Exam Flashcards
1
Q
What are the key components of tissue engineering?
A
- The right cell source for the job
- The right microenvironment/scaffold to support the cells
- The right growth factors to make cells healthy and productive
- Physical and mechanical forces to influence cell development and assembly
2
Q
Why do we need tissue engineering?
A
- Organ donor shortage
- Increasing need and number of transplants
- Increasing ageing population
- Constraints on donor matching, transportation and transplantation
3
Q
What are the 4 types of tissue?
A
- Connective tissue- Most diverse and abundant. Provide structural support as bones, ligaments and tendons bind bone and muscle to bone, nutrient storage and transportation by bones, blood, lymphatic vessels. Have few cells, have large amount of ECM, all connective tissue comes from mesenchyme.
- Epithelium tissue- cover internal and external surfaces, creating a barrier.
- Muscle tissue - allows movement by cell contraction
- Nervous tissue- coordinates many body activities
4
Q
What are the subtypes of connective tissue?
A
- ‘Connective tissue proper’ is fibroblasts, macrophages, mast cells, white blood cells. Connective tissue fibers include collagen, elastic and reticular fibers. Collagen is the strongest, elastic can stretch and recoil, reticular is short and thin
- Supporting connective tissue- bone, cartilage
- Fluid connective tissue- blood, lymph
5
Q
The key components of the cytoskeleton
A
- microtubules- long and hollow tubulin polymer, provide structure for transport
- microfilaments (actin filaments)- actin polymer double helix, role in cell movement and cytokinesis
- intermediate filaments- variety of different proteins like keratin, provide mechanical strength, support and structure.
6
Q
Components of the ECM
A
Composition is tissue specific and is a structural scaffold that regulates cell function and homeostasis. Has ‘ground substance’ which fills space between cells and connects fibers, consists of interstitial fluid, cell adhesion proteins and proteoglycans and fibrous proteins like collagen and elastin that determine biomechanical and viscoelasticity. Fibroblasts continuously secrete fibers for the ECM and components for ground substance.
7
Q
Types of collagen
A
Type 1 is most common and provide strength, rigidity and support.
Type 2 is in cartilage
Type 3 is a major structural component in hollow organs such as large blood vessels, uterus and bowel.
Type 4 is major in the basal lamina of the basement membrane, maintaining the barrier between tissue compartments and enabling cell signaling as it interacts with proteins.
8
Q
What is laminin?
A
an ECM glycoprotein in the basal laminae (a layer of the basement membrane) that provides a site for cell attachment. Laminin works in basement membrane assembly, cell proliferation, differentiation and migration.
9
Q
What is the basement membrane?
A
A thin ECM structure that separates and anchors epithelial and endothelial layers to connective tissue. Gives mechanical support to a sheet of epithelial cells, has an apical (external) and basal (internal) side and epithelial polarity. Epithelial polarity defines the cell function and ensures transport of ions across cell sheets
10
Q
What are challenges in tissue engineering?
A
Cell Source :
-suitable sources
-limitations on sources
-cell expansion is time consuming.
-Ethical objections to human embryo harvesting for embryonic stem cells
Materials
- to create a bioactive scaffold that supports different cell types and allows interaction
- tissue vascularisation requires nutrients, oxygen and waste removal to prevent cell death or necrosis in engineered tissues.
- possibility of immune rejection
- regulatory approval
- spatial constraint, physical force, biochemical cues, cell types and growth programs that vary between tissues
- patient acceptance
11
Q
The types of stem cells
A
- embryonic stem cells (ESC) - from inner mass of blastocyst (hollow ball of cells from a fertilised egg), are pluripotent so they can become every cell type
- induced pluripotent stem cells (iPSC) - from genetically reprogramed adult stem cells. Converted tissue specific cells into cells that behave like ESC
- adult stem cells (ASC)- tissue specific, found in small numbers
12
Q
What are mesenchymal stem cells?
A
ASC’s that can become other cell types and come from many sources (bone marrow, adipose tissue, umbilical cord tissue, GI tract, placenta, amniotic fluid). MSC’s are multipotent and can self renew to sustain tissue development and maintenance. Can be isolated easily and expand in-vitro, have good differentiation ability.
