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首页> 外文会议>World biomaterials congress >Characterization and evaluation of mechanical properties of injectable porous spherical nano-hydroxyapatite/chitosan particles for craniofacial bone regeneration
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Characterization and evaluation of mechanical properties of injectable porous spherical nano-hydroxyapatite/chitosan particles for craniofacial bone regeneration

机译：可注射球形球形纳米羟基磷灰石/壳聚糖颗粒用于颅面骨再生的表征和力学性能评估

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Introduction: Craniofacial bone surgery and regeneration is a major challenge in regenerative medicine. As a solution injectable microscale particles have been fabricated and characterized since they can be implanted with a minimally invasive surgery. Although craniofacial bone is not a load bearing bone, injectable particles should possess stability towards certain physiologic conditions and retain its mechanical strength at abruptly occurring external loading applications. In this study we have fabricated porous particles using chitosan biopolymer and nano-hydroxyapatite (nHA). We hypothesized that incorporating nHA into chitosan could emulate the mineralized cortical bone structure, thus improve the mechanical strength of the particles. Materials and Methods: Medium molecular weight chitosan, hydroxyapatite nanopowder (nHA, ＜200nm particle size), sodium tripolyphosphate (TPP, technical grade, 85%) and acetic acid (A.C.S reagent, 99.7%) 2% (w/v) chitosan solution was prepared by dissolving chitosan and 1 % (v/v) acetic acid. Then nHA has been added to 2% (w/v) chitosan solution to make 0.5%, 1 % and 2% (w/v) nHA/chitosan composite mixtures. These mixtures were dripped into 27.18mM TPP/Deionized water solution using 30 gauge needle and stirred at 600RPM. After 30 minutes TPP cross-linked nHA/chitosan beads filtered out from TPP solution and lyophilized at -52oC temperature and 0.02 mbar pressure for 24 hours. Portion of each batch were treated further by soaking followed by drying and named as Lyophilized Soaked and Dried (LSD) particles. Finally particles were characterized for their morphology, chemical structure, physical structure, cell cytotoxicity and attachment. Finally particles were tested for its ultimate compressive strength (UCS) at dry and wet states using ADMET's MTESTQuattro universal testing machine using 0.05mm/s cross head movement. Statistical data were analyzed using IBM SPSS Statistics version 21. Figure 1- Scanning electron microscopy images of 2% nHA/Chitosan particle surfaces Results and Discussion: Fabricated lyophilized and LSD particles had a mean diameter of 1.33 and 0.93 mm respectively which is promising for injectability of the particles via needles. SEM images showed porous surface morphology and interconnected pore structures inside the particles. Lyophilized and LSD particles had surface pores with diameters less than 10 and 2 μm respectively (Figure 1 .A and 1 .B). This highly porous surface morphology improves the osteoconductivity of scaffolds by facilitating osteoblast cell adhesion and proliferation. One-way ANOVA