用DNP的不同来源和理化性质来解释这种歧义。用于PLLA支架的DNP是氢封端的且具有疏水性，这显着增加了PLLA-DNP支架的疏水性，使其不太适合细胞定植。 DNP的直接细胞毒性也不能排除。 DNP可以通过氧化机制和增加的Na +离子流入细胞来破坏细胞（有关综述，请参见）。结论：将爆轰产生的金刚石纳米粒子加氢终止到纳米纤维支架中对人骨来源细胞的粘附，生长和成骨分化具有负面影响，而RF-PACVD方法产生的金刚石纳米粒子具有良好的作用。因此，金刚石纳米颗粒的制备方式和表面性质对于它们的生物相容性和用作生物材料的成分可能很重要。

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