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What is tissue engineering?
Regenerative medicine is an umbrella term, underneath lies a number of approaches including tissue engineering. Tissue engineering is a multidisciplinary field aimed at development of biological substitutes to restore, maintain or improve tissue functions.
Uses cells and biomaterials to treat degenerative diseases or injury.
What is the clinical need for tissue engineering?
End-stage organ failure and tissue loss are often the most devastating. Major causes of organ failure include injury, disease and aging.
Current treatments for organ failure:
-Surgical reconstruction
-Mechanical devices
-Transplantation
Limitations include: poor success rate, surgical complications, morbidity at donor sites, only mechanical support unifunction, don’t grow with the tissue (require surgery in children as they grow), need for immunosuppressants.
What are the fatal consequences of the Transplantation Crisis?
The transplantation crisis means that 3 people die a day in the UK waiting for a suitable organ. Aging population requires more and more organ donations. This crisis means that patients receive unfit unsuitable organs (subobtimal).
CASE STUDY 2 patients were each given a kidney but a couple of days later died of Meningitis. Donor died of Meningitis caused by a rare nematode which had been transferred to them.
How can tissue engineering aid the transplantation crisis?
Tissue engineering could be a new solution for treatment of organ failure.
Needed because:
-Donor tissues and organs are in short supply
-Want to minimise immune system response/need for immunosuppressants
Goals of tissue engineering involve saving and improving lives by assembling functional constructs that can be placed into the body to restore or replace a damaged tissue/organ.
What is the history of tissue engineering?
The research on what we now know to be TE emerged in 1970s and 1980s. The term was coined in 1987. In the 1990s research accelerated and industry began to emerge partly due to the parallel development in the field of biomaterials and stem cell biology.
What are the main components when building a tissue?
- Cells to make up the tissue
- Biomaterials/scaffolds to replace ECM
- Bioactive molecules to direct fate of cells and promote integration of construct into body
What are the 2 main approaches when building a tissue?
IN VITRO: combining cells with a biomaterial/scaffold then transplantation
IN VIVO: transplantation of scaffold into the body, endogenous host cells are recruited
What are the different disciplines involved in tissue engineering?
- Cell biology to cell culture
- Bioengineering to create and engineer the biomaterial
- Immunology for transplantation/immunorejection
- Surgeons to transplant
- Clinicians to perform and organise clinical trials
- Pharmacists for immunosuppressants
What did Cao et al do in 1997 and what were their limitations?
Aim to assess the feasibility of growing tissue-engineered cartilage in the shape of a human ear.
A plaster mold of a 3 yr olds ear was cast from an impression of an ear. This was used as a scaffold for cartilage cells from a calf to be seeded and grown on. Nude mouse used as a bioreactor for the scaffold and cells to grow on. After 12 weeks the constructs were explanted, sectioned and stained.
Limitations: skin misssing, bovine chrondrocytes were used, scaffolds had to be refined for mechanical stability, implications on the growth rate of the artificial ear.
Media described it as a human ear grown on a genetically engineered mouse, none of which is true!
What makes up an organ structure?
Epithelial tissue, connective tissue, muscle tissue, nerve tissue.
Requires oxygen, discharge waste via blood, EC fluid and lymph. Vasculature is required for tissue perfusion.
What are the phases of wound healing?
Ordered sequence of events that will lead to healing of an insult to the skin.
-Inflammatory phase: stop the bleeding, inflammatory cells clear up debris caused by dead cells, injured cells and microbes.
-Proliferative phase: wound trying to close up, new tissue formed but in a disorganised manner.
-Remodelling phase: remodelling new tissue to return to normal organisation.
Overlapping processes that occur over days, weeks, months, years.
What is an overview of what occurs in wound healing?
Injury cuts the blood vessels which bleed into the wound. Blood clot forms and leukocytes clean the wound. Blood vessels regrow and granulation tissue forms (fibroblasts). Epithelium regenerates and scar forms, reforming 70-80% of original tensile strength.
What is the difference between regeneration and repair?
Mild superficial injury will only affect the uppermost layer - epithelia. Skin, intestine and blood have a high turnover and have resident stem cells allowing them to regenerate quickly. However this is providing that the injury only affects cells and not ECM and tissue affected contains stem cells. Stable tissues like heart and brain cannot regenerate so repair their injury instead. It all depends on the extent of the injury (transient or not), the type of tissue and components affected.
