Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections

ObjectivesTotal joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.Materials and MethodsFor initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.ResultsAfter 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB.ConclusionWe developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments.

研究目的：全关节置换术（total joint arthroplasty）是当今临床最常见且有效的手术术式之一。尽管手术技术已得到显著改进，但仍有大量植入物相关感染（implant-associated infections）发生。当前亟需合适的体外（in vitro）模型，以评估预防此类感染的潜在方案。本研究旨在构建人原代成骨细胞（human primary osteoblasts）与表皮葡萄球菌（S. epidermidis）的体外共培养体系，模拟植入物相关感染的发病过程，并用于抗菌植入物表面及涂层的性能检测。

材料与方法：为探究初始表面黏附情况，将人原代成骨细胞（hOB）在由聚苯乙烯、钛合金Ti6Al4V、骨水泥PALACOS R®以及载抗生素PALACOS R®制成的试样圆盘上培养24小时。随后以表皮葡萄球菌对成骨细胞进行单种菌感染，感染复数（multiplicity of infection）为0.04，构建共培养体系，并在有氧条件下分别孵育2天与7天。通过离心与菌落形成单位（colony-forming units，CFU）计数，测定浮游态表皮葡萄球菌的数量；通过超声破碎与CFU计数，定量试样表面生物膜结合态的表皮葡萄球菌。采用台盼蓝染色计数法，定量试样表面黏附且存活的原代成骨细胞。利用扫描电子显微镜（scanning electron microscopy）评估试样表面的形貌与菌群组成。

研究结果：孵育2天后，在存在人原代成骨细胞的载抗生素骨水泥试样上，检测到约10⁴ CFU/ml的生物膜结合态表皮葡萄球菌（初始菌量为10³ CFU/ml），而无成骨细胞的对照组未检出细菌。两组在孵育7天后均未检出生物膜结合态细菌。孵育2天时，有无成骨细胞的两组浮游菌水平无显著差异；孵育7天后，仅无成骨细胞的组中可检出约10⁵ CFU/ml的浮游菌。此外，此时生物膜内未观察到细菌，而成骨细胞数量降至初始值的10%，而人原代成骨细胞单培养组的细胞数量则增至初始值的150%。

结论：本研究构建的共培养体系可作为更全面的植入物相关感染发病过程体外模型，同时可为抗菌植入材料及涂层提供检测方法。本研究证实，该模型可获得单培养实验无法获取的实验观察结果。