RNAⅢ抑制肽抑制葡萄球菌在人工关节材料表面粘附的实验研究

[目的]通过体外实验验证RNAⅢ抑制肽(RIP)能否抑制葡萄球菌在人工关节材料表面黏附,探讨其用于人工关节感染治疗的可行性。[方法]制作超高分子聚乙烯、钛合金和钴铬钼合金试样,葡萄球菌经FITC标记,人工关节材料试样消毒后接种FITC标记的葡萄球菌,每种试样4组,分别为空白组、RIP组、左氧沙星组和联合组,将含有细菌、试样和药物的24孔板在37℃下孵育30min后,用荧光显微镜观察。[结果]三种材料的用药组细菌光点计数(P<0.001)和覆盖面积(P<0.05)均显著低于空白组;左氧组细菌光点计数少于RIP组,但两组的细菌覆盖面积无差别;联合组光点计数和细菌覆盖面积均少于左氧组和RIP组,差别显著(P<0.01)。[结论]RIP可以抑制表皮葡萄球菌对关节假体材料表面的粘附,如果与抗生素联合应用可以起协同作用。     

大蒜素对表皮葡萄球菌在人工关节材料表面粘附的影响

目的探讨大蒜素对表皮葡萄球菌在人工关节材料超高分子聚乙烯和钛合金表面粘附的影响。方法实验分为4组(其中按大蒜素干预的浓度不同具体细分):生理盐水干预组;1 mg/L大蒜素浓度干预组;2 mg/L大蒜素浓度干预组;4 mg/L大蒜素浓度干预组。利用细菌计数和生物膜定量实验观察不同浓度大蒜素干预后对表皮葡萄球菌在超高分子聚乙烯(UHMWPE)和钛合金表面粘附及生物膜形成的影响。细菌粘附数和生物膜量等计量资料使用单因素方差分析评估,组间两两比较使用LSD法。结果在大蒜素浓度分别为0 mg/L、1mg/L、2 mg/L、4 mg/L干预下,单位面积的超高分子聚乙烯材料表面粘附的细菌数分别为(36 312±6 522)CFU/mm2、(30 624±8 728)CFU/mm2、(13 425±6 026)CFU/mm2、(681±249)CFU/mm2,组间差异有统计学意义(F=61.52,P0.05)。钛合金螺钉表面细菌数分别为(76 113±6 504)CFU、(71 155±7 241)CFU、(18 611±6 549)CFU、(7 066±1 249)CFU,组间差异有统计学意义(F=325.42,P0.05)。超高分子聚乙烯表面生物膜吸光度值分别为(4.38±0.55)、(4.17±0.67)、(2.20±0.69)、(0.62±0.38),组间差异有统计学意义(F=81.84,P0.05)。钛合金螺钉表面的生物膜量分别为(3.43±0.51)、(3.29±0.53)、(2.74±0.58)、(0.59±0.25),组间差异有统计学意义(F=66.31,P0.05)。结论亚抑菌浓度的大蒜素能够抑制表皮葡萄球菌在UHMWPE和钛合金材料表面粘附,抑制生物膜在上述材料的形成。     

结核分枝杆菌对材料粘附能力的体外实验研究

目的：通过体外实验观察结核分枝杆菌对内植物材料的粘附情况，从细菌粘附角度出发探讨脊柱结核内固定术的安全性问题，方法：以表皮葡萄球菌为对照，在表皮葡萄球菌、结核分枝杆菌菌液中分别加人不同材料，扫描电镜观察，比较不同细菌对不同材料(钛合金、不锈钢)、不同表面(光滑面、粗糙面)的粘附情况。结果：结核分枝杆菌对两种材料的粘附均较表皮葡萄球菌少，粗糙表面所粘附的细菌量较光滑面多。结论：结核分枝杆菌对内植物材料的低粘附性可能是临床脊柱结核内固定术安全的原因之一。     