How are acute and chronic injuries different?
Acute injury occurs when the stimulus is removed quickly and there is cell death but there is still an intact tissue framework. This allows for regeneration and restitution of the normal structure.
If there is an acute injury with damaged tissue framework, it can be repaired but will leave scar tissue.
If there is a chronic injury (persistent tissue damage) it leads to fibrosis like pulmonary fibrosis (tissue scar).
What is fibrous encapsulation?
Tissue response to implanted biomaterials is similar to foreign material response. The implantation of a biomaterial/medical device results in injury to the tissues and organs. Abundant deposition of extracellular matrix. Isolation of biomaterial from local tissue environment. Type of wound healing.
What are the different sources of cells for transplantation?
- Autologous (cells from the same patient)
- Allogeneic (same species, different individual)
- Xenogeic (different species)
- Syngenic or isogenic (genetically identical/twin)
What are the different types of cells used for tissue engineering?
-Differentiated mature cells
-Mixture of differentiated cells
-Stem cells
Adult stem cells: can’t be kept in culture for long, but can be autologous, need a large number, difficult to proliferate in vitro.
Embryonic stem cells: ethical issues but can give almost any cell.
Induced pluripotent stem cells: can be autologous, almost any cell, difficult to control differentiation, unknown risks.
How do autologous and allogenic cells compare?
Autologous cells don’t require tissue matching, no need for graft vs host response, faster engraftment, no disease transmission.
Allogeneic cells require tissue matching and graft vs host response, slower engraftment, possible disease transmission (HIV or hepatitis).
What are the problems with using differentiated cells in tissue engineering?
Examples of differentiated cells that may be used include fibroblasts, keratinocytes, osteoblasts, endothelial cells, chrondrocytes, preadipocytes and adipocytes.
Main problem is sourcing the cells.
Biopsies are intrusive and don’t give many cells.
Differentiated cells can’t really be expanded in vitro because they senesce after a while. If they do they change too much. Batch to batch variability.
How to culture cells?
- Growth medium: high energy, source of glucose, amino acids, GFs. Replacing blood so needs to be exchanged every day to mimic circulation.
- Laminar hood to ensure aseptic conditions and to direct the airflow.
- Incubator to keep cells at 37c and use water to humidify the environment.
What is good manufacturing practice?
Ensures that medicinal products are consistently produced and controlled to the quality standards appropriate to their intended use.
Cell manufacturing must be free from animal products, eliminate batch-to-batch variability as much as possible and meet their storage needs.
What are the roles of the ECM?
Most cells require attachment to a solid surface otherwise they undergo apoptosis.
Roles of ECM:
-Provides structural support for cells
-Contributes to the mechanical properties e.g. collagen bundles in tendon, collagen and elastin fibrils in skin for elasticity and toughness, calcified ECM in bone for strength.
-Provides bioactive cues for cells to modulate them
-Act as reservoirs of growth factors and potentiates their actions
-Scaffolding for orderly tissue renewal, avoiding scarring.
What are the components of the ECM?
Tends to differ from tissue to tissue.
- Fibrous structural proteins like collagens and elastin for tensile strength and recoil
- Water hydrated gels like proteoglycans and hyaluronon for resilience and lubrication
- Adhesive glycoproteins like fibronectin and laminin for connection to ECM components and cells
What is collagen?
Most abundant protein. 80-90% of collagen in the body is either type 1, 2 or 3. Basic collagen unit has 3 helical structures, each composed of 1050 AAs. Helical due to the increase abundance of sequence of proline, hydroproline and glycine. Assemble into fibrils end to end side to side to make fibres.
Vit C required for collagen production.
What are proteoglycans?
Composed of glycosaminoglycan chains linked to a specific protein core. Very hydrophilic, form highly hydrated compressible gels.
What are adhesive molecules?
Very diverse
e.g. fibronectin contains a RGD sequence (Arg-Gly-Asp). The sequence is recognised by cells as a cell binding domain. If cells will not adhere to biomaterial you can add this sequence onto the biomaterial (structurally integrate).
How do cells adhere to ECM/biomaterials?
Cells mechanically interact with ECM before making contact via receptors/signalling parts.
Cell flattens out to reach appropriate shape.
Can move around and explore their environment.
What are integrins?
19a and 8B subunits.
