Lec 13 Oxygen in TE PDF

Oxygen in TE Prof. Tracy Jane Puccinelli B M E 510 – Introduction to Tissue Engineering Lecture Outline Critical role that oxygen plays in tissues Different levels of oxygenation TE strategies capable of regulating oxygen Another regulatory signal – osmolarity...

Oxygen in TE Prof. Tracy Jane Puccinelli B M E 510 – Introduction to Tissue Engineering Lecture Outline Critical role that oxygen plays in tissues Different levels of oxygenation TE strategies capable of regulating oxygen Another regulatory signal – osmolarity 2 Environmental Cues in Stem Cell Niche Nature Biotechnology 2014; 32: 795-803 3 What if the earth lost oxygen for 5 seconds? 4 https://youtu.be/rdt85jgRHt0  What if we doubled the amount of oxygen on Earth? change athmosphere - colder because the waves cannot get through Pressure - but we vill probably not survive that fires - more uncontrolled Metals http://funsubstance.com/fun/45771/if-the-world-lost-oxygen-for-5-seconds/ more oxidative stress - cumulative of the oxidative stress 5 Critical Roles of Oxygen in TE Embryonic development Maintaining stem cell pluripotency Initiation of signaling pathways, such as angiogenesis Effects on cell behaviors including differentiation, migration, apoptosis, proliferation 6 Oxygen Tension in Organs and Tissues Oxygen tension is the partial pressure of oxygen Drops in circulation and again in tissues drops a bit when going into the lungs Tissues have different oxygen tensions round up to 1 from 1 to 13 oxygen in the class Eddington et al, Lab on a Chip, 2014 Sitkovsky et al., Nature Reviews Immunology 2005 7 Oxygen Tension in Organs and Tissues dont memories this Borrás et al., Intl J of Molec Sciences 2019 8 Oxygen Tension in Organs and Tissues remember this relative scale, not the exact number moderate to low Cheema et al., J of Tissue Engr. 2011 9 Various Levels of Oxygenation Normoxia – atmospheric oxygen pressure (21% O2) Physioxia or Tissue Normoxia or Physiological Hypoxia – oxygen partial pressure in different organs/tissues in a physiological condition (1-14% O2) range from 1 to 14 and will be different in different tissue types stem cell - Hypoxia is used to describe below 5 precent Hypoxia – oxygen partial pressure lower than physioxia and indicative for a lack of oxygenation in the tissue--varies in different tissues Note: term hypoxia often used in TE to describe relatively low oxygen Tissue Harvest need more cells for our applicaiton Expansion Culture done at a specific oxygen level Differentiation Culture 15 Stem Cell Aging/Senescence in Culture Challenges associated with in vitro cell expansion Poor growth kinetics Early senescence Genetic instability during expansion Poor engraftment after transplantation 16 Hypoxia and Stem Cell Biology Reduces the rate of hESC differentiation, maintains full pluripotency, and enhances formation of embryoid bodies Maintains a primitive phenotype and enhances engraftment capabilities of HSCs-hematopoietic stem cells blood cells Increases efficiency of reprogramming of iPSCs trying to make adult cells like embryonic stem cells. 17 Hypoxia and Mesenchymal Stem Cell Biology Upregulates cell proliferation and increases population doubling Upregulates osteogenic and adipogenic differentiation Maintains multi-lineage differentiation Reduces DNA damage Improves engraftment in vivo Increases expression of chemokine receptor critical to homing of MSCs 18  Small Molecules Mimicking Hypoxia Dimethyloxallyl glycine (DMOG) Non-specific inhibition of prolyl-4-hydroxylase by competition https://www.caymanchem.com/product/71210 Desferrioxamine Inhibition of prolyl hydroxylase by Fe2+ chelation of the catalytic core in hemoglobin https://www.drugfuture.com/chemdata/Deferoxamine.html Metal ions (for example, Co2+ and Cu2+) Inhibition of prolyl hydroxylase by substitution for Fe2+ of the catalytic core in hemoglobin *Prolyl hydroxylase is able to inactivate HIF-1α activity in normoxic conditions 19 Oxygen Tension in a Cell Culture Incubator An incubator provides an environment with a 95% air (79% N2/21% O2) atmosphere and 5% carbon dioxide, thus constituting 19.95% O2 20 Hypoxic Culture Set-up Hypoxia chamber Hypoxia Station Hypoxia chamber with controller 21 Proliferation and Cell Morphology The graph (A) shows the effect of oxygen levels on the proliferation of MSCs over time. Two conditions are compared Normoxia = 21% This metric reflects how many times the cell population has doubled over a given time Hypoxia = 1% looking at MSC cell number P = passage grow you need to remove them?? long and spindely MSC's in hypoxic culture reach a higher PD number and exhibit a more normal morphology than those in normoxic Higher Proliferation Under Hypoxia: MSCs cultured under 1% oxygen reach a higher accumulated culture! population doubling than those in 21% oxygen. This suggests that hypoxic conditions promote more The cells remain long and spindle-shaped, which is associated rapid and sustained proliferation of MSC with a younger, more proliferative state in MSCs Passage Points (p4 and p7): These points (p4 and p7) represent times when cells were subcultured or “passaged” (transferred to a new culture dish to prevent overcrowding and