【作者】 吴涛；

【导师】 裴国献；

【作者基本信息】 南方医科大学 ， 骨外科学， 2009， 博士

【摘要】 背景中医药治疗骨折和骨不连具有数千年的历史,用药独到、简便灵验,但由于成分复杂、基础研究落后,其优势尚未得到充分发掘。中药与生物材料的复合及其在组织工程骨构建中的应用,国外尚未见正式报导,国内在此方面的研究也处于刚刚起步的阶段。已有研究表明:中药淫羊藿来源的植物黄酮——淫羊藿苷(C₃₃H₄₀O₁₅,分子量:676.67)可促进成骨细胞BMP和Cbfa1基因的表达,抑制间充质细胞的成脂分化;可通过发挥雌激素样作用,增加去卵巢大鼠的骨形成、抑制骨吸收。这些结果都暗示:淫羊藿苷可作为一种骨诱导活性因子用于骨再生研究。此外,淫羊藿苷来源广泛、提取工艺相对简单、性质稳定、易于储存、可耐受消毒灭菌,这些特性亦为组织工程支架材料的载药提供了方便。目的1.研究传统中药淫羊藿有效药理成分——淫羊藿苷对人骨髓间充质干细胞（human bone marrow-derived mesenchymal stem cells,hBMSCs）增殖和成骨分化等生物学行为的影响,探讨其促进成骨分化的作用机制,评价淫羊藿苷作为一种新型成骨诱导信号分子替代生长因子应用于骨组织工程的可行性;2.构建仿生组装淫羊藿苷—壳聚糖/羟基磷灰石（淫羊藿苷—CS/HA）骨修复材料,探讨其理化性质和生物相容性;3.研究淫羊藿苷—CS/HA复合材料的体外释药行为和释药动力学;4.研究淫羊藿苷—CS/HA复合材料的体内成骨效能。方法1.淫羊藿苷对hBMSCs成骨分化的影响和机制研究hBMSCs的分离培养和鉴定健康志愿者经知情同意后,于髂骨抽取骨髓,经全骨髓培养、扩增后,对第二代细胞进行表型标志物鉴定和成脂、成软骨和成骨三向诱导,取第3代细胞用于试验。淫羊藿苷的细胞毒性试验将hBMSCs接种至96孔板中进行培养,5000细胞/孔,贴壁后加入150μl浓度为10^-9M～2.0×10^-4M的淫羊藿苷培养基和0.05%（v/v）DMSO培养基进行培养,用普通培养基作为对照,干预48h后采用MTT法检测各浓度淫羊藿苷对hBMSCs活力的影响。细胞增殖试验将hBMSCs接种于96孔板中进行培养,2000细胞/孔,贴壁后吸出原培养基,分别加入150μl浓度为10^-9～10^-4M的淫羊藿苷培养基进行培养,以DMSO培养基作为对照。于加液后第1、3、5、7和9d MTT法测定OD值,绘制细胞生长曲线。hBMSCs成骨分化试验将hBMSCs植入6孔板中进行培养,2×10⁷细胞/孔,贴壁后加入1.5ml浓度为10^-9M～10^-4M的淫羊藿苷培养基进行培养,以DMSO培养基作为阴性对照,rhBMP-2培养基作为阳性对照。于加液后3、7、11d裂解细胞,采用碱性磷酸酶（ALP）试剂盒和BCA试剂盒分别检测ALP和蛋白浓度,根据公式ALP（U/g）=ALP/总蛋白量换算ALP含量;同上法培养细胞,于7、14、21d裂解细胞,骨钙素（OCN）Elisa试剂盒检测OCN表达量;采用NBT/BCIP染液对培养11d的hBMSCs进行ALP染色;采用茜素红染液对培养28d的hBMSCs进行钙化结节染色并定量。淫阳藿苷促进hBMSCs成骨分化的机制研究将hBMSCs植入6孔板中进行培养,2×10⁷细胞/孔,贴壁后加入DMSO培养基、10^-6M淫羊藿苷培养基、rhBMP-2培养基和10^-6M淫羊藿苷+rhBMP-2培养基进行培养。分别于1、4、7d采用RT-PCR方法对成骨相关基因ALP、OCN、OPN、Col-Ⅰ、Cbfa1、BMP-2、BMP-4和BMP-7mRNA的表达进行检测。将hBMSCs植入φ10cm培养皿中进行培养,5×10⁷细胞/皿,贴壁后加入4ml DMSO培养基、10^-6M淫羊藿苷培养基、rhBMP-2培养基和淫羊藿苷+rhBMP-2培养基连续培养14d,经提取总蛋白后进行Westernblot检测成骨相关蛋白Cbfa1、OCN、BMPs的表达;细胞爬片后进行OCN免疫细胞荧光检测。2.仿生组装淫羊藿苷-CS/HA骨修复材料的构建仿生组装CS/HA复合材料的构建、表征和生物相容性采用原位复合和冷冻干燥方法制备CS/HA复合材料,通过扫描电镜（SEM）、切片染色观察材料的孔隙结构,采用常规方法评估材料的密度、孔隙率、孔径;采用X线衍射仪（XRD）和傅立叶红外光谱（FTIR）分析材料的理化性质;制备材料浸提液,采用MTT法观察其对hBMSCs增殖的影响,将hBMSCs接种至材料表面,分别于第3d和第10d采用SEM观察细胞的数量和形态;将CS/HA复合材料植入新西兰兔背部肌袋,分别于术后1、4、8、12 w行组织切片观察材料的组织相容性和降解情况。仿生组装淫羊藿苷-CS/HA骨修复材料的制备、表征和生物学相容性在CS/HA复合材料的制备过程中掺入淫羊藿苷,制备载药剂量分别为10^-7、10^-6、10^-5mol的淫羊藿苷-CS/HA复合材料。采用同上方法分析淫羊藿苷-CS/HA复合材料的理化性质,万能材料试验机测试材料的力学性能;制备材料浸提液,采用MTT法观察其对不同密度接种的hBMSCs增殖和成骨分化（ALP表达）的影响,采用SEM对其表面接种10d的细胞进行观察;通过溶血试验考查淫羊藿苷-CS/HA复合材料对红细胞的影响;通过热原试验考查淫羊藿苷-CS/HA复合材料热原性。3.仿生组装淫羊藿苷-CS/HA骨修复材料的体外释药行为将载药量分别为10^-7、10^-6、10^-5mol的淫羊藿苷-HA/CS材料置于盛有5mlPBS的密闭玻璃离心管中,37℃恒温振荡,分别于1、2、3、10、15、20、30、60、90 d定时收集全部溶液进行超高效液相色谱（UPLC）检测。通过精密度试验、重复性试验、加样回收率试验、稳定性试验考查设备的精密度、灵敏度和淫羊藿苷的稳定性,通过标准曲线换算供试品每次释药量,并将其进行累积绘制成释药曲线;采用Logarithmic模型对释药行为进行曲线拟合。4.仿生组装淫羊藿苷-CS/HA骨修复材料的体内成骨效能60只雄性新西兰大白兔随机分为5组（n=12）,麻醉后于右侧桡骨中段截骨制作长度为1.5 cm的骨缺损模型,将CS/HA和载10^-7、10^-6、10^-5mol淫羊藿苷—CS/HA复合材料随机植入骨缺损处,骨缺损模型组不植入材料。放射性核素骨扫描（ECT）检查:术后4 w各组随机选取4只动物于耳缘静脉注射^99mTc-MDP,3 h后置单光子核素扫描仪上检测骨缺损部位^99mTc-MDP浓聚情况,采集结束后在图像上选取相同面积的感兴趣区域（ROI）进行定量计数,ROI均值=计数/面积。大体标本观察和X线检查:术后4、8、12 w处死动物后收集右前臂尺桡骨进行大体观察和X线拍片检查。骨密度（BMD）检查:取各组术后12 w标本行骨密度检查,于电脑上选取桡骨缺损区域并计算该区域的骨矿含量（BMC）,BMD（g/cm²）=BMC/选取面积。