乳化溶剂挥发法制备纳米粒

目的：建立乳化溶剂挥发法制备纳米粒的方法。方法：采用单因素法和正交设计法考察不同影响因素对乳化溶剂挥发法所制得的纳米粒粒径、包封率和载药量的影响。结果：采用乳化溶剂挥发法,通过改变处方和工艺因素所制得的纳米粒,外观圆整,大小均匀,粒径可控,包封率多数可达50%以上。结论：优化确立了乳化溶剂挥发法制备纳米粒的处方和工艺,可以制备满足不同要求的纳米粒。     

凝聚法制备川芎嗪固体脂质纳米粒

目的:制备川芎嗪固体脂质纳米粒.方法:采用凝聚法制备,并以包封率和载药量为指标采用正交设计法优化川芎嗪固体脂质纳米粒的制备工艺,并利用透射电镜、激光粒度分析仪、Zeta电位测定仪表征了其药剂学性质结果:所得川芎嗪固体脂质纳米粒的最佳制备处方是川芎嗪45mg,卵磷脂600mg,硬脂酸450mg,0.4%的泊洛沙姆60ml 结论:该处方可用于川芎嗪固体脂质纳米粒的制备,工艺简单、可行.     

壳聚糖和帕米膦酸双修饰的固体脂质纳米粒的制备

目的:制备壳聚糖和帕米膦酸双修饰的固体脂质纳米粒。方法:首先利用课题组发表的专利合成帕米膦酸修饰Brij78的新型非离子表面活性剂(Pa-Brij78),然后以壳聚糖(CS)溶液为水相,Pa-Brij78为乳化剂,E-Wax为油相,采用微乳法,利用修饰的帕米膦酸基团与壳聚糖分子链中质子化的氨基交联反应原理,通过一系列实验条件的探索,确定了最佳实验工艺条件,成功制备了壳聚糖和帕米膦酸双修饰的固体脂质纳米粒。通过动态光散射(DLS)粒径仪测定了纳米粒的粒径大小和Zeta电位;透射电子显微镜(TEM)对CS-Pa-Brij78-SLNs的形貌结构进行了表征。结果:实验结果显示,制备壳聚糖和帕米膦酸双修饰的固体脂质纳米粒的最佳条件为:p H=6.0,壳聚糖浓度分别为0.1%,0.2%;反应温度65℃,反应时间40 min,在该条件下,制备的壳聚糖和帕米膦酸双修饰的固体脂质纳米粒(CS-Pa-Brij78-SLNs)粒径分别为97.9±6.6 nm和182.4±62.2 nm,表面电位分别为(+5.21±1.4m V);(+7.94±0.80 m V),装载姜黄素时,载药量为10%,包封率在90%以上,透射电镜下观察其形态圆整,清晰可见壳聚糖包裹的电晕。结论:本文以壳聚糖(CS)溶液为水相,合成的新型非离子表面活性剂Pa-Brij78为乳化剂,E-Wax为油相,采用微乳化法,经过最佳实验条件的探索,通过一步法成功制备了稳定的壳聚糖和帕米膦酸双修饰的固体脂质纳米粒(CS-Pa-Brij78-SLNs)。     

藤黄酸聚乳酸纳米粒的研制

以新型材料聚乳酸(PLA)为载体,研制出质量稳定的藤黄酸聚乳酸纳米粒(GA-PLA-NPs)乳液制剂,并对其安全性进行评价。采用改良的溶剂蒸发法制备藤黄酸聚乳酸纳米粒(GA-PLA-NPs);用透射电子显微镜（TEM）观察纳米粒的形态;用激光粒度分析仪测定其平均粒径大小和分布;经超速离心后用紫外分光光度计测定纳米粒的包封率与载药量;考察藤黄酸纳米粒的体外释放特性;经急性毒性实验考察藤黄酸纳米粒的安全性。得到确定处方工艺为：水相∶有机相为2∶1（v/v）,表面活性剂在有机相中的浓度为0.5%（w/v）,藤黄酸（GA）在有机相中的浓度为0.1%（w/v）,GA∶PLA为1∶4（w/w）。处方条件下制备的纳米粒平均粒径为51.36 nm;平均包封率与载药量分别为98.87%和13.3%;藤黄酸纳米粒的体外释药分为两相：突释期和缓释期;急性毒性试验测得藤黄酸纳米粒的ID50为26.3mg/kg。制备的藤黄酸聚乳酸纳米粒（GA-PLA-NPs）质量稳定、分散性良好。聚乳酸可能成为藤黄酸的新型载体。     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

