Collagen-GAG scaffold fabrication, characterization, and measurement of cell migratory and contractile behavior via confocal microscopy

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Includes bibliographical references (v. 2, leaves 371-393).Three-dimensional, collagen scaffolds are an analog of the extracellular matrix and are used for many tissue engineering applications. While material and microstructural properties significantly affect overall scaffold bioactivity, the specific influence of construct mechanical properties, composition, and pore microstructure is unknown. In this thesis, experimental and theoretical approaches are employed to systematically examine the independent effect of extracellular features on cell behavior within a series of standardized, well-characterized, collagen-glycosaminoglycan (CG) scaffolds, providing valuable information for designing biomaterials with improved physiological relevance. This thesis also aims to provide experimental and theoretical approaches appropriate for characterizing and describing a wide range of porous biomaterials and for quantifying the effect of extracellular cues on cell behavior within these biomaterials. CG scaffolds are fabricated via freeze drying. Novel thermal processing conditions were developed to produce two homologous series of uniform, mechanically isotropic CG scaffolds, one with varying pore size and constant stiffness and the other with constant pore size and varying stiffness.(cont.) The thermal processing conditions and the resultant scaffold microstructure have been modeled using an isothermal coarsening heat transfer model within a conductive mold with interface resistance, allowing fabrication of future scaffolds with engineered microstructures. The mechanical properties, specific surface area, and permeability of the scaffolds have been experimentally measured and theoretically described using a cellular solids framework appropriate for modeling many porous biomaterials. This thesis research has produced a standardized series of CG scaffolds appropriate for quantitative in vitro cell behavior assays. An experimental methodology for measuring cell-generated contractile forces and cell motility is described and implemented; the independent effect of scaffold pore size and stiffness on the magnitude and kinetics of cell motility within the scaffolds was determined via confocal microscopy. Slight changes in the extracellular environment appreciably influence cell behavior. Significant effects of cell density, cell type, scaffold microstructure, and scaffold stiffness were observed: cell migration speed increased with decreasing pore size or increasing cell density and increased asymptotically with scaffold stiffness. An improved measurement of the contraction force generated by single dermal fibroblasts (Fc = 26 + 13 nN) within the CG scaffold has also been made.by Brendan A. Harley.Sc.D

Peripheral nerve regeneration through collagen devices with different in vivo degradation characteristics

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2002.Includes bibliographical references (leaves 158-159).In the United States more than 200,000 people are treated each year for peripheral nerve injuries that require surgery. Functional recovery of motor and sensory capability is limited following autograft, the most common procedure for peripheral nerve repair. Tubulation repair of a transected peripheral nerve presents an alternative to autograft, but has not yet shown the ability to satisfactorily restore lost function. A variety of degradable and non-degradable materials have been used to fabricate the tubes used to bridge the stumps of a transected nerve, and collagen tubes have been specifically shown to improve the quality of regeneration. The optimal characteristics for the tube have yet to be identified, and a collagen device that can be manipulated and optimized to improve functional peripheral nerve recovery is needed. The overall goal of this thesis was to fabricate and characterize a new collagen tube such that the tube characteristics and their effects on peripheral nerve regeneration can be closely studied, manipulated, and optimized. Following characterization of a homologous series of collagen tubes, each produced with a different density of crosslinks, the regenerative capacity of each member of the series of tubes was studied to determine the effects of device permeability, crosslink density, and in vivo tube degradation rate on peripheral nerve regeneration. It was first observed that the device permeability to cells and large soluble regulators significantly affected the quality of peripheral nerve regeneration, with the more permeable device (pore diameter 80 - 100 tm)(cont.) showing significantly improved regenerative capacity compared to a semi-permeable tube (pore diameter - 22 nm). It was then observed that the quality of nerve regeneration increases with an increase in tube degradation rate; however, after reaching a maximum at an intermediate degradation rate, the quality of nerve regeneration decreases gradually at increasingly higher tube degradation rates. The data indicate that the morphologic properties of the regenerated axons can be optimized by varying the in vivo degradation rate of the tube, and that tube degradation has a powerful effect on peripheral nerve regeneration.by Brendan A. Harley.S.M

Transient Flow Routing in Channel Networks

The formulation of a mathematical model to predict transient flows in hydraulic networks is presented. The network formulation consists of breaking the network into a series of connected reaches; reducing the finite difference equations for each reach into two "reach" equations; forming an exterior matrix consisting of the reach equations, external boundary conditions, and interior compatibility conditions; solving the external matrix for the end values of discharge and water surface elevation for all reaches and back-substituting for all interior values. Examples presented include the James River, USA, estuary model (twenty-four nodes and twenty-six reaches), the Cork Harbour, Ireland, estuary (thirteen-reach, double-looped network), and the Rio Bayamon basin, Puerto Rico. Results are very satisfactory when compared to known data

Influence of freezing rate on pore structure in freeze-dried collagen-GAG scaffolds.

The cellular structure of collagen-glycosaminoglycan (CG) scaffolds used in tissue engineering must be designed to meet a number of constraints with respect to biocompatibility, degradability, pore size, pore structure, and specific surface area. The conventional freeze-drying process for fabricating CG scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold. In this study, the scaffold synthesis process was modified to produce more homogeneous freezing by controlling of the rate of freezing during fabrication and obtaining more uniform contact between the pan containing the CG suspension and the freezing shelf through the use of smaller, less warped pans. The modified fabrication technique has allowed production of CG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold (mean: 95.9 microm, CV: 0.128) compared to the original scaffold (mean: 132.4 microm, CV: 0.185). The pores produced using the new technique appear to be more equiaxed, compared with those in scaffolds produced using the original technique

Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds.