组织学观察:各时间点组织标本经固定、脱钙、切片后行HE染色观察。结果1.淫羊藿苷对hBMSCs成骨分化的影响和机制研究全骨髓培养的第二代hBMSCs表面标志物鉴定结果分别为:CD29（93.98±6.32）%、CD44（85.98±3.87）%、CD71（72.19±4.66）%、CD105（79.28±7.37）%、CD166（97.42±7.43）%、CD14（0.95±0.06）%、CD34（1.45±0.38）%、CD45（0.73±0.11）%;经成脂、成软骨和成骨三向诱导后全骨髓培养法分离的细胞可分别向脂肪细胞、软骨细胞和成骨细胞分化。高于10^-4M的淫羊藿苷具有一定的细胞毒性;培养基中0.05%（v/v）DMSO用量安全、无细胞毒性,可用于淫羊藿苷的助溶。浓度为10^-8M淫羊藿苷可促进hBMSCs的增殖。淫羊藿苷的促成骨分化作用（ALP表达）与剂量有关,浓度过低时(＜10^-8M)不能促进hBMSCs的成骨分化,浓度过高时(＞10^-5M)则抑制了hBMSCs的成骨分化。浓度在10^-8～10^-5M的淫羊藿苷可促进OCN的表达;在第11d的ALP染色和第28d的钙化结节染色和茜素红定量也说明10^-8～10^-5M的淫羊藿苷可促进hBMSCs向成骨方向分化。各试验均以10^-6M为最佳浓度,但是从整体上看,淫羊藿苷的成骨诱导能力不及rhBMP-2。10^-6M淫羊藿苷可促进成骨相关基因ALP、OCN、OPN、Col-Ⅰ、Cbfa1、BMP-2、BMP-4和BMP-7 mRNA的表达和成骨相关蛋白Cbfa1、OCN、BMPs的表达,与rhBMP-2具有协同作用。2.仿生组装淫羊藿苷-CS/HA骨修复材料的构建采用原位复合和冷冻干燥技术可构建出CS/HA复合材料,扫描电镜观察发现该材料表面具有均匀分散的200～700nm HA颗粒,XRD和FTIR分析表明合成的HA是含CO₃^2-弱结晶纳米晶体;该材料的孔隙率、孔径和密度分别为:88.70±2.27%、112.63±20.52μm和71.51±2.55 kg/m³;材料浸提液对细胞生长曲线无干扰,其表面接种的细胞亦可自由生长;肌袋埋植试验表明,8w后组织炎性反应消退,12w时CS/HA复合材料已基本降解,材料被纤维组织爬行替代。淫羊藿苷载药过程对CS/HA复合材料的理化性质无显著影响,对其力学性能的影响与载药剂量相关:10^-5和10^-6mol载药量降低了材料的弹性模量（与CS/HA比较,P＜0.05）;该材料细胞相容性良好,可诱导hBMSCs向成骨方向分化;溶血试验表明淫羊藿苷-CS/HA复合材料血液相容性良好,不会导致溶血;热原试验亦证明淫羊藿苷-CS/HA复合材料无热原性。3.仿生组装淫羊藿苷-CS/HA骨修复材料的体外释药行为超高效液相色谱（UPLC）的精密度试验和重现性试验的RSD分别为0.636%和3.245%;加样回收率试验的平均回收率为96.667%,RSD为2.139%;稳定性试验RSD为1.286%;在1～2000ng质量范围内,淫羊藿苷的色谱峰面积与进样量之间呈良好的线性关系,回归方程为:Y=7877.3X+202422,R²=0.9976。通过释药累积曲线可知,释药初期（0～3d）,药物从支架材料中爆发性地释放出来,约达载药量的25%,而后释药速度迅速下降,至第20d约有40%～60%左右的药物释出,之后以低速持续释放,90d后仍有部分药物存留于支架材料中。三种载药量的释药拟合方程分别为,载10^-7mol淫羊藿苷-CS/HA复合材料:Y=6.267+13.468 ln（X） R²=0.901;载10^-6mol淫羊藿苷-CS/HA复合材料:Y=5.668+16.846 ln（X）R²=0.916;载10^-5mol淫羊藿苷-CS/HA复合材料:Y=6.322+18.466 ln（X）R²=0.923,释药行为符合一级方程。4.仿生组装淫羊藿苷-CS/HA骨修复材料的体内成骨效能骨缺损模型组的各项检测结果表明:骨缺损部位自身修复能力低下,造模后两断端骨髓腔逐渐闭合,至12 w时髓腔完全封闭形成骨缺损。第4 w进行的ECT检测结果表明:4个材料植入组的ECT值均显著高于骨缺损模型组（P＜0.001）,载药量为10^-6mol和10^-5mol的淫羊藿苷-CS/HA组显著高于单纯CS/HA植入组（P＜0.01）;大体观察和X线检查结果表明,在第4w植入淫羊藿苷—CS/HA材料可观察到明显的骨痂桥接断端,植入8w后骨痂大量生长,骨缺损基本愈合,到12 w时髓腔再通,骨愈合进入塑形期。BMD检测结果:4个材料植入组的BMD值均显著高于骨缺损模型组（P＜0.001）,载药量为10^-6mol和10^-5mol的淫羊藿苷-CS/HA组显著高于单纯CS/HA植入组（P＜0.001）。从组织学切片的观察发现,淫羊藿苷-CS/HA植入骨缺损后,材料的降解速度随载药剂量的增加而明显加快,4 w时材料即发生明显的崩解、碎裂,其周围可见大量新生软骨形成并逐渐向材料的中央长入;8 w时材料进一步降解,被分割的材料间隙有大量软骨组织形成,部分发生骨化;至12 w时材料完全降解,软骨被骨组织替代,新生的骨组织排列紊乱,中央可见细小的骨髓腔结构,骨修复速度快于单纯应用CS/HA复合材料。结论1.淫羊藿苷可促进hBMSCs的增殖和成骨分化,这两种作用与淫羊藿苷的浓度相关;淫羊藿苷诱导成骨效能逊于rhBMP-2。2.淫羊藿苷可诱导hBMSCs成骨相关基因和蛋白的表达,与rhBMP-2具有协同作用。3.采用原位复合和冷冻干燥方法可制备出CS/HA复合材料,该材料具有良好的孔隙率和生物相容性,化学构成与天然骨近似。4.淫羊藿苷载药过程对CS/HA复合材料的理化性质无显著影响,对其力学性能的影响与载药剂量相关;载药过程不会影响CS/HA复合材料的生物相容性。5.淫羊藿苷-CS/HA复合材料在体外释药缓慢,释放时间可达90d以上,释药行为遵循一级方程。6.淫羊藿苷-CS/HA复合材料具有骨传导性和骨诱导活性,可促进原位骨再生。更多还原