钛铌锆β钛合金生物相容性的体外实验研究

[目的]通过体外实验对自行研发的低弹性模量、高强度新型β型钛合金人工关节假体材料生物相容性进行评价。[方法]（1）采用L-929细胞（小鼠成纤维细胞）对合金进行细胞毒性试验,将细胞相对增殖率（RGR）转换成6级材料毒性进行评级。（2）将1μm左右的钛铌锆合金（Ti-Nb-Zr）颗粒与J774A.1巨噬细胞体外共同培养24～48h后,采用倒置相差显微镜和透射电镜观察细胞变化、RT-PCR方法测定IL-6和TNF-α表达、ELISA法测定细胞培养上清TNF-α等方法对Ti-Nb-Zr的生物相容性进行评价,并与传统的人工关节假体材料进行比较。[结果]Ti-Nb-Zr的细胞毒性为0级。吞噬了Ti-Nb-Zr颗粒的J774A.1巨噬细胞形态改变明显轻于钴铬钼颗粒组和钛铝钒颗粒组。巨噬细胞与钛铝钒合金颗粒、钴铬钼合金颗粒和Ti-Nb-Zr颗粒共同培养48h后都有IL-6和TNF-αmRNA表达的增加,钴铬钼颗粒和钛铝钒颗粒引起增加更加明显。ELISA方法测定显示：巨噬细胞吞噬Ti-Nb-Zr颗粒48h后分泌TNF-α的量明显低于钛铝钒和钴铬钼（P〈0.05）。[结论]低弹性模量Ti-Nb-Zr钛合金具有优良的体外组织相容性,是一种有前途的骨科内植物材料。     

人工关节磨损微粒对体外培养外周血单核细胞的影响

目的：研究人工关节磨损微粒诱发骨溶解的机理，探讨不同微粒物质倡导骨溶解能力的差异。方法：通过体外培养人外周血单核细胞对超高分子聚乙烯微粒入钛合金（Ｔｉ６Ａ１４Ｖ）微粒反应，检测培养上清液中肿瘤坏死因子（ＴＮＦ－α）含量。结果：超高分子聚乙烯微粒和钛合金微粒均可刺激单核细胞，使之产生更多的ＴＮＦ－α，与对照组相比样差异有统计学意义（Ｐ〈０．０１）。但超高分子聚乙烯组与钛合金组的ＴＮＦ－α含量比较差异     

人工关节金属磨损颗粒体外制备分离方法的实验研究

[目的]设计一种体外制备、分离人工关节金属磨损颗粒的方法，并验证这种颗粒用于医学实验的可行性。[方法]用钛铝钒合金、钴铬钼合金材料分别制成球磨罐（国家发明专利，申请号：03142073．7），球磨罐内装有用同种材料制成的磨块；向该球磨罐内注入模拟生物体液；震动球磨得到颗粒混悬液。梯度离心获得金属颗粒。对颗粒进行：（1）元素成分鉴定；（2）颗粒大小鉴定和粒度分析；（3）扫描电镜对颗粒的表面形态观察；（4）将颗粒与J774A．1巨噬细胞共同培养，观察细胞吞噬颗粒的情况。[结果]通过此方法成功产生并分离出大量直径1μm左右的钛合金和钴铬钼合金颗粒。（1）元素分析证实整个处理过程中无杂质成分污染；（2）钛合金颗粒的平均直径Dv90：1．009，钴铬钼颗粒的平均直径Dv90：1．008，粒度分布曲线基本成正态；（3）扫描电镜图象显示颗粒大小均匀，形状多为不规则，与体内颗粒极为相似；（4）J774A．1巨噬细胞能完整吞噬2种材料的颗粒。[结论]此方法能持续大量产生人工关节假体金属材料的磨损颗粒，产生的颗粒能在各方面很好地模拟体内磨损颗粒，为今后人工关节假体松动相关研究的体内、体外研究提供可靠的颗粒来源。     

