Introduction Flashcards
What is tissue engineering and why it’s necessary
The use of living cells together with extracellular components (natural or synthetic) in the development of implantable devices for the restoration (ex. old age) or replacement (ex. missing tissue due to trauma or disease like cancer) of tissue. Can also be used instead of animals in pharmaceuticals.
Insufficient readily available transplants, current implants are acellular and natural tissue repair can be messy
Interdisciplinary approach is required in tissue engineering:
Medical sciences (anatomy, form and function, animal/human testing)
Engineering (making supports; scaffolds, implants)
Chemistry (material creation, modifying surfaces)
Physics (mechanical stability, interface analysis)
Developmental biology (principles of regeneration, differentiate cells)
Biomaterials (choice of material, support for cell adhesion)
Cell/Molecular biology (inclusion of GFs, understanding cell needs; oxygen, nutrients, niches for stem cells)
Steps in making a neo-organ
Identify problem
Assess required anatomy
Source cells (cell isolation, stem cells, GFs)
Craft material (natural, artificial, materials, shape)
Cultivation (bioreactor to keep alive. Cells out of body needs high standard of regulation for viability and prevent contamination, etc. In particularly when more than 6-8hrs out needs more tedious regulation due to environmental stress, nutrient depletion, etc)
Test hypothetical uses (animal testing)
Storage (backup, GLP)
Alternatives to tissue engineering and downsides
Cut out non-working/diseased region and all natural wound healing to repair/regenerate a working tissue.
Wound healing 3 stage:
Inflammation (1-2 days) in which there’s the formation of the initial fibrin clot to stabilise the wound, prevent further blood loss and protect from further bacteria/infection
Granulation (2days to 3weeks) in which there’s formation of new (granulation) tissue as a substrate for cell migration and proliferation of other cells into to form new tissue, vessels, and wound closure
Remodelling (3weeks to 2years) in which the new tissue is strengthened and refined to restore wound to original state (always a partial loss of complexity and functionality; healing worsens with age)
Works for bone, skin, muscle and liver (restores well enough to not be life threatening) but not for heart tissue (ex. in a heart infraction the piece of heart tissue won’t work the same), brain cells/tissue, kidney and whole organs (loss of function is fatal)
OR
Acellular replacement, ex. hip bone replacement with titanium. Different:
Thermal conductivity
Strength
Density
Surface chemistry
Biochemistry
Mechanical strength
Surrounding bone properties changes structure in response to change in surroundings. Construct also has unchangeable ECM. Ideally replace with in vitro grown bone.
In the classical wound healing process what occurs at stage 1
Inflammation/Exudative phase
Coagulation cascade:
Platelets are released from blood vessels.
Platelets release cytokines (PDGF, etc) to recall neutrophils (short-lived inflammation) and fibroblasts.
Fibroblasts are encouraged to divide and invade (polymerised into fibrin clot) and acts as a scaffold for immune cells. Endothelial cells are attracted.
In the classical wound healing process what occurs at stage 2
Proliferation (2days to 3weeks)
- Cells start proliferating (ex. Keratinocytes in skin wound)
- Cytokines released by neutrophils and platelets recall monocytes which activate into macrophages (phagocytose dead cells and debris, secrete VEGF etc) and endothelial cells (for angiogenesis). Know by studying zebrafish with cut off tail.
- Cytokines released by all the cells present attract fibroblasts that activate into myofibroblasts (high myosin content) and so can seal the wound (wound closure and contracts edges).
- Epithelial cells at the edges proliferate and migrate across the wound surface forming a cell sheet that travels ~3cm
In the classical wound healing process what occurs at stage 3
Remodelling (3weeks to years depending on tissue. Months if there was chronic inflammation)
Fibroblasts produces collagen which is a structural scaffold that helps hold wound together. Collagen recruits fibroblasts which produces more collagen and other proteins to form the scab.
Apoptosis of endothelial cells, macrophages, myofibroblasts as no longer needed (if they don’t die then a very high fibrotic scar can form which is different from original tissue). The vascular content reduces.
Granulation tissue formed has high collagen III and secretes proteases for remodelling to produce a tissue made of mostly collagen I to increase tensile strength (scar tissues has 80% strength of original)
Engineering a bladder (reason, anatomy, requirements, previous method)
In cancer, injury, congenital anomalies (ex. bladder exstrophy in which foetuses bladder grows out of body)
Anatomy:
3 layers of smooth muscle with different cell alignment/ECM orientation to allow expansion and shrinking when full/empty:
-Outer layer of longitudinal muscle fibres
-Circular muscle fibres
-Inner layer of longitudinal muscle fibres. 2 more layers of:
-Submucosal coat as a mechanical dampener (soft, flexible ECM like that below skin that allows muscle to contract and change shape)
Urothelium/Transitional epithelium (multiple layers with each cell contacting the ECM allowing stretching)
Requires:
Size to fill with liquid (0.5-1l)
Balloon like shape with two entries (each kidney) and one exit (ureter)
Blood supply (thick)
Previously:
-Cystoplasty with Gastrointestinal segments
Reconstruct or augment/enlarge bladder with intestine tissue.