叶酸-壳聚糖Prdx6 shRNA纳米粒的构建及其对胃癌细胞增殖的影响

目的:探讨叶酸-壳聚糖Prdx6 shRNA纳米粒对胃癌细胞生长的影响。方法:制备靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒,原子力显微镜观察其形态,激光粒度分析仪测定纳米粒的粒径;倒置荧光显微镜观察叶酸-壳聚糖Prdx6 shRNA纳米粒的转染效率;采用蛋白质印迹法检测胃癌细胞Prdx6蛋白的表达变化;CCK8细胞增殖实验检测胃癌细胞的存活率。结果:1制备成功叶酸-壳聚糖Prdx6 shRNA向纳米粒。2荧光显微镜下观察靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒转染胃癌细胞的效率明显高于非靶向纳米粒;胃癌细胞转染靶向组纳米粒后Prdx6蛋白的表达显著低于非靶向组。3与对照组相比,叶酸-壳聚糖Prdx6 shRNA纳米粒能够明显抑制胃癌细胞的增殖(P0.01)。结论:1叶酸-壳聚糖Prdx6 shRNA纳米粒可高效转染胃癌细胞。2转染叶酸-壳聚糖Prdx6 shRNA纳米粒后胃癌细胞的生长明显受抑制。     

包载紫杉醇PLGA纳米粒的制备工艺优化及表征

以生物可降解材料乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]为载体材料,采用乳化-溶剂挥发法制备包载紫杉醇(PTX)的PLGA微球(PTX-PLGANPs)。采用正交实验设计,考察乳化剂质量浓度、PLGA与紫杉醇质量比、油相用量、剪切速度4个因素对粒径和载药率的影响,优化纳米粒最佳制备工艺。研究结果表明:当PLGA与PTX的质量比为4∶1,聚乙烯醇PVA用量0.1%,二氯甲烷用量为4mL,剪切速度为16000r·min^-1是纳米粒的最佳制备工艺。最佳工艺条件下的PTX-PLGANPs多批次重复实验得到PLGA-NPs粒径分布为(207.1±20.5)nm,Zeta电位为(-23.8±2.5)mV,载药量为(14.45±0.04)%。制得的PTX-PLGANPs均匀圆整、理化性质稳定。冻干粉复溶溶液12h粒径变化不大,具有良好的药物稳定性,为新型抗肿瘤缓释制剂的研发提供实验基础。     

长春新碱阳离子纳米结构脂质载体及其小肠吸收的研究

目的:硫酸长春新碱作为一种细胞毒型抗肿瘤药物,临床上多用其注射剂,虽应用广泛,但存在较多缺点,如药物半衰期短,代谢速率快以及毒副作用明显。本文目的是制备包载长春新碱和十二烷基磺酸钠的阳离子纳米结构脂质载体,并对其进行评价。方法:用复乳挥发法制备出目标脂质纳米粒;利用激光粒度仪对其粒径及zeta电位进行检测;利用高效液相色谱法对其包封率和载药量进行测定;透析法检测纳米粒的体外释放行为;用小肠吸收法评价纳米粒的促进吸收作用。结果:制得的纳米粒的平均粒径为(192.4±4.14)nm,多分散系数(PDI)为0.184±0.015,包封率为32.28%,Zeta电位为(30.6±4.09)m V,载药量为(1.56±0.10)%;体外释放实验显示在pH=7.4的中性释放介质中,硫酸长春新碱脂质纳米粒表现出缓释特性;小肠吸收实验表明十二烷基磺酸钠的加入和阳离子纳米粒的修饰可提高小肠对药物的吸收。结论:阳离子硫酸长春新碱纳米结构脂质载体具有缓释效果,并可以促进小肠对药物的吸收。     

