Functional maturation of human neural stem cells in a 3D bioengineered brain model enriched with fetal brain-derived matrix.

Brain extracellular matrix (ECM) is often overlooked in vitro brain tissue models, despite its instructive roles during development. Using developmental stage-sourced brain ECM in reproducible 3D bioengineered culture systems, we demonstrate enhanced functional differentiation of human induced neural stem cells (hiNSCs) into healthy neurons and astrocytes. Particularly, fetal brain tissue-derived ECM supported long-term maintenance of differentiated neurons, demonstrated by morphology, gene expression and secretome profiling. Astrocytes were evident within the second month of differentiation, and reactive astrogliosis was inhibited in brain ECM-enriched cultures when compared to unsupplemented cultures. Functional maturation of the differentiated hiNSCs within fetal ECM-enriched cultures was confirmed by calcium signaling and spectral/cluster analysis. Additionally, the study identified native biochemical cues in decellularized ECM with notable comparisons between fetal and adult brain-derived ECMs. The development of novel brain-specific biomaterials for generating mature in vitro brain models provides an important path forward for interrogation of neuron-glia interactions.

3D extracellular matrix microenvironment in bioengineered tissue models of primary pediatric and adult brain tumors.

Dynamic alterations in the unique brain extracellular matrix (ECM) are involved in malignant brain tumors. Yet studies of brain ECM roles in tumor cell behavior have been difficult due to lack of access to the human brain. We present a tunable 3D bioengineered brain tissue platform by integrating microenvironmental cues of native brain-derived ECMs and live imaging to systematically evaluate patient-derived brain tumor responses. Using pediatric ependymoma and adult glioblastoma as examples, the 3D brain ECM-containing microenvironment with a balance of cell-cell and cell-matrix interactions supports distinctive phenotypes associated with tumor type-specific and ECM-dependent patterns in the tumor cells' transcriptomic and release profiles. Label-free metabolic imaging of the composite model structure identifies metabolically distinct sub-populations within a tumor type and captures extracellular lipid-containing droplets with potential implications in drug response. The versatile bioengineered 3D tumor tissue system sets the stage for mechanistic studies deciphering microenvironmental role in brain tumor progression.

Nondestructive, Label-Free Characterization of Mechanical Microheterogeneity in Biomimetic Materials.

We propose a novel nondestructive, label-free, mechanical characterization method for composite biomimetic materials. The method combines microscale-force measurement, bright-field microscopy based deformation analysis, and finite-element methods (FEM) to study the heterogeneity in bioengineered composite materials. The method was used to study silk fibroin protein based, donut-shaped scaffolds consisting of a shell (diameter 5 mm) and a core (diameter 2 mm) with a stiff-core or a soft-core configuration. The samples were based on our previously reported bioengineered brain tissue model. Step-wise images of sample deformation were recorded as the automated mechanical stage compressed the sample. The force-compression curves were also recorded with a load cell. A MATLAB program was used to compare and match optically measured strain distribution with that found from the FEM simulations. Iterative processes are used to determine the values that best represent the elastic moduli of the shell and the core regions. The calculated moduli found from the composite models were not significantly different from the values measured separately for each material, demonstrating the efficacy of this new approach. In addition, the method successfully measured multiple distinct regions embedded in a polydimethylsiloxane block. These results demonstrated the feasibility of our method in the microheterogeneity characterization of biomimetic composite structures.

Endogenous Two-Photon Excited Fluorescence Imaging Characterizes Neuron and Astrocyte Metabolic Responses to Manganese Toxicity.

As neurodegenerative conditions are increasingly linked to mitochondrial dysfunction, methods for studying brain cell metabolism at high spatial resolution are needed to elucidate neurodegeneration mechanisms. Two-photon excited fluorescence (TPEF) imaging is a non-destructive, high-resolution technique for studying cell metabolism via endogenous fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). We employed TPEF to study the metabolism of primary rat astrocyte and neuronal cultures under normal growth conditions and in response to manganese (Mn) treatment. Histograms of pixel-wise optical redox ratio, defined as FAD/(FAD + NAD(P)H), revealed three distinct redox distributions and significant differences in their relative weights between astrocytes and neurons. When treated with Mn, both cell types exhibited redox ratio shifts consistent with increased oxidative stress. However, the manner in which the redox distributions was affected was distinct for the two cell types. Furthermore, NAD(P)H fluorescence lifetime imaging revealed an increase in bound NAD(P)H fraction upon Mn treatment for neurons, consistent with enhanced apoptosis. Astrocytes showed a decrease in bound fraction, possibly due to a shift towards glycolytic metabolism in response to impaired respiration. These results exhibit TPEF's utility for characterizing detailed metabolic changes of different brain cell types in response to neurotoxins.