Pros: has blood supply
Cons: Infection (evolution has made secretion of its epithelium a bioreactor for bacteria to serve normal function)
-Direct exit from kidneys (constant drip, leakages and irritation)
Engineering a bladder (who, cell source, )
Atala et al., 2006 produced a neo bladder
Grew smooth muscle cells in explant culture then multiple subcultures
Grew urothelium obtained via enzyme digest. Grew in multiple subcultures. For both grew tens of millions of cells total
How are terminally differentiated cells sourced/obtained
Cell recruitment
Chemotaxis: biochemical gradient attracts cells
Topography: Create micropatterned/nanotextured surfaces (ex. on grid with micro ridges or nano pores)
Haptotaxis: grow cells on gradient of chemical cues (adhered onto surface not in solution) and/or gradient of adhesion proteins on grid/scaffold then peel off
Cell Source
Autologous cells: from patient themselves
(less chance of rejection. Typically from biopsies then expanded in cell culture, ex. fibroblasts from skin biopsies)
Allogenic cells: from donor of same species (ex. MSC from hip replacement)
Xenogeneic cells: from another species (pigs, goats, etc)
Potential issues with infection (ex. HGP revealed pigs and humans have retrovirus but with CRISPR now we can use pig for transplant) and/or rejection
How are stem cells sourced/obtained
Isolate:
-Autologous cells: from patient themselves
(for adult stem cells ex. adipose derived MSCs from liposuction. Use surface marker to confirm. Dedeifferentiate with mercapto-ethanol, then differential with chemicals into desired cell type)
-Allogenic cells: from donor of same species (ex. embryonic stem cells from blastocysts from IVF)
-Xenogeneic cells: from another species (pigs, goats, etc)
Potential issues with infection (ex. HGP revealed pigs and humans have retrovirus but with CRISPR now we can use pig for transplant) and/or rejection
Need to maintain stemness when proliferating.
Drive towards desired differentiation with GF, matrix mechanics (soft/hard) and matrix chemistry (collagen, fibronectin).
Need to retain phenotype once differentiated, else it turns into teratoma (embryonic SC) or tumour (adult SC)
How to grow/divide cells and issues
Required as can only extract limited amount
Grow on contruct
Grow in cell culture:
Explant culture:
Chop tissue into small (<1mm) pieces an leave in flask/dish with minimal medium to adhere. Meniscus forces press onto cells to push to bottom. Replace medium occasionally to feed
Adhere cell culture in plastic flasks
Subculture: Cells are connected to ECM so use enzymes (trypsin, collagenase, etc) to digest ECM and to remove cell-cell interactions, use low/no Ca with chelator (binds ion, ex. EDTA binds Ca) since cadherins are maintained by them.
Problems:
Dedifferentiation of SCs
Genetic drift
Additions to cell media
Handling
Hayflick limit
What is the essential knowledge for tissue engineering
Aims to be achieved
Target tissue/organ
What specifies normal/diseased (analysis of mechanics/biological components/histology/physiology etc)
Cell source
What to do to promote healing in the setting:
-Reduce (anti-inflammatory drugs)/Increase (vaccine, interleukins) immunity
-Incorporate cells (SCs, fibroblasts delivered by injection, cells seeded into biodegradable scaffold and implant, etc)
-Immunosuppression (reduce excessive immune response, prevent organ rejection)
Materials that encourage ideal regeneration: ECM surrounds cells and is important in support, adhesion, mechanical stability, protein binding and 2D/3D organisation
How cells/constructs can be stored
How can cells can be grown in specified environment
What is the ECM made of
Mainly collagens: matrix strength, development and physiological roles, cell attachment and differentiation
Proteoglycanes:
matrix resilience, cell adhesion, migration, proliferation e.g. aggrecan, keratan sulfate, heparan sulfate, chondroitin sulfate, hyaluronic acid, …
Cell interactive glycoproteins:
tissue cohesiveness, cell adhesion e.g. laminin, fibronectin, vitronectin, thrombospondin, tenascin (in basal lamina that touches the cells)
Elastic fibres:
elasticity of the matrix e.g. elastin
