感谢陕西省自然科学基金（批准号：2021SF-118）和国家自然科学基金（批准号：81973409,82204631）的财政支持。

这些动物是从习交通大学购买的。所有动物实验程序均经习交通大学动物伦理学委员会批准（批准号：SCXK [陕西] 2021-103）。将小鼠饲养一周，每个笼子饲养5只小鼠。他们被允许免费获得食物和水。在进行研究之前，将动物保持在室温（RT;24°C±1°C）和光/暗循环（12小时/12小时）下。
1.缓冲液和设备准备
<ol><li>白色念珠菌 细胞培养<ol><li>将酵母提取物蛋白胨葡萄糖 （YPD） 平板培养基 中的白色念珠菌 单克隆菌落 （SC5314） 接种到 5 mL YPD 液体培养基（YPD + 50 μg/mL 羧苄青霉素）中。</li>
		<li>随后在30°C下以220rpm的速度摇动 白色念珠菌 细胞过夜。</li>
		<li>在室温下以400× g 离心悬浮液5分钟，将 白色念珠菌 细胞重悬于生理盐水中，并通过目视调节浊度将细胞浓度稀释至1×106 个细胞/ mL与0.5McFarland相同。</li>
	</ol></li>
	<li>生理盐水的制备<ol><li>称取 0.9 g 氯化钠，溶于 100 mL 去离子水中，制备 0.9% 生理盐水。</li>
	</ol></li>
	<li>手术器械制备<ol><li>使用前高压灭菌（121°C，30分钟）手术器械（剪刀，镊子，止血钳，针架，缝合针）和钛镍合金丝（直径约0.5mm）。</li>
	</ol></li>
</ol>2. 小鼠PJI模型建立
<ol><li>将30只C57BL/6小鼠（雄性，15-20g）随机分为3组（10只小鼠/组），即对照组、空白植入组（钛镍丝植入无 白色念珠菌 感染）和PJI组（钛镍丝植入伴 白色念 珠菌感染）。</li>
	<li>用1-4%异氟烷吸入麻醉小鼠，然后去除左后肢上的毛发并用碘消毒。矫正反射的丧失和对脚趾刺激无反应证实了麻醉的深度。麻醉时，在双眼上涂抹眼药膏，以防止角膜干燥，并在手术和恢复期间补充热量。</li>
	<li>对于对照组中的小鼠，不提供任何治疗。为他们提供免费的水和食物。</li>
	<li>对于空白植入物组和PJI组的小鼠，用#5刀片或无菌剃须刀在每个左后肢的膝盖上做一个10毫米的纵向切口，以暴露关节。</li>
	<li>通过插入无菌注射器（26G）针头，在股骨髓内管上打一个5毫米长的孔。</li>
	<li>在用剪刀切割之前，将光滑的镍钛合金线（直径 0.5 毫米，长 5 毫米）插入孔中（图 1）。</li>
	<li>对于空白植入物组中的小鼠，在使用尼龙缝合线（直径0.15mm）逐层闭合伤口之前，沿着镍钛合金丝逐滴加入2μLYPD培养基。</li>
	<li>对于PJI组中的小鼠，将2μL白色 念珠菌 细胞（1×106个细胞 / mL）沿镍钛合金丝逐滴接种到小鼠的关节空间中，然后使用尼龙缝合线逐层闭合伤口。</li>
	<li>将小鼠饲养 14 天，免费获得水和食物。每 24 小时皮下注射 （4 mg/kg） 给药美洛昔康，最多 3 天。</li>
	<li>14天后，用3%异氟醚麻醉小鼠，然后通过颈椎脱位对小鼠实施安乐死。</li>
</ol>3. PJI模型评估
<ol><li>主要器官感染的评估<ol><li>安乐死后从小鼠身上收集肾脏、肝脏和脾脏。</li>
		<li>在每个器官中加入500μL无菌生理盐水，并在4°C的匀浆器上研磨组织。</li>
		<li>将步骤3.1.2中制备的100μL匀浆加入YPD板中，然后用弯曲的棒均匀铺开。</li>
		<li>将YPD板倒置置在37°C培养箱中48小时。</li>
		<li>目视观察并计算菌落数量。</li>
	</ol></li>
	<li>观察植入物上的 白色念珠菌 和生物膜<ol><li>在用镊子收集植入物之前，用剪刀小心地剪开小鼠关节上的皮肤。</li>
		<li>将植入物浸入2.5%戊二醛溶液中，以便在4°C下固定48小时。</li>
		<li>用无菌PBS冲洗植入物三次，然后将其浸入1%锇酸溶液中3小时。</li>
		<li>用无菌PBS冲洗植入物三次，然后将其浸入50%，70%，80%，90%和100%乙醇溶液中15分钟进行脱水。</li>
		<li>在冷冻干燥植入物之前，将植入物浸入叔丁醇中 30 分钟三次。</li>
		<li>将植入物样品固定在样品台上，用金（10 nm涂层）溅射涂覆植入物，并在高真空和1.5 kV的扫描电子显微镜（SEM）下观察它。</li>
	</ol></li>
	<li>股骨组织的病理分析<ol><li>对小鼠实施安乐死后用剪刀收集股组织。</li>
		<li>将股骨组织浸入4%多聚甲醛溶液中，在4°C下固定48小时。</li>
		<li>将股骨组织置于10%福尔马林中1周。</li>
		<li>将股组织分别浸入50%、70%、80%、90%和100%乙醇溶液中15分钟，使股组织脱水。</li>
		<li>将脱水的股组织嵌入石蜡中，然后使用切片机将组织切成4μm样品。</li>
		<li>在病理分析之前，按照标准方案用苏木精和伊红染色股骨切片21.</li>
	</ol></li>
</ol>

