壳聚糖/纳米羟基磷灰石治疗髓室底穿的实验研究

目的：研究壳聚糖／纳米羟基磷灰石（CS／n—HA）复合材料作为髓室底穿通封闭修复材料的可行性。方法：建立髓室底穿通模型,通过HE染色观察,比较CS／n—HA复合材料、n—HA、Dycal、丁香油氧化锌膏剂4种材料的组织修复能力。结果：4种材料中以CS／n—HA组织修复能力最强,且随时间延长效果更加明显。结论：4种材料中CS／n—HA组织修复能力最强,炎症反应最小,愈后最好,有牙骨质样物质将穿孔处封闭,应是理想的髓室底穿通修复材料。     

壳聚糖-纳米羟基磷灰石复合材料修复兔胫骨缺损的成骨效果

目的探讨壳聚糖-纳米羟基磷灰石复合材料(CTS-nHA)植入动物体内修复骨缺损时,观察成骨效果和材料吸收速度。方法新西兰大白兔18只,随机分为3组,实验组(A组):复合材料;实验对照组(B组):纳米羟基磷灰石(N-HA);空白对照组(C组):不植入任何材料,于兔双侧后肢胫骨制造约直径0.5 cm大小的骨缺损,分别植入相应的材料或不植入任何材料,于术后2、4、6、10周取骨缺损标本,进行大体和组织学观察。结果在各时间段CTS-nHA组新的骨基质生成量均明显高于n-HA组和空白组,其中n-HA组又高于空白组,只有在2周时n-HA组和空白组相似。同时复合材料在体内吸收速度比n-HA组快,复合材料的吸收与新骨生成速度协调性似乎欠佳。结论复合材料植入的缺损区具有较强的成骨活性,但在体内吸收速度比较快。     

纳米羟基磷灰石和纳米羟基磷灰石／聚酰胺66直接盖髓术的组织学对比观察

随着科学技术的发展,生物材料在口腔领域引起了人们的广泛关注,羟基磷灰石因与生物体硬组织如骨、牙中的无机成份相似而具有良好的生物相容性,已被广泛用于植骨和盖髓研究。而纳米仿生材料的出现为盖髓剂的研究开拓了一个全新的领域。nHA—PA66作为新型纳米仿生材料的代表成为研究的热点,它已通过前期实验表明具有较好的生物安全性、生物相容性和生物活性其有机物和无机物的组成比例及力学性能与牙本质相似,本实验通过对比纳米羟基磷灰石和纳米羟基磷灰石聚酰胺66作狗牙直接盖髓术的组织学反应,为临床筛选理想的盖髓材料提供组织学依据。     

纳米羟基磷灰石种植体的实验研究

采用冷冻干燥技术制备三种纳米羟基磷灰石支架材料:纳米羟基磷灰石(n-HA)、纳米羟基磷灰石/丝素蛋白(n-HA/SF)、纳米羟基磷灰石/丝素蛋白-壳聚糖(n-HA/SF-CS),并植入实验兔股骨下端缺损处,通过种植体在不同时期的骨结合界面情况的研究,探讨复合体的生物学行为,为建立更为理想的种植体提供理论依据。     

纳米羟基磷灰石／壳聚糖-海藻酸钠复合材料的制备及性能研究

目的研究合理人工骨三元复合生物材料的制备及性能。方法采用共沉淀法制备了纳米羟基磷灰石／壳聚糖-海藻酸钠（n-HAP/CS-ALG）三元复合材料。用XRD、SEM、TEM和IR对材料性能进行表征。结果纳米羟基磷灰石／壳聚糖．海藻酸钠的比例为70／30时形成的复合材料中．n—HAP在有机相中分散均匀。并与有机相发生作用,界面结合牢固;复合材料中的n—HAP呈弱结晶状态,有较高生物活性。壳聚糖和海藻酸钠发生聚合。聚合物对材料起到增强作用。结论该复合材料可望作为骨组织替代材料。     

纳米羟基磷灰石修复牙槽骨缺损的动物实验研究

目的：探讨纳米羟基磷灰石(Nano—HA)的生物学性质和在牙槽骨缺损修复中的成骨机制，为进一步的动物和临床实验提供理论依据。方法：在兔牙槽骨颊侧骨板做人为骨开窗，实验组填入纳米羟基磷灰石，空白对照组不做充填，羟基磷灰石对照组充填普通羟基磷灰石(HA)。于4周、8周、12周分别处死动物做组织学切片。结果：实验组无异物排斥反应，Nano—HA参与骨形成最终被降解。骨修复显著，大量新骨形成，与空白对照组的骨修复比较快速而且活跃。结论：Nano—HA具有良好的生物相容性、生物降解性、骨引导性，能加速牙槽骨的修复，为应用Nano—HA修复牙周病引起的牙槽骨缺损提供了理论依据。     