【Abstract】 BACKGROUNDEpimedium sagittatum is a traditional Chinese medical herb and widely used in the therapies of fractures,bone and joint diseases,impotence and senility in China for hundreds of years.Icariin(C₃₃H₄₀O₁₅,molecular weight:676.67),a typical flavonol glycoside,is considered to be the major pharmacological component of Epimedium sagittatum.Recent evidences have indicated icariin can improve the osteogenesis from mesenchymal stem cells and suppress the activities of osteoclasts in vitro, thereby it exerts its bone-protective functions by increasing bone formation and inhibiting bone resorption.Additional studies have demonstrated that icariin has the ability to enhance the expression of osteogenic-related mRNA level in osteoblasts, and has a direct stimulatory effect on the proliferation and differentiation of pre-osteoblastic MC3T3-El cells in a BMP-and Runx2-dependent manner.Taken together,such results indicate that icariin is a potential osteogenic inductive agent and can be used in bone repair.What is more,icariin is chemically stable,and has high melting point,thus benefiting its extraction from raw herb and combination to form artificial bone material usually used for bone defect repair and/or drug-loading scaffolds. OBJECTIVE1.To investigate effects of icariin on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells（hBMSCs）,and study the mechanism of promoting osteogenic differentiation action.2.To construct biomimetic icariin-chitosan/hydroxyapatite（icariin-CS/HA） scaffolds,and study physicochemical properties and biocompatibility.3.To study icariin release behavior from icariin-CS/HA scaffolds in vitro.4.To investigate bone repair capability of icariin-CS/HA scaffolds in vivo.METHODS1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanismIsolation and culture of hBMSCs Cells were obtained from the posterior iliac crests of 3 healthy adult volunteers following informed consent.After being cultured and expanded,the second generation cells were identified by phenotypic analysis and induced by adipogenic,osteogenic and chondrogenic media.hBMSCs in the third generation were used in the investigations.Cytotoxicity test of icariin The hBMSCs were seeded at a density of 5 000 cells/well in a 96-well plate and incubated for 24 h prior to the addition of 150μl icariin(icariin dosages were 10^-9,10^-8,10^-7,10^-6,10^-5, 10^-4and 2×10^-4M separately)media and 0.05%（v/v）DMSO media,while control cells were with fresh DMEM.After 48 h,the number of survival cells was detected by MTT method.hBMSCs proliferation test hBMSCs were cultured in 96-well plates at a density of 2 000 per well,and were treated with icariin(10^-9M～10^-4M) and DMSO media.At 1,3,5,7 and 9d,cell proliferation curves were drawn based on OD values which were measured by MTT method.Osteogenic differentiation test of hBMSCs hBMSCs were plated on 6-well plates,2×10⁷ cells/well,and were treated with icariin(10^-9～10^-4M),DMSO（negative control）and rhBMP-2（positive control） media separately.At 3,7 and 11d,cells were lysed in 100μl deionized water and homogenized by ultrasound at 4℃.Alkaline phosphatase（ALP）activity and total protein content in cell lysates were measured using an ALP activity kit and a micro-BCA Assay kit separately,and ALP activity was normalized for the