建立 白色念珠菌 生物膜相关 PJI 的小鼠模型

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作者声明，他们没有已知的相互竞争的经济利益或个人关系，这些利益或关系可能会影响本文所报告的工作。

手术过程中手术器械或手术环境的污染引起的感染是大多数植入物感染的主要原因24,25,26,27。因此，本研究构建了小鼠白色念珠菌生物膜相关PJI模型。与传统的PJI模型相比，以悬浮在盐水中的无菌不锈钢颗粒为植入物，本研究使用常用的植入物材料镍钛合金丝来模拟白色念珠菌、植入物?...

白色念珠菌（白色念珠菌）共存于人体的许多部位1，这也是最常见的机会性病原体，可引起危及生命的侵袭性真菌感染，尤其是在免疫功能低下的患者中2,3。白色念珠菌可以作为多态性真菌在酵母和菌丝体状态之间转化。菌丝体状态表现出更高的毒力、更强的粘附性以及对细胞和组织的侵袭 4,5。此外，白色念珠菌可以在假牙、导管和支架等生物医学材料的表面形成生物膜1,6,7。生物膜致密的三维结构限制了抗真菌药物的浸润，表达耐药基因，下调真菌细胞的代谢以抵抗免疫系统清除6,7。因此，与生物膜相关的感染在临床中非常具有挑战性8.
金黄色葡萄球菌、凝固酶阴性葡萄球菌和肠杆菌是引起PJI9的主要病原体。虽然真菌PJI的发生率相对较低（约1%）10，但真菌PJI的治疗成本较高11，治疗周期较长11，治疗成功率低于细菌PJI10。近年来，真菌PJI的发病率逐年增加10。念珠菌PJI占真菌PJI 10,12的77%-84%，白色念珠菌是念珠菌中最常见（54%）。因此，需要研究真菌PJI。
目前，PJI 通过翻修手术进行治疗，包括 （1） 移除受感染的植入物、（2） 清创术、（3） 抗菌治疗和 （4） 再植入。彻底清创后，放置含有骨水泥的抗生素，并对患者进行全身抗生素治疗 6 周以上，以有效控制感染，然后再放置新的植入物13。然而，这种方法不能完全消除组织中的病原体，并且用长期抗菌治疗治疗的复发性感染极有可能在耐药菌株中发展14,15,16。
建立PJI的动物模型对于PJI新药或疗法的研发具有重要意义。在PJI的发展中，假体周围形成大的死腔，导致血肿的形成，血肿进一步阻塞周围组织的血液供应并损害抗生素的作用11,15。由于难以模拟假体的周围环境，传统的动物模型无法准确模拟PJI17,18的实际情况。
在本文中，通过使用临床上广泛使用的钛镍丝来模拟关节植入物，在小鼠中构建了白色念珠菌生物膜相关的PJI模型19,20。该PJI模型具有操作简单、成功率高、重复性高、临床相关性高等优点。有望成为研究白色念珠菌生物膜相关PJI防治的重要模型。

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a periprosthetic joint candida albicans infection model in mouse

假体周围关节感染 （PJI） 是由 白色念珠菌 （C. albicans） 引起的常见感染之一， 越来越受到外科医生和科学家的关注。通常，可以在感染部位形成可以保护 白色念珠菌 免受抗生素和免疫清除的生物膜。手术包括切除受感染的植入物、清创术、抗菌治疗和再植入术是治疗 PJI 的金标准。因此，建立动物PJI模型对于PJI新药或新疗法的研发具有重要意义。在这项研究中，将一根光滑的镍钛合金丝（骨科诊所广泛使用的植入物）插入 C57BL/6 小鼠的股骨关节，然后沿线将 白色念珠菌 接种到关节腔中。14天后，在扫描电子显微镜（SEM）下在植入物表面观察到成熟和厚厚的生物膜。在感染关节标本的H&E染色中发现骨小梁明显减少。综上所述，建立了具有操作简便、成功率高、重复性高、临床相关性高等优点的小鼠PJI模型。这有望成为白色 念珠菌 生物膜相关PJI预防临床研究的重要模型。