羟基磷灰石纳米粒子的制备及分散性研究

纳米羟基磷灰石(nHA)能够抑制多种癌细胞的增殖。本文介绍了复分解法制备nHA的过程,讨论并分析了该粉体的几种主要分散技术,提出通过改善生成制备工艺获得具有高分散性和稳定性的nHA,并展望了纳米羟基磷灰石应用前景。     

羟基磷灰石纳米粒子对Hela细胞凋亡作用的研究

目的:观察不同浓度的羟基磷灰石纳米粒子对人宫颈癌Hela细胞作用的影响。方法:将羟基磷灰石纳米粒子以不同浓度和时间作用于人宫颈癌Hela细胞,采用MTT比色法观察细胞毒性;电镜技术观察细胞凋亡的形态;原位末端标记法(TUNEL法)检测细胞凋亡率。结果:羟基磷灰石纳米粒子以剂量依赖和时间依赖的方式抑制人宫颈癌Hela细胞的生长,作用48h后的半数有效抑制浓度IC50值为211.8μg/ml。透射电镜观察到凋亡特性的形态学改变。结论:HAP抑制人宫颈癌Hela细胞细胞的增殖,诱导Hela细胞凋亡,为临床治疗肿瘤提供依据。     

纳米级羟基磷灰石透射电镜鉴定方法

纳米级羟基磷灰石研究倾向于人体骨组织替代材料,目前探索工作最多的是人工材料种植牙镶复.纳米级羟基磷灰石,分两步完成,外购粗原料,在实验室分别用粗、细球磨机进行加工.经精细球磨后可达到纳米级颗粒,但需经电子显微镜(TEM)鉴定确认是否达到纳米级颗粒.     

纳米羟基磷灰石抑制变形链球菌粘附的体外研究

目的：研究纳米羟基磷灰石（Nano-HA）对变形链球菌附着力的影响。方法：将不同浓度的Nano-HA溶液加入到轻唾液体培养基中培养变形链球菌,2d后测定粘附于变形链球菌吸附板表面变形链球菌量．并进行比较;对已附着变形链球菌的吸附板放入Nano-HA溶液中,观察变形链球菌的脱落情况。结果：Nano-HA对变链菌的附着有抑制作用和对已附着的变形链球菌有解粘附的作用。结论：Nano-HA对变链菌的粘附有显著的抑制作用。     

Dual effects of heparin on BMP-2-induced osteogenic activity in MC3T3-E1 cells

Heparin displays several types of biological activities by binding to various extracellular molecules, including pivotal roles in bone metabolism. We have previously reported that heparin competitively inhibits the binding activity of bone morphogenic protein-2 (BMP-2) to BMP and the BMP receptor (BMPR) and suppresses BMP-2 osteogenic activity. In the present study, we examined whether heparin affects osteoblast differentiation induced by BMP-2 at various time points in vitro. We found that 72 h of treatment with heparin inhibited alkaline phosphatase (ALP) activity. However, 144 h of treatment enhanced the ALP activity in BMP-2-stimulated MC3T3-E1 cells. Although heparin decreased the phosphorylation of Smad1/5/8 after 0.5 h of culture, prolonged periods of culture with heparin enhanced the Smad phosphorylation. In addition, 72 h of treatment with heparin enhanced the mRNA expression of runx2 and osterix in BMP-2-stimulated MC3T3-E1 cells. Furthermore, the mRNA expression of BMP antagonists and inhibitory Smads induced by BMP-2 was preferentially blocked by heparin at the 24 and 48 h time points. These findings indicate biphasic effects of heparin on BMP-2 activity and suggest that heparin has complex effects on the BMP-2 osteogenic bioactivities. Prolonged culture with heparin stimulated BMP-2-induced osteogenic activity via down-regulation of BMP-2 antagonists and inhibitory Smads.     

骨形态蛋白2在糖尿病肾病大鼠肾内小动脉中表达的意义

目的观察骨形态蛋白2（BMP-2）在糖尿病肾病（DN）大鼠肾实质内小动脉上的表达及其变化规律,探讨其对糖尿病肾病血管病变的影响。方法设对照组（N组）、DN组两组,建立STZ诱导的DN大鼠模型。茜素红染色观察肾实质内小动脉周围钙盐沉积,并分别采用免疫组化及mRNA原位杂交法检测BMP-2在肾实质内小动脉上的蛋白表达及基因定位。结果 （1）茜素红染色DN组第4~12周时偶见肾内小动脉少许点状红色钙盐沉积,第24周时可见肾实质内小动脉及周围组织有较多红色点片状沉积;N组茜素红染色为阴性。（2）DN组肾实质内小动脉BMP-2蛋白及mRNA在4周时已有表达,随时间延长表达逐渐增强,各时间点均明显高于N组;N组各时间点表达差异无统计学意义。结论 （1）DN大鼠肾内小动脉钙化前即已有BMP-2的表达;（2）BMP-2可能是糖尿病血管钙化的重要转录因子及早期生物学标志之一;（3）肾内小动脉上BMP-2的表达变化可能参与DN进展。     