13
Q
Examples of biomaterials?
A
- Bioceramics for dental and bone replacement/implant. Can be bioactive or bioinert . Have high mechanical stiffness, low elasticity and a hard/brittle surface.
- Synthetic polymer- Can be manufactured on a large scale, can control properties. Lack biological capacity for recognition as they have no ligands
- Natural polymers like hydrogels that have similar properties to natural tissue.
- smart biomaterials that respond to changes in external stimuli or the local physiological environment
- inert biomaterial produces no toxic reaction while an active biomaterial offer uncontrolled release of therapeutics.
14
Q
What is bio fabrication?
A
→ production of complex living and non living biological product from raw materials
15
Q
Technology used in bio fabrication?
A
3D bioprinting, bioreactor, inkjet printing, laser bioprinter, Extrusion based 3d bioprinting, light based technology
16
Q
What is organogenesis?
A
a phase in early embryonic development that starts after gastrulation and continues until adulthood where organs are formed from the three primary germ layers.
17
Q
What is the process in which the three germ layers are formed and what is its importance?
A
Gastrulation is cell movement that organises an embryo from 2d cells to 3d cell layers called the gastrula, which has ectoderm (outer layer that becomes skin, nervous tissues and eye tissues), mesoderm (middle layer that becomes cardiac, skeletal, smooth muscle, connective tissue and RBC) and endoderm (innermost layer that becomes lungs, thyroid, pancreas, digestive and respiratory tract) germ layers.
18
Q
How are the three germ layers formed?
A
- Epiblast cells invaginate and migrate from their epiblast layer to the hypoblast layer to differentiate into the endoderm.
- Remaining epiblast layer cells differentiate into the ectoderm.
- Cells migrating from epiblast to the middle via the primitive streak differentiate into the mesoderm.
19
Q
What are the two major cell types of the body?
A
- Epithelial cells: Have apical-basal polarity and are stationary.
They can be squamous (flat and sheet-like), cuboidal and columnar (column-like). They can be simple (one layer), stratified (more than one layer) or pseudostratified (closely packed and look stratified but its actually simple).
They adhere tightly to each other and to the ECM at their basal surface to form a sheet of epithelium. - Mesenchymal cells: come from mesoderm layer and have enhanced migratory and invasive properties. They are stellate shaped and are scattered in the ECM. They form connective tissues and do not have apical and basal polarity, but front and back end polarity. Are mobile cells
20
Q
What is EMT and its process?
A
Epithelial to mesenchymal transition (EMT). It is needed in embryonic development and shows a loss of cell-cell adhesion and cell polarity as well as an acquisition of migration and invasive properties.
- loss of cell-cell adhesion
- loss of apical-basal polarity due to loss of adhesion complexes
- re-organisation of the actin cytoskeleton
- loss of epithelial specific markers like e-cadherin
- completion, signalled by degradation of the basement membrane and the formation of a mesenchymal cell that can migrate
21
Q
What is endochondral ossification?
A
Hyaline cartilage is used as a blueprint for ossification and is replaced by bone. This process forms long bones and begins after 2 months of embryo development. It begins when
mesenchymal cells differentiate into chondrocytes. Chondrocytes proliferate rapidly and secrete an extracellular matrix to form the cartilage model for bone.
22
Q
What is intramembranous ossification?
A
Develops compact and spongy bones from mesenchymal stem cells that differentiate into osteoblast cells in connective tissue. Primary method of bone development for first 2 months of an embryo and assists flat bone formation.
23
Q
Which cells form bone?
A
- Osteoblasts: from mesenchymal stem cells. Synthesise a collagen matrix and mineralize bones. Form bone and differentiate into osteocytes.
- Osteocytes: mature osteoblasts remodel bone.
- Osteoclasts that degrade the bone by releasing enzyme.
- Osteogenic cells, stem cells that become osteoblasts.
24
Q
What are the four major scaffolding approaches for tissue engineering?
