Use of a 3D perfusion bioreactor with osteoblasts and osteoblast/endothelial cell co-cultures to improve tissue-engineered bone

The delivery of oxygen, nutrients, and the removal of waste are essential for cellular survival. Culture systems for 3D bone tissue engineering have addressed this issue by utilizing perfusion flow bioreactors that stimulate osteogenic activity through the delivery of oxygen and nutrients by low-shear fluid flow. It is also well established that bone responds to mechanical stimulation, but may desensitize under continuous loading. While perfusion flow and mechanical stimulation are used to increase cellular survival in vitro, 3D tissue-engineered constructs face additional limitations upon in vivo implantation. As it requires significant amounts of time for vascular infiltration by the host, implants are subject to an increased risk of necrosis. One solution is to introduce tissue-engineered bone that has been pre-vascularized through the co-culture of osteoblasts and endothelial cells on 3D constructs. It is unclear from previous studies: 1) how 3D bone tissue constructs will respond to partitioned mechanical stimulation, 2) how gene expression compares in 2D and in 3D, 3) how co-cultures will affect osteoblast activity, and 4) how perfusion flow will affect co-cultures of osteoblasts and endothelial cells. We have used an integrated approach to address these questions by utilizing mechanical stimulation, perfusion flow, and a co-culture technique to increase the success of 3D bone tissue engineering. We measured gene expression of several osteogenic and angiogenic genes in both 2D and 3D (static culture and mechanical stimulation), as well as in 3D cultures subjected to perfusion flow, mechanical stimulation and partitioned mechanical stimulation. Finally, we co-cultured osteoblasts and endothelial cells on 3D scaffolds and subjected them to long-term incubation in either static culture or under perfusion flow to determine changes in gene expression as well as histological measures of osteogenic and angiogenic activity. We discovered that 2D and 3D osteoblast cultures react differently to shear stress, and that partitioning mechanical stimulation does not affect gene expression in our model. Furthermore, our results suggest that perfusion flow may rescue 3D tissue-engineered constructs from hypoxic-like conditions by reducing hypoxia-specific gene expression and increasing histological indices of both osteogenic and angiogenic activity. Future research to elucidate the mechanisms behind these results may contribute to a more mature bone-like structure that integrates more quickly into host tissue, increasing the potential of bone tissue engineering

Efficacy of Learning with Course-provided Equation Reference Sheets in Engineering Education

This paper studies the efficacy of course-provided reference sheets on student learning when allowing reference material on exams versus other methods. Moving from student-provided note sheets to course-provided note sheets reduced the course exam failure rate from 11.8% to 0%. In previous iterations of ME388 Helicopter Aeronautics exam resources varied from student-provided note sheets to open-book exams with several iterations taking some combination of the two. Another course in the department of Civil and Mechanical Engineering at the United States Military Academy, MC311 Thermal-Fluid Systems I, inspired this research with its long-standing success with a course-provided 8½ x 11” front-and-back equation reference card used for over 20 iterations with positive results, affectionately named and referred to as the RDC for Reference Data Card, although it contains almost entirely equations, not data. Thus, the ME388 course instructors piloted a two-page course-provided equation sheet to students taking ME388 Helicopter Aeronautics at the beginning of the spring semester in the 2020 Academic Year with the goal of simplifying the teaching model and attempting to help students avoid common mistakes made during previous iterations of the course that used various formats of closed- and open-book exams. This paper will introduce the concept of closed- and open-reference teaching and assessment methods including a canvas of academic literature on related research. Motivation for the inclusion of the course-provided equation reference sheet determined from course feedback collected from previous iterations is analyzed and discussed. Current students are surveyed to gain insight into the students’ comfort with the material and gain anecdotal results on the method. Next, the aspects of designing and implementing the reference material are discussed with thoughts on layout, which equations to include, which data to include, and how to incorporate the reference material into daily instruction. Student feedback is analyzed with discussion on any adjustments made thereafter along with applicable justification from student feedback. Finally, a conclusive evaluation is determined from a synthesis of anecdotal evidence, Likert scale feedback, and exam grade comparison to previous iterations. This is weighed against the literature from other academic research and a discussion of the merits and disadvantages of allowing reference material on exams. The paper concludes with a final determination on the pilot program’s efficacy on student learning when implementing a course-provided equation reference sheet and recommendations for future work