糖尿病大鼠骨折愈合过程中 VEGF、BMP-2及BMP-7的动态观察

目的：动态观察糖尿病模型大鼠骨折愈合过程中的血管内皮细胞生长因子（ VEGF）、骨形态发生蛋白-2（BMP-2）、骨形态发生蛋白-7（BMP-7）的变化趋势,探索影响糖尿病骨折愈合的机制。方法选取Wester 雄性大鼠70只,采用随机数字表法分为糖尿病组[（腹腔注射链脲佐菌素（ STZ）造模]和对照组,每组35只。采用线锯锯断2组大鼠的左侧胫骨,采用外固定复位处理,观察2组大鼠术后第1、3、5、7周的血清及骨痂中VEGF、BMP-2、BMP-7的表达变化。结果糖尿病组大鼠术后第1周、术后第3周的VEGF、BMP-2、BMP-7的表达水平均显著低于对照组（ P＜0构．05）,术后第5、第7周2组大鼠骨痂组织中的VEGF、BMP-2、BMP-7的表达水平差异无统计学意义（ P ＞0．05）。糖尿病组与对照组大鼠的血清中VEGF、BMP-2、BMP-7在术后第1～7周差异无统计学意义（ P ＞0．05）,糖尿病大鼠术后第1、第3周BMP-7的表达水平显著低于对照组大鼠,差异有统计学意义（ P ＜0．05）。结论糖尿病大鼠血清、骨痂组织中BMP-7、BMP-2及VEGF表达减少可能是其延迟愈合的一个重要原因。     

短期高浓度氟对小鼠磨牙成釉细胞内BMP-2表达的影响

目的通过研究短期高浓度氟对小鼠磨牙成釉细胞形态及骨形成蛋白-2表达的影响,探讨氟对牙釉质发育的作用机制。方法选择4日龄的ICR小鼠共32只,随机分为两组,两组中均等分配实验动物和对照动物,将20mg/k(g体重)和10mg/kg(体重)的NaF分别单次注入实验动物腹腔内,将等剂量的NaCl分别单次注入对照动物腹腔内,注射量均为10μL/g,所有动物24h后被处死。HE染色、免疫组化染色被采用以观察两种浓度氟对小鼠磨牙不同分化阶段成釉细胞形态及骨形成蛋白-2的表达。结果实验组分泌期和过渡期的成釉细胞形态紊乱,对照组动物中BMP-2的表达明显高于实验组动物,差异有统计学意义(P<0.01),而成熟期成釉细胞未见明显变化。结论短期高浓度氟使骨形成蛋白-2在分泌期和过渡期成釉细胞中的表达被抑制,使釉质形成受阻。     

盐酸二甲双胍对成骨细胞增殖、BMP-2及Cbfa-1基因表达的影响

目的初步研究二甲双胍（MF）在体外对小鼠颅盖骨成骨细胞增殖、骨形态发生蛋白一2（BMP一2）及核心结合因子（Cbfa一1）mRNA表达的影响,探讨二甲双胍对骨代谢的可能作用机制。方法（1）分离培养原代颅盖骨成骨细胞并对其进行鉴定。（2）以乳鼠成骨细胞为体外实验模型,不同浓度（0、50、100、200、400Ixmol／L）的MF干预体外培养的成骨细胞24h后,M1vr法检测成骨细胞的增殖能力,实时荧光定量PCR法检测成骨细胞BMP-2及Cbfa-1基因表达。结果二甲双胍干预成骨细胞24h后,可促进成骨细胞的增殖,在浓度400μxmol／L的OD值最大为0．298±0．047（P〈0．05）;可促进BMP-2及Cbfa-1mRNA的表达,呈剂量效应关系。结论二甲双胍可促进成骨细胞的增殖和分化,可能通过调节BMP-2及Cbfa-1的表达,从而促进骨的形成。     

Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway

Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders.     

