"Periodontal ligament-on-chip" as a Novel Tool for Studies on the Physiology and Pathology of Periodontal Tissues.

Teeth exert fundamental physiological functions, such as mastication and speech, and are a key feature of oral health that affects life quality. Teeth are anchored to the alveolar bone via the periodontal ligament, which provides stability to the teeth and absorbs mechanical stresses during mastication. Periodontal infection leads to periodontitis, a severe inflammation of the supporting soft tissues that ultimately cause tooth loss. Despite the pressing need of periodontal regeneration for improved oral care, efficient in vitro models of the periodontal tissues are still missing, thus hampering the development of novel, faster, and more effective therapy modalities. Herein, a novel "periodontal ligament (PDL)-on-chip" model that integrates patient-derived periodontal ligament cells (PDLCs) and endothelial cells is introduced. This microfluidic platform provides optimal conditions for the formation of extensive and perfusable vascular networks. Furthermore, PDLCs elicit blood vessels' development and maturation while establishing close contacts with the endothelial cells. Potential applications for inflammatory periodontal diseases are also successfully displayed in the "PDL-on-chip" by stimulating inflammation and detecting inflammatory cytokines. This work offers a cornerstone for more complex and specialized microfluidic dental models, which are necessary to unravel complex oral diseases that affect individuals' general health that go beyond the field of dentistry.

Self-assembled and perfusable microvasculature-on-chip for modeling leukocyte trafficking.

Leukocyte recruitment from blood to tissue is a process that occurs at the level of capillary vessels during both physiological and pathological conditions. This process is also relevant for evaluating novel adoptive cell therapies, in which the trafficking of therapeutic cells such as chimeric antigen receptor (CAR)-T cells throughout the capillaries of solid tumors is important. Local variations in blood flow, mural cell concentration, and tissue stiffness contribute to the regulation of capillary vascular permeability and leukocyte trafficking throughout the capillary microvasculature. We developed a platform to mimic a biologically functional human arteriole-venule microcirculation system consisting of pericytes (PCs) and arterial and venous primary endothelial cells (ECs) embedded within a hydrogel, which self-assembles into a perfusable, heterogeneous microvasculature. Our device shows a preferential association of PCs with arterial ECs that drives the flow-dependent formation of microvasculature networks. We show that PCs stimulate basement membrane matrix synthesis, which affects both vessel diameter and permeability in a manner correlating with the ratio of ECs to PCs. Moreover, we demonstrate that hydrogel concentration can affect capillary morphology but has no observed effect on vascular permeability. The biological function of our capillary network was demonstrated using an inflammation model, where significantly higher expression of cytokines, chemokines, and adhesion molecules was observed after tumor necrosis factor-alpha (TNF-α) treatment. Accordingly, T cell adherence and transendothelial migration were significantly increased in the immune-activated state. Taken together, our platform allows the generation of a perfusable microvasculature that recapitulates the structure and function of an in vivo capillary bed that can be used as a model for developing potential immunotherapies.

Multiplexed analysis of signalling proteins at the single-immune cell level.

High numbers of tumour-associated macrophages (TAMs) in the tumour microenvironment are associated with a poor prognosis. However, the effect of TAMs on tumour progression depends on the proteins secreted by individual TAMs. Here, we developed a microfluidic platform to quantitatively measure the secreted proteins of individual macrophages as well as macrophages polarized by the culture medium derived from breast cancer cells. The macrophages were captured in hydrodynamic traps and isolated with pneumatically activated valves for single-cell analysis. Barcoded and functionalized magnetic beads were captured in specially designed traps to determine the secreted proteins by immunoassay. Individual bead trapping facilitated the recording of the protein concentration since all beads were geometrically constrained in the same focal plane, which is an important requirement for rapid and automated image analysis. By determining three signaling proteins, namely interleuking 10 (IL-10), vascular endothelial growth factor (VEGF), and tumour necrosis factor alpha (TNF-α), we successfully distinguished between differently polarized macrophages. The results indicate a heterogeneous pattern, with M2 macrophages characterized by a higher secretion of IL-10, while M1 macrophages secrete high levels of the inflammatory cytokine TNF-α. The macrophages treated with the supernatant from cancer cells show a similar signalling pattern to M2 macrophages with an increased secretion of the pro-tumoural cytokine VEGF. This microfluidic method resolves correlations in signaling protein expression at the single-cell level. Ultimately, single-macrophage analysis can contribute to the development of novel therapies aimed at reversing M2-like TAMs into M1-like TAMs.

A microfluidic fully paper-based analytical device integrated with loop-mediated isothermal amplification and nano-biosensors for rapid, sensitive, and specific quantitative detection of infectious diseases.

Bacterial meningitis, an infection of the membranes (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord, is one of the major causes of death and disability worldwide. Higher case-fatality rates and short survival times have been reported in developing countries. Hence, a quick, straightforward, and low-cost approach is in great demand for the diagnosis of meningitis. In this research, a microfluidic fully paper-based analytical device (µFPAD) integrated with loop-mediated isothermal amplification (LAMP) and ssDNA-functionalized graphene oxide (GO) nano-biosensors was developed for the first time for a simple, rapid, low-cost, and quantitative detection of the main meningitis-causing bacteria, Neisseria meningitidis (N. meningitidis). The results can be successfully read within 1 hour with the limit of detection (LOD) of 6 DNA copies per detection zone. This paper device also offers versatile functions by providing a qualitative diagnostic analysis (i.e., a yes or no answer), confirmatory testing, and quantitative analysis. These features make the presented µFPAD capable of a simple, highly sensitive, and specific diagnosis of N. meningitis. Furthermore, this microfluidic approach has great potential in the rapid detection of a wide variety of different other pathogens in low-resource settings.

Assessing the aneurysm occlusion efficacy of a shear-thinning biomaterial in a 3D-printed model.

Metallic coil embolization is a common method for the endovascular treatment of visceral artery aneurysms (VAA) and visceral artery pseudoaneurysms (VAPA); however, this treatment is suboptimal due to the high cost of coils, incomplete volume occlusion, poor reendothelialization, aneurysm puncture, and coil migration. Several alternative treatment strategies are available, including stent flow diverters, glue embolics, gelfoam slurries, and vascular mesh plugs-each of which have their own disadvantages. Here, we investigated the in vitro capability of a shear-thinning biomaterial (STB), a nanocomposite hydrogel composed of gelatin and silicate nanoplatelets, for the minimally-invasive occlusion of simple necked aneurysm models. We demonstrated the injectability of STB through various clinical catheters, engineered an in vitro testing apparatus to independently manipulate aneurysm neck diameter, fluid flow rate, and flow waveform, and tested the stability of STB within the models under various conditions. Our experiments show that STB is able to withstand at least 1.89 Pa of wall shear stress, as estimated by computational fluid dynamics. STB is also able to withstand up to 10 mL s-1 pulsatile flow with a waveform mimicking blood flow in the human femoral artery and tolerate greater pressure changes than those in the human aorta. We ultimately found that our in vitro system was limited by supraphysiologic pressure changes caused by aneurysm models with low compliance.