仿生组装淫羊藿苷控释型骨修复材料的构建与应用基础研究
|
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(C_(33)H_(40)O_(15),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~(-9)M～2.0×10~(-4)M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~(-4)M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,2×10~7细胞/孔,贴壁后加入1.5ml浓度为10~(-9)M～10~(-4)M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,2×10~7细胞/孔,贴壁后加入DMSO培养基、10~(-6)M淫羊藿苷培养基、rhBMP-2培养基和10~(-6)M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,5×10~7细胞/皿,贴壁后加入4ml DMSO培养基、10~(-6)M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~(-5)mol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~(-5)mol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~(-5)mol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~(99m)Tc-MDP,3 h后置单光子核素扫描仪上检测骨缺损部位~(99m)Tc-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/cm~2)=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~(-4)M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~(-8)M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~(-8)M)不能促进hBMSCs的成骨分化,浓度过高时(＞10~(-5)M)则抑制了hBMSCs的成骨分化。浓度在10~(-8)～10~(-5)M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~(-5)M的淫羊藿苷可促进hBMSCs向成骨方向分化。各试验均以10~(-6)M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~(-6)M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CO_3~(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/m~3;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~(-6)mol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,R~2=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~(-7)mol淫羊藿苷-CS/HA复合材料:Y=6.267+13.468 ln(X) R~2=0.901;载10~(-6)mol淫羊藿苷-CS/HA复合材料:Y=5.668+16.846 ln(X)R~2=0.916;载10~(-5)mol淫羊藿苷-CS/HA复合材料:Y=6.322+18.466 ln(X)R~2=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~(-6)mol和10~(-5)mol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~(-6)mol和10~(-5)mol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。
BACKGROUND
Epimedium 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(C_(33)H_(40)O_(15),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.
OBJECTIVE
1.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.
METHODS
1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanism
Isolation 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~(-4)and 2×10~(-4)M 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~(-9)M～10~(-4)M) 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,2×10~7 cells/well,and were treated with icariin(10~(-9)～10~(-4)M),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×l0~7cells/well,and were treated with DMSO,10~(-6)M icariin,rhBMP-2 and 10~(-6)M 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 scaffolds
The 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~(-5)mol 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 vitro
Icariin-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 vivo
60 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.
RESULTS
1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanism
Phenotypic 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~(-4)M 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~(-8)M icariin could promote proliferation of hBMSCs,and osteo-induction(ALP activities)related with dose:low dose(＜10~(-8)M)icariin could not increase hBMSCs osteogenic differentiation,high dose(＞10~(-5)M),however,inhibited osteigenic differentiation. The concentration between 10~(-8)M and 10~(-5)M could promote OCN expression;ALP staining and Alizarin red S staining also indicated 10~(-8)～10~(-5)M icariin could accelerate osteodifferentiation of hBMSCs,and 10~(-6)M was the best concentration.On the whole,osteo-induction of icariin was not better than rhBMP-2.10~(-6)M 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 scaffolds
Biomimetic 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~(-5)and 10~(-6)mol icariin-CS/HA had lower fracture strength and elastic modulus;10~(-5)mol 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 vitro
RSD 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,R~2=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~(-7)mol icariin-CS/HA scaffold:Y=6.267+13.468 ln(X)R~2=0.901;10~(-6)mol icariin-CS/HA scaffold: Y=5.668+16.846 ln(X)R~2=0.916;10~(-5)mol icariin-CS/HA scaffold:Y=6.322+18.466 ln(X)R~2=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 vivo
The 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 ~(99m)Tc-MDP density.At 4 w,ROI values of three icariin-CS/HA groups(icariin dosages were 10~(-7), 10~(-6)and 10~(-5)mol each)and CS/HA group were higher than the bone defect control group(P＜0.001).Furthermore,ROIs of the 10~(-6)and 10~(-5)mol 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~(-6)and 10~(-5)mol 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.
CONCLUSION
1.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.
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(C_(33)H_(40)O_(15),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~(-9)M～2.0×10~(-4)M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~(-4)M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,2×10~7细胞/孔,贴壁后加入1.5ml浓度为10~(-9)M～10~(-4)M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,2×10~7细胞/孔,贴壁后加入DMSO培养基、10~(-6)M淫羊藿苷培养基、rhBMP-2培养基和10~(-6)M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,5×10~7细胞/皿,贴壁后加入4ml DMSO培养基、10~(-6)M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~(-5)mol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~(-5)mol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~(-5)mol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~(99m)Tc-MDP,3 h后置单光子核素扫描仪上检测骨缺损部位~(99m)Tc-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/cm~2)=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~(-4)M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~(-8)M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~(-8)M)不能促进hBMSCs的成骨分化,浓度过高时(＞10~(-5)M)则抑制了hBMSCs的成骨分化。浓度在10~(-8)～10~(-5)M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~(-5)M的淫羊藿苷可促进hBMSCs向成骨方向分化。各试验均以10~(-6)M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~(-6)M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CO_3~(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/m~3;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~(-6)mol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,R~2=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~(-7)mol淫羊藿苷-CS/HA复合材料:Y=6.267+13.468 ln(X) R~2=0.901;载10~(-6)mol淫羊藿苷-CS/HA复合材料:Y=5.668+16.846 ln(X)R~2=0.916;载10~(-5)mol淫羊藿苷-CS/HA复合材料:Y=6.322+18.466 ln(X)R~2=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~(-6)mol和10~(-5)mol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~(-6)mol和10~(-5)mol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。
BACKGROUND
Epimedium 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(C_(33)H_(40)O_(15),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.
OBJECTIVE
1.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.
METHODS
1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanism
Isolation 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~(-4)and 2×10~(-4)M 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~(-9)M～10~(-4)M) 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,2×10~7 cells/well,and were treated with icariin(10~(-9)～10~(-4)M),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×l0~7cells/well,and were treated with DMSO,10~(-6)M icariin,rhBMP-2 and 10~(-6)M 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 scaffolds
The 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~(-5)mol 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 vitro
Icariin-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 vivo
60 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.
RESULTS
1.Effects of icariin on osteogenic differentiation of hBMSCs and its mechanism
Phenotypic 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~(-4)M 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~(-8)M icariin could promote proliferation of hBMSCs,and osteo-induction(ALP activities)related with dose:low dose(＜10~(-8)M)icariin could not increase hBMSCs osteogenic differentiation,high dose(＞10~(-5)M),however,inhibited osteigenic differentiation. The concentration between 10~(-8)M and 10~(-5)M could promote OCN expression;ALP staining and Alizarin red S staining also indicated 10~(-8)～10~(-5)M icariin could accelerate osteodifferentiation of hBMSCs,and 10~(-6)M was the best concentration.On the whole,osteo-induction of icariin was not better than rhBMP-2.10~(-6)M 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 scaffolds
Biomimetic 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~(-5)and 10~(-6)mol icariin-CS/HA had lower fracture strength and elastic modulus;10~(-5)mol 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 vitro
RSD 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,R~2=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~(-7)mol icariin-CS/HA scaffold:Y=6.267+13.468 ln(X)R~2=0.901;10~(-6)mol icariin-CS/HA scaffold: Y=5.668+16.846 ln(X)R~2=0.916;10~(-5)mol icariin-CS/HA scaffold:Y=6.322+18.466 ln(X)R~2=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 vivo
The 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 ~(99m)Tc-MDP density.At 4 w,ROI values of three icariin-CS/HA groups(icariin dosages were 10~(-7), 10~(-6)and 10~(-5)mol each)and CS/HA group were higher than the bone defect control group(P＜0.001).Furthermore,ROIs of the 10~(-6)and 10~(-5)mol 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~(-6)and 10~(-5)mol 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.
CONCLUSION
1.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.
引文
[1].杨志明.加强再生医学研究,推动再生医学产业化[J].中国修复重建外科杂志.2009,23(2):129-30.
[2].王正国.再生医学——机遇与挑战[J].中华创伤杂志,2006,22(1):1-4.
[3].曹谊林.组织工程学理论与实践[M].科学技术出版社,上海,2004.3-7.
[4].Vacanti J P,Langer R,Schloo B,et al.Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation[J].Plast Reconstr Surg,1991,88(5):753-9.
[5].Cao Y L,Vacanti J P,Paige K T,et al.Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue engineered cartilage in the shape of a human ear[J].Plast Reconstr Surg,1997,100(2):297-302.
[6].张志雄,奚廷斐.冷静反思组织工程该如何发展[J].中国修复重建外科杂志.2009,23(2):131-5.
[7].曾宪立.血管化组织工程骨修复猕猴胫骨缺损的实验研究[M].南方医科大学博士学位论文,广州,南方医科大学,2007.1-3.
[8].孙明学,许文静,卢世璧.组织工程技术应用面临的问题[J].中国矫形外科杂志,2004,12(17):1285-6.
[9].[9]裴国献.面向21世纪的组织工程学研究趋势与策略[J].中华创伤骨科杂志,2004,6(7):721-3.
[10].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004,15-7.
[11].裴国献主译.洛克伍德-格林成人骨折[M].北京:人民军医出版社,2009,10.
[12].Niu X,Feng Q,Wang M,et al.Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2[J].J Control Release,2009,134(2):111-7.
[13].Chen FM,Zhao YM,Wu H,et al.Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-Ⅰ from dextran-co-gelatin microspheres[J].J Control Release,2006,114(2):209-22.
[14].Bhattarai N,Ramay HR,Gunn J,et al.PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release[J].J Control Release,2005,103(3):609-24.
[15].Cheng XM,Li YB,Zuo Y,et al.Properties and in vitro biological evaluation of nano-hydroxyapatite/chitosan membranes for bone guided regeneration[J].Materials Science and Engineering:C,2009,29:29-35.
[16].Zhang Y Venugopal JR,El-Turki A,et al.Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering[J].Biomaterials,2008,29:4314-22.
[17].Shi Z,Huang X,Cai Y,et al.Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells[J].Acta Biomater,2009,5:338-345.
[18].连芩,李涤尘,张永睿,等.三种仿生人工骨内部微细结构的对比研究[J].西安交通大学学报,2005,39(11):1211-4.
[19].王迎军,陈晓峰,赵娜如,等.纳米仿生骨组织材料的生理响应及生物矿化[J].华南理工大学学报(自然科学版),2002,30(11):149-54.
[20].Urist MR,Lietze A,Dawson E.Beta-tricalcium phosphate delivery system for bone morphogenetic protein[J].Clin Orthop Relat Res,1984,(187):277-80.
[21].Miyaji F.;Kim H.M.;Handa S.;et al.Bonelike apatite coating on organic polymers:novel nucleation process using sodium silicate solution[J].Biomaterials.1999,20:913-19.
[22].Markus A.;Michael B.;Christine G.;et al.Inorganic/organic mesostructures in the presence of double-hydrophilic block copolymers[J].Chemistry-A European Journal.1998,4[12]:2493-500.
[23].Grinnell F.;Feld M.K.Adsorption characteristics of plasma fibronectin in relationship to biological activity[J].J Biomed Mater Res,1981,15:363-81.
[24].Rieke PC,Tarasevich GJ,Bentjen SB,et al.Biomimetic Thin-film Synthesis[J].ACS Symposium Series.1992,499:61-75.
[25].Liu HW,Chen CH,Tsai CL,et al.Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation[J].Bone,2006,39(4):825-36.
[26].Suciati T,Howard D,Barry J,et al.Zonal release of proteins within tissue engineering scaffolds[J].J Mater Sci Mater Med,2006,17(11):1049-56.
[27].Ruhe PQ,Boerman OC,Russel FGM,et al.Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo[J].J Control Release,2005,106(1-2):162-71.
[28].崔建德,裴国献,金丹,等.带血管蒂筋膜瓣包裹组织工程骨修复兔桡骨缺损对降钙素基因相关肽和神经肽Y表达的影响[J].解放军医学杂志,2008,33(6):698-707.
[29].刘勇,裴国献,江汕,等.筋膜瓣包裹组织工程骨修复兔大段骨缺损的动态变化[J].中国组织工程研究与临床康复,12(24):4672-6.