Form heterodimers with extracellular domain, transmembrane domain and short intracellular domain. ECD and ICD can be used in inside-out and outside-in signalling. a and B can make up many different combinations, different affinit depending on environment. Both specificity and redundancy allowing a robust system.
How is integrin conformationally activated?
-Extrinsic ligand ‘outside-in’ (collagen, laminin, fibronectin) causes a conformational change in integrin, changing their affinities for specific ECM components, activates signalling pathways.
-Intrinsic ligand ‘inside-out’ (talin, kindins) causes changes which intiates the assembly of the actin cytoskeleton.
Mostly mediated by focal adhesion kinase which recruits a number of other proteins to the sites of focal adhesion triggering a cellular response.
What is machanotransduction?
Process by which external mechanical stimuli are transmitted into the nucleus. Cells can sense the environment and alter it accordingly.
What is a Sheffied case study of tissue engineering?
Macular degeneration clinical trial with 2 patients. Treatment looks promising from Phase 1 trials. Stem cells used were SHEF-1hESC. Using fully differentiated embryonic stem cells placed on a coated, synthetic basement membrane.
What are biomaterials?
Nonviable materials used in a medical device intended to interact with biological systems. Can be used to develop scaffolds for tissue engineering. Developed from the field of medical devices.
What is the history behind biomaterials?
Using hair, silk etc for suturing wounds.
Issues with infection and immune response.
Sir Harold Ridley examined eyes of pilots and saw that they had plastic splinters from the cockpit without an immune response. Developed intraocular lens for the treatment of cataracts.
Important development: a material can be integrated into the body without an immune response, making it biocompatible.
What is the biocompatibility of materials?
Ability of a material to perform with an appropriate host response in a specific application.
e.g. haemolyalysis membrane for patients with poor kidney function, the membrane is only in contact with blood for a couple of hours. Urinary catheter is implanted for days or weeks. Hip replacement is needed for life.
What are some examples of appropriate host response?
Resistance of blood clotting
Resistance to bacterial colonization
Normal healing
What is the evolution of the biomaterials field?
First generation: Bioinertness
Determined to be biocompatible if do not induce toxic reaction or carcinogenesis.
Second generation: Bioactivity
Bioglass is a ceramic implant that you can place in rats. Bonded with bone and could not be separated. Cannot respond to inputs.
Third generation: Functional tissue
Materials that can induce a cellular response at a molecular level.
What are polymers?
‘Poly’ (many) ‘meros’ (parts)
Large molecules made up of chains or rings of linked monomeric units. Mw 200,000 (compared to h20 18Da).
Can be linear, branched or networked.
Common polymer biomaterials include Polyethylene, Polytetrafluroethylene, Polypropylene, Polyvinylchloride, Polydimethylsiloxane, Poly (methyl methacrylate). Typically have carbon atoms plus side chains.
How can polymers be structured?
Can be arranged in different shapes -Homopolymer -Random copolymer -Alternating copolymers -Block copolymer -Graft copolymer It is possible to mix two polymers e.g. blend of homopolymer A and B. We can change their functional properties by changing how they are arranged.
What are hydrogels?
Crosslinked polymer networks that are insoluble but swell in aqueous medium. Offer an environment that resembles the highly hydrated state of natural tissues.
Can model soft tissue environments.
What are natural polymers?
Already exist in animal or plant tissue.
1) Protein-based natural polymers
Collagen - well integrated, highly common, high immunogeneity
Gelatin - less immunoggenic, denatured form of collagen
Fibrin - major component of blood clots
Elastin and Soybean
2) Polysaccharides
Chitosan - present in fungi and crustaceans
Aliginates - produced by seaweed and some bacteria
Hyaluronan - present in umblical cord, skin, cartiliage
Chondroitin sulfate - present in cartilage
Obtain the plant/animal tissue by extraction. Purify them from the tissue. Concentrate them to get good enough sample.
What are synthetic polymers?
Synthesise from scrath.
Polylactic acid
Polyglycolic acid
Poly (lactic-co-glycolic) acid
What are semi-synthetic polymers?
Hybrid molecules made by incorporation of biologically active macromolecules onto the balance of synthetic polymers. e.g. semi-synthetic PEG (polyethylene glycol)-fibrinogen. PEG fives density, stiffness and biodegradability and fibrinogen gives biofunctional domains.
What are the advantages and disadvantages of natural polymers?