碳增强的聚醚醚酮作为髋臼假体材料的实验研究

目的：评价碳纤维增强的聚醚醚酮（CFPEEK）作为髋臼假体材料的生物相容性和耐磨损性。方法：（1）采用国产材料和工艺研制出碳纤CFPEEK复合材料，在模拟体液环境下对其本身以及与不同磨擦偶的耐磨损性能进行测试，并与传统髋臼假体材料超高分子聚乙烯（UHMWPE）作比较。（2）体外研究CFPEEK磨屑对巨噬细胞ANA-1释放白细胞介素1(IL-1β)和肿瘤坏死因子α（TNF-α）的影响，并与传统的人工关节材料比较，以确定各材料间生物学反应的差异。结果：（1）CFPEEK的体积磨损率只为UHMWPE的1/2，耐磨损性能明显优于UHMWPE(Alpha=0.01水平)。CFPEEK与钴铬钼合金（CoCrMo）对磨将产生最少的磨屑（Alpha=0.01水平）。CFPEEK与CoCrMo对磨时的摩擦系数最低。（2）各实验组IL-1β和TNF-α的释放都有增高，实验结果总体差异有显著性意义（P=0.0001），UHMWPE组明显高于CFPEEK组（Alpha=0.01水平）。结论：CFPEEK材料具耐磨损、生物相容性好的特点，是一种未来髋臼假体的理想材料。     

改良大鼠气囊模型在人工关节假体松动研究中的应用

[目的]设计并改良大鼠气囊模型（the air pouch model），证实该模型在人工关节松动相关研究中的应用价值。[方法]（1）改良大鼠气囊模型的建立：健康成年SD大鼠18只，背部皮下注入空气20ml，48h后补充注入空气10ml形成气囊。形成气囊24h后气囊穿刺，注入PBS液5ml，6、24、48h分别穿刺抽液，收集囊内液体采用ELISA方法测定IL-6和TNF—Ot；（2）改良大鼠气囊模型对不同关节材料磨损颗粒生物反应的比较：健康成年SD大鼠24只，皮下充气造模后将钴铬钼、钛铝钒和钛铌锆合金（TNZ）颗粒混悬液注入气囊，收集囊内液体采用ELISA方法测定IL-6和TNF—α，用囊壁组织学切片进行炎症细胞反应分级并测量囊壁厚度。[结果]（1）在SD大鼠皮下注入空气20ml可以形成饱满的气囊，大鼠术后活动正常。48h后气囊由于气体吸收体积减小，张力下降。再次注射10ml空气后张力恢复。PBS能使囊内液体的TNF—α在6h左右出现一个高峰达239．30pg／ml，与24h和48h比较，差异有显著统计学意义（P〈0．05）。IL-6的测定值没有TNF—α那样的6h分泌高峰。6、24、48h之间无显著性差异（P〉0．05）；（2）钻铬钼、钛铝钒、TNZ3种颗粒注入气囊48h后都能引起TNF—α分泌量显著升高（P〈0．05），两两比较，钛铝钒组明显高于钴铬钼组和TNZ组（P〈0．05）。3种材料均不能引起IL-6分泌的显著增加（P〉0．05）。TNZ组气囊囊壁厚度明显小于钴铬钼组和钛铝钒组（P〈0．001）。[结论]改良大鼠气囊模型具有造模简单、对人工关节颗粒反应快速敏感的特点，是一种人工关节松动相关实验研究有效的动物模型。     

去甲万古霉素载药涤纶血管材料体外抗菌活性测定

目的：评价去甲万古霉素（NV）载药涤纶血管材料体外抗菌活性。方法：血管材料剪成直径6mm圆形。分别制成空白涤纶材料和载药（NV）涤纶材料，分别于0，7，14，28d取样观察体外缓释性能。选用耐甲氧西林表皮葡萄球菌（MRSE）和耐甲氧西林金黄色葡萄球菌（MRSA）作为试验菌，分别观察空白和载药涤纶血管材料0．7，14，28d和快慢相的抗菌效果。结果：两种细菌0，7，14，28d载药涤纶材料-细菌混合培养液的菌落数显著少于空白涤纶材料-细菌混合培养液（P均〈0．001）。结论：体外试验证实载NV涤纶材料MRSE和MRSA具有抗菌作用，且可持续维持28d。提示该载药材料具有持久抗菌活性，可用于制备抗感染血管移植物。     