[30].王秋实,裴国献,江汕,等.感觉、运动神经匀浆对兔成骨细胞增殖与成骨功能的影响[J].南方医科大学学报,2008,28(5):774-8.
[31].裴国献,金丹,陈滨,等.骨组织工程研究中的血管、神经化问题[J].中国创伤骨科杂志,2000,2(4):311-3.
[32].张元平,崔继秀,裴国献,等.神经化组织工程骨构建的初步观察[J].中华创伤骨科杂志,2005,7(1):60-5.
[33].戴金良,裴国献,刘勇,等.神经植入对大段组织工程骨成骨效果的一年观察[J].中华创伤骨科杂志,2008,10(4):354-8.
[34].Yang F,Murugan R,Ramakrishna S,Wang X,Ma Y X,Wang S.Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering[J].Biomaterials,2004,25:1891-900.
[35].Ramay H R,Zhang M.Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods[J].Biomaterials,2003,24:3293-302.
[36].Murugan R,Ramakrishna S.Bioresorbable composite bone paste using potysaccharide based nano hydroxyapatite[J].Biomaterials,2004,25:3829-35.
[37].Komori T.Regulation of osteoblast differentiation by transcription factors[J].J Cell Biochem,2006,99(5):1233-9.
[38].王和鸣,王力,李楠.巴戟天对骨髓基质细胞向成骨细胞分化过程Cbfal表达的影响[J].中国中医骨伤科杂志,2004,12(6):22-26.
[39].冯伟,傅文彧,魏义勇,等.单味中药对成骨相关基因表达的影响[J].中医正骨,2004,16(3):6-8.
[40].Kazuhisa Nakashima,Xin Zhou,Gary Kunkel,et al.The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J].Cell,2002,108,17-29.
[41].Wu L,Wu Y,Lin Y,et al.Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix[J].Mol Cell Biochem.2007.301(1-2):83-92.
[42].李淑梅,宋利格,张秀珍.淫羊藿甙对大鼠成骨细胞功能及Osterix表达的影响[J].同济大学学报(医学版),2005,26(6):8-11.
[43].孙奋勇,潘秋辉,洪岸.牛膝促进成骨细胞增殖的作用与机理研究[J].中药材,2004,27(4):264-6.
[44].孙奋勇,潘秋辉,洪岸.反义封闭PKAR Iβ基因表达对双龙接骨丸含药血清体外促成骨细胞增殖作用的影响[J].中国病理生理杂志,2004,20(12):2316-9.
[45].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J].Endocrinology,2007,148(4):1629-37.
[46].Widmann C.Gibson S,Jarpe MB,et al.Mitogen-activated protein kinase:conservation of a three——kinase module from yeast to human[J].PhysioI Rev,1999,79(1):143-80.
[47].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J1.Endocrinology,2007,148(4):1629-37.
[48].刘尚全,杨颖,周丽斌等.淫羊藿甙逆转地塞米松抑制成骨细胞分化及其机制[J].中华内分泌代谢杂志,2006,22(3):218-21.
[49].廖清船,肖洲生,秦艳芳等.植物雌激素金雀异黄酮通过p38MAPK通路促进骨髓间充质干细胞向成骨细胞分化[J].中国药理学通报,2006,22(6):683-7.
[50].Hsu YL,Chang JK,Tsai CH,et al.Myricetin induces human osteoblast differentiation through bone morphogenetic protein-2/p38 mitogen-activated protein kinase pathway[J]. Biochem Pharmacol,2007,73(4):504-14.
[51].Masahiro Kawabatal,Kohei Miyazono.Signal Transduction of the TGF-β Superfamily by Smad Proteins[J].J Biochem,1999,125:9-16.
[52].Li X,Cao X.BMP Signaling and Skeletogenesis[J].Ann N.Y.Acad Sci,2006,1068:26-40.
[53].Lee KS,Hong SH,Bae SC.Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta an d bone morphogeneticprotein[J].Oncogene,2002,21(47):7156-63.
[54].Yamamoto T,Saatcioglu,Matsuda T.Cross-talk between bone morphogenic proteins and estrogen receptor signaling[J].Endocrinology,2002,143(7):2635-42.
[55].雪原,王沛,齐清会等.淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究[J].中华骨科杂志,2005,25(2):119-23.
[56].雪原,齐清会,王沛等.淫羊藿甙对OVX大鼠成骨细胞Smad4 mRNA的作用[J].天津医药,2006,34(4):256-8.
[57].Pan W,Quarles LD,Song LH,et al.Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture[J].J Cell Biochem,2005,94(2):307-16.
[58].鞠大宏,赵宏艳,刘梅洁,等.左归丸含药血清对成骨细胞IL-1、IL-6和COX-2表达的影响[J].中国实验动物学报,2006,14(2):96-9.
[59].傅淑平,张荣华,徐立群.益骨胶囊含药血清在共育体系中对大鼠成骨细胞增殖、ALP活性及IL-11 mRNA表达的影响[J].中国病理生理杂志,2006,22(6):1171-3.
[60].Yuk TH,Kang JH,Lee SR,et al.Inhibitory effect of Carthamus tinctorius L seed extracts on bone resorption mediated by tyrosine kinase,COX-2(cyclooxygenase)and PG (prostaglandin)E2[J].Am J Chin Med,2002,30(1):95-108.
[61].[61]Jin UH,Kim DI,Lee TK,et al.Herbal formulation,Yukmi-jihang-tang-Jahage,regulates bone resorption by inhibition of phosphorylation mediated by tyrosine kinase Src and cyclooxygenase expression[J].J Ethnopharmacol,2006,106(3):333-43.
[62].Jeong JC,Lee JW,Yoon CH,et al.Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells[J].J Ethnopharmacol,2005,96(3):489-95.
[63].周丽珍,王淑丽,徐赫男等.复方仙贞汤抑制成骨细胞凋亡的实验研究[J].中国骨质疏松杂志,2004,10(4):451-5.
[64].陈虹,张秀珍.淫羊藿甙对大鼠成骨细胞分泌细胞因子的影响[J].同济大学学报(医学版),2005,26,(2):5-7.
[65].李万里,田玉慧,杨献军等.大豆异黄酮对成骨细胞转化生长因子-β_1及其受体Ⅰ、Ⅱ的影响[J].中医药学刊,2004,22(1):47-8.
[66].李楠,王和鸣,林旭,等.巴戟天对成骨细胞生物学特性影响的实验研究[J].中国医药学报,2004,19(12):726-8.
[67].程杰,王永清,彭锐等.生骨注射液对大鼠成骨细胞VEGF mRNA表达的影响[J].中国矫形外科杂志,2005,13(5):362-4.
[68].沈冯君,刘日光,杨述华等.活血补肾中药对培养成骨细胞VEGF活性的影响[J].中国骨伤,2004,17(5):260-2.
[69].Huh JE,Yang HR,Park DS,et al.Puerariae radix promotes differentiation and mineralization in human osteoblast-like SaOS-2 cells[J].J Ethnopharmacol,2006,104(3):345-50.
[70].程志安,吴燕峰,曾志勇等.健骨二仙丸对成骨细胞分化及IGF-I mRNA表达的影响[J].中国中医骨伤科杂志,2004,12(2):4-9.
[71].隋福革,张达,符强等.骨松康含药血清对成骨细胞生物学活性的影响[J].中国骨肿瘤骨病,2005,4(4):234-6.
[72].张荣华,朱晓峰,蔡宇等.益骨胶囊含药血清对大鼠成骨细胞IGF-ImRNA及其蛋白表达 的影响[J].中国病理生理杂志,2004,20(7):1222-5.
[73].Liu HC,Chen RM,Jian WC,et al.Cytotoxic and antioxidant effects of the water extract of the traditional Chinese herb gusuibu(Drynaria fortunei)on rat osteoblasts[J].J Formos Med Assoc.2001.100(6):383-8.
[74].张荣华,王廷春,朱晓峰等.益骨胶囊含药血清对SD大鼠成骨细胞分泌NO、NOS的影响[J].中药材,2004,27(8):593-6.
[75].肖辉,牛涛,齐锦生等.丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响[J].大然产物研究与开发,2005,17(6):754-7.
[76].Theoleyre S,Wittrant Y,Tat SK,et al.The molecular triad OPG/RANK/RANKL:involvement in the orchestration of pathophysiological bone remodeling[J].Cytokine Growth Factor Rev,2004,15(6):457-75.
[77].Wittrant Y,Theoleyre S,Chipoy C,et al.RANKL/RANK/OPG:new therapeutic targets in bone tumours and associated osteolysis[J].Biochim Biophys Acta,2004,1704(2):49-57.
[78].王运林,刘晓晴,夏秦等.金雀异黄素通过雌激素受体β增加人成骨细胞护骨素与破骨细胞分化因子表达比值[J].华中科技大学学报(医学版),2006,35(3):321-3.
[79].余增丽.金雀异黄酮对成骨细胞增殖的影响及其可能的分子生物学机制[J].中华老年医学杂志,2005,24(9):645-7.
[80].胡彬,吴翠环,陈璐璐.蛇床子素对大鼠成骨细胞中OPG和RANKL基因mRNA表达的影响[J].中国骨质疏松杂志,2004,10(4):415-9.
[81].吴乃中,李红丽,崔淑云.葛根对成骨细胞OPG、RANKL mRNA表达的影响[J].中华医学研究杂志,2006,6(1):6-9.
[82].唐井钢,李娟,吴贺勇,等.饲补肾中药大鼠血清对成骨细胞、破骨细胞共育系中破骨细胞功能的影响[J].白求恩军医学院学报,2006,4(2):68-9.
[83].吴丽萍,陶天遵,石义刚,等.三七总甙对成骨细胞增殖、分化及OPG表达影响的研究[J].中国骨质疏松杂志,2004,10(2):239-43.
[84].张秀珍,杨黎娟.淫羊藿甙对大鼠成骨细胞护骨素、RANKL表达的影响.中华内分泌代谢杂志[J],2006,22(3):222-5.
[85].刘钰瑜,崔燎,吴铁,等.大黄素对体外培养新生大鼠颅骨成骨细胞的影响[J].中国药理学通报,2005,21(2):235-40.
[86].杨亚军,杨中林,王冬春等.芦丁与槲皮素对成骨细胞代谢影响的比较研究[J].中药材,2006,29(5):467-70.
[87].Chen WF,Wong MS.Genistein modulates the effects of parathyroid hormone in human osteoblastic SaOS-2 cells[J].Br J Nutr,2006,95(6):1039-47.
[88].Lee KH,Choi EM.Biochanin A stimulates osteoblastic differentiation and inhibits hydrogen peroxide-induced production of inflammatory mediators in MC3T3-E1 cells[J].Biol Pharm Bull,2005,28(10):1948-53.
[89].屈树新,翁杰,沈如,等.含中药的磷酸钙生物材料制备方法的研究[J].生物医学工程与临床,2005,9(2):65-8.
[90].李茂红,屈树新,姚宁,等.含丹参磷酸钙骨水泥的制备和表征[J].生物医学工程与临床,2005,9(3):132-5.
[91].许春姣,翦新春,成洪泉,等.黄芪对兔骨髓基质细胞增殖和向成骨细胞分化的影响[J].中南大学学报(医学版),2004,29(4):489-91.
[92].许春姣,翦新春,彭解英,等.黄芪-聚乳酸/壳聚糖复合材料的体外细胞相容性实验研究[J].口腔医学研究,2005,21(2):142-6.
[93].许春姣,翦新春,彭解英,等.黄芪-壳聚糖/聚乳酸多孔支架对犬骨髓基质细胞生物学行为的影响[J].中南大学学报(医学版),2005,30(3):283-7.
[94].许春姣,郭峰,高清平,等.骨髓基质干细胞与黄芪—壳聚糖/聚乳酸支架对犬牙周 骨缺损再生的影响[J].中南大学学报(医学版),2006,31(4):512-7.
[95].邹琴,张利,左奕,等.载黄芪多糖骨水泥的理化性能及体外细胞相容性研究[J].功能材料,2008,39(9):1515-21.
[1].Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-7.
[2].Pountos I,Giannoudis PV.Biology ofmesenchymal stem cells[J].Injury,2005,36:S8-S12.