A
- Pre-made porous scaffold
- Decellularised ECM- mimic non-immune environment. There is autogenous dECM, allogenic dECM and xenogenic dECM.
- Cell sheets with secreted ECM
- Cells encapsulated in self assembled hydrogel
25
What are the design requirements for 3D scaffold, and their importance?
- solid
- biocompatible: doesn’t elicit undesired effects in patient
- biodegradable: can be absorbed by tissue without surgical removal with good balance between degradation and secretion of new ECM
- mechanical properties: provide stability and integrity
- cost effective, scaling up appropriate manufacturing technology
- porous with interconnected pores. Higher porosity allows more surface binding. Pores must be large enough for cell migration
26
Compare synthetic and natural polymers in scaffolds:
- Synthetic polymers: have FDA approval. Hydrogels are in this category and are used for creating soft tissue. Disadvantage in there is no natural binding site for cells so they need surface functionalisation. Can control degradation, strength, mechanical and physical characteristics. Lack cell-interactive character due to bioinert nature.
- Natural polymers: collagen, biodegradable, biocompatible, poor mechanical characteristics, low cell adhesion. Has natural binding sites for cells to attach and grow. Allow host cells to produce ECM. An example is alginate, which has negative charge and can retain biological structure + function, is biocompatible, biodegradable, hydrophilic, has tunable cross linking and can be used for cartilage regeneration. Disadvantages are poor mechanical characteristics and low cell adhesion.
27
Pros of hydrogel?
The elasticity of hydrogel affects stem cell differentiation, cell shape, gene transfection efficiency, the immune response. Can contain a lot of water.
Can manipulate polymer chemistry and cross linking density to change physical properties to match native tissue.
Bioactive molecules can be integrated to hydrogel by covalent and non covalent interactions to mimic ECM but these can be difficult to manufacture and can have complex regulatory pathways.
Cell controlled degradation can be done with enzyme sensitive crosslinks.
Hydrogels are biocompatible and biodegradable, have high water content, promote cell differentiation, similar to ECM, are porous and are tunable, minimally invasive, can be injected.
28
Types of environmentally responsive hydrogels?
- Photo responsive hydrogel: Light could trigger biological signals or the weakening or strengthening of structural crosslinks. Light is beneficial as it is non invasive. These polymers may use chromophores like azobenzene that can switch between rans or cis conformation, converting light to chemical signal. Photo-responsive part can be in crosslinking points, polymer backbone, along side chain or in the aqueous medium.
- Thermal responsive hydrogels can undergo reversible, dramatic shifts in their conformation like swelling/deflating. Can also transition between soluble and non soluble depending on temperature.
- pH responsive hydrogels are polyelectrolytes with weak acidic or basic groups that accept or release protons in response to pH changes. Become negatively charged when they are basic, causing swelling from repulsion occurring. With positive charges, deswelling occurs.
29
What is cell sheet engineering?
‘Scaffold free cell sheet engineering’ can regenerate tissue using thermoresponsive polymers to form a cell sheet that can be detached at a certain temp. Can assemble 3d tissues without a scaffold using ECM on a cell sheet.
30
Why is nitinol important?
When the body heats up this smart material 'remembers' its initial programmed shape.
31
Steps of wound healing
**Key steps of wound healing**
1. hemostasis- close the wound, lasting 5-10 minutes. Primary hemostasis forms the platelet plug and secondary hemostasis causes coagulation of the blood in the area.
2. acute inflammation- wound cleaning to prevent infection. Surface sensors such as toll like receptors on immune cells sense tissue damage or infection and will be activated to release inflammatory cytokines. This causes vasodilation, vascular permeability and the overall ‘vascular response’. This means that blood flow slows, preventing spread of pathogen in the blood and there will also be increased transport of oxygen, nutrients and immune cells. Permeability allows cells and proteins to leave blood vessel and move to tissue space by the margination effect. The cellular response then occurs, in which macrophages migrate to the site of infection by three stages: vasodilation and margination, extravasation into tissue and then chemotaxis toward infection.
