Homeostatic pressure-driven compaction is a ubiquitous mechanical force in multicellular organisms and is proposed to be important in the maintenance of multicellular tissue integrity and function. Previous cell-free biochemical models have demonstrated that there are cross-talks between compaction forces and tissue structural functions, such as cell-cell adhesion. However, its involvement in physiological tissue function has yet to be directly demonstrated. Here, we use the bile canaliculus (BC) as a physiological example of a multicellular functional structure in the liver, and employ a novel 3D microfluidic hepatocyte culture system to provide an unprecedented opportunity to experimentally modulate the compaction states of primary hepatocyte aggregates in a 3D physiological-mimicking environment. Mechanical compaction alters the physical attributes of the hepatocyte aggregates, including cell shape, cell packing density and cell-cell contact area, but does not impair the hepatocytes' remodeling and functional capabilities. Characterization of structural and functional polarity shows that BC formation in compact hepatocyte aggregates is accelerated to as early as 12 hours post-seeding; whereas non-compact control requires 48 hours for functional BC formation. Further dynamic immunofluorescence imaging and gene expression profiling reveal that compaction accelerated BC formation is accompanied by changes in actin cytoskeleton remodeling dynamics and transcriptional levels of hepatic nuclear factor 4α and Annexin A2. Our report not only provides a novel strategy of modeling BC formation for in vitro hepatology research, but also shows a first instance that homeostatic pressure-driven compaction force is directly coupled to the higher-order multicellular functions.

In this study, we are introducing a new drug-delivery approach to demineralized dentin substrates through microsized dentinal tubules in the form of drug-loaded nanocapsules. Chlorhexidine (CHX) is widely used in adhesive dentistry due to its nonspecific matrix metalloproteinase inhibitory effect and antibacterial activities. Poly(ε-caprolactone) nanocapsules (nano-PCL) loaded with CHX were fabricated by interfacial polymer deposition at PCL/CHX ratios of 125:10, 125:25, and 125:50. Unloaded nanocapsules (blank) were fabricated as control. The fabricated nanocapsules were characterized in vitro in terms of particle size, surface charges, particle recovery, encapsulation efficiency, and drug loading. Nanocapsule morphology, drug inclusion, structural properties, and crystallinity were investigated by scanning and transmission electron microscopes (SEM/TEM), energy-dispersive x-ray analysis, Fourier transform infrared spectroscopy, and x-ray diffraction. Initial screening of the antibacterial activities and the cytotoxicity of the nanocapsules were also conducted. Nanocapsules, as carried on ethanol/water solution, were delivered to demineralized dentin specimens connected to an ex vivo model setup simulating the pulpal pressure to study their infiltration, penetration depth, and retention inside the dentinal tubules by SEM/TEM. Nanocapsules were Ag labeled and delivered to demineralized dentin, followed by the application of a 2-step etch-and-rinse dentin adhesive. CHX-release profiles were characterized in vitro and ex vivo up to 25 d. Spherical nanocapsules were fabricated with a CHX core coated with a thin PCL shell. The blank nanocapsules exhibited the largest z-average diameter with negatively charged ζ-potential. With CHX incorporation, the nanocapsule size was decreased with a positive shift in ζ-potential. Nano-PCL/CHX at 125:50 showed the highest drug loading, antibacterial effect, and CHX release both in vitro and ex vivo. SEM and TEM revealed the deep penetration and retention of the CHX-loaded nanocapsules inside dentinal tubules and their ability to be gradually degraded to release CHX in vitro and ex vivo. Ag-labeled nanocapsules revealed the close association and even distribution of nanocapsules throughout the resin tag structure. This study demonstrated the potential of introducing this novel drug-delivery approach to demineralized dentin substrates and the resin-dentin interface with nanosized CHX-loaded nanocapsules through the microsized dentinal tubules.

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Previous studies reported that grapeseed extract (GSE), which is rich in proanthocyanidins (PAs), improves the biodegradation resistance of demineralized dentin. This study aimed to investigate the effect of a new GSE delivery strategy to demineralized dentin through loading into biodegradable polymer poly-[lactic-co-glycolic acid] (PLGA) nanoparticles on the biodegradation resistance in terms of structural stability and surface/bulk mechanical and biochemical properties with storage time in collagenase-containing solutions. GSE-loaded nanoparticles were synthetized by nanoprecipitation at PLGA/GSE (w/w) ratios of 100:75, 100:50, and 100:25 and characterized for their morphological/structural features, physicochemical characteristics, and drug loading, entrapment, and release. Nanoparticle suspensions in distilled water (12.5% w/v) were applied (1 min) to demineralized dentin specimens by simulating pulpal pressure. The nanoparticle delivery was investigated by scanning electron microscopy (SEM)/transmission electron microscopy (TEM), and the GSE release from the delivered nanoparticles was further characterized. The variations in surface and bulk mechanical properties were characterized in terms of reduced elastic-modulus, hardness, nanoindentation testing, and apparent elastic-modulus with a storage time up to 3 mo. Hydroxyproline release with exposure to collagenase up to 7 d was estimated. An etch-and-rinse dentin adhesive was applied to investigate the morphology of the resin-dentin interface after nanoparticle delivery. Treatment with the GSE-loaded nanoparticles enhanced the collagen fibril structural resistance, reflected from the TEM investigation, and improved the biomechanical and biochemical stability of demineralized dentin. Nanoparticles having PLGA/GSE of 100:75 (w/w) showed the highest cumulative GSE release and were associated with the best improvement in biodegradation resistance. TEM/SEM showed the ability of the nanoparticles to infiltrate dentinal tubules' main and lateral branches. SEM revealed the formation of a uniform hybrid layer and well-formed resin tags with the presence of numerous nanoparticles located within the dentinal tubules and/or attached to the resin tag. This study demonstrated the potential significance of delivering collagen crosslinkers loaded into biodegradable polymer nanoparticles through the dentinal tubules of demineralized dentin on the biodegradation resistance.

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