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首页> 外文会议>World biomaterials congress >Fabrication and characterization of zirconium cross-linked carboxymethyl cellulose-chitosan microparticles for bone tissue engineering application
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Fabrication and characterization of zirconium cross-linked carboxymethyl cellulose-chitosan microparticles for bone tissue engineering application

机译：骨组织工程应用锆交联羧甲基纤维素-壳聚糖微粒的制备与表征

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Introduction: Carboxymethyl cellulose (CMC) and chitosan (CS) are natural biodegradable and biocompatible polymers. CMC is an anionic polymer obtained through chemical modification of cellulose and CS is a cationic polymer derived from chitin and hence being oppositely charged these two polymers can undergo ionotropic gelation. Zirconium (Zr) is a tetravalent cationic metal extensively used in orthopedic implants to promote osseointegration in the form of zirconia and has recently been found to stimulate proliferation and differentiation of human osteoblasts by stimulating different signaling pathways. This study was aimed at developing injectable, non-toxic polyelectrolyte polymer complex microparticles based on Zr as a novel cross-linker. We hypothesize the use of Zr as cross-linker will not induce any cytotoxicity and promote adhesion of osteoblasts into microparticle. Methods: Aqueous solution of CMC (3% w/v) was prepared and added dropwise into the homogenous mixture of zirconium tetrachloride solution (5% w/v) and CS solution (1 % w/v) and kept under stirring for 1 h. The volume ratio of CMC to CS was optimized at 1:2 for improved complexation between two polymers. The particles thus obtained were then dried at room temperature for 48 h before further analysis. The surface morphology of microparticles was characterized by scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) was done for elemental analysis. Fourier transform-infrared (FTIR) spectrometer in ATR mode was used to characterize the intramolecular interaction between the components in micropartide. In vitro cytotoxicity was assessed using live/dead cell assay kit after culturing the murine pre-osteoblasts (OEMS) in the presence of microparticles up to 10 days. In order to specifically determine the viability of cells attached to micropartide, the microparticles were transferred into separate well of 24 well-plate and then live/dead cell assay was performed on day 5 and 10 and imaged under fluorescence microscope. The percent area of micropartide covered with ceils was determined using ImageJ taking visible portion of micropartide. Results and Discussion: The microparticles were spherical in shape with an average diameter of 787±0.045 μm. The surface of the microparticles contained uniform grooves and ridges creating rougher surface as seen on SEM image (Fig. 1B). Few intact cracks were also observed on the surface of microparticles which might be due to the excessive evaporation of solvent and hence high shrinkage during drying process. EDS data obtained showed the presence of Zr along the surface of microparticles. The comparison of FTIR spectrum of CMC, CS and micropartide showed the presence of all characteristic peaks of both CMC and CS on micropartide. Moreover, absorbance band at 1580 cm~(-1) corresponding to the characteristic band of NH_3~+ and COO~-suggested strong electrostatic interaction and hence complexation between two polymers (Fig. 2). The use of Zr as a cross-linker did not induce any cytotoxicity as most of the cells were viable up to 10 days of culture with microparticles. Fluorescence images of cells seeded micropartide showed adhesion of cells to the surface of micropartide (Fig. 3). The area percent of cells covering micropartide was significantly higher at day 10 (42%) compared to that at day 5 (15%) suggesting the proliferation of cells on micropartide. Conclusion: In this study, an injectable CMC-CS polyelectrolyte complex microparticles with Zr as a novel crosslinker was developed. Based on the results obtained, it can be concluded that polyelectrolyte complexation occurs between CS and CMC which can be developed into injectable microparticles by crosslinking with Zr. Zr used in this study was not cytotoxic and provided a suitable surface for cell adhesion and proliferation by enhancing the surface roughness of microparticle.
机译：简介：羧甲基纤维素（CMC）和壳聚糖（CS）是天然的可生物降解和生物相容性聚合物。 CMC是通过对纤维素进行化学修饰而获得的阴离子聚合物，CS是衍生自几丁质的阳离子聚合物，因此，这两种聚合物带相反电荷，可以进行离子凝胶化。锆（Zr）是一种四价阳离子金属，广泛用于整形外科植入物中，以促进氧化锆形式的骨整合，最近发现它可以通过刺激不同的信号传导途径来刺激人类成骨细胞的增殖和分化。这项研究旨在开发基于Zr作为新型交联剂的可注射，无毒的聚电解质聚合物复合物微粒。我们假设使用Zr作为交联剂不会引起任何细胞毒性，也不会促进成骨细胞粘附到微粒中。方法：制备CMC水溶液（3％w / v），滴加到四氯化锆溶液（5％w / v）和CS溶液（1％w / v）的均匀混合物中，并搅拌1小时。将CMC与CS的体积比优化为1：2，以改善两种聚合物之间的络合度。然后将如此获得的颗粒在室温下干燥48小时，然后进行进一步分析。通过扫描电子显微镜（SEM）表征了微粒的表面形态，并进行了能量色散光谱（EDS）进行元素分析。使用ATR模式的傅立叶红外光谱仪（FTIR）表征微粒子中各组分之间的分子内相互作用。在存在微粒的情况下培养鼠前成骨细胞（OEMS）长达10天后，使用活/死细胞测定试剂盒评估了体外细胞毒性。为了具体确定附着在微粒上的细胞的活力，将微粒转移到24孔板的单独孔中，然后在第5天和第10天进行活/死细胞测定，并在荧光显微镜下成像。使用ImageJ确定可见部分的微粒来确定覆盖有细胞的微粒的面积百分比。结果与讨论：微粒呈球形，平均直径为787±0.045μm。如在SEM图像上所见，微粒的表面包含均匀的凹槽和脊，从而产生了更粗糙的表面（图1B）。在微粒表面上也没有观察到完整的裂纹，这可能是由于溶剂的过度蒸发以及干燥过程中的高收缩率所致。获得的EDS数据表明，沿着微粒表面存在Zr。对CMC，CS和微颗粒的FTIR光谱的比较表明，微颗粒上同时存在CMC和CS的所有特征峰。此外，在1580 cm〜（-1）处的吸收带对应于NH_3〜+和COO〜的特征带，表明强烈的静电相互作用并因此导致了两种聚合物之间的络合（图2）。 Zr作为交联剂的使用不会引起任何细胞毒性，因为大多数细胞在用微粒培养10天后仍可存活。接种了微粒的细胞的荧光图像显示细胞粘附到微粒的表面（图3）。与第5天（15％）相比，第10天覆盖微颗粒的细胞面积百分比（42％）显着更高，表明细胞在微颗粒上增殖。结论：在这项研究中，开发了一种以Zr作为新型交联剂的可注射CMC-CS聚电解质复合微粒。根据获得的结果，可以得出结论，CS和CMC之间发生了聚电解质络合，可以通过与Zr交联将其发展为可注射的微粒。本研究中使用的Zr无细胞毒性，并通过增强微粒的表面粗糙度为细胞粘附和增殖提供了合适的表面。

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