中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2014年
12期
758-762
,共5页
滕伟%王琴梅%陈盈%黄洪章
滕偉%王琴梅%陳盈%黃洪章
등위%왕금매%진영%황홍장
钛%骨形态生成蛋白2%聚电解质膜%脂多糖胺纳米囊泡
鈦%骨形態生成蛋白2%聚電解質膜%脂多糖胺納米囊泡
태%골형태생성단백2%취전해질막%지다당알납미낭포
Titanium%Bone morphogenetic protein 2%Polyelectrolyte multilayer films%Lipopolysaccharide-amine nanopolymersomes
目的 探讨聚电解质多层膜(polyelectrolyte multilayer films,PEM)对钛表面细胞生物学效应的影响,以期为PEM用于钛种植体表面改性提供依据.方法 利用含骨形成蛋白2(plasmid of bone morphogenetic protein-2,pBMP-2)基因的脂多糖胺纳米囊泡(lipopolysaccharide-amine nanopolymersomes,LNP)(简称pLNP)作为阳离子聚电解质,透明质酸(hyaluronic acid,HA)作为阴离子聚电解质,通过层层自组装技术在碱热处理后的钛表面构建PEM,HA和pLNP在钛表面依次组装1次为1个组装循环.采用扫描电镜观察组装前后钛表面形貌.通过紫外光谱分析、表面接触角测量对不同组装循环PEM进行表征.检测大鼠骨髓间充质干细胞在自组装A组(4个组装循环,外表面为pLNP聚电解质层)、自组装B组(4.5个组装循环,外表面为透明质酸聚电解质层)、空白对照组(抛光钛)和碱热处理组(碱热处理钛)表面接种0.5和1h的黏附情况及1、3、5d的增殖情况(每组每个时间点样本量为6).检测空白对照组、阴性对照组(膜中不含pBMP-2)和自组装A组7d的碱性磷酸酶活性及原位转染能力(每组样本量为6).结果 扫描电镜可见自组装后钛表面被PEM覆盖,变得相对平滑.紫外光谱显示PEM在260 nm处出现DNA吸收峰,其吸光度值随组装循环数增加而增加.抛光钛经碱热处理后接触角从(62.6±4.9)°骤降至(8.1±2.2)°,组装过程中表面接触角呈锯齿状交替上升,随自组装循环数增加而增势变缓,至5.5个循环时达到(49.3±1.3)°.接种0.5和1h后自组装A组细胞黏附A值(0.415±0.085、0.426±0.048)均显著高于自组装B组(0.299±0.012、0.355±0.022)、空白对照组(0.225±0.007、0.260±0.010)和碱热处理组(0.302±0.056、0.339±0.028)(P＜0.01).培养1、3和5d后自组装A和B组细胞增殖均显著高于空白对照组和碱热处理组(P＜0.01).7d时自组装A组碱性磷酸酶活性(261±58)显著高于空白对照组和阴性对照组(P＜0.01).自组装A组表面具有原位转染骨髓间充质干细胞的能力.结论 钛表面可成功构建pLNP-透明质酸PEM并具有良好的细胞生物学效应.
目的 探討聚電解質多層膜(polyelectrolyte multilayer films,PEM)對鈦錶麵細胞生物學效應的影響,以期為PEM用于鈦種植體錶麵改性提供依據.方法 利用含骨形成蛋白2(plasmid of bone morphogenetic protein-2,pBMP-2)基因的脂多糖胺納米囊泡(lipopolysaccharide-amine nanopolymersomes,LNP)(簡稱pLNP)作為暘離子聚電解質,透明質痠(hyaluronic acid,HA)作為陰離子聚電解質,通過層層自組裝技術在堿熱處理後的鈦錶麵構建PEM,HA和pLNP在鈦錶麵依次組裝1次為1箇組裝循環.採用掃描電鏡觀察組裝前後鈦錶麵形貌.通過紫外光譜分析、錶麵接觸角測量對不同組裝循環PEM進行錶徵.檢測大鼠骨髓間充質榦細胞在自組裝A組(4箇組裝循環,外錶麵為pLNP聚電解質層)、自組裝B組(4.5箇組裝循環,外錶麵為透明質痠聚電解質層)、空白對照組(拋光鈦)和堿熱處理組(堿熱處理鈦)錶麵接種0.5和1h的黏附情況及1、3、5d的增殖情況(每組每箇時間點樣本量為6).檢測空白對照組、陰性對照組(膜中不含pBMP-2)和自組裝A組7d的堿性燐痠酶活性及原位轉染能力(每組樣本量為6).結果 掃描電鏡可見自組裝後鈦錶麵被PEM覆蓋,變得相對平滑.紫外光譜顯示PEM在260 nm處齣現DNA吸收峰,其吸光度值隨組裝循環數增加而增加.拋光鈦經堿熱處理後接觸角從(62.6±4.9)°驟降至(8.1±2.2)°,組裝過程中錶麵接觸角呈鋸齒狀交替上升,隨自組裝循環數增加而增勢變緩,至5.5箇循環時達到(49.3±1.3)°.接種0.5和1h後自組裝A組細胞黏附A值(0.415±0.085、0.426±0.048)均顯著高于自組裝B組(0.299±0.012、0.355±0.022)、空白對照組(0.225±0.007、0.260±0.010)和堿熱處理組(0.302±0.056、0.339±0.028)(P＜0.01).培養1、3和5d後自組裝A和B組細胞增殖均顯著高于空白對照組和堿熱處理組(P＜0.01).7d時自組裝A組堿性燐痠酶活性(261±58)顯著高于空白對照組和陰性對照組(P＜0.01).自組裝A組錶麵具有原位轉染骨髓間充質榦細胞的能力.結論 鈦錶麵可成功構建pLNP-透明質痠PEM併具有良好的細胞生物學效應.