Inhibition of osteoblast differentiation by aluminum trichloride exposure is associated with inhibition of BMP-2/Smad pathway component expression

Bone morphogenetic protein-2 (BMP-2)/Smad signaling pathway plays an important role in regulating osteoblast (OB) differentiation. OB differentiation is a key process of bone formation. Aluminum (Al) exposure inhibits bone formation and causes Al-induced bone disease. However, the mechanism is not fully understood. To investigate whether BMP-2/Smad signaling pathway is associated with OB differentiation in aluminum trichloride (AlCl₃)-treated OBs, the primary rat OBs were cultured and exposed to 0 (control group, CG), 1/40 IC₅₀ (low-dose group, LG), 1/20 IC₅₀ (mid-dose group, MG), and 1/10 IC₅₀ (high-dose group, HG) of AlCl₃ for 24 h, respectively. We found that the expressions of OB differentiation markers (Runx-2, Osterix and ALP) and BMP-2/Smad signaling pathway components (BMP-2, BMPR-IA, p-BMPR-IA, BMPR-II, p-Smad1/5/8 and p-Smad1/5/8/4) were all decreased in AlCl₃-treated OBs compared with the CG. These results indicated that inhibition of OB differentiation by AlCl₃ was associated with inhibition of BMP-2/Smad pathway component expression. Our findings provide a novel insight into the mechanism of AlCl₃-induced bone disease.     

Synthesis and characterization of chitosan quaternary ammonium salt and its application as drug carrier for ribavirin

Abstract

N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) is hydro-soluble chitosan (CS) derivative, which can be obtained by the reaction between epoxypropyl trimethyl ammonium chloride (ETA) and CS. The preparation parameters for the synthesis of HTCC were optimized by orthogonal experimental design. ETA was successfully grafted into the free amino group of CS. Grafting of ETA with CS had great effect on the crystal structure of HTCC, which was confirmed by the XRD results. HTCC displayed higher capability to form nanoparticles by crosslinking with negatively charged sodium tripolyphosphate (TPP). Ribavrin- (RIV-) loaded HTCC nanoparticles were positively charged and were spherical in shape with average particle size of 200?nm. More efficient drug encapsulation efficiency and loading capacity were obtained for HTCC in comparison with CS, however, HTCC nanoparticles displayed faster release rate due to its hydro-soluble properties. The results suggest that HTCC is a promising CS derivative for the encapsulation of hydrophilic drugs in obtaining sustained release of drugs.     

Fabrication of hybrid scaffold based on hydroxyapatite-biodegradable nanofibers incorporated with liposomal formulation of BMP-2 peptide for bone tissue engineering

In the present study, we fabricated an efficient, simple biomimetic scaffold to stimulate osteogenic differentiation of mesenchymal stem cells (MSCs). Electrospun poly L-lactic acid nanofibers were employed to mimic the nanofibrillar structure of bone proteins and coated with hydroxyapatite nanoparticles to simulate bone minerals. Thereafter, we regulated the release pattern of BMP-2 peptide through covalent attachment of an optimized liposomal formulation to the scaffold. The fabricated platform provided a sustained release profile of BMP-2 peptide up to 21?days while supporting cellular attachment and proliferation without cytotoxicity. In-vitro results confirmed the superiority of the scaffold containing liposomes through enhancement of growth and differentiation of MSCs. Ectopic bone formation model exhibited significant localized initiation of bone formation of liposome incorporated scaffold. Consequently, these findings demonstrated that our designed platform with modified release properties of BMP-2 peptide considerably promoted osteogenic differentiation of MSCs making it a unique candidate for bone regeneration therapeutics.     

Noninvasive imaging of c(RGD)2‐9R as a potential delivery carrier for transfection of siRNA in malignant tumors

The purpose of our study was to develop and evaluate a novel integrin α_vβ₃‐specific delivery carrier for transfection of siRNA in malignant tumors. We adopted arginine‐glycine‐aspartate (RGD) motif as a tissue target for specific recognition of integrin α_νβ₃. A chimaeric peptide was synthesized by adding nonamer arginine residues (9‐arginine [9R]) at the carboxy terminus of cyclic‐RGD dimer, designated as c(RGD)₂‐9R, to enable small interfering RNA (siRNA) binding. To test the applicability of the delivery carrier in vivo, c(RGD)₂‐9R was labeled with radionuclide of technetium‐99m. Biodistribution and γ‐camera imaging studies were performed in HepG2 xenograft‐bearing nude mice. As results, an optimal 10:1 molar ratio of ^99mTc‐c(RGD)₂‐9R to siRNA was indicated by the electrophoresis on agarose gels. ^99mTc‐c(RGD)₂‐9R/siRNA remained stable under a set of conditions in vitro. For in vivo study, tumor radioactivity uptake of ^99mTc‐c(RGD)₂‐9R/siRNA in nude mice bearing HepG2 xenografts was significantly higher than that of control probe (P  < .05). The xenografts were clearly visualized at 4 hours till 6 hours noninvasively after intravenous injection of ^99mTc‐c(RGD)₂‐9R/siRNA, while the xenografts were not visualized at any time after injection of control probe. It was concluded that c(RGD)₂‐9R could be an effective siRNA delivery carrier. Technetium‐99m radiolabeled‐delivery carrier represents a potential imaging strategy for RNAi‐based therapy.