Poster AA18 - Peri-implant Tissue on a Chip Reveals New Mechanisms in Bacteria-Metal-Host Interactions

Introduction: Biomedical devices and implants have transformed medicine and dentistry, playing a crucial role in the treatment and management of many diseases. However, implant-associated infection (IAI) remains a persistent complication. Specifically, pathogens colonize and form biofilms on implant surfaces leading to inflammation and infection.

Dental implants are particularly vulnerable to peri-implantitis, a pathological condition characterized by inflammation of the soft tissue, bacterial infiltration, and progressive bone loss around the osseointegrated implant eventually leading to implant failure. Although surgical and non-surgical (i.e., antibiotics) approaches have been adopted for peri-implantitis treatment, they are limited due to large heterogeneity in the clinical response. Thus, the development of new treatments and prevention strategies is essential. To achieve that, it is necessary to better understand the complex biological interactions between the implant, bacteria, and host tissue.

Current experimental in vitro models fall short of accurately recapitulating the 3D microphysiological environment of peri-implant tissue. Herein, we aim to develop a new 3D preclinical platform based on organ-on-a-chip technology to investigate the biomechanical and inflammatory mechanisms that regulate the peri-implant soft tissue. Specifically, our objective is to probe into the changes in epithelial cell integrity in response to mechanical disruptions caused by the presence of the implant material and interstitial stress.

Materials and

Methods: To address this, we developed a 3D microfluidic platform named peri-implant mucosa on a chip by integrating a range of physiological hydrostatic pressures (1-10 kPa), titanium, bacteria, and major oral mucosa extracellular matrix components.

The 3D platform was fabricated with a 3D printed scaffold forming cylindrical channels (160 μm diameter) within a Collagen I gel and titanium rods in which human oral keratinocytes were adhered.

The titanium rods’ surface were modified by hydrofluoric acid (HF) at 0, 60, 120 and 180 sec. Titanium rods’ surface morphology was characterized via Scanning Electron Microscopy and Atomic Force Microscopy.

Porphyromonas gingivalis (P. gingivalis), a predominant pathogen associated with peri-implantitis, was introduced into the perfusion media at a multiplicity of infection (MOI) of 50 for 15 minutes.

The epithelial barrier function was evaluated in response to interstitial stress, the presence of treated and HF-treated titanium, and exposure to P.gingivalis. Epithelial cell leakiness was measured by capturing the extravasation of fluorescently labeled 40 kDa dextran from the epithelium to the interstitial area.

Cell-cell interactions were evaluated by immunostaining for the adherens junction protein, E-cadherin and the tight junction protein, Zonula Occludens-1 (ZO-1). The devices were imaged using a confocal microscope and were analyzed by Image J.

Results, Conclusions, and Discussions: Since inflammation in peri-implantitis leads to an increase of tissue pressure, we initially tested the effect of interstitial pressure on barrier function. Our results showed that prolonged exposure to high levels of interstitial hydrostatic pressure (10 kPa) increases epithelial leakiness (by ~ 3-fold) compared to 1kPa (p < 0.05) indicating that tissue pressure controls barrier function. Additionally, we examined the role of the titanium implant on the epithelial barrier function. Our data showed that the presence of metal increased epithelial leakiness by ~ 3-fold (p < 0.05). Also, immunofluorescent staining for adherens and tight junction molecules, namely E-cadherin and ZO-1 showed a decrease in E-cadherin and ZO-1 junctional intensity by ~ 4-fold (p < 0.05) indicating that disrupted cell-cell junction formation likely regulates this decline in barrier integrity. Interestingly, the presence of the bacteria further induces epithelial leakiness by ~ 4-fold (p < 0.05).

Next, we aimed to test the effect of implant surface morphology on barrier function. SEM and AFM imaging showed that HF treatment elicited changes to titanium surface morphology and increased surface roughness (RMS) by ~ 4 fold (p < 0.05). This change in surface roughness led to restored barrier integrity by ~ 3-fold (p < 0.05) and increased intraepithelial interaction via E-cadherin and ZO-1.

Overall, the 3D peri-implant mucosa on a chip platform permits us to fine-tune biomechanical cues that epithelial cells sense and to measure dynamic changes to epithelial integrity in vitro, avoiding animal studies and conventional transwell assays. As a result, this platforms will enable us to identify new molecular mechanisms involved in mucosal diseases and implant-associated infections. Additionally, this device can be used to test potential treatment and preventative strategies for IAIs, such as altering the mechanical properties of the implant surface. Future applications of this device include incorporating immune cells to further explore host-material-pathogen interactions, as the immunological response to bacterial infiltration is a key characteristic of peri-implantitis.

Acknowledgements (Optional): This work is supported by NIH/NIDCR grant (R01DE031046) to Dr. Stella Alimperti. We gratefully acknowledge Alimperti Lab members and Microscopy and Imaging Shared Resource for access to Nikon Sora Microscope and Dr. Xinran Zhang from the I(SM)2 for AFM.