No abstract

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.

Idiopathic pulmonary fibrosis (IPF) is a complex disease of unknown etiology with no current curative treatment. Modeling pulmonary fibrotic (PF) tissue has the potential to improve our understanding of IPF disease progression and treatment. Rodent animal models do not replicate human fibroblastic foci (Hum-FF) pathology, and current iterations of in vitro model systems (e.g., collagen hydrogels, polyacrylamide hydrogels, and fibrosis-on-chip systems) are unable to replicate the three-dimensional (3D) complexity and biochemical composition of human PF tissue. Herein, we fabricated a 3D bioengineered pulmonary fibrotic (Eng-PF) tissue utilizing cell laden silk collagen type I dityrosine cross-linked hydrogels and Flexcell bioreactors. We show that silk collagen type I hydrogels have superior stability and mechanical tunability compared to other hydrogel systems. Using customized Flexcell bioreactors, we reproduced Hum-FF-like pathology with airway epithelial and microvascular endothelial cells. Eng-PF tissues can model myofibroblast differentiation and permit evaluation of antifibrotic drug treatments. Further, Eng-PF tissues could be used to model different facets of IPF disease, including epithelial injury with the addition of bleomycin and cellular recruitment by perfusion of cells through the hydrogel microchannel.

Phenol red entrapped within hydrogels facilitates pH sensing in phenol red free environments. Leak-proof phenol red based pH sensors require covalent binding techniques, but are complicated due to the lack of amino or carboxyl groups on phenol red. Currently, there is no simple, reliable technique to covalently link phenol red to hydrogel matrices, for real-time pH sensing in cell culture environments. Herein, we take advantage of phenolic groups for covalent linkage of phenol red to silk tyrosine in the presence of HRP and H2O2. The novelty of the current system stems from its simplicity and the use of silk protein to create a cytocompatible, degradable sensor capable of real-time pH sensing in cell culture microenvironments.

In 1999 we reported an important demonstration of a working brain-machine interface (BMI), in which recordings from multiple, single neurons in sensorimotor cortical areas of rats were used to directly control a robotic arm to retrieve a water reward. Subsequent studies in monkeys, using a similar approach, demonstrated that primates can use a BMI device to control a cursor on a computer screen and a robotic arm. Recent studies in humans with spinal cord injuries have shown that recordings from multiple, single neurons can be used by the patient to control the cursor on a computer screen. The promise is that one day it will be possible to use these control signals from neurons to re-activate the patient’s own limbs. However, the ability to record from large populations of single neurons for long periods of time has been hampered because either the electrode itself fails or the immunological response in the tissue surrounding the microelectrode produces a glial scar, preventing single-neuron recording. While we have largely solved the problem of mechanical or electrical failure of the electrode itself, much less is known about the long term immunological response to implantation of a microelectrode, its effect on neuronal recordings and, of greatest importance, how it can be reduced to allow long term single neuron recording. This article reviews materials approaches to resolving the glial scar to improve the longevity of recordings. The work to date suggests that approaches utilizing bioactive interventions that attempt to alter the glial response and attract neurons to the recording site are likely to be the most successful. Importantly, measures of the glial scar alone are not sufficient to assess the effect of interventions. It is imperative that recordings of single neurons accompany any study of glial activation because, at this time, we do not know the precise relationship between glial activation and loss of neuronal recordings. Moreover, new approaches to immobilize bioactive molecules on microelectrode surfaces while maintaining their functionality may open new avenues for very long term single neuron recording. Finally, it is important to have quantitative measures of glial upregulation and neuronal activity in order to assess the relationship between the two. These types of studies will help rationalize the study of interventions to improve the longevity of recordings from microelectrodes.