3. proliferation- close the wound by three stages: filling the wound bed by tissue granulation (by fibroblasts that migrate and proliferate) and angiogenesis (vessels that provide new tissue with nutrients), contraction of the wound and scar formation by myofibroblasts and covering of the wound by epithelialization.
4. remodeling by: re-epithelialization with new epithelium and maturation.
32
What is the foreign body reaction and steps?
an unavoidable process which takes place whenever any material becomes implanted into the body. The process of implantation injures the tissue around the foreign object, which triggers an inflammatory process.
1. blood-material interactions: host plasma proteins adsorb to the implant, depending on the surface properties of the biomaterial
2. acute inflammation: immune cell infiltration and recruitment. Neutrophils arrive first to the protein layer and secrete chemokines.
3. acute inflammation: Blood neutrophils arrive via: vasodilation and margination, extravasation and chemotaxis
4. Chronic fibrotic inflammation at the implant site: macrophages are here, foreign body giant cells form, fibroblasts become myofibroblasts, macrophages proliferating.
5. Encapsulation of implanted biomaterial in fibrous tissue: fibrotic layer is a barrier between implant and host tissue and will remain active until implant degrades or is removed. Macrophages here can either be M1 (pro inflammatory and impaired tissue repair) or M2 (anti inflammatory and tissue generation)
33
Embryonic stem cell properties?
- potency: number of possible cell fates
- lineage: cell types that rise from a stem cell or progenitor cell
34
Types of stem cell differentiation?
Totipotent: can become all cell types
Pluripotent: can become almost any cell except zygote - distinction between this and totipotent cell takes place between the 4 cell and morula stages
Multipotent: all cell types of a particular tissue or organ
Unipotent: one type but are stem cells as they can self renew
35
Types of stem cell?
- Embryonic stem cells come from inner cell mass of blastocyte, are pluripotent. Can become the three germ layers and every cell type. Can expand indefinitely when they are unspecialised and provide a continuous supply of new cells.
- Adult stem cells(tissue specific or somatic stem cells) come from bone marrow
- Induced pluripotent stem cells are products of somatic cell reprogramming to an embryonic state. From adult somatic cells that are reprogrammed to be like embryonic stem cells.
36
What are the 3 ways to establish human embryonic stem cell lines?
1. Immunosurgery- selectively removes the outer cell layer (trophoblast) which envelops the inner cell mass of a blastocyst
2. Somatic cell nuclear transfer- nucleus of somatic cell is placed into an egg with no nucleus. This is used in reproductive cloning (cloning an organism) and therapeutic cloning (harvesting stem cells from inner cell mass to make tissues and organs). Nucleus of donor cell is introduced to cytoplasm of an unfertilized egg that has been removed of genetic material- this becomes a blastocyst.
3. Embryo biopsy- growing cells in culture instead of a living organism (as seen in IVF)
37
What are adult stem cells and what are iPSCs?
- Adult stem cells are more specialised than embryonic, more scarce, donor-dependent and typically can differentiate in cell types for specific native tissues or organs. Are multipotent and tissue specific.
- Induced pluripotent stem cells are adult cells that are reprogrammed to become pluripotent using transcription factors (Yamanaka/OKSM factors). Equivalent to embryonic cells but not identical in morphology or gene expression. Can self renew.
38
What are the OKSM factors that reprogrammed iPSCs?
OX2 (transcription factor essential for self renewal and pluripotency in undifferentiated embryonic stem cell), Oct4 (transcription factor in self renewal of undifferentiated embryonic cells), KLF 4 (transcription factor in cell proliferation, differentiation, apoptosis) and C-Myc (proto oncogene that codes for transcription factors)
39
Methods of reprogramming iPSC:
1. Integrative methods of reprogramming cells: reprogramming factors are permanently inserted to host cell genome as continuous expression of reprogramming factors is crucial for successful iPSCs. Viral vectors are most efficient but have less safety than non viral reprogramming.
2. Non integrative reprogramming: the preferred method for iPSC generation. No genomic integration needed so there’s less chance of harmful mutations occurring.
40
What are additive and subtractive manufacturing?
- Additive manufacturing is a 3D printing approach that can build objects in layers using a computer aided design. Freedom in design.