Candida albicans (C. albicans) commensally reside in many parts of the human body1, which is also the most common opportunistic pathogen that causes life-threatening invasive fungal infections, especially in immunocompromised patients2,3. C. albicans can transform between yeast and mycelium states as a polymorphic fungus. The mycelium state exhibits higher virulence, stronger adhesion, and invasion of cells and tissues4,5. Besides, C. albicans can form biofilms on the surfaces of biomedical materials such as dentures, catheters, and stents1,6,7. The dense three-dimensional structure of biofilms restricts the infiltration of antifungal drugs, expresses drug-resistant genes, and down-regulates the metabolism of fungal cells to resist immune system clearance6,7. Therefore, biofilms-related infections&#160;are quite challenging in clinics8.
Staphylococcus aureus, coagulase-negative staphylococcus, and enterobacter are the main pathogens causing PJI9. Although the incidence of fungal PJI is relatively low (about 1%)10, the treatment cost of fungal PJI is higher11, the treatment cycle is longer11, and the treatment success rate is lower10 than bacterial PJI. In recent years, the incidence of fungal PJI has been increasing year-by-year10. Candida PJI accounts for 77%-84% of fungal PJI10,12, and C. albicans is the most common in Candida (54%). Therefore, fungal PJI needs to be studied.
Currently, PJI is treated via revision surgery by (1) removing the infected implant, (2) debridement, (3) antimicrobial treatment, and (4) reimplantation. After thorough debridement, an antibiotic containing bone cement is placed, and the patient is treated with antibiotics systemically for more than 6 weeks to effectively control the infection before a new implant is placed13. However, this method cannot fully eliminate pathogens in tissues, and recurrent infections treated with long-term antimicrobial therapy are highly likely to develop in drug-resistant strains14,15,16.
Establishing animal models of PJI is important for the research and development of new drugs or therapeutics for PJI. In the development of PJI, large dead spaces are formed around the prosthesis, leading to the formation of hematomas, which further block the blood supply of the surrounding tissues and impair the effect of antibiotics11,15. Due to the difficulty in mimicking the surrounding environment of the prosthesis, traditional animal models cannot accurately simulate the actual situation of PJI17,18.
In this paper, a C. albicans biofilm-associated PJI model in mice was constructed by using a clinically widely used titanium-nickel wire to simulate joint implants19,20. This PJI model exhibits the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation. It is expected to be an important model for studying the prevention and treatment of C. albicans biofilm-related PJI.

作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

小鼠假体周围关节 白色念珠菌 感染模型

In this study of Candida albicans biofilm-associated peri-prosthetic joint, Candida albicans infection model in mice was constructed, using titanium-nickel wire, commonly used to simulate joint implants. This PJI model is easy to operate, has a high success rate, and is clinically relevant. It will help understanding the prevention and treatment of Candida albicans biofilm-related PJI.Traditional animal models cannot accurately simulate the actual situation of PJI, due to the complex environment surrounding the processes. Candida albicans biofilm-related mouse PJI model, can address the research gap. The establishment of PJI animal model is very important for the research and the development of new drugs or therapeutics for PJI.The Candida albicans biofilm-associated mouse PJI model established in this paper is expected to be an important model for the prevention and treatment of Candida albicans biofilm-associated PJI.

将样品转移到平板培养基上并在孵育过夜后计数菌落通常用于评估病灶附近的局部病原体载量22,23。在我们的研究中，肝脏、肾脏和脾脏样本的微生物培养呈阴性，表明本研究中的模型仅导致小鼠的局部感染而不是全身感染23。
植入物的SEM图像如 图2所示。在空白植入物组中，没有白色 念<...

Watch this Scientific Journal Video about Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections at JoVE.com

由危险病原体引起的假体周围关节感染 （PJI） 在临床骨科中很常见。现有的动物模型无法准确模拟PJI的实际情况。在这里，我们建立了一个 白色念珠菌 生物膜相关的PJI小鼠模型，以研究和开发PJI的新疗法。

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

在这项白色念珠菌生物膜相关假体周围关节的研究中，使用通常用于模拟关节植入物的钛镍丝构建了小鼠白色念珠菌感染模型。该 PJI 模型易于操作，成功率高，并且具有临床相关性。它将有助于了解白色念珠菌生物膜相关 PJI 的预防和治疗。传统的动物模型由于过程周围的复杂环境，无法准确模拟PJI的实际情况。白色念珠菌生物膜相关小鼠PJI模型，可以弥补研究空白。PJI动物模型的建立对于PJI新药或新疗法的研究和开发具有重要意义。本文建立的白色念珠菌生物膜相关小鼠PJI模型有望成为预防和治疗白色念珠菌生物膜相关PJI的重要模型。

a-periprosthetic-joint-candida-albicans-infection-model-in-mouse

Research

JoVE Journal

Immunology and Infection

A Periprosthetic Joint Candida albicans Infection Model in Mouse

498 次观看.  Xi’an Jiaotong University. 在这项白色念珠菌生物膜相关假体周围关节的研究中，使用通常用于模拟关节植入物的钛镍丝构建了小鼠白色念珠菌感染模型。该 PJI 模型易于操作，成功率高，并且具有临床相关性。它将有助于了解白色念珠菌生物膜相关 PJI 的预防和治疗。传统的动物模型由于过程周围的复杂环境，无法准确模拟PJI的实际情况。白色念珠菌生物膜相关小鼠PJI模型，可以弥补研究空白。...

视频: 作者聚焦：推进白色念珠菌生物膜相关人工关节感染的研究 

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.