Advantages: -Already exist, easily obtainable -Existing bioactivity -Can make the most of naturally-occuring properties Disadvantages: -May be difficult to harvest -Potential ethics -Batch-to-batch variability -Shelf-life -Immune response
What are the advantages and disadvantages of synthetic polymers?
Advantages: -Can be tailored to specific properties -Can control process and structure Disadvantages: -Diffferent structures that may not interact with tissues -Difficult to predict bioactivity
What are the essential properties of biomaterials?
- Physical/mechanical - strength, elasticity, architecture
- Chemical - degradability, resorbtion, water content
- Biological - interactions with cells, release of biologically acitve signals
What is the difference between degradable and resorbabale materials?
Degradable materials:
Hydrolysis of a polymer require H20 to break down covalent bonds to give degradation products. For it to be biocompatible degradation products should be non-toxic.
Resorbable materials:
Total elimination of the initial foreign material and its byproducts. Broken down by the body, metabolised leaving no trace.
What are the bulk properties of biomaterials?
Strength, toughness, fatigue resistance, stability.
What are the surface properties of biomaterials?
- Chemically or physically altering the atoms/molecules in the existing surface
- Overcoating the existing surface with a material having a different composition
- Creating surface textures or patterns
How does matrix direct stem cell fate?
Engler et al (2006) Matrix elasticity directs stem cell lineage specification. Mesenchymal stem cells were placed on substrates of different elasticity (soft tissue, muscle, collagenous bone resemblance). The cells were directed to different lineages (neural, muscle, bone) just based on stiffness, can sense mechanical environment,
What are the consideration for creating surface modifications?
- Thin surface modifications (3-10nm) to avoid changing the marker properties of the material
- Delamination resistance
- Surface analysis
How do cells interact with biomaterials?
Cells do not interact directly with materials.
- A later of protein (from growth or plasma) adheres to the surface of biomaterials
- Cells recognise these proteins and adsorb to them depending on the surface material properties.
What are non-fouling surfaces?
Surfaces that resist adsorption of proteins and/or adhesion of cells e.g. PEG and Zwitterionic. In medical devices this is good because may inhibit bacterial colonization.
How to functionalise a surface?
Attachment of biomolecules to polymer surfaces
Immobilisation of integrin-binding peptides or entire proteins (RGD domains added).
How do cells respond to substrate chemistry?
Cell adhesion to materials occurs through receptors in the cell membrane
Arginine-glycine-aspartic acid (RGD) domain in fibronectin and vitronectin
Cellular responses can vary with the surface density of RGD peptides immobilised
What are Micrometer-scale chemical patterns?
Can be created using microstamping or microcontact printing (µCP)
- Prepolymer is poured onto a structured master
- Prepolymer is cured and the stamp is peeled off the mamster
- Stamp is cut into smaller pieces using an enzyme
- Stamp is inked by soaking in ink solution
- Ink printed on suitable surface
- A pattern of substrate is obtained, cells adhere to ECM area stamp
How does cell shape control cell fate of endothelial cells?
Chen et al 1997
It was already known that angiogenesis relies on interplay of chemical and mechanical signals. Endothelial cell growth is also critical, early experiments suggest that increases in spread area are accompanied by an increase in cell proliferation.
Hypothesis: cell shape per se controls cell fate of endothelial cells.
Approach: Micropatterning of fibronectin islands on a non-fouling surface of lots of different sizes
Result: The extent of cell spreading determined whether a cell underwent proliferation or apoptosis. Same GF, same genes, same ECM but different geometry, different fate outcome. Cells can filter the same set of chemical inputs to produce different functional outputs.
Warmflash et al (2014) used this to recapitulate gastrulation,
Teixeira et al (2006) used microstamping or microcontact printing. Thin lines, cells were perpendicular, thick lines cells were vertical.
What is the need for an artificial scaffold?
Scaffolds provide the support for cells to proliferate and maintain their functions.
They deliver and retain cells and bioactive molecules. Therefore when you are tissue engineering you need to develop an artificial extracellular matrix which would usually perform these functions in vivo.
What are the design considerations for a scaffold?
- Material science: is it biodegradable?
- Scaffold architecture: does it have adequate mechanical properties?
- Scaffold-cell interaction: does it allow cell attachment/function?
- Up-scaling production: can it be mass produced to meet clinical need?
- 3D
- Nutrient supply: is there capability for it to be vascularised to allow for O2 perfusion and tissue integration?
How can a scaffold be degradable?
3D scaffold can be added to two types of cells to support the cells. If the scaffold is degradable, it will gradually degrade and the cells put down their own ECM, allowing for stability at all times. The scaffold itself and the degradation process should be non-toxic.
Degradation kinetics should be matched to the tissue you are trying to regenerate.
How must a scaffold interact with cells?
The surface properties of the scaffold must allow for cell attachment. If they don’t (e.g synthetic materials) they can be made functional by adding RDG domains which create cell binding domains.
The scaffold mustn’t hinder the ability of cells to attach to the matrix and start signalling (functionality).
What are the different materials that can be used for scaffold fabrication?
Naturally derived materials
Synthetic materials
Semi-synthetic materials
Acellular tissue matrices
What is acellular tissue matrices?
Can be considered as natural. However, all the cellular material is removed from the extracted tissue, either by physical scrapping, although this is often not enough. Chemical methods (acids/bases) or biological methods (enzymes) can be used instead. Removing acellular components also removes antibodies to prevents an immunogenic reaction to seeded cells.
What are the advantages and limitations of decellularising a tissue to make a scaffold?
Decellularisation maintains intricate structures that would otherwise be difficult to manufacture.
Exploiting the intact 3D structure of the ECM
Some are commercially available (alloderm)
Up-scaling may be a problem.
Acessibility is a potential issue.
However it is important to fully decellularize a tissue to avoid initiating the host response, bacteria contamination or crosslinking which will affect the tissue’s normal morphology.
What are the mechanics and architecture of a scaffold?
Macroscopically the final shape of the tissue engineered is defined by the scaffold.
Microscopically human skin and bone are usually very porous for infiltration of O2 and nutrients. However, there may be limited interconnectivity and some pores may not be accessible. Further more too many pores will decrease the mechanical stability of the tissue, however this depends on the tissue being engineered.
What is porogen leaching?
Porogen leaching is a method used to create an artificial porous scaffold. It involves mixing a polymer solution with salt particles and placing it into a mold. Solvent evaporation causes polymer to solidify which when placed in water causes the salt to leach out. This is then freeze-dried to create a porous scaffold where the salt particles used to be. You can control the size of the salt particles to control the pore size.
What is phase separation?
Phase separation is a method used to create an artificial porous scaffold based on properties of a mixed system. It involves mixing the polymer solution with a solvent, placing into a mold. Using temperature, you can separate a polymer-rich phase and a solvent-rich phase. Evaporate the solvent to cause the polymer to solidify. Pores are formed where the solvent used to be.
What is electrospinning?
Electrospinning is a method used to create an artificial porous scaffold. Polymer solution is placed into a syringe. It is linked to a high voltage power supply. An electromagnetic field is created between the tip of the syringe and the grounded collection plate. As the polymer is released from the syringe the electromagnetic field causes the polymer fibres to splay. Very fine micronanofibres are collected onto the grounded plate.
What is additive manufacturing?
Additive manufacturing (aka rapid prototyping or free form fabrication) is a method used to create an artificial porous scaffold. It is a collection of methods. Process of joining materials to make objects from 3D model data usually layer upon layer. 3d scaffold design is converted to particular file that makes virtual slices in 2D. This is then transferred to a rapid prototyping machine. Roller spreads polymer across the table, inkjet head secretes a polymer binder to specific areas according to the shape determined by the computer.
What are the advantages of additive manufacturing?
High control of architecture
Production of scaffolds with precise morphologies
Combines medical imagine like MRI to fabricate anatomically shaped implants.
Main limitation is that there is a limited number of biomaterials that can be used.
What is cell encapsulation?
Usually when you combine the artificial scaffold and seeded cells, it may be the case that the cells are not integrated or that they clog the pores.
Cell encapsulation provides an alternative way to provide a better integration in 3D printing. In this method, scaffolds and cells are already combined and gaps are free to act as pores.
How was ITOP developed?
Kang et al (2016) sought to address the challenge of producing 3D, vascularised cellular constructs of clinically relevant size, shape and structural integrity. Designed the ITOP Integrated Tisuue-Organ Printer to overcome the limitation of currently used bioprinting technologies (structural integrity, mechanical stability, printability). Contains different bioinks: cells combined with hydrogels which are non-toxic and bio-compatible. New idea to print cell-laden hydrogels with sacrificial scaffold to provide initial mechanical integrity.