- Subtractive manufacturing removes materials to create parts.
41
Process of 3d bioprinting?
1. Bioink is created by taking cells from a patient and expanding them in growth media before they are mixed in hydrogel and loaded into nozzle of printer
2. Printer seeds the bioink in a pattern layer by layer
3. When printing is done it is placed in growth media to mature. Hydrogel will begin to biodegrade allowing cells to interact more
4. Solid tissue is ready for transplantation
42
Types of bio inks
- Synthetic hydrogel based bio ink
Pro: are reproducible, controlled biodegradability and low cytotoxicity, can tune biomechanical properties
Con: biologically inactive, minimising cell interaction. Can not be printing simultaneously with cells as organic solvents and high temps are used to print the hydrogel ink.
Natural hydrogel based bio ink
- Pro: biocompatible, high water content to mimic ECM, low immune response, can encapsulate cells
- Con: low viscosity, so must be blended with other polymers
Scaffold free bio ink
- micron scale hydrogel beads allow cells to be printed without scaffold
43
Types of 3d bioprinting
- Inkjet
- Thermal inkjet
- Piezoelectric inkjet
- Layer based stereolithography
- Micro extrusion- based bioprinting
44
Inkjet bioprinting
- first 3d bioprinting technology that used droplets dispensed drop by drop.
Pros in that it is non contact based to reduce contamination, is high speed, can fabricate vasculature like structures, bio ink deposition is achieved by placing nozzle in desired location first.
Cons in bio ink must have low viscosity, drops are non uniform, forcing through nozzle causes cell damage, higher cell concentration will clog nozzle
Step 1: Discrete droplets are formed and directed to desired location of the substrate
Step 2: they will interact with the substrate. Is non contact based to reduce contamination, is high speed, can fabricate vasculature like structures. Bio ink must have low viscosity, drops are non uniform, forcing through nozzle causes cell damage
45
Thermal inkjet printer:
uses voltage pulses to heat a thermal actuator, causing partial vaporization
46
Piezoelectric inkjet printer:
uses piezoelectric actuator to make and eject bio ink drops
47
Laser based stereolithography (SLA):
layer by layer fabrication. UV laser selectively cures a photosensitive polymer into a hardened polymer. Can get high resolution printing with no clogging but is slow.
48
Micro extrusion-based bioprinting:
- most well known, sequential deposition of bio ink as continuous string. Have pneumatic driven, positron driven and screw driven dispensing systems. Has high printing speed, can print high viscosity inks with high cell density, versatile in printing ability, gives good structural integrity.
Has inadequate control and resolution of bio ink deposition, creates complex tissue micro-microenvironment, high pressure when dispensing reduces cell viability.
49
What is 4D bioprinting?
When 3D printed structures undergo transformation when exposed to a stimulus like temp, light, magnetism. The fourth dimension is time, meaning that this is achieved when the tissue is showing dynamic properties in response to stimulus. We use stimuli responsive material
50
3 limitations to field of organ transplantation
1. using immunosuppressive drugs to transplant a graft without rejection will increase the patient’s risk of cancer, infection, diabetes, hypertension
2. chronic rejection by host immune response
3. organ demand is higher than supply
51
Types of transplant
- Autograft: donor is recipient
- Isograft: identical twin donor
- Allograft: transfer from genetically different individual of same species
- Xenograft: transfer from a different species
52
What is hematopoietic stem cell transplantation?
- transplant of multipotent hematopoietic stem cells from bone marrow (preferred but painful), peripheral blood (donor treated with drug to trigger bone marrow stem cells to move into blood) or umbilical cord blood (high number of stem cells but have slowest time of cell engraftment. Also immunologically naïve). Helps reestablish healthy blood cell production in patients with damaged bone marrow
- Needs to be replaced because of: diseases like leukemia, cell dysfunctionality, high dose chemo or immunodeficiency.
53
Which type of transplant is best for hematopoietic transplantation?
Autologous transplant is better for immunocompromised patients as the cells are recognised as self and have less risk of chronic immune rejection. Don’t need immunosuppressive drugs. We collect peripheral blood stem cells, harvest hematopoietic stem cells, freeze them, give chemotherapy to suppress immune HSCs and then transplant the cells by transfusion where they will engraft (home to bone marrow where they will find good conditions to survive and proliferate in, forming new white and red cells and platelets)
Allogeneic: done after chemotherapy or radiation. Donors healthy stem cells replace the unhealthy blood stem cells. Need to consider an immune response from the self antigens that are already present on the cells. If recognised as non self, Host vs graft response can occur when the host rejects the cells and T/NK cells start working. Graft vs host response could occur too, where donor cells reject host cells.
54
Tests before a transplant?
1. ABO blood matching: blood typing determines compatibility between donor and recipient. Blood type depends on A or B antigens on blood cells. Blood type O has neither antigen and can donate to all blood types = universal donor. AB blood types are universal recipients.
2. Human leukocyte antigens matching: determines if donor is compatible with recipient. Uses HLA markers to determine if the cells are self or non self. We want a high degree of HLA matching to give the longest half-life.
55
Host vs Graft:
immune response of host rejects donor transplanted cells
- hyperacute: happens minutes after due to ABO mismatch
- acute: t cell mediated rejection that takes weeks or months. Donor antigen presenting cells migrate from transplant to lymph node. APC interact with host T cells and graft will be rejected
- chronic: antibody mediated, slow progression. Allograft APC will be phagocytosed in lymph node of patient. Recipient APCs will phagocytose parts of dying allograft APCs. Host dendritic cells present allopeptide on their MHC type II. Naïve T cell binds to allo peptide causing T cell activation into CD4 cells. These activate macrophages that slowly reject the transplant
56
Graft vs Host:
donors T cells reject the host cells and attack. Acute causes inflammation while chronic causes scarring and fibrosis
57
Immune cells function to:
- recognise self and non self
- react
- regulate by signaling
- remember pathogens for specific responses
58
Describe neutrophils
- most abundant immune cell and WBC
- phagocytes with multilobe nucleus
- first responders
- agranular
- phagocytose bacteria or debris
- pro and anti inflammatory responses
59
Describe eosinophils
- toxic
- granular
- phagocytes with bi lobe nucleus
- attack large pathogen by cytokine release and subsequent phagocytosis
60
Describe macrophages
- tissue resident phagocytes and antigen presenting cells that differentiate from monocytes
- high cell plasticity and can change phenotype
- Type 1 Macrophage M1: pro inflammatory, eliminates pathogens and cell debris
- Type 2 Macrophage M2: anti inflammatory, essential for tissue repair, promote angiogenesis and tissue remodeling, suppress inflammation through cytokine and chemokines
- Can change phenotype between the two types
- Biomaterial can be modified to manipulate the immune response, moving toward and M2 phenotype. We want to modulate the M2:M1 ratio for tissue remodeling.
61
Describe basophils
- granulocyte
- tissue resident with three lobe nucleus
- not phagocyte
62
Describe dendritic cells
- antigen presenting cells
- initiate adaptive immune responses
63
T and B cells
- both associated with graft acceptance or rejection
- antibody formation
- immunologic memory
- play role in resolving inflammation and promoting wound healing
- helper and cytotoxic T, plasma and memory B
64
Chimeric antigen receptor T cell (CART) therapy
- Cellular immunotherapy that gets a patients own T cells to fight cancer by manipulating them ex vivo (adding CAR receptor) and then reinfusing them
- CAR receptor recognises cancer cells and causes their death by releasing cytokine. Target is CD19
65
Issues with CAR T cell therapy
- lengthy vein to vein time- harvesting, transportation, production of CAR T cells, quality assurance and shipment back takes time
- lentivector platform- using virus means possible immune reaction
- production cost- shipment to manufacturing facility, specialised conditions, manpower, time all takes money
- availability and access- very expensive therapy, not available for general population
66
What are the 4 strategies being used for CAR delivery?