The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration

The effect of pore size on permeability and cell attachment in collagen scaffolds for tissue engineering.

The permeability of scaffolds and other three-dimensional constructs used for tissue engineering applications is important as it controls the diffusion of nutrients in and waste out of the scaffold as well as influencing the pressure fields within the construct. The objective of this study was to characterize the permeability/fluid mobility of collagen-GAG scaffolds as a function of pore size and compressive strain using both experimental and mathematical modeling techniques. Scaffolds containing four distinct mean pore sizes (151, 121, 110, 96 microns) were fabricated using a freeze-drying process. An experimental device was constructed to measure the permeability of the scaffold variants at different levels of compressive strain (0, 14, 29 and 40% while a low-density open-cell foam cellular solids model utilizing a tetrakaidecahedral unit cell was used to accurately model the permeability of each scaffold variant at all level of applied strain. The results of both the experimental and the mathematical analysis revealed that scaffold permeability increases with increasing pore size and decreases with increasing compressive strain. The excellent comparison between experimentally measured and predicted scaffold permeability suggests that cellular solids modelling techniques can be utilized to predict scaffold permeability under a variety of physiological loading conditions as well as to predict the permeability of future scaffolds with a wide variety of pore microstructures

Biodiversity 2020: climate change evaluation report

In 2011, the government published Biodiversity 2020: A strategy for England’s wildlife and ecosystem services [1]. This strategy for England builds on the 2011 Natural Environment White Paper - NEWP [2] and provides a comprehensive picture of how we are implementing our international and EU commitments. It sets out the strategic direction for biodiversity policy between 2011-2020 on land (including rivers and lakes) and at sea, and forms part of the UK’s commitments under the ‘the Aichi targets’ agreed in 2010 under the United Nations Convention of Biological Diversity’s Strategic Plan for Biodiversity 2011-2020 [3]. Defra is committed to evaluating the Biodiversity 2020 strategy and has a public commitment to assess climate change adaptation measures. This document sets out the information on assessing how action under Biodiversity 2020 has helped our wildlife and ecosystems to adapt to climate change. Biodiversity 2020 aims to halt the loss of biodiversity and restore functioning ecosystems for wildlife and for people. The outcomes and actions in Biodiversity 2020, although wider in scope, aimed to increase resilience of our wildlife and ecosystems in the face of a changing climate. In order to inform the assessment, we have defined which of the measurable outputs under Biodiversity 2020 contribute to resilience. Biodiversity 2020 included plans to develop and publish a dedicated set of indicators to assess progress towards the delivery of the strategy. The latest list (at the time of writing), published in 2017, contains 24 biodiversity indicators [4] that would help inform progress towards achieving specific outcomes, they are also highly relevant to the outputs (detailed below) that form the basis for this evaluation. The Adaptation Sub-Committee’s 2017 UK Climate Change Risk Assessment Evidence Report [5] sets out the priority climate change risks and opportunities for the UK. The ASC also produced a review of progress in the National Adaptation Programme - “Progress in preparing for climate change” [6], which highlights adaptation priorities and progress being made towards achieving them. The UK Government’s response to the ASC [7] review includes a set of recommendations, of which Recommendation 6 states that “Action should be taken to enhance the condition of priority habitats and the abundance and range of priority species”. The recommendation further iterated that “This action should maintain or extend the level of ambition that was included in Biodiversity 2020” and that “An evaluation should be undertaken of Biodiversity 2020 including the extent to which goals have been met and of the implications for resilience to climate change.” To this, end an evaluation process has been put in place to define: a. What worked and why? Which actions or activities have had the greatest benefit in terms of delivering the desired outcomes? And, conversely, what prevented progress? b. Where are the opportunities? What are the financial, political, scientific and social opportunities for furthering the desired outcomes in the future? These objectives underpin the evaluation process for actions to date, and will also inform future actions and the iteration of a new nature strategy for England

Return of Genomic Results to Research Participants: The Floor, the Ceiling, and the Choices In Between

As more research studies incorporate next-generation sequencing (including whole-genome or whole-exome sequencing), investigators and institutional review boards face difficult questions regarding which genomic results to return to research participants and how. An American College of Medical Genetics and Genomics 2013 policy paper suggesting that pathogenic mutations in 56 specified genes should be returned in the clinical setting has raised the question of whether comparable recommendations should be considered in research settings. The Clinical Sequencing Exploratory Research (CSER) Consortium and the Electronic Medical Records and Genomics (eMERGE) Network are multisite research programs that aim to develop practical strategies for addressing questions concerning the return of results in genomic research. CSER and eMERGE committees have identified areas of consensus regarding the return of genomic results to research participants. In most circumstances, if results meet an actionability threshold for return and the research participant has consented to return, genomic results, along with referral for appropriate clinical follow-up, should be offered to participants. However, participants have a right to decline the receipt of genomic results, even when doing so might be viewed as a threat to the participants’ health. Research investigators should be prepared to return research results and incidental findings discovered in the course of their research and meeting an actionability threshold, but they have no ethical obligation to actively search for such results. These positions are consistent with the recognition that clinical research is distinct from medical care in both its aims and its guiding moral principles

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure