Junka Adam Feliks, Szymczyk Patrycja, Smutnicka Danuta, Kos Marcin, Smolina Iryna, Bartoszewicz Marzenna, Chlebus Edward, Turniak Michal, Sedghizadeh Parish P
Researcher, Department of Pharmaceutical Microbiology and Parasitology, Medical University of Wroclaw, Wrocław, Poland.
Researcher, Centre for Advanced Manufacturing Technologies, Wroclaw University of Technology, Wrocław, Poland.
J Oral Maxillofac Surg. 2015 Mar;73(3):451-64. doi: 10.1016/j.joms.2014.09.019. Epub 2014 Dec 24.
It is widely thought that inflammation and osteoclastogenesis result in hydroxyapatite (HA) resorption and sequestrum formation during osseous infections, and microbial biofilm pathogens induce the inflammatory destruction of HA. We hypothesized that biofilms associated with infectious bone disease can directly resorb HA in the absence of host inflammation or osteoclastogenesis. Therefore we developed an in vitro model to test this hypothesis.
Customized HA discs were manufactured as a substrate for growing clinically relevant biofilm pathogens. Single-species biofilms of Streptococcus mutans, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans and mixed-species biofilms of C albicans plus S mutans were incubated on HA discs for 72 hours to grow mature biofilms. Three different non-biofilm control groups also were established for testing. HA discs were then evaluated by means of scanning electron microscopy, micro-computed tomography metrotomography, x-ray spectroscopy, and confocal microscopy with planimetric analysis. In addition, quantitative cultures and pH assessment were performed. Analysis of variance was used to test for significance between treatment and control groups.
All investigated biofilms were able to cause significant (P < .05) and morphologically characteristic alterations in HA structure as compared with controls. The highest number of alterations observed was caused by mixed biofilms of C albicans plus S mutans. S mutans biofilm incubated in medium with additional sucrose content was the most detrimental to HA surfaces among single-species biofilms.
Our findings suggest that direct microbial resorption of bone is possible in addition to immune-mediated destruction, which has important translational implications for the pathogenesis of chronic bone infections and for targeted antimicrobial therapeutics.
人们普遍认为，在骨感染过程中，炎症和破骨细胞生成会导致羟基磷灰石（HA）吸收和死骨形成，并且微生物生物膜病原体可诱导HA的炎性破坏。我们推测，与感染性骨病相关的生物膜在不存在宿主炎症或破骨细胞生成的情况下可直接吸收HA。因此，我们建立了一个体外模型来验证这一假设。
定制HA盘作为培养临床相关生物膜病原体的底物。将变形链球菌、金黄色葡萄球菌、铜绿假单胞菌和白色念珠菌的单菌种生物膜以及白色念珠菌加变形链球菌的混合菌种生物膜在HA盘上孵育72小时以形成成熟生物膜。还设立了三个不同的非生物膜对照组进行测试。然后通过扫描电子显微镜、显微计算机断层扫描、X射线光谱以及带有平面测量分析的共聚焦显微镜对HA盘进行评估。此外，还进行了定量培养和pH评估。采用方差分析来检验治疗组和对照组之间的差异是否具有显著性。
与对照组相比，所有研究的生物膜均能导致HA结构发生显著（P < .05）且具有形态学特征的改变。观察到的改变数量最多的是白色念珠菌加变形链球菌的混合生物膜。在含有额外蔗糖的培养基中孵育的变形链球菌生物膜在单菌种生物膜中对HA表面的损害最大。
我们的研究结果表明，除了免疫介导的破坏外，微生物对骨的直接吸收也是可能的，这对于慢性骨感染的发病机制和靶向抗菌治疗具有重要的转化意义。