corresponding total protein concentration（U/g）.At 7,14 and 21d,osteocalcin（OCN） content in cell lysates were measured using an OCN Elisa kit.In addition,ALP staining of hBMSCs cultured for 11d were exerted by NBT/BCIP,and calcified nodules of hBMSCs cultured for 28d were stained with Alizarin red S,then Quantitive analyses of Alizarin red S were exerted.The mechanism of icariin promoting hBMSCs osteogenic differentiation Cells were cultured on 6-well plates at density of 2×l0⁷cells/well,and were treated with DMSO,10^-6M icariin,rhBMP-2 and 10^-6M icariin+rhBMP-2 media separately.At 1,4 and 7d,RT-PCR was used to examined mRNA expression of osteogenic genes（ALP,OCN,OPN,Col-Ⅰ,Cbfa1, BMP-2,BMP-4 and BMP-7）in hBMSCs.After being seeded onΦ10cm dishes and treated as above for 14d,osteogenic proteins（Cbfa1,OCN and BMPs）in hBMSCs were detected by Westernblot,at the same time,the OCN protein expression of climbing-cells was examined by immunofluorescence detection.2.Construction of biomimetic icariin-CS/HA scaffoldsThe construction,characterization and biocompatibility of biomimetic CS/HA scaffolds CS/HA scaffolds were prepared by in situ hybridization and freeze-dried methods.The micro-structure of scaffolds was examined by scanning electron microscopy（SEM）and HE staining,the density,porosity and pore diameter of CS/HA scaffolds were evaluated with the normal methods,and the physicochemical properties were detected by X-ray diffraction（XRD）examination and Fourier transformed infrared spectroscopy（FTIR）.Effects of CS/HA conditional media,prepared with normal method,on proliferation of hBMSCs were tested by MTT method.At 3d and 10d,SEM was used to observe hBMSCs which were seeded on the surface of CS/HA.After being implanted in dorsal muscle pockets of New Zealand rabbits,CS/HA scaffolds were obtained at 1,4,8 and 12w,and histocompatibility and degradation of scaffolds were observed by histomorphology. The construction,characterization and biocompatibility of biomimetic icariin-CS/HA scaffolds We prepared 10^-7、10^-6、10^-5mol icariin-CS/HA scaffolds with the same procedures as the preparation of CS/HA scaffolds but without touching icariin.Physicochemical properties of icariin-CS/HA were analyzed as above.And the mechanical properties of scaffolds in wet state were detected with universal testing machine.Influences of icariin-CS/HA on proliferation and osteodifferentiation （ALP activity）of hBMSCs were tested by MTT and ALP kit.And cells were observed using SEM after seeding for 10d.Biocompatibility of icariin-CS/HA in vitro and in vivo was evaluated by hemolysis test and pyrogen test separately.3.Icariin release behavior of icariin-CS/HA scaffolds in vitroIcariin-CS/HA scaffolds were soaked in 5 ml phosphate-buffered solution（PBS） and maintained at 37℃and kept shaking gentally at 10 rpm.The samples of 1,2,3, 5,10,15,20,30,60 and 90 d were analyzed by ultra performance liquid