1. retroviral and lentiviral methods- good for ex vivo transduction
2. transposon systems- ex vivo electroporation
3. mRNA encoding CAR- ex vivo electroporation
4. CRISPR Cas9- ex vivo electroporation
67
What is multiple myeloma and how could it be treated?
cancer that forms in plasma B cells. Can target cells against CD19. Plasma cells make antibodies against pathogens that are encountered.
Treating with CAR T: harvest blood cells from patient, separate blood components, extract immune cells and reinfuse the rest, cryopreserve cells being used. These are shipped to a facility where they will be re-engineered (~5 weeks). Retroviral transfection will be delivered with the gene for CAR into T cells. These will be expanded in vitro and then reinfused into the patient.
68
What is cellular nanoinjection?
Intracellular access with a nanoneedle that could be used for drug delivery, manipulate cell function and behaviour, delivery of cargo to cell, re-engineering immune cells outside the body, cellular nanobiopsy. Has minimal invasiveness.
Need to consider material designs, types and fabrication. Smooth, porous or not, polymer or not, chemical modification, cell type that is used.
Can be used in creating CAR T cells. Can replace viral vector.
69
What are other ways to do nano injection?
Nano injection can also be done by nano electroporation, laser assisted optoporation and mechanical force applications. Can adhere cells to nanoneedles by spontaneous penetration, centrifugation, electroporation, pressing into cells or optoporation. Mechanism of delivery could be penetration, endocytosis or permeabilisation.
Electroactive nano injection can deliver cargo by delivering voltage through nanoelectrodes that will open pores in cell membrane, allowing cargo inside.
70
What characterises nano injection?
Scanning electron microscopy: can assess efficacy of nanoinjection
Transmission electron microscopy: can imprint interaction
Focused ion beam scanning electron microscopy: characterise efficacy
Fluorescence confocal microscopy: assesses delivery and efficacy
Flow cytometry: assessed delivery and efficacy
71
Bottom up nanofabrication of nanoparticles
1. self assembly of hard colloidal particles
2. (dry etching) to produce nanowires
3. Near field laser ablation and MACE to create nanowires
4. soft nanoparticle templating
72
What are immunoisolation devices?
- Can transplant cells for treating disease. Cells are encapsulated in a device isolated from the body by a semipermeable porous membrane that restricts host immune cells from infiltrating and allows transport of therapeutic proteins and nutrients.
- This tech is based on hydrogel microcapsules and macroscopic devices.
73
Benefits of immunoisolation devices
Immunosuppressant drugs are needed for patients receiving organ transplants, but increase risk of infection/cancer/other adverse side effects. Immunoisolation devices could avoid the use of these drugs.
74
Designs of immunoisolation devices
- Spherical: porous hydrogel used as it has high SA:V ratio, numerous spheres are required and they are difficult to retrieve. Molecular exchange can occur in x,y,z directions
- Cylindrical: molecular exchange is limited to x and y direction
- Planar: molecular exchange is limited to two z directions
75
Nutrient/waste pathway in immunoisolation
Nutrients need to cross the membrane and waste needs to be removed. Consider the porosity of membrane, transplantation site, density of cells in the device, metabolic requirements of the transplanted cells.
76
What needs to be considered for immunoisolation devices?
To choose the transplantation site, consider the disease being dealt with, device geometry, cell type being transplanted but SPECIFICALLY: number of cells needed, volume of space for the device, metabolic requirements of the encapsulated cells, immune cell activity at transplantation site, ease of transplantation.
Must account for transport of therapeutic into the device and waste out, cells in the device can stay alive and will be protected from host immune cell infiltration
- Tuning pore size to optimise nutrient intake, preventing the direct activation pathway. Pore size being at nanoscale prevents T cell entry and escape of donor cells from the device. We want to prevent direct contact between donor cells and host immune cells.
77
Challenges of immunoisolation devices
Challenges are the immune response, scalability of the device, inducing vascularisation, ease of transplantation and retrieval, capacity to conduct clinical management post transplantation
78
Diabetes
une attack of insulin producing beta cells, reducing significantly insulin production.
Type 2= lifestyle related
Pancreatic islets of Langerhans have alpha cells that make glucagon (raises glucose in blood), beta cells that make insulin (lowers glucose).