results showed that, there is significant increase in UCS of 1 % and 2% nHA compared to 0% nHA lyophilized particles at (p＜0.001) significance level (Figure 2.A). Further increase in nHA percentage affects the dripping process due to high clogging factor in the needle and higher viscosity of the solution. Moreover, 2% nHA LSD particles had increased their mean UCS by 120% compared to 2% nHA lyophilized particles at (p＜0.005) significance level. In a drawback, all particles have lost their mechanical properties by 95% at (p＜0.001) significance level on the 2nd day when fully immersed in phosphate buffered saline (PBS). However, particles preserved its initial shape and showed minimal degradation at the end of 100th day in PBS solution. Additionally live and dead cell assay showed zero cytotoxicity and good osteoblast cell attachment to both lyophilized and LSD particles at the end of 6th day of in vitro studies (Figure 2.B and 2.C). Figure 2- Ultimate compressive strengths of nHA/Chitosan lyophilized particles at different nHA compositions are shown in 2.A. Live and Dead cell assay results are shown in 2.B and 2.C. Live cells proliferation can be seen on 2.B FITC image. Presence of dead cells in the same region is captured using TRITC filter as shown in 2.C Conclusion: Experiments results shows that incorporating nHA can improve the mechanical properties of lyophilized chitosan particles. This result is consistent with a study that HA can improve the mechanical properties of electrospun fibers. No cell cytotoxicity and higher cell attachment shows improved osteoconductivity of the particles. However further modifications on particles is required to improve to wet state mechanical properties.
机译：简介：颅面骨手术和再生是再生医学的主要挑战。作为溶液，已经制备并表征了可注射的微米级颗粒，因为它们可以通过微创手术植入。尽管颅面骨不是承重骨，但可注射颗粒应在某些生理条件下具有稳定性，并在突然发生的外部负荷应用中保持其机械强度。在这项研究中，我们使用壳聚糖生物聚合物和纳米羟基磷灰石（nHA）制备了多孔颗粒。我们假设将nHA掺入壳聚糖可以模拟矿化的皮质骨结构，从而提高颗粒的机械强度。材料与方法：中等分子量的壳聚糖，羟基磷灰石纳米粉（nHA，＜200nm粒度），三聚磷酸钠（TPP，工业级，85％）和乙酸（ACS试剂，99.7％）2％（w / v）壳聚糖溶液通过溶解脱乙酰壳多糖和1％（v / v）乙酸制备。然后将nHA添加到2％（w / v）的壳聚糖溶液中，制成0.5％，1％和2％（w / v）的nHA /壳聚糖复合混合物。使用30号针将这些混合物滴入27.18mM TPP /去离子水溶液中，并在600RPM下搅拌。 30分钟后，TPP交联的nHA /壳聚糖珠从TPP溶液中滤出，并在-52oC温度和0.02 mbar压力下冻干24小时。通过浸泡然后干燥进一步处理每批的部分，并命名为冻干浸泡并干燥（LSD）颗粒。最后，对颗粒的形态，化学结构，物理结构，细胞毒性和附着进行了表征。最终，使用ADMET的MTESTQuattro万能试验机以0.05mm / s的十字头运动测试了颗粒在干燥和潮湿状态下的极限抗压强度（UCS）。使用IBM SPSS Statistics版本21分析统计数据。图1- 2％nHA /壳聚糖颗粒表面的扫描电子显微镜图像结果与讨论：制备的冻干颗粒和LSD颗粒的平均直径分别为1.33和0.93 mm，有望实现注射通过针的颗粒。 SEM图像显示了颗粒内部的多孔表面形态和相互连接的孔结构。冻干和LSD颗粒的表面孔直径分别小于10和2μm（图1.A和1.B）。这种高度多孔的表面形态通过促进成骨细胞的粘附和增殖来改善支架的骨传导性。单向方差分析结果表明，与显着性水平为（p ＜0.001）的0％nHA冻干颗粒相比，UCS分别增加了1％和2％nHA（图2.A）。由于针中的高堵塞因子和溶液的更高粘度，nHA百分比的进一步增加会影响滴液过程。此外，与显着性水平为（p ＜0.005）的2％nHA冻干颗粒相比，2％nHA LSD颗粒的平均UCS增加了120％。一个缺点是，当完全浸入磷酸盐缓冲盐水（PBS）中时，所有颗粒在第2天的（p ＜0.001）显着性水平都丧失了95％的机械性能。然而，在PBS溶液中，颗粒保持其初始形状并在第100天结束时表现出最小的降解。此外，在体外研究的第6天结束时，活细胞和死细胞分析显示出零的细胞毒性和良好的成骨细胞附着于冻干和LSD颗粒（图2.B和2.C）。图2-在2.A中示出了在不同nHA组成下的nHA /壳聚糖冻干颗粒的极限抗压强度。活细胞和死细胞测定结果显示在2.B和2.C中。在2.B FITC图像上可以看到活细胞增殖。如2.C所示，使用TRITC过滤器捕获了同一区域中的死细胞。结论：实验结果表明，掺入nHA可以改善冻干的壳聚糖颗粒的机械性能。该结果与HA可以改善电纺纤维的机械性能的研究一致。没有细胞的细胞毒性和较高的细胞附着性表明颗粒的骨传导性改善。然而，需要对颗粒进行进一步的改性以改善至湿态的机械性能。

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