The effect of choline-stabilized orthosilicic acid in patients with peri-implantitis: an exploratory randomized, double-blind, placebo controlled study.

BACKGROUND: Choline-stabilized orthosilicic acid (CS-OSA) was previously found to stimulate bone collagen formation in osteopenia and to improve biomarkers of cartilage degradation in knee osteoarthritis. The aim of the present study was to investigate the effect of oral administration of CS-OSA on clinical symptoms of peri-implantitis and the associated bone loss. METHODS: Twenty-one patients with peri-implantitis were randomized in CS-OSA or placebo groups. After initial clinical and cone beam computed tomography (CBCT) measurements [probing pocket depth (PPD), bleeding on probing (BOP), mucosal recession (REC), distance from implant shoulder to alveolar crest (IS-AC) and distance from implant shoulder to first bone-to-implant contact (IS-BIC)], flap operations were performed at the peri-implantitis sites. All patients were instructed to use either placebo or CS-OSA capsules twice a day for 1 year. Measurements were repeated 6 and 12 months after randomization. RESULTS: The data of 18 patients (36 implants) were used in the per protocol analysis. PPD and BOP improved significantly (p < 0.05) compared to baseline for both groups after 6 and 12 months. However, REC significantly increased in the placebo group but not in the CS-OSA group. The change in REC over 6 and 12 months was significantly different between groups (p < 0.01). IS-BIC and IS-AC measurements remained stable in the CS-OSA group whereas in the placebo group, both parameters increased significantly after 6 and 12 months. The change in IS-BIC over 12 months was significantly different between groups (p < 0.05). CONCLUSION: The results of this preliminary study suggest that CS-OSA may stabilize and even prevent further bone loss after surgical peri-implantitis treatment and support mucosal tissue healing. Trial registration The trial was retrospectively registered at ISRCTN registry, registration number: ISRCTN14348802, registration date: 24/06/2020.

Sonoprinting of nanoparticle-loaded microbubbles: Unraveling the multi-timescale mechanism.

Ultrasound-triggered microbubble-assisted drug delivery is a promising tool for localized therapy. Several studies have shown the potential of nanoparticle-loaded microbubbles to effectively enhance the delivery of therapeutic agents to target tissue. We recently discovered that nanoparticle-carrying microbubbles can deposit the nanoparticles in patches onto cell membranes, a process which we termed 'sonoprinting'. However, the biophysical mechanisms behind sonoprinting are not entirely clear. In addition, the question remains how the ultrasound parameters, such as acoustic pressure and pulse duration, influence sonoprinting. Aiming for a better understanding of sonoprinting, this report investigates the behavior of nanoparticle-loaded microbubbles under ultrasound exposure, making use of three advanced optical imaging techniques with frame rates ranging from 5 frames per second to 10 million frames per second, to capture the biophysical cell-bubble interactions that occur on a multitude of timescales. We observed that non-spherically oscillating microbubbles release their nanoparticle payload in the first few cycles of ultrasound insonation. At low acoustic pressures, the released nanoparticles are transported away from the cells by microstreaming, which does not favor uptake of the nanoparticles by the cells. However, higher acoustic pressures (>300 kPa) and longer ultrasound pulses (>100 cycles) lead to rapid translation of the microbubbles, due to acoustic radiation forces. As a result, the released nanoparticles are transported along in the wake of the microbubbles, which eventually leads to the deposition of nanoparticles in elongated patches on the cell membrane, i.e. sonoprinting. We conclude that a sufficiently high acoustic pressure and long pulses are needed for sonoprinting of nanoparticles on cells.

Engineered 3D Microvascular Networks for the Study of Ultrasound-Microbubble-Mediated Drug Delivery.

Localized and targeted drug delivery can be achieved by the combined action of ultrasound and microbubbles on the tumor microenvironment, likely through sonoporation and other therapeutic mechanisms that are not well understood. Here, we present a perfusable in vitro model with a realistic 3D geometry to study the interactions between microbubbles and the vascular endothelium in the presence of ultrasound. Specifically, a three-dimensional, endothelial-cell-seeded in vitro microvascular model was perfused with cell culture medium and microbubbles while being sonicated by a single-element 1 MHz focused transducer. This setup mimics the in vivo scenario in which ultrasound induces a therapeutic effect in the tumor vasculature in the presence of flow. Fluorescence and bright-field microscopy were employed to assess the microbubble-vessel interactions and the extent of drug delivery and cell death both in real time during treatment as well as after treatment. Propidium iodide was used as the model drug while calcein AM was used to evaluate cell viability. There were two acoustic parameter sets chosen for this work: (1) acoustic pressure: 1.4 MPa, pulse length: 500 cycles, duty cycle: 5% and (2) acoustic pressure: 0.4 MPa, pulse length: 1000 cycles, duty cycle: 20%. Enhanced drug delivery and cell death were observed in both cases while the higher pressure setting had a more pronounced effect. By introducing physiological flow to the in vitro microvascular model and examining the PECAM-1 expression of the endothelial cells within it, we demonstrated that our model is a good mimic of the in vivo vasculature and is therefore a viable platform to provide mechanistic insights into ultrasound-mediated drug delivery.

Microvascular Injury and Perfusion Changes Induced by Ultrasound and Microbubbles in a Machine-Perfused Pig Liver.