[3].Connolly JF.Injectable bone marrow preparations to stimulate osteogenic repair[J].Clin Orthop,1995,313:8-18.
[4].Aubin JE.Regulation of osteoblast formation and function[J].Rev Endocr Metab Disord,2001,2:81-94.
[5].Rollins BJ.Chemokines[J].Blood,1997,90(3):909-28.
[6].王亦璁.骨与关节损伤(第3版)[M].北京:人民卫生出版社,2001:150.
[7].胥少汀,葛宝丰,徐印坎.实用骨科学(第3版)[M].北京:人民军医出版社,2005:330-7.
[8].Zhou S,Greenberger JS,Epperly MW,et al.Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts[J].Aging Cell,2008,7:335-43.
[9].Seebach C,Henrich D,Tewksbury R,et al.Number and proliferative capacity of human mesenchymal stem cells are modulated positively in multiple trauma patients and negatively in atrophic nonunions[J].Calcif Tissue Int,2007,80:294-300.
[10].Hernigou P,Poignard A,Beaujean F,et al.Percutaneous autologous bone-marrow grafting for nonunions.Influence of the number and concentration of progenitor cells[J].J Bone Joint Surg Am,2005,87:1430-7.
[11].Siwach RC,Sangwan SS,Singh R,et al.Role of percutaneous bone marrow grafting in delayed unions,non-unions and poor regenerates[J].Indian J Med Sci,2001,55:326-36.
[12].Wilkins RM,Chimenti BT,Rifkin RM.Percutaneous treatment of long bone nonunions:the use of autologous bone marrow and allograft bone matrix[J].Orthopedics,2003,26:549-54.
[13].周志玲,袁进国,刘英飞,等、白体骨髓干细胞移植治疗骨不连[J].中国临床康复,2006,10(29):22-3.
[14].曹宜林主编,刘伟,崔磊副主编.组织工程学理论与实践[M].上海:上海科学技术出版社,2004:114.
[15].叶成,史鹏.骨髓问充质干细胞与成骨[J].中国矫形外科杂志,2008.16(5):356-8.
[16].Ippokratis P,Diane C,Paul E,et al.Mesenchymal stem cell tissue engineering:Techniques for isolation,expansion and application[J].Injury,2007,38S4:S23-S33.
[17].Wildemann B,Kadow-Romacker A,Haas NP,et al.Quantification of various growth factors in different demineralized bone matrix preparations[J].J Biomed Mater Res A,2006,81A (2):437-442
[18].Alt V,Heissel A.Economic considerations for the use of recombinant human bone morphogenetic protein-2 in open tibial fractures in Europe:the German model[J].Curr Med Res Opin 2006,22 Suppl 1:S19-22
[19].Hu ZM,Peel SA,Sandor GK,et al.The osteoinductive activity of bone morphogenetic protein(BMP)purified by repeated extracts of bovine bone[J].Growth Factors 2004,22(1):29-33
[20].Montesano R.Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth[J].Biochem Biophys Res Commun 2007,353(3):817-822
[21].Miriyala S,Gongora Nieto MC,Mingone C,et al.Bone morphogenic protein-4 induces hypertension in mice:role of noggin,vascular NADPH oxidases,and impaired vasorelaxation[J].Circulation 2006,113(24):2818-2825
[22].Deng H,Makizumi R,Ravikumar TS,et al.Bone morphogenetic protein-4 is over expressed in colonic adenocarcinomas and promotes migration and invasion of HCT116 cells[J].Exp Cell Res 2007,313(5):1033-1044
[23].Feldman GJ,Billings PC,Patel RV,et al.Over-expression of BMP4 and BMP5 in a child with axial skeletal malformations and heterotopic ossification:A new syndrome[J].Am J Med Genet A 2007,143(7):699-706
[24].Zhao J,Ohba S,Shinkai M,et al.Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner[J].Biochem Biophys Res Commun 2008,369:444-448
[25].Yin X X,Chen Z Q,Liu Z J,et al.Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2[J].Chin Med J (Engl)2007,120:204-210
[26].Qian G,Zhang X,Lu L,et al.Regulation of Cbfal expression by total flavonoids of Herba epimedii[J].Endocr J 2006,53:87-94
[27].Sheng H,Zhang G,Wang X L,et al.Phytochemical molecule icariin stimulates osteogenic but inhibits adipogenic differentiation of mesenchymal stem cells[J].Bone 2008(43):S42-S43
[28].Qin L,Han T,Zhang Q,et al.Antiosteoporotic chemical constituents from Er-Xian Decoction,a traditional Chinese herbal formula[J].J Ethnopharmacol,2008 118:271-279
[29].国家药典委员会.中华人民共和国药典一部[M].2005年版.北京:化学工业出版社,2005:229.
[30].Xu W,Zhang YP,Yang M,et al.LC-MS/MS method for the imultaneous determination of icariin and its major metabolites in rat plasma[J].Journal of Pharmaceutical and Biomedical Analysis,2007,45:667-72.
[31].刘铁汉,王毅,王本祥,等.淫羊藿苷的肠菌代谢研究Ⅰ[J].肠内细菌对淫羊藿苷的代谢转化.中草药,2000,31(11):834-837
[32].徐文,张亚萍,张卫东,等.离体大鼠肠道菌群对淫羊藿苷的代谢研究[J].世界科学技术,2006(6):98-100.
[33].Bao JR,Yang JW,Li SF,et al.Effects of Icariin on ovariectomized osteoporotic rats[J].Wei Sheng Yan Jiu,2005,34:191-3.
[34].Li L,Zhou QX,Shi JS.Protective effects of icariin on neurons injured by cerebral ischemia/reperfusion[J].Chin Med J(Engl),2005,118:1637-43.
[35].Wang YK,Huang ZQ.Protective effects of icariin on human umbilical vein endothelial cell injury induced by H2O2 in vitro[J].Pharmacol Res,2005,52:174-82.
[36].He W,Sun H,Yang B,et al.Immunoregulatory effects of the herba Epimediia glycoside icariin[J].Arzneimittelforschung 1995,45:910-3.
[37].Zhang ZB,Yang QT.The testosterone mimetic properties of icariin[J].Asian J Androl,2006,8(5):601-5.
[38].Pan Y,Kong L,Xia X,et al.Antidepressant-like effect of icariin and its possible mechanism in mice[J].Pharmacol Biochem Behav,2005,82:686-94.
[39].H.Y.Ye,Y.J.Lou.Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells[J].Phytomedicine,2005,12:735-741.
[40].张京伟,周云峰,文显梅,等.淫羊藿甙逆转胃癌细胞恶性表型的研究[J].中华实验外科杂志,2006,23(10):1213-4.
[41].Zheng XX,Hu YH,Liu JH,et al.Screening of active compounds as neuromedin U2 receptor agonist from natural products[J].Bioorg Med Chem Lett,2005,15:4531-5.
[42].Meng FH,Li YB,Xiong ZL,et al.Osteoblastic proliferative activity of Epimedium brevicornum Maxim[J].Phytomedicine,2005,12:189-93.
[43].Chen KM,Ma HP,Ge BF,et al.Icariin enhances the osteogenic differentiation of bone marrow stromal cells but has no effects on the differentiation of newborn alvarial osteoblasts of rats[J].Pharmazie,2007,62:785-9.
[44].Yin XX,Chen ZQ,Liu Z J,et al.Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2[J].Chin Med J (Engl),2007,120:204-10.
[45].Zhao J,Ohba S,Shinkai M,et al.Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner[J].Biochem Biophys Res Commun.2008,369:444-8.
[46].Xiao QB,Chen AM,Guo FJ.Effects of Icariin on expression of OPN mRNA and type I collagen in rat osteoblasts in vitro[J].J Huazhong Univ Sci Technolog Med Sci,2005,25,690-2.
[47].Zhang DW,Cheng Y,Wang NL,et al.Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts[J].Phytomedicine,2008,15:55-61.
[48].谢玲玲,邹移海,刘炫斯.淫羊藿苷对骨髓问质干细胞增殖及分泌血管内皮生长因子、碱性成纤维生长因子的影响[J].实用医学杂志,2008,24(6):908-10.
[49].张秀珍,杨黎娟.淫羊藿甙对大鼠成骨细胞护骨素、RANKL表达的影响[J].中华内分泌代谢杂志,2006,22(3):222-5.
[50].雪原,王沛,齐清会,等.淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究[J].中华骨科杂志,2005,25(2):119-23.
[51].Santos,NC,Figueira-Coelho J,Martins-Silva J.et al.Multidisciplinary utilization of dimethyl sulfoxide:pharmacological,cellular,and molecular aspects[J].Biochem.Pharmacol,2003,65,1035-41.
[52].Xing L,Remick DG.Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production[J].J Immunol,2005,174:6195-202.
[53].Melchior D,Packer CS,Johnson TC,et al.Dimethyl sulfoxide:does it change the functional properties of the bladder wall?[J].J Urol,2003,170:253-8.
[54].[54]Cheung WMW,Ng WW,Kung AWC.Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-El cells[J].FEBS Letters.2006.580:121-6.
[55].陈其昕,袁中兴,刘清,等.骨折愈合中修复性细胞增殖与骨形成的关系[J].中华骨科杂志,1992,12(4):290-2.
[56].刘大鹏,艾合麦提,古丽,等.体外培养骨髓基质细胞增殖和分化的相互关系[J].中国临床康复,2005,9(38):9-12.
[57].Javazon EH,Colter DC,Schwarz EJ,et al.Rat Marrow Stromal Cells are More Sensitive to Plating Density and Expand More Rapidly from Single-Cell-Derived Colonies than Human Marrow Stromal Cells[J].STEM CELLS,2001,19:219-25.
[58].Peister A,Mellad J A,Larson B L,et al.Adult stem cells from bone marrow(MSCs)isolated from different strains of inbred mice vary in surface epitopes,rates of proliferation,and differentiation potential[J].Blood,2004,103(5):1662-8.
[59].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004,36-41.
[60].Schmitt B,Ringe J,H(a|¨)upl T,et al.BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture[J].Differentiation,2003,71(9-10):567-77.
[61].唐林,林珠,李永明,等.不同大小机械牵张力对成骨细胞增殖及碱性磷酸酶的影响[J]解放军医学杂志,2006,31(6):580-1.
[62].叶凌,李江宁,李联,等.骨钙素在体内的表达[J].国外医学口腔医学分册,2002,29(4):228-9.
[63].Komori T.Regulation of osteoblast differentiation by transcription factors[J].J Cell Biochem.2006,99(5):1233-9.
[64].Komori T.Regulation of skeletal development by the Runx family of transcription factors[J].J Cell Biochem,2005,95(3):445-53.
[65].Ducy P,Zhang R,Geoffroy V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation[J].Cell,1997,89:747-54.
[66].Wai PY,Mi Z,Gao C,et al.Ets-1 and runx2 regulate transcription of a metastatic gene,osteopontin,in murine colorectal cancer cells[J].J Biol Chem,2006,281(28):18973-82.
[67].Hernan Roca,Mattabhorn Phimphilai,Rajaram Gopalakrishnan,et al.Cooperative Interactions between RUNX2 and Homeodomain Protein-binding Sites Are Critical for the Osteoblast-specific Expression of the Bone Sialoprotein Gene[J].J Biol Chem,2005,280(35):30845-55.
[68].Kazuhisa Nakashima,Xin Zhou,Gary Kunkel,et al.The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J].Cell,2002,108:17-29.
[69].Wu L.Wu Y,Lin Y,et al.Osteogenic differentiation of adipose derived stem cells promoted by overexpression ofosterix[J].Mol Cell Biochem,2007,301(1-2):83-92.
[70].Nicolas H,St(?)phanie M,Martine B,et al.Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme[J].Developmental Biology,2007,304:860-74.
[71].LI X,CAO X.BMP Signaling and Skeletogenesis[J].Ann N.Y.Acad Sci,2006,1068:26-40.
[72].Widmann C.Gibson S,Jarpe MB,et al.Mitogen-activated protein kinase:conservation of a three-kinase module from yeast to human[J].PhysioI Rev,1999,79(1):143-80.