分枝PEI 修饰的PLGA 纳米粒转染DNA 的研究*

目的:建立基于聚(乳酸-羟基乙酸)纳米粒(PLGA)载DNA的基因转染体系,比较用空白聚(乳酸-羟基乙酸)纳米粒(PLG-A-E)吸附质粒DNA和用分枝PEI修饰后的PLGA纳米粒(PLGA-BPEI)吸附质粒DNA优缺点。方法:用乳化蒸发法制备纳米粒,对纳米粒进行表征研究,包括包封率、Zeta电位、粒径大小、稳定性,用荧光显微镜观察它们对NIH3T3和HEK293细胞的转染效率,用MTT检测对它们细胞的毒性。结果:制备了两种基于PLGA的纳米粒,PLGA-E和PLGA-BPEI粒径大小为200-270nm,zeta电位为0-30mV,在血清和不同的pH值时两者均较稳定,转染效率PLGA-BPEI较PLGA-E高,且释放时间早,但前者较后者对细胞毒性大。结论:这两种基于PLGA纳米粒均能有效转染质粒DNA,它们存在不同的优缺点,应根据不同需要进行选择。     

牛血红蛋白PLGA纳米粒的制备及其体外功能的研究

目的:利用poly(lactide-co-glycolide)(PLGA)和poly(styrene-co-4-styrene-sulfonate)(PSS)制备带负电荷的牛血红蛋白纳米粒,并对其载氧性能和体外性能进行评价.方法:采用溶剂蒸发法制备出空白的PLGA-PSS的空白纳米粒,通过改变pH值,吸附牛血红蛋白,从而制备出牛血红蛋白PLGA纳米粒.通过TEM、粒径、Zeta电位、包裹率和载药量、体外释放及其携氧能力对该纳米粒进行了综合分析.结果:Hb-PLGA-PSS-NPsTEM电镜下呈类球形,平均粒径为226.8±23.4nm,Zeta电位为-68.62 mV,优化条件后最大包裹率约为99.3%,载药量约为28.6%,在37℃,pH7.4的PBS溶液中释放缓慢.通过对Hb结构的分析表明此工艺未对蛋白的结构造成影响,体外携氧实验测量了P50(29mmHg)和Hill系数(2.036),结果说明该Hb纳米微囊具有良好携氧功能.结论:成功制备了一种Hb-PLGA-PSS-NPs纳米粒,稳定性好,具有很好的携氧能力.     

Characterization and in vitro cytotoxicity of doxorubicin-loaded γ-polyglutamic acid-chitosan composite nanoparticles

In this study, γ-polyglutamic acid (γ-PGA) and chitosan (CS) nanoparticles were characterized as a carrier for the anti-cancer drug doxorubicin (DOX). Using ionic complexation between the positively charged DOX and the negatively charged polyelectrolyte γ-PGA, DOX:γ-PGA complexes were produced with an efficiency of approximately 99%. SEM micrographs demonstrated that the complexation of γ-PGA and DOX alone does not lead to the formation of nanoparticles and that the addition of a third component, chitosan, is required. Drug-loaded DOX:γ-PGA:CS nanoparticles were produced with particle sizes ranging from ~150 to ~630 nm. The stability of the DOX:γ-PGA:CS nanoparticles was examined by suspending the nanoparticles in different kinds of aqueous media. For the first time, in vitro studies with DOX-loaded nanoparticles demonstrated the cytotoxicity of the nanoparticles against a human oral squamous cell carcinoma cell line (HN-5a). Non-drug-loaded γ-PGA:CS nanoparticles did not display cytotoxic effects. It was shown that the encapsulated or surface-bound DOX did not lose its bioactivity and the prepared drug-loaded particles exhibited a considerable anti-proliferative activity against the human cancer cell line.     