A closer look at ARSA activity in a patient with metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease mainly caused by a deficiency of arylsulfatase A activity. The typical clinical course of patients with the late infantile form includes a regression in motor skills with progression to dysphagia, seizures, hypotonia and death. We present a case of a 4-year-old female with rapidly progressive developmental regression with loss of motor milestones, spasticity and dysphagia. MRI showed volume loss and markedly abnormal deep white matter. Enzymatic testing in one laboratory showed arylsulfatase A activity in their normal range. However, extraction of urine showed a large increase in sulfatide excretion in a second laboratory. Measurement of arylsulfatase A in that laboratory showed a partial decrease in arylsulfatase A activity measured under typical conditions (about 37% of the normal mean). When the concentration of substrate in the assay was lowered to one quarter of that normally used, this individual had activity \u3c10% of controls. The patient was found to be homozygous for an unusual missense mutation in the arylsulfatase A gene confirming the diagnosis of MLD. This case illustrates the importance of careful biochemical and molecular testing for MLD if there is suspicion of this diagnosis

Impaired burrowing is the most prominent behavioral deficit of aging htau mice

htau mice are deficient of murine tau but express all six human tau isoforms, leading to gradual tau misprocessing and aggregation in brain areas relevant to Alzheimer's disease. Whilst histopathological changes in htau mice have been researched in the past, we focused here on functional consequences of human tau accumulation. htau mice and their background controls - murine tau knock-out (mtau-/-) and C57Bl/6J mice - underwent a comprehensive trial battery to investigate species-specific behaviour, locomotor activity, emotional responses, exploratory traits, spatial and recognition memory as well as acquisition, retention and extinction of contextual fear at two-, four-, six-, nine- and twelve-months-of-age. In htau mice, tau pathology was already present at two-months-of-age, whereas deficits in food burrowing and spatial working memory were first noted at four-months-of-age. At later stages the presence of human tau on a murine tau knockout background appeared to guard cognitive performance; as mtau-/- but not htau mice differed from C57Bl/6J mice in the food burrowing, spontaneous alternation and object discrimination tasks. Aging mtau-/- mice also exhibited increased body mass and locomotor activity. This data highlights that reduced food burrowing performance was the most robust aspect of the htau phenotype with ageing. htau and mtau-/-deficits in food burrowing pointed at the necessity of intact tau systems for daily life activities. Whilst some htau and mtau-/- deficits overlap, age differences between the two genotypes may reflect distinct functional effects and compared to C57Bl/6J mice, the htau phenotype appeared stronger than the mtau-/- phenotype at young ages but milder with ageing

Modulatory effect of mild inflammation on tau phosphorylation in a human tau mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is the most common form of dementia and involves the pathological hyperphosphorylation and aggregation of the microtubule associated protein tau. Aetiological evidence, and evidence from post-mortem studies, suggests neuroinflammation to be an early mechanistic driver of AD. In murine tau models, systemic inflammation induced by either chronic or acute lipopolysaccharide (LPS) administration has been reported to be a strong inducer of early tau pathology through augmenting tau hyperphosphorylation. However, the method by which LPS has been utilised to model inflammation in these studies has not always been representative of the underlying mild inflammation occurring in AD. For example, following chronic LPS administration, rapid tolerance occurs which is not representative of AD. While acute LPS administration results in a rapid pro-inflammatory response, the doses which have generally been utilised are more analogous with sepsis rather than the underlying inflammatory response observed in AD. This thesis is aimed at furthering clarifying the contribution of systemic inflammation to early tau pathology through systemic administration of low doses of LPS in the hTau model. The hTau model is thought to be the most relevant AD tau model for the expression of all 6, non-mutated, human tau isoforms on a murine tau (mTau) knockout (KO) background. However, hTau mice are associated with systemic pathologies and an increased ratio of 3R:4R tau isoforms which is not representative of AD. As the systemic pathologies are linked to the KO of mTau which consists solely of 4R tau isoforms, hTau mice were bred on a partial mTau background in the hope to avert the systemic pathologies, improve the isoform ratio and conserve the development of tau pathology. Heterozygous mTau expression in hTau/mTau+/- mice resulted in ablation of systemic pathologies, an increase in 4R tau isoforms and augmented tau hyperphosphorylation compared to hTau/mTau-/- mice, indicating the model ideal for understanding early pathological tau alterations. To determine the effect mild inflammation might have on early tau pathology in AD, tau phosphorylation, localisation and aggregation were assessed after 0, 100, 250 and 330 µg/kg (i.v.) LPS administration in 3 month old hTau/mTau+/- and hTau/mTau-/- mice during the height of the pro-inflammatory response at 4h following administration. LPS administration resulted in dose-dependent decreases in the pre-tangle associated phosphorylation epitope; pS202 in both genotypes while the post-tangle associated epitope; pS396/404 levels were selectively decreased in hTau/mTau+/- mice. On the other hand, LPS did not induce tau aggregation. To determine whether less immediate effects on tau pathology occurred, tau pathology was assessed 24h following 250 µg/kg (i.v.) LPS administration in hTau/mTau+/- mice. Tau dephosphorylation persisting at both pS202 and pS396/404 epitopes at this time point. These results suggest that systemic inflammation might play a beneficial role on tau pathology in AD