[73].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J].Endocrinology.2007.148(4):1629-37.
[74].王茸影,易静.骨形成蛋白调控成骨分化的信号机制[J].生命科学,2005,17(1):34-9.
[75].Cheng HW,Jiang W,Phillips FM,etal.Ostcogcnicactivity ofthe fourteen types of human bone morphogenetic proteins(BMPs)[J].J Bone Joint Surg Am,2003,85:1544-52.
[76].Li SH,Guo DZ,Li B,et al.The stimulatory effect of insulin-like growth factor-1 on the proliferation,differentiation,and mineralisation of osteoblastic cells from Holstein cattle[J].J Veterinary,2009,179:430-6.
[77].[77]Koch H,Jadlowiec JA,Campbell PG.Insulin-like growth factor-Ⅰ induces early osteoblast gene expression in human mesenchymal stem cells[J].Stem Cells Dev,2005,14(6):621-31.
[78].Alliston T.TGF-beta regulation of osteoblast differentiation and bone matrix properties[J].J Musculoskelet Neuronal Interact,2006,6(4):349-50.
[79].Bodo M,Venti G,Pezzetti F,et al.Interleukin-1 alpha:regulation of cellular proliferation and collagen synthesis in cultured human osteoblast-like cells[J].Cell Mol Biol (Noisy-le-grand),1992,38(5-6):679-86.
[80].Nakayama Y,Kato N,Nakajima Y,et al.Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2osteoblast-like cells[J].Cell Biol Int,2004,28(10):653-60.
[81].Mukai T,Otsuka F,Otani H,et al.TNF-alpha inhibits BMP-induced osteoblast differentiation through activating SAPK/JNK signaling[J].Biochem Biophys Res Commun,2007,356(4):1004-10.
[82].李淑梅,宋利格,张秀珍.淫羊藿甙对大鼠成骨细胞功能及Osterix表达的影响[J].同济大学学报(医学版),2005,26(6):8-11.
[83].刘尚全,杨颖,周丽斌等.淫羊藿甙逆转地塞米松抑制成骨细胞分化及其机制[J].中华内分泌代谢杂志,2006,22(3):218-21.
[84].雪原,齐清会,王沛等.淫羊藿甙对OVX大鼠成骨细胞Smad4 mRNA的作用[J].天津医药,2006,34(4):256-58.
[85].陈虹,张秀珍.淫羊藿甙对大鼠成骨细胞分泌细胞因子的影响[J].同济大学学报(医学版),2005,26,(2):5-7.
[86].Theoleyre S,Wittrant Y,Tat SK,et al.The molecular triad OPG/RANK/RANKL:involvement in the orchestration of pathophysiological bone remodeling[J].Cytokine Growth Factor Rev,2004,15(6):457-75.
[87].Chen KM,Ge BF,Liu XY,et al.Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture[J].Pharmazie,2007,62(5):388-91.
[88].Wang ZQ,Lou YJ.Proliferation-stimulating effects of icaritin and desmethylicaritin in MCF-7 cells[J].Eur J Pharmacol.2004;504(3):147-53.
[89].McCarthy TL,Clough ME,Gundberg CM,et al.Expression of an estrogen receptor agonist in differentiating osteoblast cultures[J].Proc Natl Acad Sci U S A,2008,105(19):7022-7.
[90].Zhang G,Qin L,Sheng H,et al.Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition[J].Bone,2007,40(3):685-92.
[91].Orciani M,Trubiani O,Vignini A,et al.Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells[J].Acta Histochem,2009,111(1):15-24.
[92].Hitoshi K,Pascale VG,Chan J,et al.Osterix Induces Osteogenic Gene Expression but not Differentiation in Primary Human Fetal Mesenchymal Stem Cells.Tissue Engineering,2007,13(7):1513-23.
[93].Morsczeck C.Gene expression of runx2,Osterix,c-fos,DLX-3,DLX-5,and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.Calcif Tissue,2006,78(2):98-102.
[94].Zhou Y,Chen F,Ho ST,et al.Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials,2007,28(5):814-24.
[95].Yang S,Zhong C,Frenkel B,et al.Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells.Cancer Res,2005,65(13):5769-77.
[96].Sano H,Hosokawa K,Kidoya H,et al.Negative regulation of VEGF-induced vascular leakage by blockade of angiotensin Ⅱ type 1 receptor.Arterioscler Thromb Vasc Biol,2006,26(12):2673-80.
[1].缪旭东,裴国献,黎健伟,等.脱蛋白骨复合纤维蛋白胶构建骨组织工程支架的体外实验研究[J].中国矫形外科杂志,2006,14(18):1413-1414.
[2].Ruksudjarit A,Pengpat K,Rujijanagul G,et al.Synthesis and characterization of nanocrystalline hydroxyapatite from natural bovine bone[J].Current Applied Physics,2008,8(3-4):270-272.
[3].Liu CZ,Han ZW,Czernuszka JT.Gradient collagen/nanohydroxyapatite composite scaffold:Development and characterization[J].Acta Biomaterialia,2009,5(2):661-669.
[4].Zhang PB,Hong ZK,Yu T,et al.In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide)and hydroxyapatite surface-grafted with poly(l-lactide)[J].Biomaterials,2009,30(1):58-70.
[5].Nejati E,Mirzadeh H,Zandi M.Synthesis and characterization of nano-hydroxyapatite rods/poly(l-lactide acid)composite scaffolds for bone tissue engineering[J].Applied Science and Manufacturing,2008,39(10):1589-1596.
[6].Hu Q,Li B,Wang M,et al.Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization:a potential material as internal fixation of bone fracture[J].Biomaterials,2004,25(5):779-785.
[7].南开辉.仿生组装淫羊藿苷控释型骨组织工程支架及其对骨髓基质干细胞生物学行为干预研究[M].南方医科大学博士后出站报告.广州:南方医科大学,2007.30-56
[8].赵丽东,张华峰,董静洲.等.淫羊藿苷的超声波强化提取及其稳定性初探[J].西北农业学报.2007,16(6):285-288.
[9].刘铁汉,王毅,王本祥,等.淫羊藿苷的肠菌代谢研究Ⅰ.肠内细菌对淫羊藿苷的代谢转化[J].中草药,2000,31(11):834-837.
[10].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004.36-41.
[11].刘大鹏,艾合麦提,古丽,等.体外培养骨髓基质细胞增殖和分化的相互关系[J].中国临床康复,2005,9(38):9-12.
[12].国家药典委员会.中华人民共和国药典二部[M].2005年版.北京:化学工业出版社,2005:附录85.
[1].Enescu D,Hamciuc V,Ardeleanu R,et al.Polydimethylsiloxane modified chitosan.Part Ⅲ:Preparation and characterization of hybrid membranes[J].Carbohydrate Polymers,2009,76:268-278.
[2].Berrada M,Serreqi A,Dabbarh F,et al.A novel non-toxic camptothecin formulation for cancer chemotherapy[J].Biomaterials,2005,26(14):2115-2120.
[3].Park JH,Cho YW,Son YJ,et al.Preparation and characterization of self-assembled nanoparticles based on glycol chitosan bearing adriamycin[J].Colloid Polymer Sci,adriamycin,2006,284(7):763-770.
[4].郑彩虹,梁文权,虞和永.海藻酸-壳聚糖-聚乳酸羟乙醇酸复合微球的制备及其对蛋白释放的调节[J].药学学报.2005.40(2):182-186.
[5].Sonaje K,Lin YH,Juang JH,et al.In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery[J].Biomaterials,2009,30(12):2329-2339.
[6].Bhattarai N,Ramay HR,Gunn J,et al.PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release[J].J Control Release,2005,103(3):609-624.
[7].张维颖,樊东辉,徐政,等.温敏性壳聚糖凝胶的阿霉素药物体外释放研究[J].中国海洋药物,2005,24(5):50-53.
[8].Rodrigues LB,Leite HF,Yoshida MI,et al.In vitro release and characterization of chitosan films as dexamethasone cartier[J].Int J Pharm,2009,368(1-2):1-6.
[9].滕利荣,王博,刘艳,等.羟基磷灰石纳米粒子作为蛋白类缓释药物载体的应用[J].吉林大学学报(工学版),2007,37(5):1093-1096.
[10].Zhou H,Qian J,Wang J,et al.Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N,6-O-sulfated chitosan in vitro and in vivo[J].Biomaterials,2009,30(9):1715-1724.
[11].Niu X,Feng Q,Wang M,et al.Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2[J].J Control Release,2009,134(2):111-117.
[12].Tan H,Chu CR,Payne KA,et al.Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering[J].Biomaterials,2009,30(13):2499-2506.
[13].Michalska M,Kozakiewicz M,Bodek KH.Polymer angiogenic factor carrier.Part Ⅰ.Chitosan-alginate membrane as carrier PDGF-AB and TGF-beta[J].Polim Med,2008,38(4):19-28.
[14].Abarrategi A,Moreno-Vicente C,Ramos V,et al.Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application[J].Tissue Eng Part A,2008,14(8):1305-1319.
[15].Cai DZ,Zeng C,Quan DP,et al.Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-betal delivery for cartilage tissue engineering[J].Chin Med J(Engl),2007,120(3):197-203.
[16].南开辉.仿生组装淫羊藿苷控释型骨组织工程支架及其对骨髓基质干细胞生物学行为干预研究.南方医科大学博士后出站报告[M].广州:南方医科大学,2007.80-89.
[17].罗华丽,鲁在君.壳聚糖作为药物载体的缓释机理的研究进展[J].大中学刊,2006,21(2):38-40.
[18].[18]屠锡德,张钧寿,朱家璧.药剂学(第三版)[M].北京:人民卫生出版社,2002.附录ⅩⅨ D.
[1].Kondo N,Ogose A,Tokunaga K,et al.Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation[J].Biomaterials,2006,27(25):4419-4427.
[2].Duan YR,Zhang ZR,Wang CY,et al.Dynamic study of calcium phosphate formation on porous HA/TCP ceramics[J].J Mater Sci Mater Med,2005,16(9):795-801.
[3].Hoshino M,Egi T,Terai H,et al.Repair of long intercalated rib defects using porous beta-tricalcium phosphate cylinders containing recombinant human bone morphogenetic protein-2 in dogs[J].Biomaterials,2006,27(28):4934-4940.
[4].Kamakura S,Nakajo S,Suzuki O,et al.New scaffold for recombinant human bone morphogenetic protein-2 [J].J Biomed Mater Res A,2004,71(2):299-307.
[5].Seeherman HJ,Bouxsein M,Kim H,et al.Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model [J].J Bone Joint Surg Am,2004,86-A(9):1961-1972.
[6].Liu HW,Chen CH,Tsai CL,et al.Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation [J].Bone, 2006,39(4):825-836.
[7].Hsieh CY,Hsieh HJ,Liu HC,et al.Fabrication and release behavior of a novel freeze-gelled chitosan/gamma-PGA scaffold as a carrier for rhBMP-2 [J].Dent Mater,2006,22(7):622-629.
[8].Suciati T,Howard D,Barry J,et al.Zonal release of proteins within tissue engineering scaffolds [J].J Mater Sci Mater Med,2006,17(11):1049-1056.
[9].Keskin DS, Tezcaner A, Korkusuz P, et al.Collagen-chondroitin sulfate-based PLLA-SAIB-coated rhBMP-2 delivery system for bone repair [J].Biomaterials,2005,26(18):4023-4034.
[10].Rai B,Teoh SH,Hutmacher DW,et al.Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2 [J].Biomaterials,2005,26(17):3739-3748.
[11].Ruhe PQ,Boerman OC,Russel FGM,et al.Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo [J].J Control Release,2005,106 (1-2):162-171.
[12].Lee YM,Nam SH,Seol YJ,et al.Enhanced bone augmentation by controlled release of recombinant human bone morphogenetic protein-2 from bioabsorbable membranes [J].J Periodontol,2003,74(6):865-872.
[13].Chu TM,Warden SJ,Turner CH,et al.Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2 [J].Biomaterials,2007,28(3):459-467.
[14].Yoneda M,Terai H,Imai Y,et al.Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant [J].Biomaterials,2005,26 (25):5145-5152.