Tutorials for Electrophysiological Recordings in Neuronal Tissue Engineering.

Electrophysiology is a powerful tool to examine cellular functions, but the use of the techniques remains challenging outside of physiology and neuroscience fields. We aim to provide a practical methods guide for electrophysiological recordings to nonexperts in the field to help with the utility of these important research tools. We focus on two techniques that are critical in the context of tissue engineering, whole-cell patch clamp recording for assessing cellular functions and extracellular field potential recording for evaluating network activities. Specific examples are presented to demonstrate how the techniques were applied to tissue engineering studies.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues.

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.

Fetal brain extracellular matrix boosts neuronal network formation in 3D bioengineered model of cortical brain tissue.

The extracellular matrix (ECM) constituting up to 20% of the organ volume is a significant component of the brain due to its instructive role in the compartmentalization of functional microdomains in every brain structure. The composition, quantity and structure of ECM changes dramatically during the development of an organism greatly contributing to the remarkably sophisticated architecture and function of the brain. Since fetal brain is highly plastic, we hypothesize that the fetal brain ECM may contain cues promoting neural growth and differentiation, highly desired in regenerative medicine. Thus, we studied the effect of brain-derived fetal and adult ECM complemented with matricellular proteins on cortical neurons using in vitro 3D bioengineered model of cortical brain tissue. The tested parameters included neuronal network density, cell viability, calcium signaling and electrophysiology. Both, adult and fetal brain ECM as well as matricellular proteins significantly improved neural network formation as compared to single component, collagen I matrix. Additionally, the brain ECM improved cell viability and lowered glutamate release. The fetal brain ECM induced superior neural network formation, calcium signaling and spontaneous spiking activity over adult brain ECM. This study highlights the difference in the neuroinductive properties of fetal and adult brain ECM and suggests that delineating the basis for this divergence may have implications for regenerative medicine.

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Living donor liver transplantation for giant cavernous hemangioma of liver in a child.

A 10-yr-old girl presented with a seven-month history of upper abdominal discomfort and weight loss. Physical examination revealed an abdominal lump in the right hypochondrium and epigastrium. Ultrasound examination and a computerized tomographic scan showed a large lobulated mass arising from segments I, 1V, and VIII of liver with arteriovenous shunting and multiple small masses in segments VI and VII. An initial diagnosis of hemangioendothelioma with metastasis was made elsewhere following which she received chemotherapy. She had persistent abdominal discomfort because of which she became dependent on narcotics. The patient had fever because of tumor necrosis and also developed peripheral neuropathy. Finally, owing to progressively worsening of symptoms, she underwent left lobe living donor liver transplantation. Histopathological examination showed the mass to be a cavernous hemangioma, and the patient is now well.

Esophageal tuberculosis mimicking submucosal leiomyoma- report of a case.

We present a case of primary esophageal tuberculosis presenting as solitary submucosal mass in cervical esophagus with clinical features of dysphagia, thus mimicking a submucosal leiomyoma on imaging in a 39 year old lady. Excision of this mass revealed features suggestive of tuberculosis on histopathology.

Liver hemangioma mimicking cholangiocarcinoma--a diagnostic dilemma.

A 55-year-old man presented with a liver mass that had been diagnosed on ultrasonography, carried out in response to the patient's complaint of non-specific abdominal pain. Triphasic computed tomography (CT) revealed a lesion involving segments 1, 4, 5 and 8 of the liver. It was centrally hypodense with peripheral enhancement in the arterial phase suggesting a cholangiocarcinoma. The middle hepatic vein was encased and the tumour was present near the junction of the left hepatic and middle hepatic veins. We planned a right hepatic trisegmentectomy including resection of the caudate lobe but since the estimated volume of the liver remnant was only 17% of the total, we first embolised the right portal vein. CT scan repeated 5 weeks later revealed that the lesion was still resectable and that the left lateral segment had hypertrophied to 27% of the liver volume. We performed a right trisegmentectomy including caudate lobe resection using intra-operative ultrasonography to establish that the left hepatic vein was not involved. The removed lesion was hard with ill-defined margins. Histopathological examination revealed a hemangioma.