Beta cells in alginate microspheres can be injected to mice with diabetes, helping them to be healthy for a few days. Can correct diabetes long term.
79
Advances in islet encapsulation
Must account for transporting therapuetic into the device and waste out. Cells must stay alive and do their job. Cells must be protected from host immune cells.
- Natural hydrogel like alginate, which is semipermeable and porous to make an artificial pancreas that encapsulates beta cells for type 1 diabetics. Can transport therapeutic, prevent escape of islet cells, restrict host immune cell entry and transplant the cells without chronic rejection or using immunosuppressive drugs.
80
Creation and challenges of semipermeable capsule membranes
1. islet cells and hydrogel are mixed to create a gel
2. gel fed through droplet generator to form alginate spheres
3. alginate droplets are immersed in a gelling bath that causes them to solidify
- Challenges are in lack of control over cell positions in the capsules and the biocompatibility upon transplantation. Immune system tries to cover transplanted cells with pericapsular fibrotic overgrowth, depositing ECM and building a cellular network around the transplant. This affects viability and integration of immunoisolation device, starving encapsulated islet cells as they cannot get nutrients.
81
Direct rejection
cell-cell contact between host immune cells and transplanted donor cells activate CD4+ T cells and naive CD8+ T cells of host immune system. Cd8 cytotoxic cells initiate an inflammatory immune response to kill transplanted cells. CD4 helpers secrete factors to activate B cells for the humoral response (antibodies).
82
Indirect rejection
1. Small antigen crosses barrier that protects transplanted cells by diffusion.
2. This is processed by host antigen presenting cells
3. CD4+ recognise this on MHC II and are activated.
4. CD4+ activates CD8+ and B cells.
83
3 functions of skin
1. Protects organs and tissues
2. Regulates homeostasis and prevnts dehydration
3. Senses pathogens and can generate an immune response
84
The three layers of skin?
1. Outermost- **epidermis** that forms a hydrophobic barrier. This has 4 avascular layers
2. Middle layer- **dermis**, which has two layers of papillary dermis that gives nutrients by blood vessels and reticular dermis that gives elasticity and strength. Gives sensory and mechanical properties and anchors hair, sweat and sebaceous glands.
3. Inner layer- **hypodermis** that has blood vessels, lymphatic vessels and collagen. Mostly adipose and collagen. Gives thermal insulation and an energy reservoir.
85
Layers of the epidermis
- The stratum corneum (horny layer) that has matured, dead keratinocytes. Layer is replaced every 2 weeks. Forms a protective barrier
- The granular layer with flatter and dying cells. Prevents pathogen infiltration and has more dead cells. Keratinocytes migrate from spiny layer to here.
- The spiny layer which has desmosome junctions, Langerhans's cells and keratinocytes are here.
- The deepest basal layer that has young keratinocytes. The basal layer is a single cell, regenerative layer where keratinocytes are born, melanocytes give skin colour and Merkle cells mediate mechanotransduction for pressure, temp. and pain.
86
Hair follicle growth phases
Have an Anagen growing phase for 3-6 years where hair is formed by rapidly proliferating matrix keratinocytes, Catagen degeneration phase for 10 days where hair detaches from papilla, Telogen rest phase for 3 months and an Exogen shedding phase.
87
Epithelial renewal models
- In the epithelial proliferative unit model, stem cells give rise to new cells identical to itself and more differentiated cells. Transit amplifying cells come from basal stem cells and divide a finite number of times until they are differentiated to give upper layers of epidermis.
- Committed progenitor model says all stem cells in basal layer have same potential to make identical progeny. Basal cell can divide by either symmetric division or asymmetric division
88
Immune response in skin
Key immune cells in epidermis are epidermal Langerhans cells and keratinocytes. In dermis, key cells are dermal dendritic cells, lymphocytes (T, B, NK)and mast cells.
Epidermal Langerhans cells determine adaptive immune response- inflammation or tolerance through their dendrites to sense the microenvironment. Langerhans cells coordinate immune tolerance to prevent unnecessary immune activation. When they sense something is wrong they will instruct adaptive immune response to coordinate T cell response.