[15].Chen B,Lina H,Wang JH,et al.Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2 [J].Biomaterials,2007,28 (6):1027-1035.
[16].Santana RB,Trackman PC.Controlled release of fibroblast growth factor 2 stimulates bone healing in an animal model of diabetes mellitus [J].Int J Oral Maxillofac Implants.2006,21(5):711-718.
[17].Kleinheinz J,Stratmann U,Joos U,et al.VEGF-activated angiogenesis during bone regeneration [J].J Oral Maxillofac Surg,2005,63 (9):1310-1316.
[18].Lee JY,Nam SH,Im SY,et al.Enhanced bone formation by controlled growth factor delivery from chitosan-based biomaterials [J].J Control Release,2002,78 (1-3):187-197.
[19].Raiche AT,Puleo DA.In vitro effects of combined and sequential delivery of two bone growth factors [J].Biomaterials,2004,25(4):677-685.
[20].Takahashi Y,Yamamoto M,Tabata Y Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate [J].Biomaterials,2005,26(23):4856-4865.
[21].Ueda H,Nakamura T,Yamamoto M,et al.Repairing of rabbit skull defect by dehydrothermally crosslinked collagen sponges incorporating transforming growth factor β_1 [J].J Control Release,2003,88(1):55-64.
[22].Chen FM,Zhao YM,Wu H,et al.Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-Ⅰ from dextran-co-gelatin microspheres [J].J Control Release,2006,114(2):209-222.
[23].Wei G,Jin Q,Giannobile WV,et al.The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres [J].Biomaterials,2007,28(12):2087-2096.
[24].Lu SS,Zhang X,Soo C,et al.The osteoinductive properties of Nell-1 in a rat spinal fusion model.Spine J,2007,7(1):50-60.
[25].Hosseinkhani H,Hosseinkhani M,Khademhosseini A,et al.Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold [J].J Control Release,2007,117(3):380-386.
[26].Lutolf MP,Lauer-Fields JL,Schmoekel HG,et al.Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration:engineering cell-invasion characteristics[J].Proc Natl Acad Sci U S A,2003,100(9):5413-5418.
[27].Chen FM,Zhao YM,Sun HH,et al.Novel glycidyl methacrylated dextran (Dex-GMA)/gelatin hydrogel scaffolds containing microspheres loaded with bone morphogenetic proteins:Formulation and characteristics[J].J Control Release,2007,118(1):65-77.
[28].Yuan H,Kurashina K,de Bruijn JD,et al.A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics[J].Biomaterials,1999,20(19):1799-1806.
[29].Habibovic P,Yuan H,van der Valk CM,et al.3D microenvironment as essential element for osteoinduction by biomaterials[J].Biomaterials,2005,26(17):3565-7355.
[30].Kondo N,Ogose A,Tokunaga K,et al.Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation[J].Biomaterials,2006,27(25):4419-4427.
[2].王正国.再生医学——机遇与挑战[J].中华创伤杂志,2006,22(1):1-4.
[3].曹谊林.组织工程学理论与实践[M].科学技术出版社,上海,2004.3-7.
[4].Vacanti J P,Langer R,Schloo B,et al.Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation[J].Plast Reconstr Surg,1991,88(5):753-9.
[5].Cao Y L,Vacanti J P,Paige K T,et al.Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue engineered cartilage in the shape of a human ear[J].Plast Reconstr Surg,1997,100(2):297-302.
[6].张志雄,奚廷斐.冷静反思组织工程该如何发展[J].中国修复重建外科杂志.2009,23(2):131-5.
[7].曾宪立.血管化组织工程骨修复猕猴胫骨缺损的实验研究[M].南方医科大学博士学位论文,广州,南方医科大学,2007.1-3.
[8].孙明学,许文静,卢世璧.组织工程技术应用面临的问题[J].中国矫形外科杂志,2004,12(17):1285-6.
[9].[9]裴国献.面向21世纪的组织工程学研究趋势与策略[J].中华创伤骨科杂志,2004,6(7):721-3.
[10].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004,15-7.
[11].裴国献主译.洛克伍德-格林成人骨折[M].北京:人民军医出版社,2009,10.
[12].Niu X,Feng Q,Wang M,et al.Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2[J].J Control Release,2009,134(2):111-7.
[13].Chen FM,Zhao YM,Wu H,et al.Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-Ⅰ from dextran-co-gelatin microspheres[J].J Control Release,2006,114(2):209-22.
[14].Bhattarai N,Ramay HR,Gunn J,et al.PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release[J].J Control Release,2005,103(3):609-24.
[15].Cheng XM,Li YB,Zuo Y,et al.Properties and in vitro biological evaluation of nano-hydroxyapatite/chitosan membranes for bone guided regeneration[J].Materials Science and Engineering:C,2009,29:29-35.
[16].Zhang Y Venugopal JR,El-Turki A,et al.Electrospun biomimetic nanocomposite nanofibers of hydroxyapatite/chitosan for bone tissue engineering[J].Biomaterials,2008,29:4314-22.
[17].Shi Z,Huang X,Cai Y,et al.Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells[J].Acta Biomater,2009,5:338-345.
[18].连芩,李涤尘,张永睿,等.三种仿生人工骨内部微细结构的对比研究[J].西安交通大学学报,2005,39(11):1211-4.
[19].王迎军,陈晓峰,赵娜如,等.纳米仿生骨组织材料的生理响应及生物矿化[J].华南理工大学学报(自然科学版),2002,30(11):149-54.
[20].Urist MR,Lietze A,Dawson E.Beta-tricalcium phosphate delivery system for bone morphogenetic protein[J].Clin Orthop Relat Res,1984,(187):277-80.
[21].Miyaji F.;Kim H.M.;Handa S.;et al.Bonelike apatite coating on organic polymers:novel nucleation process using sodium silicate solution[J].Biomaterials.1999,20:913-19.
[22].Markus A.;Michael B.;Christine G.;et al.Inorganic/organic mesostructures in the presence of double-hydrophilic block copolymers[J].Chemistry-A European Journal.1998,4[12]:2493-500.
[23].Grinnell F.;Feld M.K.Adsorption characteristics of plasma fibronectin in relationship to biological activity[J].J Biomed Mater Res,1981,15:363-81.
[24].Rieke PC,Tarasevich GJ,Bentjen SB,et al.Biomimetic Thin-film Synthesis[J].ACS Symposium Series.1992,499:61-75.
[25].Liu HW,Chen CH,Tsai CL,et al.Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation[J].Bone,2006,39(4):825-36.
[26].Suciati T,Howard D,Barry J,et al.Zonal release of proteins within tissue engineering scaffolds[J].J Mater Sci Mater Med,2006,17(11):1049-56.
[27].Ruhe PQ,Boerman OC,Russel FGM,et al.Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo[J].J Control Release,2005,106(1-2):162-71.
[28].崔建德,裴国献,金丹,等.带血管蒂筋膜瓣包裹组织工程骨修复兔桡骨缺损对降钙素基因相关肽和神经肽Y表达的影响[J].解放军医学杂志,2008,33(6):698-707.
[29].刘勇,裴国献,江汕,等.筋膜瓣包裹组织工程骨修复兔大段骨缺损的动态变化[J].中国组织工程研究与临床康复,12(24):4672-6.
[30].王秋实,裴国献,江汕,等.感觉、运动神经匀浆对兔成骨细胞增殖与成骨功能的影响[J].南方医科大学学报,2008,28(5):774-8.
[31].裴国献,金丹,陈滨,等.骨组织工程研究中的血管、神经化问题[J].中国创伤骨科杂志,2000,2(4):311-3.
[32].张元平,崔继秀,裴国献,等.神经化组织工程骨构建的初步观察[J].中华创伤骨科杂志,2005,7(1):60-5.
[33].戴金良,裴国献,刘勇,等.神经植入对大段组织工程骨成骨效果的一年观察[J].中华创伤骨科杂志,2008,10(4):354-8.
[34].Yang F,Murugan R,Ramakrishna S,Wang X,Ma Y X,Wang S.Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering[J].Biomaterials,2004,25:1891-900.
[35].Ramay H R,Zhang M.Preparation of porous hydroxyapatite scaffolds by combination of the gel-casting and polymer sponge methods[J].Biomaterials,2003,24:3293-302.
[36].Murugan R,Ramakrishna S.Bioresorbable composite bone paste using potysaccharide based nano hydroxyapatite[J].Biomaterials,2004,25:3829-35.
[37].Komori T.Regulation of osteoblast differentiation by transcription factors[J].J Cell Biochem,2006,99(5):1233-9.
[38].王和鸣,王力,李楠.巴戟天对骨髓基质细胞向成骨细胞分化过程Cbfal表达的影响[J].中国中医骨伤科杂志,2004,12(6):22-26.
[39].冯伟,傅文彧,魏义勇,等.单味中药对成骨相关基因表达的影响[J].中医正骨,2004,16(3):6-8.
[40].Kazuhisa Nakashima,Xin Zhou,Gary Kunkel,et al.The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J].Cell,2002,108,17-29.
[41].Wu L,Wu Y,Lin Y,et al.Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix[J].Mol Cell Biochem.2007.301(1-2):83-92.
[42].李淑梅,宋利格,张秀珍.淫羊藿甙对大鼠成骨细胞功能及Osterix表达的影响[J].同济大学学报(医学版),2005,26(6):8-11.
[43].孙奋勇,潘秋辉,洪岸.牛膝促进成骨细胞增殖的作用与机理研究[J].中药材,2004,27(4):264-6.
[44].孙奋勇,潘秋辉,洪岸.反义封闭PKAR Iβ基因表达对双龙接骨丸含药血清体外促成骨细胞增殖作用的影响[J].中国病理生理杂志,2004,20(12):2316-9.
[45].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J].Endocrinology,2007,148(4):1629-37.
[46].Widmann C.Gibson S,Jarpe MB,et al.Mitogen-activated protein kinase:conservation of a three——kinase module from yeast to human[J].PhysioI Rev,1999,79(1):143-80.
[47].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J1.Endocrinology,2007,148(4):1629-37.
[48].刘尚全,杨颖,周丽斌等.淫羊藿甙逆转地塞米松抑制成骨细胞分化及其机制[J].中华内分泌代谢杂志,2006,22(3):218-21.
[49].廖清船,肖洲生,秦艳芳等.植物雌激素金雀异黄酮通过p38MAPK通路促进骨髓间充质干细胞向成骨细胞分化[J].中国药理学通报,2006,22(6):683-7.
[50].Hsu YL,Chang JK,Tsai CH,et al.Myricetin induces human osteoblast differentiation through bone morphogenetic protein-2/p38 mitogen-activated protein kinase pathway[J]. Biochem Pharmacol,2007,73(4):504-14.
[51].Masahiro Kawabatal,Kohei Miyazono.Signal Transduction of the TGF-β Superfamily by Smad Proteins[J].J Biochem,1999,125:9-16.
[52].Li X,Cao X.BMP Signaling and Skeletogenesis[J].Ann N.Y.Acad Sci,2006,1068:26-40.
[53].Lee KS,Hong SH,Bae SC.Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta an d bone morphogeneticprotein[J].Oncogene,2002,21(47):7156-63.
[54].Yamamoto T,Saatcioglu,Matsuda T.Cross-talk between bone morphogenic proteins and estrogen receptor signaling[J].Endocrinology,2002,143(7):2635-42.
[55].雪原,王沛,齐清会等.淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究[J].中华骨科杂志,2005,25(2):119-23.
[56].雪原,齐清会,王沛等.淫羊藿甙对OVX大鼠成骨细胞Smad4 mRNA的作用[J].天津医药,2006,34(4):256-8.
[57].Pan W,Quarles LD,Song LH,et al.Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture[J].J Cell Biochem,2005,94(2):307-16.
[58].鞠大宏,赵宏艳,刘梅洁,等.左归丸含药血清对成骨细胞IL-1、IL-6和COX-2表达的影响[J].中国实验动物学报,2006,14(2):96-9.
[59].傅淑平,张荣华,徐立群.益骨胶囊含药血清在共育体系中对大鼠成骨细胞增殖、ALP活性及IL-11 mRNA表达的影响[J].中国病理生理杂志,2006,22(6):1171-3.