Facile preparation of well-defined near-monodisperse chitosan/sodium alginate polyelectrolyte complex nanoparticles (CS/SAL NPs) via ionotropic gelification: A suitable technique for drug delivery systems

Polymeric nanoparticles have emerged as a promising approach for drug delivery systems. We prepared chitosan (CS)/sodium alginate (SAL) polyelectrolyte complex nanoparticles (CS/SAL NPs) via a simple and mild ionic gelation method by adding a CS solution to a SAL solution, and investigated the effects of molecular weight of the added CS, and the SAL:CS mass ratio on the formation of the polyelectrolyte complex nanoparticles. The well-defined CS/SAL NPs with near-monodisperse particle size of about 160 nm exhibited a pH stable structure, and pH responsive properties with a negatively or positively charged surface. The so-called “electrostatic sponge” structure of the polyelectrolyte complex nanoparticles enhanced their drug-loading capacity towards the differently charged model drug molecules, and favored controlled release. We also found that the drug-loading capacity was influenced by the nature of the drugs and the drug-loading media, while drug release was affected by the solubility of the drugs in the drug-releasing media. The biocompatibility and biodegradability of the polyelectrolytes in the polyelectrolyte complex nanoparticles were maintained by ionic interactions. These results indicate that CS/SAL NPs can represent a useful technique for pH-responsive drug delivery systems.     

Chitosan graft copolymer nanoparticles for oral protein drug delivery: preparation and characterization

Several novel functionalized graft copolymer nanoparticles consisting of chitosan (CS) and the monomer methyl methacrylate (MMA), N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC), and N-trimethylaminoethyl methacrylate chloride (TMAEMC), which show a higher solubility than chitosan in a broader pH range, have been prepared by free radical polymerization. The nanoparticles were characterized in terms of particle size, zeta potential, TEM, and FT-IR. These nanoparticles were 150-280 nm in size and carried obvious positive surface charges. Protein-loaded nanoparticles were prepared, and their maximal encapsulation efficiency was up to 100%. In vitro release showed that these nanoparticles provided an initial burst release followed by a slowly sustained release for more than 24 h. These graft copolymer nanoparticles enhanced the absorption and improved the bioavailability of insulin via the gastrointestinal (GI) tract of normal male Sprague-Dawley (SD) strain rats to a greater extent than that of the phosphate buffer solution (PBS) of insulin.     

Preparation of nanoparticles composed of chitosan/poly-gamma-glutamic acid and evaluation of their permeability through Caco-2 cells

In this study, a novel nanoparticle system for paracellular transport was prepared using a simple and mild ionic-gelation method upon addition of a poly-gamma-glutamic acid (gamma-PGA) solution into a low-molecular-weight chitosan (low-MW CS) solution. The particle size and the zeta potential value of the prepared nanoparticles can be controlled by their constituted compositions. The results obtained by the TEM and AFM examinations showed that the morphology of the prepared nanoparticles was spherical in shape. Evaluation of the prepared nanoparticles in enhancing intestinal paracellular transport was investigated in vitro in Caco-2 cell monolayers. It was found that the nanoparticles with CS dominated on the surfaces could effectively reduce the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers. After removal of the incubated nanoparticles, a gradual increase in TEER was noticed. The confocal laser scanning microscopy observations confirmed that the nanoparticles with CS dominated on the surface were able to open the tight junctions between Caco-2 cells and allowed transport of the nanoparticles via the paracellular pathways.     