What are the different biomolecules that can be used for inducing tissue regeneration?
Small molecules (corticosteroids and hormones)
Proteins and peptides
Oligonucleotides
What are bone morphogenetic proteins?
Initially extracted from bone matrix.
Members of the TGFb family.
Made by osteoblasts (bone-forming cells)
Osteoinductive - able to recruit osteoblasts and initiate differentiation of mesenchymal stem cells.
What are the problems with using BMPs in tissue engineering?
The efficient clinical use of BMPs depends on the delivery strategy.
BMPs act locally so they easily dissipate.
The use of biomaterials that can retain and sequester BMPs will enhance efficacy in bone regeneration.
What method was developed to effectively use BMPs in tissue engineering?
Lutolf et al (2003) Polyethylene glycol (PEG) is non-fouling so it is functionalised using RDG domain. Crosslinked to create a network mesh using MMP substrate sites. Cells in the vicinity recognise sites and bind to the scaffold. Cells secrete MMPs that degrade MMP cleavable bonds serving as crosslinks for the matrix. rhBMP2 is physically entrapped into the pores of the PEG gel by mixing it with PEG precursor before gelation. BMP is liberated from the matrix and can diffuse from the site signalling osteoblast precursor cells to secrete bone marrow and regenerate the bone.
How was ITOP used for skeletal muscle reconstruction?
3D muscle construct 15mm x 5mm x 1mm dimension containing mouse myoblasts.
Designed a fibre bundle structure for muscle organisation. PCL pillars were used to maintain the structure and to induce cell alignment.
The printed construct was cross-linked with thrombin solution to induce gelation of fibrinogen and uncross-linked sacrificial material was removed by dissolving in cold medium.
TE construct matured into a functional muscle in vivo in rats. High cell viability after the printing process
What are the steps involved with ITOP?
Medical imaging 3D CAD model Visualised motion program 3D printing process 3D bioprinted tissue product
What were the results of Lutolf et al (2003) research to effectively use BMPs in tissue engineering?
First they tested the sensitivity of the gels to proteolysis by cell-secreted MMPs using an in vitro model system for cell invasion. Fibroblasts recognised the cleavage site and could invade.
Control: no MMP cleavage site or MMP inhibitor
Monitored by BMP release
Functional proof cam from testing it on critical injuries (large enough so that the bone cannot regenerate).
MMP-sensitive, no BMP
MMP-insensitive, BMP present
No regeneration however MMP-sensitive with BMP showed good regeneration of the bone.
How does using bioreactors compare in industry and tissue engineering?
Classical bioreactors are fermentors used in industry to grow high numbers of eukaryotic and prokaryotic cells for the production of antibiotics, recombinant genes and metabolic products under controlled conditions.
In tissue engineering bioreactors enable us to direct, maintain and intiate 3D structures which would be very difficult otherwise.
What are the challenges associates with tissue engineering that are tackled with the use of a bioreactor?
- The ability to grow 3D tissue structures of relevant clinical sizes, same as in vivo
- The growth and 3D assembly of multiple cell types that are requires for more complex functional tissue.
What is static culturing and what are the associated issues?
Static culture is usually performed in a culture flask, no special equipment is required, it is relatively cheap and easy. Cells are grown statically without any mixture, causing concentration gradients to occur. Fed with medium every day/few days - no control over pH and environment. Diffusion of O2 and nutrients is inefficient so cells on the inside will become necrotic.
What is a dynamic culture system?
A dynamic culture is the mixing of the cells in culture. Gives rise to homogenous concentrations of nutrients, toxins and other components. Can control environment, pH, temperature etc. Cells are placed in the bioreactors, medium is pumped in and out from medium tank by peristatic pump providing perfusion.
What are the key roles of bioreactors?
- To establish spatially uniform cell distributions on 3D scaffolds
- To overcome mass transport limitations in 3D constructs
- To expose the developing tissue to physical stimuli (physical conditioning)
What are the requirements for cell distribution on 3D scaffolds (bioreactors)?
Usually you just place cells of choice on scaffold using a pipette, relying on gravity to pull the cells through the porous structure and distribute through 3D. This is an inefficient process so cells will ususally just stay on the top and many will be lost. This is a problem especially when many biopsies are painful.
