Exam Flashcards
What are the key components of tissue engineering?
- 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
Why do we need tissue engineering?
- Organ donor shortage
- Increasing need and number of transplants
- Increasing ageing population
- Constraints on donor matching, transportation and transplantation
What are the 4 types of tissue?
- 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
What are the subtypes of connective tissue?
- ‘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
The key components of the cytoskeleton
- 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.
Components of the ECM
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.
Types of collagen
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.
What is laminin?
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.
What is the basement membrane?
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
What are challenges in tissue engineering?
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
The types of stem cells
- 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
What are mesenchymal stem cells?
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.
Examples of biomaterials?
- 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.
What is bio fabrication?
→ production of complex living and non living biological product from raw materials
Technology used in bio fabrication?
3D bioprinting, bioreactor, inkjet printing, laser bioprinter, Extrusion based 3d bioprinting, light based technology
What is organogenesis?
a phase in early embryonic development that starts after gastrulation and continues until adulthood where organs are formed from the three primary germ layers.
What is the process in which the three germ layers are formed and what is its importance?
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.
How are the three germ layers formed?
- 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.
What are the two major cell types of the body?
- 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
What is EMT and its process?
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
What is endochondral ossification?
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.
What is intramembranous ossification?
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.
Which cells form bone?
- 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.
What are the four major scaffolding approaches for tissue engineering?
- 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
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
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.
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.
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.
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.
Why is nitinol important?
When the body heats up this smart material ‘remembers’ its initial programmed shape.
Steps of wound healing
Key steps of wound healing
- 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.
- 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.
- 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.
- remodeling by: re-epithelialization with new epithelium and maturation.
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)
Embryonic stem cell properties?
- potency: number of possible cell fates
- lineage: cell types that rise from a stem cell or progenitor cell
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
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.