Serratiopeptidase Loaded Chitosan Nanoparticles by Polyelectrolyte Complexation: In Vitro and In Vivo Evaluation

The aim of the present study was to formulate serratiopeptidase (SER)-loaded chitosan (CS) nanoparticles for oral delivery. SER is a proteolytic enzyme which is very sensitive to change in temperature and pH. SER-loaded CS nanoparticles were fabricated by ionic gelation method using tripolyphosphate (TPP). Nanoparticles were characterized for its particle size, morphology, entrapment efficiency, loading efficiency, percent recovery, and in vitro dissolution study. SER-CS nanoparticles had a particle size in the range of 400–600 nm with polydispersity index below 0.5. SER association was up to 80 ± 4.2%. SER loading and CS/TPP mass ratio were the primary parameters having direct influence on SER-CS nanoparticles. SER-CS nanoparticles were freeze dried using trehalose (20%) as a cryoprotectant. In vitro dissolution showed initial burst followed by sustained release up to 24 h. In vivo anti-inflammatory activity was carried out in rat paw edema model. In vivo anti-inflammatory activity in rat paw edema showed prolonged anti-inflammatory effect up to 32 h relative to plain SER.KEY WORDS: anti-inflammatory activity, chitosan, nanoparticle, serratiopeptidase, TPP     

Comparative Studies on Chitosan and Polylactic-co-glycolic Acid Incorporated Nanoparticles of Low Molecular Weight Heparin

This study was performed to test the feasibility of chitosan and polylactic-co-glycolic acid (PLGA) incorporated nanoparticles as sustained-release carriers for the delivery of negatively charged low molecular weight heparin (LMWH). Fourier transform infrared (FTIR) spectrometry was used to evaluate the interactions between chitosan and LMWH. The shifts, intensity, and broadening of the characteristic peaks for the functional groups in the FTIR spectra indicated that strong interactions occur between the positively charged chitosans and the negatively charged LMWHs. Three types of LMWH nanoparticles (NP-1, NP-2, and NP-3) were prepared using chitosan with or without PLGA: NP-1 nanoparticles were formed by polyelectrolyte complexation after single mixing, NP-2 nanoparticles were prepared by polyelectrolyte complexation after single emulsion–diffusion–evaporation, and NP-3 nanoparticles were optimized by double emulsion–diffusion–evaporation. NP-3 nanoparticles of LMWH prepared by the emulsion–diffusion–evaporation method showed significant differences in particle morphology, size, zeta potential, and drug release profile compared to NP-1 nanoparticles formed by polyelectrolyte complexation. Another ionic complex of LMWH with chitosan-incorporated PLGA nanoparticles (NP-2) showed lower drug entrapment efficiency than that of NP-1 and NP-3. The drug release rate of NP-3 was slower than the release rates of NP-1 and NP-2, although particle morphology of NP-3 was similar to that of NP-2. Cell viability was not adversely affected when cells were treated with all three types of nanoparticles. The data presented in this study demonstrate that nanoparticles formulated with chitosan–PLGA could be a safe sustained-release carrier for the delivery of LMWH.Key words: chitosan, low molecular weight heparin, nanoparticles, PLGA     

Chitosan-dextran Sulfate Nanoparticles for Delivery of an Anti-angiogenesis Peptide

Summary A novel nanoparticle delivery system has been developed by employing the oppositely charged polymers chitosan (CS) and dextran sulfate (DS), and a simple coacervation process. Under the conditions investigated, the weight ratio of the two polymers is identified as a determining factor controlling particle size, surface charge, entrapment efficiency and release characteristics of the nanoparticles produced. Particles of 223 nm mean diameter were produced under optimal conditions with a zeta potential of approximately −32.6 mV. A maximum of 75% anti-angiogenesis peptide entrapment efficiency was achieved with a CS:DS weight ratio of 0.59∶1. The same nanoparticle formulation also showed slow and sustained peptide release over a period of 6 days. In contrast, the formulation containing a lower ratio of CS:DS (0.5∶1) was found to have reduced entrapment efficiency and more rapid peptide release characteristics. The results of this study suggest that physicochemical and release characteristics of the CS-DS nanoparticles can be modulated by changing ratios of two ionic polymers. The novel CS-DS nanoparticles prepared by the coacervation process have potential as a carrier for small peptides.     