[60].Yuk TH,Kang JH,Lee SR,et al.Inhibitory effect of Carthamus tinctorius L seed extracts on bone resorption mediated by tyrosine kinase,COX-2(cyclooxygenase)and PG (prostaglandin)E2[J].Am J Chin Med,2002,30(1):95-108.
[61].[61]Jin UH,Kim DI,Lee TK,et al.Herbal formulation,Yukmi-jihang-tang-Jahage,regulates bone resorption by inhibition of phosphorylation mediated by tyrosine kinase Src and cyclooxygenase expression[J].J Ethnopharmacol,2006,106(3):333-43.
[62].Jeong JC,Lee JW,Yoon CH,et al.Stimulative effects of Drynariae Rhizoma extracts on the proliferation and differentiation of osteoblastic MC3T3-E1 cells[J].J Ethnopharmacol,2005,96(3):489-95.
[63].周丽珍,王淑丽,徐赫男等.复方仙贞汤抑制成骨细胞凋亡的实验研究[J].中国骨质疏松杂志,2004,10(4):451-5.
[64].陈虹,张秀珍.淫羊藿甙对大鼠成骨细胞分泌细胞因子的影响[J].同济大学学报(医学版),2005,26,(2):5-7.
[65].李万里,田玉慧,杨献军等.大豆异黄酮对成骨细胞转化生长因子-β_1及其受体Ⅰ、Ⅱ的影响[J].中医药学刊,2004,22(1):47-8.
[66].李楠,王和鸣,林旭,等.巴戟天对成骨细胞生物学特性影响的实验研究[J].中国医药学报,2004,19(12):726-8.
[67].程杰,王永清,彭锐等.生骨注射液对大鼠成骨细胞VEGF mRNA表达的影响[J].中国矫形外科杂志,2005,13(5):362-4.
[68].沈冯君,刘日光,杨述华等.活血补肾中药对培养成骨细胞VEGF活性的影响[J].中国骨伤,2004,17(5):260-2.
[69].Huh JE,Yang HR,Park DS,et al.Puerariae radix promotes differentiation and mineralization in human osteoblast-like SaOS-2 cells[J].J Ethnopharmacol,2006,104(3):345-50.
[70].程志安,吴燕峰,曾志勇等.健骨二仙丸对成骨细胞分化及IGF-I mRNA表达的影响[J].中国中医骨伤科杂志,2004,12(2):4-9.
[71].隋福革,张达,符强等.骨松康含药血清对成骨细胞生物学活性的影响[J].中国骨肿瘤骨病,2005,4(4):234-6.
[72].张荣华,朱晓峰,蔡宇等.益骨胶囊含药血清对大鼠成骨细胞IGF-ImRNA及其蛋白表达 的影响[J].中国病理生理杂志,2004,20(7):1222-5.
[73].Liu HC,Chen RM,Jian WC,et al.Cytotoxic and antioxidant effects of the water extract of the traditional Chinese herb gusuibu(Drynaria fortunei)on rat osteoblasts[J].J Formos Med Assoc.2001.100(6):383-8.
[74].张荣华,王廷春,朱晓峰等.益骨胶囊含药血清对SD大鼠成骨细胞分泌NO、NOS的影响[J].中药材,2004,27(8):593-6.
[75].肖辉,牛涛,齐锦生等.丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响[J].大然产物研究与开发,2005,17(6):754-7.
[76].Theoleyre S,Wittrant Y,Tat SK,et al.The molecular triad OPG/RANK/RANKL:involvement in the orchestration of pathophysiological bone remodeling[J].Cytokine Growth Factor Rev,2004,15(6):457-75.
[77].Wittrant Y,Theoleyre S,Chipoy C,et al.RANKL/RANK/OPG:new therapeutic targets in bone tumours and associated osteolysis[J].Biochim Biophys Acta,2004,1704(2):49-57.
[78].王运林,刘晓晴,夏秦等.金雀异黄素通过雌激素受体β增加人成骨细胞护骨素与破骨细胞分化因子表达比值[J].华中科技大学学报(医学版),2006,35(3):321-3.
[79].余增丽.金雀异黄酮对成骨细胞增殖的影响及其可能的分子生物学机制[J].中华老年医学杂志,2005,24(9):645-7.
[80].胡彬,吴翠环,陈璐璐.蛇床子素对大鼠成骨细胞中OPG和RANKL基因mRNA表达的影响[J].中国骨质疏松杂志,2004,10(4):415-9.
[81].吴乃中,李红丽,崔淑云.葛根对成骨细胞OPG、RANKL mRNA表达的影响[J].中华医学研究杂志,2006,6(1):6-9.
[82].唐井钢,李娟,吴贺勇,等.饲补肾中药大鼠血清对成骨细胞、破骨细胞共育系中破骨细胞功能的影响[J].白求恩军医学院学报,2006,4(2):68-9.
[83].吴丽萍,陶天遵,石义刚,等.三七总甙对成骨细胞增殖、分化及OPG表达影响的研究[J].中国骨质疏松杂志,2004,10(2):239-43.
[84].张秀珍,杨黎娟.淫羊藿甙对大鼠成骨细胞护骨素、RANKL表达的影响.中华内分泌代谢杂志[J],2006,22(3):222-5.
[85].刘钰瑜,崔燎,吴铁,等.大黄素对体外培养新生大鼠颅骨成骨细胞的影响[J].中国药理学通报,2005,21(2):235-40.
[86].杨亚军,杨中林,王冬春等.芦丁与槲皮素对成骨细胞代谢影响的比较研究[J].中药材,2006,29(5):467-70.
[87].Chen WF,Wong MS.Genistein modulates the effects of parathyroid hormone in human osteoblastic SaOS-2 cells[J].Br J Nutr,2006,95(6):1039-47.
[88].Lee KH,Choi EM.Biochanin A stimulates osteoblastic differentiation and inhibits hydrogen peroxide-induced production of inflammatory mediators in MC3T3-E1 cells[J].Biol Pharm Bull,2005,28(10):1948-53.
[89].屈树新,翁杰,沈如,等.含中药的磷酸钙生物材料制备方法的研究[J].生物医学工程与临床,2005,9(2):65-8.
[90].李茂红,屈树新,姚宁,等.含丹参磷酸钙骨水泥的制备和表征[J].生物医学工程与临床,2005,9(3):132-5.
[91].许春姣,翦新春,成洪泉,等.黄芪对兔骨髓基质细胞增殖和向成骨细胞分化的影响[J].中南大学学报(医学版),2004,29(4):489-91.
[92].许春姣,翦新春,彭解英,等.黄芪-聚乳酸/壳聚糖复合材料的体外细胞相容性实验研究[J].口腔医学研究,2005,21(2):142-6.
[93].许春姣,翦新春,彭解英,等.黄芪-壳聚糖/聚乳酸多孔支架对犬骨髓基质细胞生物学行为的影响[J].中南大学学报(医学版),2005,30(3):283-7.
[94].许春姣,郭峰,高清平,等.骨髓基质干细胞与黄芪—壳聚糖/聚乳酸支架对犬牙周 骨缺损再生的影响[J].中南大学学报(医学版),2006,31(4):512-7.
[95].邹琴,张利,左奕,等.载黄芪多糖骨水泥的理化性能及体外细胞相容性研究[J].功能材料,2008,39(9):1515-21.
[1].Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells[J].Science,1999,284:143-7.
[2].Pountos I,Giannoudis PV.Biology ofmesenchymal stem cells[J].Injury,2005,36:S8-S12.
[3].Connolly JF.Injectable bone marrow preparations to stimulate osteogenic repair[J].Clin Orthop,1995,313:8-18.
[4].Aubin JE.Regulation of osteoblast formation and function[J].Rev Endocr Metab Disord,2001,2:81-94.
[5].Rollins BJ.Chemokines[J].Blood,1997,90(3):909-28.
[6].王亦璁.骨与关节损伤(第3版)[M].北京:人民卫生出版社,2001:150.
[7].胥少汀,葛宝丰,徐印坎.实用骨科学(第3版)[M].北京:人民军医出版社,2005:330-7.
[8].Zhou S,Greenberger JS,Epperly MW,et al.Age-related intrinsic changes in human bone-marrow-derived mesenchymal stem cells and their differentiation to osteoblasts[J].Aging Cell,2008,7:335-43.
[9].Seebach C,Henrich D,Tewksbury R,et al.Number and proliferative capacity of human mesenchymal stem cells are modulated positively in multiple trauma patients and negatively in atrophic nonunions[J].Calcif Tissue Int,2007,80:294-300.
[10].Hernigou P,Poignard A,Beaujean F,et al.Percutaneous autologous bone-marrow grafting for nonunions.Influence of the number and concentration of progenitor cells[J].J Bone Joint Surg Am,2005,87:1430-7.
[11].Siwach RC,Sangwan SS,Singh R,et al.Role of percutaneous bone marrow grafting in delayed unions,non-unions and poor regenerates[J].Indian J Med Sci,2001,55:326-36.
[12].Wilkins RM,Chimenti BT,Rifkin RM.Percutaneous treatment of long bone nonunions:the use of autologous bone marrow and allograft bone matrix[J].Orthopedics,2003,26:549-54.
[13].周志玲,袁进国,刘英飞,等、白体骨髓干细胞移植治疗骨不连[J].中国临床康复,2006,10(29):22-3.
[14].曹宜林主编,刘伟,崔磊副主编.组织工程学理论与实践[M].上海:上海科学技术出版社,2004:114.
[15].叶成,史鹏.骨髓问充质干细胞与成骨[J].中国矫形外科杂志,2008.16(5):356-8.
[16].Ippokratis P,Diane C,Paul E,et al.Mesenchymal stem cell tissue engineering:Techniques for isolation,expansion and application[J].Injury,2007,38S4:S23-S33.
[17].Wildemann B,Kadow-Romacker A,Haas NP,et al.Quantification of various growth factors in different demineralized bone matrix preparations[J].J Biomed Mater Res A,2006,81A (2):437-442
[18].Alt V,Heissel A.Economic considerations for the use of recombinant human bone morphogenetic protein-2 in open tibial fractures in Europe:the German model[J].Curr Med Res Opin 2006,22 Suppl 1:S19-22
[19].Hu ZM,Peel SA,Sandor GK,et al.The osteoinductive activity of bone morphogenetic protein(BMP)purified by repeated extracts of bovine bone[J].Growth Factors 2004,22(1):29-33
[20].Montesano R.Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth[J].Biochem Biophys Res Commun 2007,353(3):817-822
[21].Miriyala S,Gongora Nieto MC,Mingone C,et al.Bone morphogenic protein-4 induces hypertension in mice:role of noggin,vascular NADPH oxidases,and impaired vasorelaxation[J].Circulation 2006,113(24):2818-2825
[22].Deng H,Makizumi R,Ravikumar TS,et al.Bone morphogenetic protein-4 is over expressed in colonic adenocarcinomas and promotes migration and invasion of HCT116 cells[J].Exp Cell Res 2007,313(5):1033-1044
[23].Feldman GJ,Billings PC,Patel RV,et al.Over-expression of BMP4 and BMP5 in a child with axial skeletal malformations and heterotopic ossification:A new syndrome[J].Am J Med Genet A 2007,143(7):699-706
[24].Zhao J,Ohba S,Shinkai M,et al.Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner[J].Biochem Biophys Res Commun 2008,369:444-448
[25].Yin X X,Chen Z Q,Liu Z J,et al.Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2[J].Chin Med J (Engl)2007,120:204-210
[26].Qian G,Zhang X,Lu L,et al.Regulation of Cbfal expression by total flavonoids of Herba epimedii[J].Endocr J 2006,53:87-94
[27].Sheng H,Zhang G,Wang X L,et al.Phytochemical molecule icariin stimulates osteogenic but inhibits adipogenic differentiation of mesenchymal stem cells[J].Bone 2008(43):S42-S43
[28].Qin L,Han T,Zhang Q,et al.Antiosteoporotic chemical constituents from Er-Xian Decoction,a traditional Chinese herbal formula[J].J Ethnopharmacol,2008 118:271-279
[29].国家药典委员会.中华人民共和国药典一部[M].2005年版.北京:化学工业出版社,2005:229.