脊柱结核内固定的生物学基础

目的观察结核杆菌与不同内固定材料的粘附情况,以期初步探讨在脊柱结核病灶清除后植入内固定选择何种材料的问题。方法在结核杆菌的菌液分别加入四种不同的内固定材料（不锈钢、钛、钛合金、聚醚醚酮）,通过碘-125标记测量,比较细菌与材料之间的粘附情况。结果四种材料中,细菌在聚醚醚酮上的粘附最多,在纯钛上粘附最少,在不锈钢的粘附多于钛合金（P〈0.05）。结论在脊柱结核患者内固定材料的选择上,以纯钛为首选,钛合金次之,碳纤维材料慎用。     

Bacterial adherence to tantalum versus commonly used orthopedic metallic implant materials

OBJECTIVES: Evaluation of bacterial adhesion to pure tantalum and tantalum-coated stainless steel versus commercially pure titanium, titanium alloy (Ti-6Al-4V), and grit-blasted and polished stainless steel. DESIGN: Experimental in vitro cell culture study using Staphylococcus aureus and Staphylococcus epidermidis to evaluate qualitatively and quantitatively bacterial adherence to metallic implants. METHODS: A bacterial adhesion assay was performed by culturing S. aureus (ATCC 6538) and S. epidermidis (clinical isolate) for one hour with tantalum, tantalum-coated stainless steel, titanium, titanium alloy, grit-blasted and polished stainless steel metallic implant discs. Adhered living and dead bacteria were stained using a 2-color fluorescence assay. Adherence was then quantitatively evaluated by fluorescence microscopy and digital image processing. Qualitative adherence of the bacteria was analyzed with a scanning electron microscope. The quantitative data were related to the implant surface roughness (Pa-value) as measured by confocal laser scanning microscopy. RESULTS: Bacterial adherence of S. aureus varied significantly (p = 0.0035) with the type of metallic implant. Pure tantalum presented with significantly (p < 0.05) lower S. aureus adhesion compared to titanium alloy, polished stainless steel, and tantalum-coated stainless steel. Furthermore, pure tantalum had a lower, though not significantly, adhesion than commercially pure titanium and grit-blasted stainless steel. Additionally, there was a significantly higher S. aureus adherence to titanium alloy than to commercially pure titanium (p = 0.014). S. epidermidis adherence was not significantly different among the tested materials. There was no statistically significant correlation between bacterial adherence and surface roughness of the tested implants. CONCLUSIONS: Pure tantalum presents with a lower or similar S. aureus and S. epidermidis adhesion when compared with commonly used materials in orthopedic implants. CLINICAL IMPLICATION: Because bacterial adhesion is an important predisposing factor in the development of clinical implant infection, tantalum may offer benefits as an adjunct or alternative material compared with current materials commonly used for orthopedic implants.     

Bacterial adherence to bioinert and bioactive materials studied in vitro

In vitro, bioinert stainless steel and titanium alloy, and bioactive sintered hydroxyapatite and hydroxyapatite-coated titanium materials were exposed to Staphylococcus epidermidis to study bacterial adhesion. Scanning electron microscopy showed that fibrous strands interconnected the adherent bacteria, and that background matrix enclosed bacterial colonies. This adherent mode of growth may reduce the susceptibility of the bacteria to host clearance mechanisms and antibiotic therapy. Adherence assays revealed that bacterial adherence to sintered hydroxyapatite was higher than to the other 3 materials.     