chromatography（UPLC）.The precision and sensitivity of UPLC were evaluated by precision test,repeatability test,icariin recovery test and stability test.Icariin released from scaffolds was calculated according to standard curve and the percentage of icariin released was accumulated.4.Bone repair capability of icariin-CS/HA scaffolds in vivo60 Male White New Zealand rabbits were allocated into groups of icariin-CS/HA,CS/HA and control（no treatment）randomly（n=12）.After anesthetized by pentobarbitol sodium,a 1.5 cm segment defect was made in the right radius of the animals.The bone defect areas were filled with icariin-CS/HA with different icariin dosages（the icariin-CS/HA groups）,CS/HA scaffolds only（the CS/HA group）or no scaffolds（the control group）.Emission computed tomography （ECT）Four weeks after surgery,four rabbits in every group were selected randomly for ECT examination.3 h after administration of mTc-MDP,the right forelimb was scanned with a single photon emission computed tomography.Thereafter,region of interesting（ROI）of the same size was chosen and quantitative counting was performed,the mean of ROI=value/area_selected.Gross specimen observation and X-ray examination Gross specimen observation and X-ray images of right forelimb were taken 4,8 and 12 weeks after implantation.Histological observation All of radius specimens were fixed in buffered formalin,and decalcified in 10%（v/v）nitric acid solution.Following routine histological processing 5-um-thick tissue slices were obtained and stained with haematoxylin and eosin（H&E）and observed under a light microscope.RESULTS1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanismPhenotypic analysis of hBMSCs in the second generation showed that CD29 （93.98±6.32）%,CD44（85.98±3.87）%,CD71（72.19±4.66）%,CD105（79.28±7.37）%, CD166（97.42±7.43）%,CD14（0.95±0.06）%,CD34（1.45±0.38）%and CD45 （0.73±0.11）%;Cells could be inducted into osteoblasts,chondrocytes and adipocytes by induction of osteo-,chondro-and adipogenesis.The data suggested that with no higher than 10^-4M concentration,cytotoxicity of icariin was low.0.05%（v/v）DMSO was safe for cell and could be used as a cosolvent for icariin.10^-8M icariin could promote proliferation of hBMSCs,and osteo-induction（ALP activities）related with dose:low dose(＜10^-8M)icariin could not increase hBMSCs osteogenic differentiation,high dose(＞10^-5M),however,inhibited osteigenic differentiation. The concentration between 10^-8M and 10^-5M could promote OCN expression;ALP staining and Alizarin red S staining also indicated 10^-8～10^-5M icariin could accelerate osteodifferentiation of hBMSCs,and 10^-6M was the best concentration.On the whole,osteo-induction of icariin was not better than rhBMP-2.10^-6M icariin could increase osteogenic genes mRNA（ALP,OCN,OPN,Col-Ⅰ,Cbfa1,BMP-2, BMP-4 and BMP-7）and proteins（Cbfa1,OCN and BMPs）expression,and synergistic effects were observed when combined rhBMP-2 with