목적 탐토취전해질다층막(polyelectrolyte multilayer films,PEM)대태표면세포생물학효응적영향,이기위PEM용우태충식체표면개성제공의거.방법 이용함골형성단백2(plasmid of bone morphogenetic protein-2,pBMP-2)기인적지다당알납미낭포(lipopolysaccharide-amine nanopolymersomes,LNP)(간칭pLNP)작위양리자취전해질,투명질산(hyaluronic acid,HA)작위음리자취전해질,통과층층자조장기술재감열처리후적태표면구건PEM,HA화pLNP재태표면의차조장1차위1개조장순배.채용소묘전경관찰조장전후태표면형모.통과자외광보분석、표면접촉각측량대불동조장순배PEM진행표정.검측대서골수간충질간세포재자조장A조(4개조장순배,외표면위pLNP취전해질층)、자조장B조(4.5개조장순배,외표면위투명질산취전해질층)、공백대조조(포광태)화감열처리조(감열처리태)표면접충0.5화1h적점부정황급1、3、5d적증식정황(매조매개시간점양본량위6).검측공백대조조、음성대조조(막중불함pBMP-2)화자조장A조7d적감성린산매활성급원위전염능력(매조양본량위6).결과 소묘전경가견자조장후태표면피PEM복개,변득상대평활.자외광보현시PEM재260 nm처출현DNA흡수봉,기흡광도치수조장순배수증가이증가.포광태경감열처리후접촉각종(62.6±4.9)°취강지(8.1±2.2)°,조장과정중표면접촉각정거치상교체상승,수자조장순배수증가이증세변완,지5.5개순배시체도(49.3±1.3)°.접충0.5화1h후자조장A조세포점부A치(0.415±0.085、0.426±0.048)균현저고우자조장B조(0.299±0.012、0.355±0.022)、공백대조조(0.225±0.007、0.260±0.010)화감열처리조(0.302±0.056、0.339±0.028)(P＜0.01).배양1、3화5d후자조장A화B조세포증식균현저고우공백대조조화감열처리조(P＜0.01).7d시자조장A조감성린산매활성(261±58)현저고우공백대조조화음성대조조(P＜0.01).자조장A조표면구유원위전염골수간충질간세포적능력.결론 태표면가성공구건pLNP-투명질산PEM병구유량호적세포생물학효응.
Objective To provide a basis for surface modification of polyelectrolyte multilayer films (PEM) on implants by exploring the effects of immobilization of PEM on titanium surfaces on their cell biological effects.Methods By using plasmid of bone morphogenetic protein-2(pBMP-2)-loaded lipopolysaccharide-amine nanopolymersomes(pLNP) as cationic polyelectrolytes and hyaluronic acid(HA) as anionic polyelectrolytes.PEM were constructed on alkaline-heat treated titanium surfaces via layer by layer self-assembly(LbL) technique.A successive deposition of HA and pLNP on titanium surfaces was defined as one cycle of assembly.The morphology of titanium surface before and after assembly treatment was observed by scanning electron microscopy (SEM).The ultraviolet (UV) spectra and surface contact angles of PEM with different self-assembly cycles were measured.The adhesion and proliferation of mesenchymal stem cell (BMSC) on surfaces of group A (4 cycles of assembly,with outermost layer of pLNP),group B (4.5 cycles of assembly,with outermost layer of HA),blank control (polished titanium,Ti group) and alkaline-heat treated titanium (Ti-OH group) were investigated.Cell differentiation indexed by alkaline phosphatase activity (ALP) and in situ transfection of BMSC on surfaces of group A,Ti,negative control [4 cycles of assembly without pBMP-2] were evaluated.Results Self assembly of PEM made the titanium surface become relatively smooth.DNA absorption peaked appear at 260 nm in UV spectra,and the absorption intensity increased with assembly,suggesting the successful construction of PEM.After alkali-heat treatment,the surface contact angle of titanium decreased from (62.6±4.9) ° to (8.1 ±2.2) °.During LbL,with alternately introducing pLNP and HA,the contact angle increased in a jagged mode at a gradually decreased rate,which further proved the success of assembly.Cell adhesion for group A at 0.5 and 1 h was 0.415±0.085 and 0.426±0.048,which was significantly higher than those for group B (0.299±0.012,0.355±0.022),Ti-OH group (0.225±0.007,0.260±0.010) and Ti group (0.302±0.056,0.339±0.028) (P＜0.01).Cell proliferation for group A and B at 3,5 and 7 d were significantly higher than those for Ti and Ti-OH group (P＜0.01).ALP in group A at day 7 was 261±58,which was significantly higher than those in group B and Ti group.Group A had in situ transfection capability for BMSC.Conclusions PEM with good cell biological effects can be constructed successfully on titanium surfaces using gene-loaded lipopolysaccharide-amine nanopolymersomes and hyaluronie acid.