Therefore you need a high seeding efficiency, short inoculation period and uniform distribution of cells within the scaffold since this is what will determine the biochemical activity and mechanical strength of final TE construct.
Give an example of a study that measured cell distribution on 3D scaffolds.
Bone marrow stromal cells were seeded onto scaffolds and after 18 hours MTT assay was used. MTT is converted by the mitochondria from a soluble yellow salt into an insoluble purple formazan salt. Used to see if cells are alive and metabolically active and where they are distributed on the scaffold. Compared between static (white area present) and perfused (even distribution).
What is mass transfer in 3D structures (bioreactors)?
Once cells are delivered to a scaffold you have to make sure that they are viable and functional.
External mass transfer: delivery of nutrients and O2 from the inner bulk to exterior
Internal mass transfer: delivery of nutrients and O2 from the exterior to the inner bulk
Also involves the removal of metabolites and CO2
Give an example of a study where mass transfer was measured in 3D structures.
Wendt et al 2008
Chrondrocytes seeded using perfusion cell seeding. Cultured for 2 weeks with OR without perfusion. In statically cultured scaffold, there is a large amount of empty space where cells have undergone necrosis. Cells are only at the edges. Whereas the perfused culture has lots of cells all spread across.
What is physical conditioning (bioreactors)?
Tissues and organs in the body are subject to complex biomechanical environment. These physical forces include hydrodynamic/hydrostatic, mechanical and electrical. The tissue engineered construct needs to be able to maintain these too.
Eg. Pulsatile flow in blood vessels causes erthrocytes mature to their appropriate phenotype.
Why are there different types of bioreactors?
Diversity in bioreactor design reflects the range of signals needed for formation of various tissues e.g. heart, bone, valves.
Biomaterials making up bioreactors must not induce toxocity or unwanted effects, they must be biocompatible and non-fouling (don’t stick).
They must also be sterile so are either single use or can be sterilised in an autoclave.
Withstand 37 degrees.
What are Spinner Flask Bioreactors?
Magnetic stirrer creates dynamic culture, the turbulence can cause damage to cells. Scaffold is dispensed from needles into the bulk media. Internal transfer is still quite limited for centre.
Relatively cheap and simple.
Mass transfer in the flasks is not good enough to deliver homogeneous cell distribution throughout scaffolds and cells predominantly reside on the construct periphery.
What are Rotator Wall Bioreactors?
Centrifugal forces generated due to the rotation of the cylinder counterbalance the gravitational pull on the scaffolds. As tissue grows in the bioreactor, the rotational speed must be increased in order to balance the gravitational force and ensure the scaffold remains in suspension.
What are Perfusion Bioreactors?
The culture medium continually circulates through the TE construct which stays static. Most mass transfer limitations are mitigated. The effects of direct perfusion can be highly dependent on the medium flow rate.
What are Compression Bioreactors?
Used to apply mechanical stimulus to cell-seeded constructs. This can be acting constantly, intermittently or cyclically. Apply mechanical pressure and examine the effects of this.
What are the applications of bioreactors in decellarisation?
Complex architecture of tissues makes decelluarising them difficult. Perfusion bioreactor has been used for the heart, lung, liver and pancreas, mostly used for whole organs.
Porcine heart
The barbed end of the tubing is inserted into the aorta of the native heart. The tubing must be secured with hose clamps or zip ties above the aortic valve to ensure perfusion through the coronary arteries. The hear is then submerged in a water in a 4L beaker and air bubbles must be removed from the tubing using a pump. As solutions are perfused through the coronary arteries, the heart will lose its native colour and become white. It can then be re-seeded with cells again.
What are the applications of bioreactors in cartilage tissue engineering?
Articular cartilage is a load-bearing tissue, it is exposed to cyclical stress. Bioreactors with mechanical loading have been used for tissue engineering of cartilage.
Mauck et al (2000) Loading applied in cycles improved biomechanical properties of engineered articular cartilage.
What are the applications of bioreactors in microgravity?
Space flights represent the best environment to investigate near-zero gravity effects but there are major limitations for setting up experimental analysis. Rotating wall bioreactors have been developed by NASA.
Tamma et al (2009) studied osteoclasts and the effects of near-zero gravity, found there was a decrease in bone density.
Where is the clinical need in tissue engineering of heart valves?