Synthesis and characterization of folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29 cells

Folate–chitosan (FA–CS) conjugates synthesized by coupling FA with CS render new and improved functions because the original properties of CS are maintained and the targeting ligand of FA is incorporated. In this work, FA–CS conjugates were synthesized based on chemical linking of carboxylic group of FA with amino group of CS as confirmed by Fourier transform spectroscopy (FTIR) and nuclear magnetic resonance (¹H NMR). FA–CS conjugates displayed less crystal nature when compared to CS. The FA–CS nanoparticles (NPs) were prepared by crosslinking FA–CS conjugates with sodium tripolyphosphate (STPP). Positively charged FA–CS nanoparticles were spherical in shape with a particle size of about 100 nm. Cellular uptake of CS or FA–CS nanoparticles was assayed by fluorescent microscopy using calcein as fluorescent marker in colon cancer cells (HT-29). The FA–CS nanoparticles exhibited improved uptake of HT-29 and could become a potential targeted drug delivery system for colorectal cancer.     

Enhanced Antibacterial Effect of Ceftriaxone Sodium-Loaded Chitosan Nanoparticles Against Intracellular Salmonella typhimurium

The aim of the present study was to utilize chitosan (CS) nanoparticles for the intracellular delivery of the poorly cell-penetrating antibiotic, ceftriaxone sodium (CTX). In vitro characterization of (CTX-CS) nanoparticles was conducted leading to an optimized formula that was assessed for its biocompatibility to blood (hemolysis test) and cells (MTT assay). Progressively, confocal laser scanning microscopy (CLSM), cellular uptake (microfluorimetry), and antibacterial activity of the nanoparticles were investigated in two cell lines: Caco-2 and macrophages J774.2 pre-infected with Salmonella typhimurium. Results showed that the optimized formula had size 210 nm, positive zeta potential (+30 mV) and appreciable entrapment efficiency for CTX (45%) and included a biphasic release pattern. The nanoparticles were biocompatible and were internalized by cells as verified by CLSM whereas microfluorimetry indicated substantial cellular uptake. Moreover, the CTX–chitosan nanoparticles showed a significant reduction in the count of intracellular S. typhimurium in Caco-2 and macrophages J774.2. This reduction was significantly higher than that obtained in case of placebo nanoparticles, CTX, and CTX–chitosan solutions and might be attributed to enhanced endocytic uptake of the nanoaprticles and antibacterial effect of the chitosan polymer. In conclusion, the results provide evidence for the potential use of chitosan nanoparticles to enhance the intracellular delivery and antibacterial effect of CTX in enterocytes and macrophages.Key words: ceftriaxone sodium, chitosan nanoparticles, enterocytes, intracellular delivery, macrophages     

Enhanced Healing of Rat Calvarial Critical Size Defect with Selenium-Doped Lamellar Biocomposites

A 3D porous lamellar selenium-containing nano-hydroxyapatite (SeHAN)/chitosan (CS) biocomposite was synthesized. The selenium-containing hydroxyapatite (HA) grains of 150～200 nm in length and 20～30 nm in width were observed by dynamic light scattering and transmission electron microscopy. A combination of X-ray diffraction, Fourier-transform infrared spectroscopy, and SEM indicated that HA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and HA. Then, a standard critical size calvarial bone defect was created in Wistar rats. In group 1, no implant was made in the defect. In groups 2 and 3, HA nanoparticles (HAN)/CS biocomposite and SeHAN/CS biocomposite were implanted into the defect, respectively. After 4 weeks, the histological assessment clearly exhibited no significant changes, only found some living cells anchored in the periphery of the implants. After 8 and 12 weeks, most newly formed osteoid tissue was found in the SeHAN/CS implant group. Additionally, the newly formed osteoid tissue, both at the edge and in the center of implants, was bioactive and neovascularized. Microfocus computerized tomography measurements also confirmed the much better quality of the newly formed bone tissue in SeHAN/CS implant group than that in HAN/CS implant group (p?<?0.01). Collectively, the SeHAN/CS biocomposite, as a bioactive bone grafting substitute, significantly enhanced the repair of bone defect.