In vitro adherence and accumulation of Staphylococcus epidermidis RP 62 A and Staphylococcus epidermidis M7 on four different bone cements

Bacterial resistance of Staphylococcus epidermidis, a serious pathogen of implant-related infections, to antibiotics is related to the production of a glycocalyx slime that impairs antibiotic access and the killing by host defense mechanisms. In vitro studies of different bone cements containing antibiotics, developed for the prevention of biomaterial-associated infection, could not always demonstrate complete eradication of biomaterial-adherent bacteria. We have investigated four different bone cements in regard to bacterial accumulation of a slime-producing strain RP 62 A and its isogenic mutant M7 lacking the ability to produce exopolysaccharide slime using a bacterial adhesion assay and modified Kirby-Bauer technique. A significant effect of exopolysaccharide production for the accumulation on bone cement could be demonstrated. The gentamicin/clindamycin bone cement was the only tested biomaterial that produced a large zone of bacterial inhibition in the inoculated area adjacent to the biomaterial. The bacterial adhesion was not reduced significantly and there was no correlation between zones of inhibition on blood agar plates and the quantitative adhesion assay. The clinical efficacy of the gentamicin/clindamycin bone cement must be proven in vivo.     

The effect of apo-transferrin on bacterial adhesion to biomaterials

Apo-transferrin (apo-Tf), the iron deficient form of Tf, has been identified previously as a potent inhibitor of Staphylococcus epidermidis adhesion to polyurethane surfaces. In this study, the ability of apo-Tf to suppress the adhesion of two other strains of bacteria, namely a Gram-positive Staphylococcus aureus and a Gram-negative Pseudomonas aeruginosa to several biomaterials, including polystyrene, polymethylmethacrylate, and silicone, is documented. The presence of apo-Tf in the medium at 20 microg/ml lowered bacterial adhesion to all tested biomaterials more than fourfold. Moreover, apo-Tf exerted its inhibitory activity even when protein coated surfaces were used as substrates for bacterial adhesion. To emphasize the importance of apo-Tf in the prevention of bacterial adhesion, human serum was depleted of Tf, employing affinity chromatography, and was shown to lose its inhibitory activity toward bacterial adhesion. Upon addition of apo-Tf to Tf-depleted serum, the activity was reestablished, resulting in a marked reduction in the number of bacteria adhered to the surfaces. Following the enzymatic deglycosylation, apo-Tf retained its ability to prevent bacterial adhesion. These results indicate that the carbohydrate moiety does not seem to play a role in this activity. The presented data provide the evidence that the inhibitory activity of apo-Tf is not bacterial strain specific and that the presence of apo-Tf in the medium results in a significant reduction of bacterial adhesion to a variety of neat and/or protein coated surfaces.     

Role of fibronectin in staphylococcal adhesion to metallic surfaces used as models of orthopaedic devices

Staphylococcal infection of various prosthetic and internal fixation devices is a major complication associated with orthopaedic surgery. This study investigated the role of the host protein fibronectin in promoting adhesion of Staphylococcus aureus and Staphylococcus epidermidis to metallic surfaces representing materials used for orthopaedic devices. Pure human fibronectin was adsorbed in vitro onto coverslips (0.8 × 0.8 cm) of stainless steel, pure titanium, or titanium-aluminum-niobium alloy. In vitro bacterial adhesion was promoted more strongly by the metallic surfaces coated with fibronectin than by albumin-coated controls for two strains of S. aureus and one strain of S. epidermidis. Furthermore, with the fibronectin-coated coverslips, bacterial adhesion to titanium alloy was significantly greater than adhesion to stainless steel. Adhesion of the three staphylococcal strains was promoted more strongly by coverslips explanted from the subcutaneous space of guinea pigs and tested under similar conditions than by albumin-coated controls. Incubation of either in vitro fibronectin-coated or explanted metallic coverslips with anti-fibronectin antibodies produced a significant decrease in staphylococcal adhesion. These results suggest that the presence of fibronectin on the surface of implanted metallic devices is an important determinant of colonization of orthopaedic biomaterials by staphylococi.     