Heart valves are required for directing the flow of blood. May be causes that valves cannot open or close, causing reverse flow. Current treatment options are surgical repair or valve replacement. Prosthetic valve replacement isn’t ideal because you need anti-thrombotic drugs and they don’t grow with children.
Tissue engineering offers the potential of creating a valve that will grow and adapt with growth.
Sourcing cells for this is difficult as you need smooth muscle cells and endothelial cells - stem cells may be the main choice. Must sustain the biophysical forces that cells experience in vivo: mechanical strech and hydrodynamic shear.
What is a Flex-Stretch-Flow bioreactor?
Developed by Engelmayr et al (2008)
Hypothesised that mimicking the mechanical stimulation and perfusion in vitro could lead to further improvement of the engineered tissues.
Flex-stretch-slow bioreactor
Place a scaffold seeded with cells which is fixed onto 2 posts, one which is fixedd and another which is movable which is linked to a linear motor to drive its movement. Enables flexing and stretching of scaffolds and allows media flow over the scaffold. Culture media can be recirculated within the bioreactor chamber via a magnetically coupled paddle-wheal to provide a laminar flow and associated fluid shear stresses to scaffold specimens.
How have mesenchymal stem cells been used in a flex-stretch-flow bioreactor?
Mesenchymal stem cells derived from sheep bone marrow were cultured on polyglycolic acid/polylactic acid scaffolds.
Test group under mechanical stimuli and hydrodynamic shear had increased collagen content and effective stiffness of engineered valves after 3 weeks in culture compared to controls.
By the end of the 3 weeks engineered valves had a modulus comparable to those generated from smooth muscle cells in previous studies. Cyclic flexure and laminar flow can synergistically accelerate MSC-mediated tissue formation.
Give examples of how tissue engineering can be used in blood vessels and vocal folds.
Nikiason et al (1999) Pulsatile bioreactors/pump mimic pulses found in blood vessels.
Vocal fold are subject to mechanical stimuli so bioreactor incorporate speakers to mimic vocal fold maturation.
What are the current challenges to bioreactor design?
- Mimicking native cell behaviour - we need further understanding of tissue development and regeneration
- Scaling up. Most bioreactors are specialised devices with a low volume output. Time consuming and labour intensive.
What is the history of skin substitutes?
Skin substitutes were the first tissue engineered created ex vivo. There is a huge clinical need for skin substitutes and keratinocytes have been well cultured since the mid 1970s.
What are the different types of tissues according to their structure?
-Flat e.g. epidermis, cornea
One dominant cell type, relatively simple
-Tube e.g. blood vessels and urethras
Several cell types, serve as conduits
-Hollow non-tubular organs e.g. bladder, stomach
-Solid organs e.g. kidneys, lungs, heart and liver
Complexity in structure, histology, function
More complex - more difficult to recreate. Need a good knowledge of their structure and function first.
What are the properties and function of skin?
Largest organ in the body by surface area
About 10% of body mass
Functions of the skin:
-Protection against chemicals, UV light and microbes
-Regulation of water, temperature, sweating etc
-Sensation, contains sensory nerves
3 main layers: epidermis (neuroectoderm), dermis (mesoderm) and hypodermis.
What is the epidermis?
A thin layer that varies in thickness according to location.
Protects the body from environmental factors.
Consists only of cells:
-Keratinocytes (95%) produce keratin for toughness
-Melanocytes - produce melanin, located at the bottom
-Langerhans’ cells - dendritic cells, antigen properties
-Merkel cells - touch sensation, near neurons
Mature epidermis is a multilatered epithelium, upper layer is shed. Cells in basal layer are mitotic so will divide and move up and differentiate. Basal cells will secrete ECM matrix to create basement membrane.
What is the dermis?
Bulk of the skin
Composed of the skin with some elastin and glycosaminoglycans (mainly molecular).
Main cell types are fibroblasts - important for wound healing.
Also contains blood vessels, hair follicles, sebaceous and sweat glands.
What is the hypodermis?
A network of adipose cells and collagen
Functions as a thermal insulator and shock absorber and stores fat as an energy reserve.
When are skin substitutes needed?
There is a huge clinical need for skin substitues, namely in 4 instances:
- Acute trauma (most common)
- Chronic wounds
- Surgery
- Genetic diseases (bullous conditions)
Thermal trauma is one of the most common reasons for skin loss. Burns and scalds cause rapid and extensive wounds. Damaging large areas of skin can lead to death.
