Targeting damaged collagen for intra-articular delivery of therapeutics using collagen hybridizing peptides.

The objective of this study was to investigate the potential of collagen hybridizing peptides (CHPs), which bind to denatured collagen, to extend the retention time of near-infrared fluorophores (NIRF) following intra-articular (IA) injection in rat knee joints. CHPs were synthesized with a NIRF conjugated to the N-terminus. Male Sprague-Dawley rats were assigned to one of four experimental groups: healthy, CHP; osteoarthritis (OA), CHP; healthy, scrambled-sequence CHP (sCHP), which has no collagen binding affinity; or OA, sCHP. Animals in the OA groups received an IA injection of monosodium iodoacetate to induce OA. All animals then received the corresponding CHP injection. Animals were imaged repeatedly over 2 weeks using an in vivo fluorescence imaging system. Joint components were isolated and imaged to determine CHP binding distribution. Safranin-O and Fast Green histological staining was performed to confirm the development of OA. CHPs were found to be retained within the joint following IA injection in both healthy and OA animals for the full study period. In contrast, sCHP signal was negligible by 24-48 h. CHP signal was significantly greater (p < 0.05) in OA joints when compared to healthy joints. At the 2-week end point, multiple joint components retained CHPs, including cartilage, meniscus, and synovium. CHPs dramatically extended the retention time of NIRFs following IA injection in healthy and OA knee joints by binding to multiple collagenous tissues in the joint. These results support the pursuit of further research to develop CHP based therapeutics for IA treatment of OA.

Mechanical characterization of the human pia-arachnoid complex.

Traumatic brain injury (TBI) is a significant problem in global health that affects a wide variety of patients. Mild forms of TBI, commonly referred to as concussion, are a result of rapid accelerations of the head from either direct or indirect impacts. Kinetic energy from the impact is transferred into deformation of the brain, leading to cellular disruption. This transfer of energy is in part mediated by the pia-arachnoid complex (PAC), a layer of anatomical structures that forms the physical connection between the brain and the skull. The importance of properly quantifying the mechanics of the PAC for use in computational models of TBI has been understood for some time, but data from human subjects has been unavailable. In this study, we quantify the normal traction modulus of the PAC in five post-mortem human subjects using hydrostatic fluid pressurization in combination with optical coherence tomography. Testing at multiple locations across each brain reveals that brain-skull stiffness is heterogeneously distributed. The material response to traction loading was linear, with a mean normal traction modulus of 12.6 ± 4.8 kPa. Modulus was 21% greater in superior regions of the brain compared to inferior regions. Comparisons with regional microstructural data suggests a potential relationship between the volume fraction of arachnoid trabeculae and modulus. Comparisons to coincident measurements of microstructural properties showed a positive correlation between arachnoid membrane thickness and normal traction modulus. This study is the first to characterize the mechanics of the human pia-arachnoid complex and quantify material properties in situ. These findings suggest implementing a heterogeneous model of the brain-skull interface in computational models of TBI may lead to more realistic injury prediction.

Spatial distribution of human arachnoid trabeculae.

Traumatic brain injury (TBI) is a common injury modality affecting a diverse patient population. Axonal injury occurs when the brain experiences excessive deformation as a result of head impact. Previous studies have shown that the arachnoid trabeculae (AT) in the subarachnoid space significantly influence the magnitude and distribution of brain deformation during impact. However, the quantity and spatial distribution of cranial AT in humans is unknown. Quantification of these microstructural features will improve understanding of force transfer during TBI, and may be a valuable dataset for microneurosurgical procedures. In this study, we quantify the spatial distribution of cranial AT in seven post-mortem human subjects. Optical coherence tomography (OCT) was used to conduct in situ imaging of AT microstructure across the surface of the human brain. OCT images were segmented to quantify the relative amounts of trabecular structures through a volume fraction (VF) measurement. The average VF for each brain ranged from 22.0% to 29.2%. Across all brains, there was a positive spatial correlation, with VF significantly greater by 12% near the superior aspect of the brain (p < .005), and significantly greater by 5%-10% in the frontal lobes (p < .005). These findings suggest that the distribution of AT between the brain and skull is heterogeneous, region-dependent, and likely contributes to brain deformation patterns. This study is the first to image and quantify human AT across the cerebrum and identify region-dependencies. Incorporation of this spatial heterogeneity may improve the accuracy of computational models of human TBI and enhance understanding of brain dynamics.

Ultrathin Silicon Membranes for in Situ Optical Analysis of Nanoparticle Translocation across a Human Blood-Brain Barrier Model.

Here we present a blood-brain barrier (BBB) model that enables high-resolution imaging of nanoparticle (NP) interactions with endothelial cells and the capture of rare NP translocation events. The enabling technology is an ultrathin silicon nitride (SiN) membrane (0.5 µm pore size, 20% porosity, 400 nm thickness) integrated into a dual-chamber platform that facilitates imaging at low working distances (∼50 µm). The platform, the µSiM-BBB (microfluidic silicon membrane-BBB), features human brain endothelial cells and primary astrocytes grown on opposite sides of the membrane. The human brain endothelial cells form tight junctions on the ultrathin membranes and exhibit a significantly higher resistance to FITC-dextran diffusion than commercial membranes. The enhanced optical properties of the SiN membrane allow high-resolution live-cell imaging of three types of NPs, namely, 40 nm PS-COOH, 100 nm PS-COOH, and apolipoprotein E-conjugated 100 nm SiO2, interacting with the BBB. Despite the excellent barrier properties of the endothelial layer, we are able to document rare NP translocation events of NPs localized to lysosomal compartments of astrocytes on the "brain side" of the device. Although the translocation is always low, our data suggest that size and targeting ligand are important parameters for NP translocation across the BBB. As a platform that enables the detection of rare transmission across tight BBB layers, the µSiM-BBB is an important tool for the design of nanoparticle-based delivery of drugs to the central nervous system.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

A silicon nanomembrane platform for the visualization of immune cell trafficking across the human blood-brain barrier under flow.

Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood-brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer.

The role of viral infections in exacerbations of chronic obstructive pulmonary disease and asthma.

Asthma and chronic obstructive pulmonary disease (COPD) are major causes of global morbidity and mortality worldwide. The clinical course of both asthma and COPD are punctuated by the occurrence of exacerbations, acute events characterized by increased symptoms and airflow obstruction. Exacerbations contribute most of the morbidity, mortality and excess healthcare costs associated with both asthma and COPD. COPD and asthma exacerbations are frequently associated with respiratory virus infections and this has led to an intense research focus into the mechanisms of virus-induced exacerbations over the past decade. Current therapies are effective in reducing chronic symptoms but are less effective in preventing exacerbations, particularly in COPD. Understanding the mechanisms of virus-induced exacerbation will lead to the development of new targeted therapies that can reduce the burden of virus-induced exacerbations. In this review we discuss current knowledge of virus-induced exacerbations of asthma and COPD with a particular focus on mechanisms, human studies, virus-bacteria interactions and therapeutic advances.