icariin.2.Construction of biomimetic icariin-CS/HA scaffoldsBiomimetic CS/HA scaffolds could be constructed by in situ hybridization and freeze-dried method.CS/HA composite had abundant homogeneous pores with the diameter（112.63±20.47）μm and porosity（88.65±2.34）%.HA parcels were distributed on the pore walls homogeneously with nanoscale（200～700nm）.The XRD and FTIR results showed that the HA crystals were carbonate-substituded and not well-crystallized.The cytocompatibility test showed that the seeded hBMSCs could adhere the scaffolds,and the proliferation ability was not effected by CS/HA composite and its leaching liquor.In addition,histocompability test found that tissue inflammatory reactions of CS/HA composite implanted were decreased significantly at 4 w,the composite was degraded mostly and was substituted by new tissue at 12 w.As we expected,icariin-CS/HA composite had abundant homogeneous pores with the diameter arround 110μm,which provided appropriate 3-demensional micro-structure for cells.Icariin loading did not change physical structure of CS/HA composite significantly,but decreased mechanical properties of CS/HA composite with higher dosage,10^-5and 10^-6mol icariin-CS/HA had lower fracture strength and elastic modulus;10^-5mol icariin-CA/HA has decreased elastic modulus（P＜0.05 vs blank CS/HA scaffolds）.icariin-CS/HA had favorable cell compatibility and promoted osteogenic differentiation of hBMSCs;hemolysis test and pyrogen test separately showed that icariin-CS/HA had food biocompatibility.3.Icariin release behavior of icariin-CS/HA scaffolds in vitroRSD of precision test and repeatability test was 0.636%and 3.245% respectively;average recovery rate of recovery test was 96.667%,RSD was 2.139%; the RSD of stability test was 1.286%.There was a good linear relationship between icariin dose（l～2000ng）and chromatographic peak area,and the regression equation was:Y=7877.3X+202422,R²=0.9976.Icariin releasing from scaffolds was calculated based on standard curve and demonstrated as the accumulated percentage of icariin.At day 1 to day 3,approximately 25%icariin was released;then the speed decreased and about 40%～60%icariin was released by 20 d.90 d later,there was still a certain amount of icariin remained in CS/HA scaffolds.Fitting equations of icariin release from CS/HA scaffolds were as follows:10^-7mol icariin-CS/HA scaffold:Y=6.267+13.468 ln（X）R²=0.901;10^-6mol icariin-CS/HA scaffold: Y=5.668+16.846 ln（X）R²=0.916;10^-5mol icariin-CS/HA scaffold:Y=6.322+18.466 ln（X）R²=0.923.And the icariin release behavior could be fitted with the first-order equation.4.Bone repair capability of icariin-CS/HA scaffolds in vivoThe self-repair ability of bone defect control was low,therefore medullary cavity at both ends of defect site was closed during 4～8 w,and the defect was obviously visible 12 weeks postoperatively.Investigated by ECT,a sensitive index of bone formation