Chemistry and Biochemistry
Mihaela Rita Mihailescu
Titanium aluminum vanadium, osteoblasts, vancomycin, spermine NONOate, KRSR, cell adhesion
Orthopedic implant surgeries are on the rise in the United States, with well over a million surgeries performed annually. Common materials used for these applications include titanium and its alloy, titanium aluminum vanadium (Ti-6Al-4V). Ti-6Al-4V is chosen for this application due to its mechanical strength and corrosion resistance. However, Ti-6Al-4V does not support osseointegration and is susceptible to bacterial colonization. Therefore, these implants suffer from aseptic loosening and infection, necessitating removal and replacement. Revision surgery and treatment is financially burdensome and taxing on the patient. Current approaches focus on modifying the surface of Ti-6Al-4V through a variety of means to allow for improved osseointegration and limited bacterial adhesion.
In this work, self-assembled monolayers (SAMs) were used as linkers to immobilize bioactive molecules to the Ti-6Al-4V surface that would help encourage osteoblast attachment while limiting bacterial adhesion. This was accomplished by forming phosphonic acid head group SAMs on the surface of Ti-6Al-4V with different tail groups. These tail groups were then used to perform chemical reactions at the interface, using orthogonal chemistry, to covalently link the bioactive molecules. Bioactive molecules were chosen that would address both osteoblast attachment as well as bacterial adhesion. Vancomycin, an antibiotic effective against Gram positive bacteria as well as spermine NONOate, a nitric oxide releasing molecule, were chosen to address bacterial adhesion and colonization of Ti-6Al-4V. A cell adhesion peptide, KRSR was selected to encourage osteoblast anchoring and viability on the Ti-6Al-4V surface.
The single, dual, and triple immobilization of these molecules was confirmed through diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. The covalent attachment of these molecules resulted in hydrophilic surfaces with contact angle values less than 90°. Nitric oxide release from functionalized Ti-6Al-4V was assessed through the Griess assay, with single and triple functionalized surfaces releasing comparable amounts of nitric oxide, 77.0 ± 1.8 and 74.7 ± 1.6 nanomoles respectively. Dual functionalized substrates with co-immobilized spermine NONOate exhibited higher nitric oxide release of 107.6 ± 1.5 nanomoles for spermine NONOate with KRSR(C) and 117.6 ±1.4 nanomoles for spermine NONOate with vancomycin.
The activity of the antimicrobial molecules immobilized alone and in conjunction with the other bioactive molecules was determined via challenges with Staphylococcus epidermidis (S. epidermidis), a Gram positive species and Escherichia coli (E. coli), a Gram negative species through fluorescence staining and imaging of the bacteria. Vancomycin immobilized alone was able to reduce the viability of S. epidermidis by 64 ± 27%. Spermine NONOate was able to reduce viability of E. coli by 27 ± 20% but not that of S. epidermidis. The selectivity observed was attributed to Gram positive species’ ability to generate their own nitric oxide, leading to tolerance of nitric oxide’s effects. Spermine NONOate was able to prevent the adhesion of both species to single functionalized surfaces which has previously been observed in the literature.27-29
Antimicrobial effectiveness was only maintained for one of the dual functionalizations, when KRSR(C) was co-immobilized with vancomycin. This dual functionalized surface showed a reduction in S. epidermidis viability of 36 ± 28%. This was approximately half the reduction in viability observed for vancomycin immobilized alone. The loss in effectiveness may be due to interactions between vancomycin and KRSR(C) or lower surface loading. Surfaces possessing all three bioactive molecules were not able to limit viability of either bacterial species. Local concentrations were either not sufficient or interactions between molecules negatively impacted their effectiveness.
Finally, osteoblast adhesion and viability on functionalized Ti-6Al-4V was characterized through fluorescence staining and microscopy. Covalent attachment of these bioactive molecules should not elicit cytotoxic effects within osteoblast cells. Furthermore, attachment of the KRSR(C) cell adhesion peptide to Ti-6Al-4V was expected to encourage osteoblast adhesion to Ti-6Al-4V and improve cell viability. While individual immobilization of bioactive molecules did not negatively affect adhesion or viability, KRSR(C) functionalized substrates showed no statistically significant increase in live osteoblast adhesion or viability. Surface loading and distribution of the cell adhesion peptide dictate its effectiveness at recruiting osteoblast adhesion. These factors may contribute to the lack of a positive, observed effect.
Co-immobilization of spermine NONOate with either vancomycin or KRSR(C) negatively impacted osteoblast viability. Dual functionalized substrates exhibited higher concentrations of nitric oxide release than single or triple functionalized. The larger, local concentrations are thought to be the source of observed cytotoxicity. The lack of observed cytotoxicity on triple functionalized substrates supports this, as these substrates showed nitric oxide release consistent with single functionalized Ti-6Al-4V.
In this work, SAMs were utilized as a platform to chemically immobilize bioactive molecules to the Ti-6Al-4V surface aimed at addressing osteoblast adhesion and bacterial colonization. This flexible platform allows for the immobilization of a variety of molecules including antibiotics, peptides, and nitric oxide releasing compounds. Although in this work immobilized molecules did not retain their bioactivity, alternative immobilization strategies can be employed. The functional groups within these molecules used in the formation of covalent bonds are prevalent in other classes of bioactive molecules. This permits control of the interfacial properties of Ti-6Al-4V with bioactive molecules specific to the metal’s application. Furthermore, as SAMs form on other metal oxide surfaces, this transferable platform can be used to alter their interfacial properties as well.
Blystone, A. (2019). Triple Functionalization of Titanium Aluminum Vanadium for Orthopedic Implant Applications (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1815