As peri-prosthetic infection is one of the most devastating complications associated with implant placement, we have reasoned that such infection can be largely subverted by development of antibacterial implants. Our previous work demonstrated that covalent coupling of vancomycin to titanium alloy prevented colonization by the gram-positive pathogens, Staphylococcus aureus and Staphylococcus epidermidis. Some orthopaedic devices, including permanent prosthesis anchors, and most dental implants are transcutaneous or transmucosal and can be prone to colonization by Gram-negative pathogens. We report here the successful covalent coupling of the broad-spectrum antibiotic, tetracycline (TET), to titanium surfaces (Ti-TET) to retard Gram-negative colonization. Synthetic progress was followed by changes in water contact angle, while the presence of TET was confirmed by immunofluorescence. Ti-TET actively prevented colonization in the presence of bathing Escherichia coli, both by fluorescence microscopy and direct counting. Finally, the Ti-TET surface supported osteoblastic cell adhesion and proliferation over a 72 hour period. Thus, this new surface offers a powerful means to protect transcutaneous implants from adhesion of Gram-negative pathogens, decreasing the need for replacement of this hardware.

Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.

While Gram‐positive organisms are the most common causative agent of initial bone infections, the percentage of Gram‐negative species increases in reoccurring bone infections. As bacterial internalization has been suggested as one cause of reoccurring bone infection, we tested the hypothesis that Gram‐negative species of bacteria can be internalized into bone cells. Using the MLO‐A5 and the MLO‐Y4 cell lines as our cell models, we demonstrated that the Gram‐negative species, Proteus mirabilis and Serratia marcescens, can be internalized in these cells using an internalization assay. This rate at which these two species were internalized was both time‐ and initial concentration‐dependent. Confocal analysis demonstrated the presence of internalized bacteria within both cell types. Inhibition of the cellular uptake with methyl‐β‐cyclodextrin and chloroquine both reduced internalized bacteria, indicating that this process is, at least in part, cell mediated. Finally, we demonstrated that the presence of internalized P. mirabilis did not impact cell viability, measured either by lactate dehydrogenase (LDH) release or 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) activity, while the presence of S. marcescens, on the other hand, both increased LDH release and reduced MTT activity, indicating a loss of cell viability in response to the organism. These results indicated that both species of Gram‐negative bacteria can be internalized by bone cells and that these internalized bacteria could potentially result in reoccurring bone infections. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:861‐870, 2020

Peri‐prosthetic infection (PPI) poses a serious threat to the continued health of any patients implanted with foreign material. Tethering anti‐microbials to the surface of implanted materials for the prevention of PPI might ultimately prove to be the safest and most cost‐effective strategy. PPIs can be the result of infection by either gram‐positive bacteria (GPB) or gram‐negative bacteria (GNB), or both. Thus, broad spectrum coverage will be ultimately required. Previous work has shown the efficacy of anti‐microbial tethered surfaces. This study sought to show that antibiotics effective against both GPB and GNB could be tethered in combination to morselized bone. Vancomycin (Vanc) and tetracycline (Tet) were covalently bound to demineralized, morselized rat bone. Immunohistochemistry showed that both antibiotics were attached to allograft individually as well as in combination. To test the hypothesis that covalently attached Vanc and Tet will prevent bacterial colonization by either type of bacteria, surfaces were challenged by both S. aureus and E. coli. The antibacterial properties and efficacy were determined using confocal microscopy and colony counting. Vanc tethered bone prevented colonization by S. aureus, and Tet tethered bone prevented colonization by E. coli. A combination of both antibiotics prevented colonization by both species. These results indicate that covalent attachment of combinations of broad spectrum antibiotics to bone allograft could potentially prevent PPIs.

Our previous work demonstrated the sensitivity of osteoblasts to the presence of microenvironmental apoptogens. As osteoblasts change their functional orientation with maturation, it follows that the sensitivity of these cells to apoptosis must change as well. It is the goal of this study to exam the relationship between the maturation of osteoblasts and the susceptibility of these cells to apoptosis. For this study the Human Fetal Osteoblast (hFOB) cell line was used. These cells exhibit an early osteoblastic phenotype when cultured at 34ºC while above 37ºC, the cells mature. Cells were cultured for 2, 7 and 14 days. Phenotypic changes were confirmed. Cells cultured at both 34ºC and 39.5ºC were exposed to increasing levels of the calcium and phosphate ion pair. Percentages of viable cells were measured using the MTT assay. Apoptosis was confirmed by evaluating Caspase‐3 activation and TUNEL staining. Results indicated that hFOB cells incubated at 39.5ºC exhibited a greater sensitivity to the activation of apoptosis by the calcium and phosphate ion pair. These results suggest that with maturation, osteoblasts become more sensitive to the activation of apoptosis. This work was supported by a grant from the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine.