(A-6) Nanomatrix Coating Applications to Improve Orthopedic Implant Outcomes

Introduction:: Approximately 2 million people in the U.S. currently live with an amputation, and over 2.2 million hip and knee replacement procedures have been performed in the past decade. These procedures are often performed to treat osteoarthritis, peripheral artery disease, infection, etc., and thus improve patient outcomes. The most common orthopedic implants are percutaneous osseo-integrated implants (POP). Despite surgical advancements, the implants themselves fail to enhance osseointegration and vascularization, and inflammation, fibrosis, and infection are still prominent, thus reducing long-term clinical success. To target these critical factors, we propose the application of nitric oxide (NO) releasing nanomatrix coating functionalized with antibiotics-encapsulated liposomes (ABLipo) onto orthopedic implants to improve implant success and bone ingrowth. In this study, we hypothesize the nanomatrix coating and ABLipo can promote osteogenic differentiation and endothelial cell function while reducing inflammation, infection, and adhesion onto the implant material.


Materials and Methods:: To create the implant coating, a peptide amphiphile (PA) with a polylysine (KKKKK, K5) NO-donor group was charged to create PA-K5-NO. To prepare the ABLipo, DPPC:DOTAP:Cholesterol liposomes were synthesized (M_R 14:3:3) and pre-loaded with vancomycin and gentamicin antibiotics. To assess antibacterial efficacy, broth microdilution and agar diffusion methods were used to evaluate the ABLipo minimal inhibitory concentrations (MIC) against S. aureus and S. epidermidis (Fig.1). To evaluate anti-inflammatory potential and macrophage adhesion, lipopolysaccharide (LPS)-stimulated macrophages were seeded onto nanomatrix-coated titanium disks and the medium and cells harvested at 1, 3, and 7 days for quantification by ELISA and cell counts, respectively. Endothelial cells seeded onto nanomatrix-coated titanium were stained with DAF-FM to quantify nitric oxide production. RT-PCR was used to assess the osteogenic differentiation of mesenchymal stem cells (MSC) when seeded onto nanomatrix-coated 6-well plates.

Results, Conclusions, and Discussions:: The ABLipo demonstrated high antibacterial efficacies against S. aureus and S. epidermidis, which are common strains found in orthopedic infection cases. The MICs found were far below the cytotoxic level for MSCs (based on MTT assay). In the agar diffusion assay, ABLipo demonstrated similar MICs to stock antibiotics, which was evaluated by zone of inhibition sizes after 24 hours. The nanomatrix coating on titanium improved endothelial cell function as a measure of nitric oxide production via DAF-FM staining. Furthermore, the nanomatrix coating reduced macrophage adhesion onto titanium in a NO-concentration dependent manner. Also, the nanomatrix reduced IL-1B cytokine secretion from pro-inflammatory macrophages in a dose-dependent manner for 7 days. Lastly, the nanomatrix coating significantly increased BMP2 and reduced ALP gene expressions, indicating improved MSC differentiation potential compared to bare titanium.

As demonstrated, our nanomatrix platform can reduce inflammation and macrophage adhesion onto the implant surface while promoting endothelial cell functionality and osteogenic differentiation. Furthermore, the ABLipo has superior antibacterial efficacy against S. aureus and S. epidermidis. Thus, we believe our coating platform and ABLipo can enhance orthopedic implant success clinically and in an in vivo animal model.

As demonstrated, our nanomatrix platform can reduce inflammation and macrophage adhesion onto the implant surface while promoting endothelial cell functionality and osteogenic differentiation. Furthermore, the ABLipo has superior antibacterial efficacy against S. aureus and S. epidermidis. Thus, we believe our coating platform and ABLipo can enhance orthopedic implant success clinically and in an in vivo animal model.


Acknowledgements (Optional): : This project was supported by the grant of 1ARDEF22-09.


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