Prospective clinical study on the efficacy of bacterial removal with mechanical debridement in and around chronic leg ulcers assessed with fluorescence imaging

Bacterial colonisation in wounds delays healing, mandating regular bacterial removal through cleaning and debridement. Real‐time monitoring of the efficacy of mechanical debridement has recently become possible through fluorescence imaging. Red fluorescence, endogenously produced during bacterial metabolism, indicates regions contaminated with live bacteria (>10⁴ CFU/g). In this prospective study, conventional and fluorescence photos were taken of 25 venous leg ulcers before and after mechanical debridement, without use of antiseptics. Images were digitally segmented into wound bed and the periwound regions (up to 1.5 cm outside bed) and pixel intensity of red fluorescence evaluated to compute bacterial area. Pre‐debridement, bacterial fluorescence comprised 10.4% of wound beds and larger percentages of the periwound area (~25%). Average bacterial reduction observed in the wound bed after a single mechanical debridement was 99.4% (p<0.001), yet periwound bacterial reduction was only 64.3%. On average, across bed and periwound, a single mechanical debridement left behind 29% of bacterial fluorescence positive tissue regions. Our results show the substantial effect that safe, inexpensive, mechanical debridement can have on bacterial load of venous ulcers without antiseptic use. Fluorescence imaging can localise bacterial colonised areas and showed persistent periwound bacteria post‐debridement. Fluorescence‐targeted debridement can be used quickly and easily in daily practice.     

钽涂层对骨髓间充质干细胞的黏附增殖及成骨分化的影响

目的探讨钽涂层金属在体外促进大鼠骨髓间充质干细胞(BMSCs)黏附、增殖及成骨分化方面的作用。方法在体外取6只6周龄SD大鼠BMSCs进行原代培养至第3代,使用化学气相沉积系统自行制备钽涂层金属。然后进行流式细胞术鉴定、BMSCs三向诱导、荧光染色、细胞增殖检测及实时荧光定量聚合酶链反应技术(Q-PCR)试验。观察比较BMSCs在钛合金圆片(Ti6Al4V组)和钽金属圆片(Ta组)表面黏附的BMSCs数量、增殖速率、成骨细胞特异性转录因子(OSX)、Runt相关转录因子2(RUNX2)、骨粘连蛋白(OSN)和骨桥蛋白(OPN)的表达情况。结果流式细胞术结果显示CD44(94.55%)、CD90(95.01%)、CD34(0.06%)。诱导成骨分化14 d后碱性磷酸酶(ALP)染色阳性;诱导成骨分化21 d后出现茜素红钙化结节;成脂诱导21 d后油红O染色呈阳性;成软骨诱导21 d后阿利新蓝染色评估有软骨形成能力。激光共聚焦显微镜结果显示BMSCs在Ti6Al4V组和Ta组金属圆片表面贴附生长,细胞间互相接触、聚集成片,Ta组金属圆片上黏附的BMSCs数量明显多于Ti6Al4V组,并且具有更好的延展性能。细胞增殖检测结果发现,分别共培养1、3、5、7 d后Ta组BMSCs的增殖速率明显快于Ti6Al4V组,差异均有统计学意义(P<0.05)。Q-PCR结果发现,与Ti6Al4V组相比,体外培养7 d后Ta组金属圆片更能促进OSN和OPN的表达,差异有统计学意义(P<0.05);体外共培养21 d后,Ta组金属圆片更能促进OSX、RUNX2、OSN和OPN的表达,差异均有统计学意义(P<0.05)。结论相比于传统的骨科植入物钛合金而言,钽涂层金属能够更好地促进BMSCs的黏附,增殖和成骨分化。     

Titanium Surface Chemical Composition Interferes in the Pseudomonas aeruginosa Biofilm Formation

Bacterial adhesion on three different surfaces: untreated Ti, plasma nitriding, and plasma carbonitriding Ti substrates were investigated. The samples were placed in bacterial cultures of Pseudomonas aeruginosa to assess biofilm formation. The correlation between the amount of bacteria attached to the surface after a lapse of time with nanotopography and physicochemical properties was performed. TiN showed the highest capacity to avoid bacterial adhesion, while presenting intermediate roughness and wettability. Although the surface of TiCN had the highest surface roughness and low contact angle (high wettability), bacterial adhesion was intermediate on this sample. Untreated Ti, even though presenting a smooth surface and low wettability, had the highest tendency to form biofilms.