Functional Consequences of Dendritic Inhibition in the Hippocampus

The ability to store and recall memories is an essential function of nervous systems, and at the core of subjective human experience. As such, neuropsychiatric conditions that impair our memory capacity are devastating. Learning and memory in mammals have long been known to depend on the hippocampus, which has motivated widespread research efforts that converge on two broad themes: determining how different cell types in the hippocampus interact to generate neural activity patterns (structure), and determining how neural activity patterns implement learning and memory (function). Central to both these pursuits are pyramidal cells (PCs) in CA1, the primary hippocampal output, which transform excitatory synaptic inputs into the action potential output patterns that encode information about locations or events relevant for memory. CA1 PCs are embedded in a network of diverse inhibitory (GABA-releasing) interneurons, which may play unique roles in sculpting the activity patterns of PCs that implement memory functions. As a consequence, investigating the functional impact of defined GABAergic interneurons can provide an experimental entry point for linking neural circuit structure to defined computations and behavioral functions in the hippocampal memory system. In this thesis I have applied a panel of novel methodologies to the mouse hippocampus in vitro and in vivo to link structure to function and behavior, and determine 1) how hippocampal inhibitory cell types shape distinct patterns of PC activity, and 2) how these inhibitory cell types contribute to the encoding of contextual fear memories. To first establish the means by which interneuron subtypes contribute to PC activity patterns, I used optogenetic techniques to activate spatiotemporally distributed synaptic excitation to CA1 in vitro, and recorded from PCs to quantify the frequency of output spikes relative to input levels. I subsequently used a dual viral and transgenic approach to combine this technique with selective pharmacogenetic inactivation of identified interneurons during synaptic excitation. I found that inactivating somatostatin-expressing (Som+) dendrite-targeting interneurons increased the gain of PC input-output transformations by causing more output spikes, while inactivating parvalbumin-expressing (Pvalb+) soma-targeting interneurons did not. Inactivating Som+ inhibitory interneurons allowed the dendrites of PCs to generate local NMDA receptor-mediated electrogenesis in response to synaptic input, resulting in high frequency bursts of output spikes. This discovery suggests neuronal coding via hippocampal burst spiking output can be regulated by Som+ dendrite-targeting interneurons in CA1. Specific types of neural codes are believed to have different functional roles. Neural coding with burst spikes is known to support hippocampal contributions to classical contextual fear conditioning (CFC). In CFC the hippocampus encodes the multisensory context as a conditioned stimulus (CS), whose burst spiking output is paired with the aversive unconditioned stimulus (US) in the amygdala, allowing for fear memory recall upon future exposure to the CS. To investigate the contribution of Som+ interneurons to this behavior, I designed a CFC task for head-fixed mice, allowing for optical recording and manipulation of activity in defined CA1 cell types during learning. Pharmacogenetic inactivation of CA1 Som+ interneurons, but not Pvalb+ interneurons, prevented the encoding of CFC. 2-photon Ca2+ imaging revealed that during CFC the US activated CA1 Som+ interneurons via cholinergic input from the medial septum, driving inhibition to the PC distal dendrites that receive coincident excitatory input from the entorhinal cortex. Inactivating Som+ interneurons increases PC population activity, and suppressing dendritic inhibition during the US alone is sufficient to prevent fear learning. These results suggest sensory features of the US reach CA1 PCs through entorhinal inputs, and thus require active inhibitory filtering by Som+ interneurons to ensure hippocampal output exclusively encodes the CS during CFC. In conclusion, I found that Som+ interneurons in CA1 are an effective regulator of PC burst spiking because they inhibit dendritic electrogenesis. This function is used by the hippocampus to prevent the US from influencing the burst spike output of PCs that encode the CS, ensuring successful CFC. This work bridges the gap between cells, circuits, and behavior, and provides mechanistic insight into one of our most essential cognitive functions - the ability to learn and remember