[30].Xu W,Zhang YP,Yang M,et al.LC-MS/MS method for the imultaneous determination of icariin and its major metabolites in rat plasma[J].Journal of Pharmaceutical and Biomedical Analysis,2007,45:667-72.
[31].刘铁汉,王毅,王本祥,等.淫羊藿苷的肠菌代谢研究Ⅰ[J].肠内细菌对淫羊藿苷的代谢转化.中草药,2000,31(11):834-837
[32].徐文,张亚萍,张卫东,等.离体大鼠肠道菌群对淫羊藿苷的代谢研究[J].世界科学技术,2006(6):98-100.
[33].Bao JR,Yang JW,Li SF,et al.Effects of Icariin on ovariectomized osteoporotic rats[J].Wei Sheng Yan Jiu,2005,34:191-3.
[34].Li L,Zhou QX,Shi JS.Protective effects of icariin on neurons injured by cerebral ischemia/reperfusion[J].Chin Med J(Engl),2005,118:1637-43.
[35].Wang YK,Huang ZQ.Protective effects of icariin on human umbilical vein endothelial cell injury induced by H2O2 in vitro[J].Pharmacol Res,2005,52:174-82.
[36].He W,Sun H,Yang B,et al.Immunoregulatory effects of the herba Epimediia glycoside icariin[J].Arzneimittelforschung 1995,45:910-3.
[37].Zhang ZB,Yang QT.The testosterone mimetic properties of icariin[J].Asian J Androl,2006,8(5):601-5.
[38].Pan Y,Kong L,Xia X,et al.Antidepressant-like effect of icariin and its possible mechanism in mice[J].Pharmacol Biochem Behav,2005,82:686-94.
[39].H.Y.Ye,Y.J.Lou.Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells[J].Phytomedicine,2005,12:735-741.
[40].张京伟,周云峰,文显梅,等.淫羊藿甙逆转胃癌细胞恶性表型的研究[J].中华实验外科杂志,2006,23(10):1213-4.
[41].Zheng XX,Hu YH,Liu JH,et al.Screening of active compounds as neuromedin U2 receptor agonist from natural products[J].Bioorg Med Chem Lett,2005,15:4531-5.
[42].Meng FH,Li YB,Xiong ZL,et al.Osteoblastic proliferative activity of Epimedium brevicornum Maxim[J].Phytomedicine,2005,12:189-93.
[43].Chen KM,Ma HP,Ge BF,et al.Icariin enhances the osteogenic differentiation of bone marrow stromal cells but has no effects on the differentiation of newborn alvarial osteoblasts of rats[J].Pharmazie,2007,62:785-9.
[44].Yin XX,Chen ZQ,Liu Z J,et al.Icariine stimulates proliferation and differentiation of human osteoblasts by increasing production of bone morphogenetic protein 2[J].Chin Med J (Engl),2007,120:204-10.
[45].Zhao J,Ohba S,Shinkai M,et al.Icariin induces osteogenic differentiation in vitro in a BMP-and Runx2-dependent manner[J].Biochem Biophys Res Commun.2008,369:444-8.
[46].Xiao QB,Chen AM,Guo FJ.Effects of Icariin on expression of OPN mRNA and type I collagen in rat osteoblasts in vitro[J].J Huazhong Univ Sci Technolog Med Sci,2005,25,690-2.
[47].Zhang DW,Cheng Y,Wang NL,et al.Effects of total flavonoids and flavonol glycosides from Epimedium koreanum Nakai on the proliferation and differentiation of primary osteoblasts[J].Phytomedicine,2008,15:55-61.
[48].谢玲玲,邹移海,刘炫斯.淫羊藿苷对骨髓问质干细胞增殖及分泌血管内皮生长因子、碱性成纤维生长因子的影响[J].实用医学杂志,2008,24(6):908-10.
[49].张秀珍,杨黎娟.淫羊藿甙对大鼠成骨细胞护骨素、RANKL表达的影响[J].中华内分泌代谢杂志,2006,22(3):222-5.
[50].雪原,王沛,齐清会,等.淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究[J].中华骨科杂志,2005,25(2):119-23.
[51].Santos,NC,Figueira-Coelho J,Martins-Silva J.et al.Multidisciplinary utilization of dimethyl sulfoxide:pharmacological,cellular,and molecular aspects[J].Biochem.Pharmacol,2003,65,1035-41.
[52].Xing L,Remick DG.Mechanisms of dimethyl sulfoxide augmentation of IL-1 beta production[J].J Immunol,2005,174:6195-202.
[53].Melchior D,Packer CS,Johnson TC,et al.Dimethyl sulfoxide:does it change the functional properties of the bladder wall?[J].J Urol,2003,170:253-8.
[54].[54]Cheung WMW,Ng WW,Kung AWC.Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-El cells[J].FEBS Letters.2006.580:121-6.
[55].陈其昕,袁中兴,刘清,等.骨折愈合中修复性细胞增殖与骨形成的关系[J].中华骨科杂志,1992,12(4):290-2.
[56].刘大鹏,艾合麦提,古丽,等.体外培养骨髓基质细胞增殖和分化的相互关系[J].中国临床康复,2005,9(38):9-12.
[57].Javazon EH,Colter DC,Schwarz EJ,et al.Rat Marrow Stromal Cells are More Sensitive to Plating Density and Expand More Rapidly from Single-Cell-Derived Colonies than Human Marrow Stromal Cells[J].STEM CELLS,2001,19:219-25.
[58].Peister A,Mellad J A,Larson B L,et al.Adult stem cells from bone marrow(MSCs)isolated from different strains of inbred mice vary in surface epitopes,rates of proliferation,and differentiation potential[J].Blood,2004,103(5):1662-8.
[59].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004,36-41.
[60].Schmitt B,Ringe J,H(a|¨)upl T,et al.BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture[J].Differentiation,2003,71(9-10):567-77.
[61].唐林,林珠,李永明,等.不同大小机械牵张力对成骨细胞增殖及碱性磷酸酶的影响[J]解放军医学杂志,2006,31(6):580-1.
[62].叶凌,李江宁,李联,等.骨钙素在体内的表达[J].国外医学口腔医学分册,2002,29(4):228-9.
[63].Komori T.Regulation of osteoblast differentiation by transcription factors[J].J Cell Biochem.2006,99(5):1233-9.
[64].Komori T.Regulation of skeletal development by the Runx family of transcription factors[J].J Cell Biochem,2005,95(3):445-53.
[65].Ducy P,Zhang R,Geoffroy V,et al.Osf2/Cbfa1:a transcriptional activator of osteoblast differentiation[J].Cell,1997,89:747-54.
[66].Wai PY,Mi Z,Gao C,et al.Ets-1 and runx2 regulate transcription of a metastatic gene,osteopontin,in murine colorectal cancer cells[J].J Biol Chem,2006,281(28):18973-82.
[67].Hernan Roca,Mattabhorn Phimphilai,Rajaram Gopalakrishnan,et al.Cooperative Interactions between RUNX2 and Homeodomain Protein-binding Sites Are Critical for the Osteoblast-specific Expression of the Bone Sialoprotein Gene[J].J Biol Chem,2005,280(35):30845-55.
[68].Kazuhisa Nakashima,Xin Zhou,Gary Kunkel,et al.The Novel Zinc Finger-Containing Transcription Factor Osterix Is Required for Osteoblast Differentiation and Bone Formation[J].Cell,2002,108:17-29.
[69].Wu L.Wu Y,Lin Y,et al.Osteogenic differentiation of adipose derived stem cells promoted by overexpression ofosterix[J].Mol Cell Biochem,2007,301(1-2):83-92.
[70].Nicolas H,St(?)phanie M,Martine B,et al.Dlx5 drives Runx2 expression and osteogenic differentiation in developing cranial suture mesenchyme[J].Developmental Biology,2007,304:860-74.
[71].LI X,CAO X.BMP Signaling and Skeletogenesis[J].Ann N.Y.Acad Sci,2006,1068:26-40.
[72].Widmann C.Gibson S,Jarpe MB,et al.Mitogen-activated protein kinase:conservation of a three-kinase module from yeast to human[J].PhysioI Rev,1999,79(1):143-80.
[73].Wang X,Goh CH,Li B.p38 mitogen-activated protein kinase regulates osteoblast differentiation through osterix[J].Endocrinology.2007.148(4):1629-37.
[74].王茸影,易静.骨形成蛋白调控成骨分化的信号机制[J].生命科学,2005,17(1):34-9.
[75].Cheng HW,Jiang W,Phillips FM,etal.Ostcogcnicactivity ofthe fourteen types of human bone morphogenetic proteins(BMPs)[J].J Bone Joint Surg Am,2003,85:1544-52.
[76].Li SH,Guo DZ,Li B,et al.The stimulatory effect of insulin-like growth factor-1 on the proliferation,differentiation,and mineralisation of osteoblastic cells from Holstein cattle[J].J Veterinary,2009,179:430-6.
[77].[77]Koch H,Jadlowiec JA,Campbell PG.Insulin-like growth factor-Ⅰ induces early osteoblast gene expression in human mesenchymal stem cells[J].Stem Cells Dev,2005,14(6):621-31.
[78].Alliston T.TGF-beta regulation of osteoblast differentiation and bone matrix properties[J].J Musculoskelet Neuronal Interact,2006,6(4):349-50.
[79].Bodo M,Venti G,Pezzetti F,et al.Interleukin-1 alpha:regulation of cellular proliferation and collagen synthesis in cultured human osteoblast-like cells[J].Cell Mol Biol (Noisy-le-grand),1992,38(5-6):679-86.
[80].Nakayama Y,Kato N,Nakajima Y,et al.Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2osteoblast-like cells[J].Cell Biol Int,2004,28(10):653-60.
[81].Mukai T,Otsuka F,Otani H,et al.TNF-alpha inhibits BMP-induced osteoblast differentiation through activating SAPK/JNK signaling[J].Biochem Biophys Res Commun,2007,356(4):1004-10.
[82].李淑梅,宋利格,张秀珍.淫羊藿甙对大鼠成骨细胞功能及Osterix表达的影响[J].同济大学学报(医学版),2005,26(6):8-11.
[83].刘尚全,杨颖,周丽斌等.淫羊藿甙逆转地塞米松抑制成骨细胞分化及其机制[J].中华内分泌代谢杂志,2006,22(3):218-21.
[84].雪原,齐清会,王沛等.淫羊藿甙对OVX大鼠成骨细胞Smad4 mRNA的作用[J].天津医药,2006,34(4):256-58.
[85].陈虹,张秀珍.淫羊藿甙对大鼠成骨细胞分泌细胞因子的影响[J].同济大学学报(医学版),2005,26,(2):5-7.
[86].Theoleyre S,Wittrant Y,Tat SK,et al.The molecular triad OPG/RANK/RANKL:involvement in the orchestration of pathophysiological bone remodeling[J].Cytokine Growth Factor Rev,2004,15(6):457-75.
[87].Chen KM,Ge BF,Liu XY,et al.Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture[J].Pharmazie,2007,62(5):388-91.
[88].Wang ZQ,Lou YJ.Proliferation-stimulating effects of icaritin and desmethylicaritin in MCF-7 cells[J].Eur J Pharmacol.2004;504(3):147-53.
[89].McCarthy TL,Clough ME,Gundberg CM,et al.Expression of an estrogen receptor agonist in differentiating osteoblast cultures[J].Proc Natl Acad Sci U S A,2008,105(19):7022-7.
[90].Zhang G,Qin L,Sheng H,et al.Epimedium-derived phytoestrogen exert beneficial effect on preventing steroid-associated osteonecrosis in rabbits with inhibition of both thrombosis and lipid-deposition[J].Bone,2007,40(3):685-92.
[91].Orciani M,Trubiani O,Vignini A,et al.Nitric oxide production during the osteogenic differentiation of human periodontal ligament mesenchymal stem cells[J].Acta Histochem,2009,111(1):15-24.