Localized drug delivery and uptake can benefit from the combined action of ultrasound and microbubbles at a specific site. Some of the possible mechanisms suggested are vessel poration and/or cell poration, but the exact acoustic parameters that trigger those phenomena remain unknown. Ex vivo machine perfusion of human-sized organs is a technique that provides an ideal environment for pre-clinical investigations with high physiologic relevance not possible with in vitro experiments. In this work, ex vivo machine-perfused pig livers were combined with an image-guided therapy system to investigate microvascular flow changes caused by the interaction of ultrasound-driven microbubbles with the vasculature. The effects of acoustic pressure (1.7-4 MPa peak negative pressures) and number of cycles (1000 or 20 cycles) were examined. Perfusion changes caused by the action of ultrasound on microbubbles in the microcirculation were qualitatively and quantitatively assessed with contrast-enhanced ultrasound and used as a metric of the extent of vessel perforation, thus, extravasation. Areas that were exposed to peak negative pressures above 1.7 MPa underwent a detectable and irreversible perfusion change. Complete devascularization of the area exposed to ultrasound was observed at much larger acoustic pressures (∼4 MPa). Shorter acoustic pulses (20 cycles) produced markedly fewer perfusion changes than longer pulses (1000 cycles) under the same acoustic amplitude exposure.

In vitro methods to study bubble-cell interactions: Fundamentals and therapeutic applications.

Besides their use as contrast agents for ultrasound imaging, microbubbles are increasingly studied for a wide range of therapeutic applications. In particular, their ability to enhance the uptake of drugs through the permeabilization of tissues and cell membranes shows great promise. In order to fully understand the numerous paths by which bubbles can interact with cells and the even larger number of possible biological responses from the cells, thorough and extensive work is necessary. In this review, we consider the range of experimental techniques implemented in in vitro studies with the aim of elucidating these microbubble-cell interactions. First of all, the variety of cell types and cell models available are discussed, emphasizing the need for more and more complex models replicating in vivo conditions together with experimental challenges associated with this increased complexity. Second, the different types of stabilized microbubbles and more recently developed droplets and particles are presented, followed by their acoustic or optical excitation methods. Finally, the techniques exploited to study the microbubble-cell interactions are reviewed. These techniques operate over a wide range of timescales, or even off-line, revealing particular aspects or subsequent effects of these interactions. Therefore, knowledge obtained from several techniques must be combined to elucidate the underlying processes.

Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles.

In the last years, research on ultrasound mediated drug delivery using microbubbles is vastly expanding. While some groups simply mix drugs and microbubbles (co-administration), other researchers have a major interest in the potential of drug-loaded microbubbles. However, today, little is known on the pros and cons of these two strategies. In this study we evaluated the delivery of nanoparticles (polystyrene nanospheres and mRNA-lipoplexes) to cells in vitro, in case the nanoparticles were mixed with unloaded microbubbles versus loaded onto the microbubbles. Flow cytometry experiments demonstrated that unloaded microbubbles did not enhance the cellular delivery of the nanospheres and mRNA-lipoplexes. However, upon loading the nanoparticles onto the microbubbles, their delivery to cells substantially improved. Real-time swept field confocal microscopy imaging of the microbubbles and cells during ultrasound radiation revealed that nanoparticle-loaded microbubbles directly deposited the nanoparticles in patches onto the cell membrane, a process that we termed 'sonoprinting'. This phenomenon resulted in the delivery of large amounts of nanoparticles to the cells and is suggested to be different from the creation of cell membrane pores and enhanced endocytosis, which have been reported before as mechanisms behind the improved delivery of drugs to cells by ultrasound.

Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis.

Although promising results are achieved in ultrasound mediated drug delivery, its underlying biophysical mechanisms remain to be elucidated. Pore formation as well as endocytosis has been reported during ultrasound application. Due to the plethora of ultrasound settings used in literature, it is extremely difficult to draw conclusions on which mechanism is actually involved. To our knowledge, we are the first to show that acoustic pressure influences which route of drug uptake is addressed, by inducing different microbubble-cell interactions. To investigate this, FITC-dextrans were used as model drugs and their uptake was analyzed by flow cytometry. In fluorescence intensity plots, two subpopulations arose in cells with FITC-dextran uptake after ultrasound application, corresponding to cells having either low or high uptake. Following separation of the subpopulations by FACS sorting, confocal images indicated that the low uptake population showed endocytic uptake. The high uptake population represented uptake via pores. Moreover, the distribution of the subpopulations shifted to the high uptake population with increasing acoustic pressure. Real-time confocal recordings during ultrasound revealed that membrane deformation by microbubbles may be the trigger for endocytosis via mechanostimulation of the cytoskeleton. Pore formation was shown to be caused by microbubbles propelled towards the cell. These results provide a better insight in the role of acoustic pressure in microbubble-cell interactions and the possible consequences for drug uptake. In addition, it pinpoints the need for a more rational, microbubble behavior based choice of acoustic parameters in ultrasound mediated drug delivery experiments.

Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells.

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.

Polymer-coated nanoparticles interacting with proteins and cells: focusing on the sign of the net charge.

To study charge-dependent interactions of nanoparticles (NPs) with biological media and NP uptake by cells, colloidal gold nanoparticles were modified with amphiphilic polymers to obtain NPs with identical physical properties except for the sign of the charge (negative/positive). This strategy enabled us to solely assess the influence of charge on the interactions of the NPs with proteins and cells, without interference by other effects such as different size and colloidal stability. Our study shows that the number of adsorbed human serum albumin molecules per NP was not influenced by their surface charge. Positively charged NPs were incorporated by cells to a larger extent than negatively charged ones, both in serum-free and serum-containing media. Consequently, with and without protein corona (i.e., in serum-free medium) present, NP internalization depends on the sign of charge. The uptake rate of NPs by cells was higher for positively than for negatively charged NPs. Furthermore, cytotoxicity assays revealed a higher cytotoxicity for positively charged NPs, associated with their enhanced uptake.