Exogenous Protein Delivery of Ionic Liquid-Mediated HMGB1 Coating on Titanium Implants

Abstract

Strategies for modifying titanium (Ti) implant surfaces are becoming increasingly popular to enhance osseointegration during acute and inflammatory healing stages. In this study, two dicationic imidazolium-based ionic liquids (IonLs) containing phenylalanine and methionine anions (IonL-Phe(1,10-bis(3methylimidazolium-1-yl)decane diphenylalanine) and IonL-Met-(1,10-bis(3-methylimidazolium-1-yl)decane dimethionine)) were investigated to stably deliver exogenous proteins on Ti to promote osseointegration. The protein selected for this study is High-Mobility Group Box 1 (HMGB1), which recruits inflammatory and mesenchymal stem cells to the implantation site, contributing to healing. To explore IonL−Ti interactions and HMGB1 stability on the IonL-coated surface, experimental characterization techniques including X-ray photoelectron spectroscopy, scanning electron microscopy, dynamic scanning calorimetry (DSC), and liquid chromatography mass spectrometry (LC−MS) were used along with molecular dynamics (MD) computer simulations to provide a detailed molecular level description. Results show well-structured IonL molecules on the Ti surface that impact protein crystallization and coating morphology. IonL cations and anions were found to bind strongly to oppositely charged residues of the protein. LC−MS/MS reveals that HMGB1 B-box lysine residues bind strongly to the IonLs. Stronger interactions of HMGB1 with Ion-Phe in contrast to IonL-Met results in greater retention capacity of HMGB1 in the IonL-Phe coating. Overall, this study provides evidence that the selected IonLs strongly interact with HMGB1, which can be a potential surface treatment for bone-implantable Ti devices.

Biological Effects of New Titanium Surface Coatings Based on Ionic Liquids and HMGB1: A Cellular and Molecular Characterization in Lewis Rats

“…Previous in vitro and in silico research from our lab validated that the IonL used in this study could pre-immobilize HMGB1 in a biologically active conformation . Considering the different HMGB1 coating formulations, Ti-IonL-DS samples demonstrated the thickest fibrous encapsulation after 14 days compared to all other treatment groups (Figure ).…”

“…Previous in vitro and in silico research from our lab validated that the IonL used in this study could pre-immobilize HMGB1 in a biologically active conformation. 15 Considering the different HMGB1 coating formulations, Ti-IonL-DS samples demonstrated the thickest fibrous encapsulation after 14 days compared to all other treatment groups (Figure 1). Correspondingly, histological results showed an increased area density % of inflammatory cells for Ti-IonL-DS samples at both 2 and 14 days and demonstrated that these tissues were potentially exposed to a particular irritant (Figure 2) compared to all other treatment groups.…”

“…Peptide coatings have been used to functionalize surfaces and harness the immune system . Thus, a well-characterized, multifunctional IonL ,,,− , was used as a method to immobilize key immunomodulatory proteins, such as HMGB1, which was previously studied in vitro and in silico by our group. , However, the behavior of this coating after in vivo implantation is still not well known.…”

“…14 Additionally, IonL cations and anions were shown to strongly bind to oppositely charged residues of the High Mobility Group Box 1 (HMGB1) protein in silico and in vitro. 15 The purpose of this study is to further evaluate the host biological response of HMGB1 as immunomodulatory coatings that can be beneficial for constructive healing responses. HMGB1 has been demonstrated as a key component in Ti osseointegration and bone healing post-trauma.…”

“…18,20 Recently, however, a recombinant fully reduced and non-oxidizable isoform of HMGB1 became commercially available (HMGBiotech, Milano, Italy) in which the three cysteines were replaced by serines (3-Serine HMGB1 (3S)), theoretically preventing oxidation from ROS. 21 Although these IonL and HMGB1 isoform interactions have been previously characterized, 15 the previous results were limited by the scope of in vitro and in silico models. There has been no investigation on how the presence of IonL-HMGB on Ti affects inflammation or early healing outcomes in vivo and the biological effects of using different HMGB1 isoforms in the new coating.…”