at early stage,osteogenesis in situ could be monitored by ^99mTc-MDP density.At 4 w,ROI values of three icariin-CS/HA groups(icariin dosages were 10^-7, 10^-6and 10^-5mol each)and CS/HA group were higher than the bone defect control group（P＜0.001）.Furthermore,ROIs of the 10^-6and 10^-5mol icariin-CS/HA groups were higher than that of the CS/HA group（P＜0.01）.At the same time,obvious bony callus at defect sites with icariin-CS/HA scaffolds could be seen by gross specimens observation and X-ray.Considering both ECT and X-ray results,icariin-CS/HA composites had osteoinduction functions at early stage.The X-ray examination thereafter showed a large amount of bony callus formed and the healing of defect at 8 w and bone marrow cavity reappeared at 12 w,in icariin-CS/HA groups.The bone mass density（BMD）values of three icariin-CS/HA groups and the CS/HA group were higher than the bone defect control group（P＜0.01）,and the 10^-6and 10^-5mol icariin-CS/HA groups were higher than CS/HA group（P＜0.01）.Histological observations at different intervals showed that after being implanted in defect site for 4 w,CS/HA composite were degraded partially with microporous structure lost and inflammatory cell infiltration.Scaffolds kept on degrading at 8 w,with newborn fibrous and cartilage tissues creeping along scaffolds.Residual CS/HA scaffolds were segmented and encapsulated by newborn tissues and bone defect site was substituted by cartilage and bone tissues at 12 w.As icariin dosage increased,the degradation speed of icariin-CS/HA composite was increased and the disintegration and fragmentation could be seen earlier at 4 weeks,and there were large amount of newborn cartilage around the scaffolds.At 8 w,scaffolds degraded mostly and some of the cartilage tissues transformed into bone tissues.And at 12 w,icariin-CS/HA scaffolds were degraded completely and cartilage tissues were substituted by bone tissues which arranged in disorder and small medullary cavities reformed in the center. CONCLUSION1.Icariin could promote proliferation and osteodifferentiation of hBMSCs, osteoinduction function of icariin,however,was inferior to that of rhBMP-2.2.Icariin could induce osteogenic genes and proteins expression,and had synergisitic effect with rhBMP-2.3.CS/HA scaffolds prepared by in situ hybridization and freeze-dried method had satisfactory porosity and biocompatibility.4.Icariin loading did not change physical structure of CS/HA composite significantly,but decreased mechanical properties of CS/HA composite with higher dosage.5.The controlled release of icariin from CS/HA scaffolds were satisfactory and the release retained after 90 d in vitro.And the icariin release behavior could be fitted with the first-order equation.6.Icariin-CS/HA scaffolds had favorable osteoconduction and osteoinduction in vivo,and could fill bone defect sites and stimulate newborn bone tissues formation at early stage.更多还原