[92].Hitoshi K,Pascale VG,Chan J,et al.Osterix Induces Osteogenic Gene Expression but not Differentiation in Primary Human Fetal Mesenchymal Stem Cells.Tissue Engineering,2007,13(7):1513-23.
[93].Morsczeck C.Gene expression of runx2,Osterix,c-fos,DLX-3,DLX-5,and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.Calcif Tissue,2006,78(2):98-102.
[94].Zhou Y,Chen F,Ho ST,et al.Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials,2007,28(5):814-24.
[95].Yang S,Zhong C,Frenkel B,et al.Diverse biological effect and Smad signaling of bone morphogenetic protein 7 in prostate tumor cells.Cancer Res,2005,65(13):5769-77.
[96].Sano H,Hosokawa K,Kidoya H,et al.Negative regulation of VEGF-induced vascular leakage by blockade of angiotensin Ⅱ type 1 receptor.Arterioscler Thromb Vasc Biol,2006,26(12):2673-80.
[1].缪旭东,裴国献,黎健伟,等.脱蛋白骨复合纤维蛋白胶构建骨组织工程支架的体外实验研究[J].中国矫形外科杂志,2006,14(18):1413-1414.
[2].Ruksudjarit A,Pengpat K,Rujijanagul G,et al.Synthesis and characterization of nanocrystalline hydroxyapatite from natural bovine bone[J].Current Applied Physics,2008,8(3-4):270-272.
[3].Liu CZ,Han ZW,Czernuszka JT.Gradient collagen/nanohydroxyapatite composite scaffold:Development and characterization[J].Acta Biomaterialia,2009,5(2):661-669.
[4].Zhang PB,Hong ZK,Yu T,et al.In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide)and hydroxyapatite surface-grafted with poly(l-lactide)[J].Biomaterials,2009,30(1):58-70.
[5].Nejati E,Mirzadeh H,Zandi M.Synthesis and characterization of nano-hydroxyapatite rods/poly(l-lactide acid)composite scaffolds for bone tissue engineering[J].Applied Science and Manufacturing,2008,39(10):1589-1596.
[6].Hu Q,Li B,Wang M,et al.Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization:a potential material as internal fixation of bone fracture[J].Biomaterials,2004,25(5):779-785.
[7].南开辉.仿生组装淫羊藿苷控释型骨组织工程支架及其对骨髓基质干细胞生物学行为干预研究[M].南方医科大学博士后出站报告.广州:南方医科大学,2007.30-56
[8].赵丽东,张华峰,董静洲.等.淫羊藿苷的超声波强化提取及其稳定性初探[J].西北农业学报.2007,16(6):285-288.
[9].刘铁汉,王毅,王本祥,等.淫羊藿苷的肠菌代谢研究Ⅰ.肠内细菌对淫羊藿苷的代谢转化[J].中草药,2000,31(11):834-837.
[10].金岩.组织工程学原理与技术[M].西安:第四军医大学出版社,2004.36-41.
[11].刘大鹏,艾合麦提,古丽,等.体外培养骨髓基质细胞增殖和分化的相互关系[J].中国临床康复,2005,9(38):9-12.
[12].国家药典委员会.中华人民共和国药典二部[M].2005年版.北京:化学工业出版社,2005:附录85.
[1].Enescu D,Hamciuc V,Ardeleanu R,et al.Polydimethylsiloxane modified chitosan.Part Ⅲ:Preparation and characterization of hybrid membranes[J].Carbohydrate Polymers,2009,76:268-278.
[2].Berrada M,Serreqi A,Dabbarh F,et al.A novel non-toxic camptothecin formulation for cancer chemotherapy[J].Biomaterials,2005,26(14):2115-2120.
[3].Park JH,Cho YW,Son YJ,et al.Preparation and characterization of self-assembled nanoparticles based on glycol chitosan bearing adriamycin[J].Colloid Polymer Sci,adriamycin,2006,284(7):763-770.
[4].郑彩虹,梁文权,虞和永.海藻酸-壳聚糖-聚乳酸羟乙醇酸复合微球的制备及其对蛋白释放的调节[J].药学学报.2005.40(2):182-186.
[5].Sonaje K,Lin YH,Juang JH,et al.In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery[J].Biomaterials,2009,30(12):2329-2339.
[6].Bhattarai N,Ramay HR,Gunn J,et al.PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release[J].J Control Release,2005,103(3):609-624.
[7].张维颖,樊东辉,徐政,等.温敏性壳聚糖凝胶的阿霉素药物体外释放研究[J].中国海洋药物,2005,24(5):50-53.
[8].Rodrigues LB,Leite HF,Yoshida MI,et al.In vitro release and characterization of chitosan films as dexamethasone cartier[J].Int J Pharm,2009,368(1-2):1-6.
[9].滕利荣,王博,刘艳,等.羟基磷灰石纳米粒子作为蛋白类缓释药物载体的应用[J].吉林大学学报(工学版),2007,37(5):1093-1096.
[10].Zhou H,Qian J,Wang J,et al.Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N,6-O-sulfated chitosan in vitro and in vivo[J].Biomaterials,2009,30(9):1715-1724.
[11].Niu X,Feng Q,Wang M,et al.Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2[J].J Control Release,2009,134(2):111-117.
[12].Tan H,Chu CR,Payne KA,et al.Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering[J].Biomaterials,2009,30(13):2499-2506.
[13].Michalska M,Kozakiewicz M,Bodek KH.Polymer angiogenic factor carrier.Part Ⅰ.Chitosan-alginate membrane as carrier PDGF-AB and TGF-beta[J].Polim Med,2008,38(4):19-28.
[14].Abarrategi A,Moreno-Vicente C,Ramos V,et al.Improvement of porous beta-TCP scaffolds with rhBMP-2 chitosan carrier film for bone tissue application[J].Tissue Eng Part A,2008,14(8):1305-1319.
[15].Cai DZ,Zeng C,Quan DP,et al.Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-betal delivery for cartilage tissue engineering[J].Chin Med J(Engl),2007,120(3):197-203.
[16].南开辉.仿生组装淫羊藿苷控释型骨组织工程支架及其对骨髓基质干细胞生物学行为干预研究.南方医科大学博士后出站报告[M].广州:南方医科大学,2007.80-89.
[17].罗华丽,鲁在君.壳聚糖作为药物载体的缓释机理的研究进展[J].大中学刊,2006,21(2):38-40.
[18].[18]屠锡德,张钧寿,朱家璧.药剂学(第三版)[M].北京:人民卫生出版社,2002.附录ⅩⅨ D.
[1].Kondo N,Ogose A,Tokunaga K,et al.Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation[J].Biomaterials,2006,27(25):4419-4427.
[2].Duan YR,Zhang ZR,Wang CY,et al.Dynamic study of calcium phosphate formation on porous HA/TCP ceramics[J].J Mater Sci Mater Med,2005,16(9):795-801.
[3].Hoshino M,Egi T,Terai H,et al.Repair of long intercalated rib defects using porous beta-tricalcium phosphate cylinders containing recombinant human bone morphogenetic protein-2 in dogs[J].Biomaterials,2006,27(28):4934-4940.
[4].Kamakura S,Nakajo S,Suzuki O,et al.New scaffold for recombinant human bone morphogenetic protein-2 [J].J Biomed Mater Res A,2004,71(2):299-307.
[5].Seeherman HJ,Bouxsein M,Kim H,et al.Recombinant human bone morphogenetic protein-2 delivered in an injectable calcium phosphate paste accelerates osteotomy-site healing in a nonhuman primate model [J].J Bone Joint Surg Am,2004,86-A(9):1961-1972.
[6].Liu HW,Chen CH,Tsai CL,et al.Targeted delivery system for juxtacrine signaling growth factor based on rhBMP-2-mediated carrier-protein conjugation [J].Bone, 2006,39(4):825-836.
[7].Hsieh CY,Hsieh HJ,Liu HC,et al.Fabrication and release behavior of a novel freeze-gelled chitosan/gamma-PGA scaffold as a carrier for rhBMP-2 [J].Dent Mater,2006,22(7):622-629.
[8].Suciati T,Howard D,Barry J,et al.Zonal release of proteins within tissue engineering scaffolds [J].J Mater Sci Mater Med,2006,17(11):1049-1056.
[9].Keskin DS, Tezcaner A, Korkusuz P, et al.Collagen-chondroitin sulfate-based PLLA-SAIB-coated rhBMP-2 delivery system for bone repair [J].Biomaterials,2005,26(18):4023-4034.
[10].Rai B,Teoh SH,Hutmacher DW,et al.Novel PCL-based honeycomb scaffolds as drug delivery systems for rhBMP-2 [J].Biomaterials,2005,26(17):3739-3748.
[11].Ruhe PQ,Boerman OC,Russel FGM,et al.Controlled release of rhBMP-2 loaded poly(dl-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo [J].J Control Release,2005,106 (1-2):162-171.
[12].Lee YM,Nam SH,Seol YJ,et al.Enhanced bone augmentation by controlled release of recombinant human bone morphogenetic protein-2 from bioabsorbable membranes [J].J Periodontol,2003,74(6):865-872.
[13].Chu TM,Warden SJ,Turner CH,et al.Segmental bone regeneration using a load-bearing biodegradable carrier of bone morphogenetic protein-2 [J].Biomaterials,2007,28(3):459-467.
[14].Yoneda M,Terai H,Imai Y,et al.Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant [J].Biomaterials,2005,26 (25):5145-5152.
[15].Chen B,Lina H,Wang JH,et al.Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2 [J].Biomaterials,2007,28 (6):1027-1035.
[16].Santana RB,Trackman PC.Controlled release of fibroblast growth factor 2 stimulates bone healing in an animal model of diabetes mellitus [J].Int J Oral Maxillofac Implants.2006,21(5):711-718.
[17].Kleinheinz J,Stratmann U,Joos U,et al.VEGF-activated angiogenesis during bone regeneration [J].J Oral Maxillofac Surg,2005,63 (9):1310-1316.
[18].Lee JY,Nam SH,Im SY,et al.Enhanced bone formation by controlled growth factor delivery from chitosan-based biomaterials [J].J Control Release,2002,78 (1-3):187-197.
[19].Raiche AT,Puleo DA.In vitro effects of combined and sequential delivery of two bone growth factors [J].Biomaterials,2004,25(4):677-685.
[20].Takahashi Y,Yamamoto M,Tabata Y Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate [J].Biomaterials,2005,26(23):4856-4865.
[21].Ueda H,Nakamura T,Yamamoto M,et al.Repairing of rabbit skull defect by dehydrothermally crosslinked collagen sponges incorporating transforming growth factor β_1 [J].J Control Release,2003,88(1):55-64.
[22].Chen FM,Zhao YM,Wu H,et al.Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-Ⅰ from dextran-co-gelatin microspheres [J].J Control Release,2006,114(2):209-222.
[23].Wei G,Jin Q,Giannobile WV,et al.The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres [J].Biomaterials,2007,28(12):2087-2096.
[24].Lu SS,Zhang X,Soo C,et al.The osteoinductive properties of Nell-1 in a rat spinal fusion model.Spine J,2007,7(1):50-60.
[25].Hosseinkhani H,Hosseinkhani M,Khademhosseini A,et al.Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold [J].J Control Release,2007,117(3):380-386.
[26].Lutolf MP,Lauer-Fields JL,Schmoekel HG,et al.Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration:engineering cell-invasion characteristics[J].Proc Natl Acad Sci U S A,2003,100(9):5413-5418.
[27].Chen FM,Zhao YM,Sun HH,et al.Novel glycidyl methacrylated dextran (Dex-GMA)/gelatin hydrogel scaffolds containing microspheres loaded with bone morphogenetic proteins:Formulation and characteristics[J].J Control Release,2007,118(1):65-77.
[28].Yuan H,Kurashina K,de Bruijn JD,et al.A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics[J].Biomaterials,1999,20(19):1799-1806.
[29].Habibovic P,Yuan H,van der Valk CM,et al.3D microenvironment as essential element for osteoinduction by biomaterials[J].Biomaterials,2005,26(17):3565-7355.
[30].Kondo N,Ogose A,Tokunaga K,et al.Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation[J].Biomaterials,2006,27(25):4419-4427.