maintain growth). Passage is common in cell culture to keep cells in optimal growing conditions Palumbo et al., Stem Cells and Development 2014 22  Oxygen Tension and Diffusion Limit diffusion limit for oxygen pO2 is decreased with an increasing distance away from the vessel wall Lack of oxygen leads to cell apoptosis and tissue necrosis dramatically decreases chondrosites in a scaffold They require very little oxygen (look at 1 slide) very evaskulair = low oxygen The graph on the right shows angiogenisis the oxygen concentration as a function of distance from the blood vessel wall. It demonstrates a rapid drop in oxygen levels as you move further away from the vessel, leveling off at very low levels This diffusion limit can pose a challenge in tissue engineering, where cells in scaffolds, such as chondrocytes (cartilage cells), often require very low oxygen levels but still need some oxygen for survival Malda et al., Biotechnology and Bioengineering 2004 Carmeliet et al., Nature 2000 23  TE Model to Study O2 Gradients and Cell Response VEGF expression in core cells increases over time, indicating that cells in low-oxygen Bone marrow stromal environments respond by upregulating VEGF, likely to promote angiogenesis and improve cells in collagen scaffolds oxygen availability. roled up and exposed to oxygen VEGF expression in cells measured the oxygen located at different positions exposed to after several days they unrolled it and looked at the expression varying O2 tensions looking at VEGF (vascular endothelial growth factor) expression, which is a key protein involved in angiogenesis (the formation of new blood vessels). The model uses bone marrow stromal cells embedded in collagen scaffolds to simulate different oxygen conditions in a controlled 3D environment. inconclusive Image edited for clarity angiogenesis happens when we have very low oxygen 24 Cheema et al., Eur Cells Mater 2010 TE Strategies to Increase Oxygen Supply Promoting vascularization in tissue-engineered constructs Incorporating oxygen-releasing biomaterials in tissue-engineered constructs 25 TE Strategies to Promote Vascularization with factors that can promote angiogenesis A: scaffold functionalization depending on muscle types B: cell-based techniques promote mass transport mimics vasculature C: bioreactor designs D: microelectromechanical systems E: modular assembly impant device and hope it connects with in vivo F: in vivo systems 26 Oxygen-generating Materials Common oxygen-releasing materials: sodium percarbonate dont remeber calcium peroxide magnesium peroxide hydrogen peroxide actually used as a disinfectant fluorinated compounds Byproduct concerns: Solids (Ca) Peroxide Basic products 27 Other Regulator Signal - Osmolarity 28 Osmolarity regulatory signal dont remeber the equations 29 Osmolarity Osmolarity imbalance can affect cells especially during processing (bottom-up approaches), and culturing cells in scaffolds Can compromise integrity of cell wall succrose = regulater Can decrease cell viability Methods to control osmolarity of the system Physiological buffers during processing (crosslinking chemistry of polymers can be a limiting factor) Chemically inert osmotic regulator (such as sucrose) Cell/tissue culture in scaffolds—bioreactors with flow and sensors 30  important that chondocytes produce GAG Chondrocyte Response to Osmotic Pressure chondrocytes = CARTILAGE Chondrocyte Activity: Under these osmotic conditions, chondrocytes actively produce GAGs, which contribute to the elasticity and resilience of cartilage Normal cartilage has an osmolarity range of around 360–500 mOsm, with blood vessels supplying oxygen, glucose, and other nutrients In osteoarthritic conditions, cartilage deteriorates, blood vessel support diminishes, and the osmolarity in the joint decreases. With decreased blood supply, the levels of oxygen, pH, and osmolarity drop, creating a less supportive environment for chondrocytes. Lower osmolarity in OA cartilage leads to reduced GAG production, weakening the cartilage structure and exacerbating joint degradation. osmolarities goes down with At higher osmolarities (470–570 mOsm), GAG production is generally higher, osteoarhritis + blood vessels degrades indicating that chondrocytes thrive and produce more GAGs in higher osmotic conditions. Lower osmolarity (270 mOsm) leads to significantly less GAG production, highlighting the negative impact of low osmotic conditions on chondrocyte function There is a peak in GAG production at around 400–500 mOsm, especially noticeable on day 4. This indicates that chondrocytes have an optimal osmolarity range where they produce the highest amounts of GAGs. At very low (200 mOsm) or high (600 mOsm) osmolarities, GAG production decreases, showing that both too low and too high osmotic pressures can impair chondrocyte function. Shigeru Kobayashi, Regenerative Medicine and Tissue Engineering 2013 31 Summary Oxygen tension plays a critical role in regulation of cell activities and maintenance of tissue homeostasis Different TE strategies have been developed to engineer a microenvironment with physioxic conditions Osmotic pressure is a critical but often neglected regulatory signal in tissue engineering studies 32