BMS354 Principles of Regenerative Medicine and Tissue Engineering Flashcards
What is the difference between regenerative medicine and tissue engineering?
- Regenerative medicine is an umbrella term that includes tissue engineering and cell therapy.
- Tissue engineering differs from cell therapy through the use of biomaterials. Biomaterial can be used in both but in tissue engineering it is used more as a scaffold
- Tissue engineering also has some applications that are distinct from regenerative medicine. For example, lab grown food
Wy is there a clinical need for tissue engineering?
Organ failure is one of the most costly challenges faced in healthcare and the current treatments have many limitations
What are the available treatments for organ failure?
Surgical reconstruction Mechanical devices - Dialysis - Pace maker - Hip replacements Transplantation - Skin grafts - Organs from donors
What are the limitations of surgical reconstruction as a treatment for organ failure?
Invasive, dangerous
Surgical complications
Morbidity at the donor sites
What are the limitations of mechanical devises as a treatment for organ failure?
- Only mechanical support
- Do not grow with the tissue – children with these will need multiple surgeries to replace as they grow
What are the limitations of transplantation as a treatment for organ failure?
Rejection
- Must be on immunosuppressors
Limited supply
- Supply and demand – due to the limited supply, doctors are forced to use organs that may not be ideal e.g. from an elderly person
- E.g. in 2014, two patients who had a kidney transplant died sue to the kidney being infected with a parasitic worm
When did the tissue engineering field emerge?
- This research emerged in the 1970s and 1980s and was coined Tissue engineering in 1987
- In the 1990s research accelerates and industry begins to emerge - In 1998, human embryonic stem cells were isolated
What are the building blocks of tissue engineering?
Cells Biomaterial scaffold (provide physical support) Bioactive molecules (direct cellular behaviour)
Regenerative medicine and tissue engineering are multidisciplinary research fields. What other disciplines participate?
- Material science
- Cell, physiology, anatomy, molecular, computational biology
- Robotics
- Engineering
- Biochemistry
What tissues form an organ?
Epithelial, connective, muscle and nerve tissue form an organ
What is the role of the different tissues that make up an organ?
Epithelial tissue carry out the function while muscle and connective tissue provides structure
What are the three stages of wound healing?
Inflammatory phase
Proliferative phase
Remodelling phase
Explain the inflammatory phase of wound healing
- Clears out dead cells from the injury and limiting the extent of tissue damage to prepare the environment for healing
- Arterioles and venules near the site of injury constrict briefly and then dilate. This increases capillary permeability which moves fluid into the effected tissue. Blood clots occur due to increased blood viscosity
- Leukocytes engulf bacteria and cellular debris through phagocytosis to clean the wound and secrete growth factors which recruits fibroblasts
Explain the proliferative phase of wound healing
- New tissue formation occurs to fill the wound space
- Fibroblasts secrete collagen and growth factors which promote angiogenesis and endothelial cell proliferation and migration.
- Granulation tissue is formed by fibroblasts and endothelial cells. It forms 2-4 days post the wound. Microscopically looks very granular due to newly formed blood vessels and forms the scaffold for remodelling
- The newly formed blood vessels are leaky and allow blood cells and plasma cells to leak into the tissues
- Epithelisation – when there is proliferation and migration of epithelial cells to form the new surface
Explain the remodelling phase of wound healing
- Begins after three weeks
- Remodels the new connective tissue. This can take months and sometimes years
- Final scar tissue formed by collagen synthesis and becomes avascular
- Scar tissue can achieve 70-80% tensile strength by the end of three months
What is the difference between regeneration and repair?
Regeneration
- If the injury is only mild and superficial then the epithelium will be regenerated and there will be no scar
Repair
- If the injury is severe then a scar will form
What decides whether an injury will result in regeneration or repair?
The outcome of the injury is dependent on the severity, tissue damaged and length of injury
What is fibrosis and how is it different to repair?
Persistent tissue damage that results in a tissue scar
The process of scar formation is the same as repair but this term refers to when a stimulus is persistent
Give an example of fibrosis
Pulmonary fibrosis
What is meant by Fibrous encapsulation?
The body will try to protect itself from biomaterials by producing lots of collagen leading to its isolation from the bodies environment
What cell sources are used in regenerative medicine?
]Autologous: From same person
Allogenic: Same species but different people
Xenogeneic: Different species
Syngeinc or isogenic: Genetically identical but different person (twins)
What different cell types can be used in regenerative medicine?
Differentiated mature cells
Mixture of differentiated cells
Stem cells
What are the advantages and disadvantages of using differentiated mature cells in regenerative medicine?
- These cells cannot self-renew meaning they can’t expand into large numbers
- However, these cells are already functional. This is an advantage over stem cells as differentiation of stem cells is sometimes difficult to get mature functional stem cell
What kind of differentiated mature cells are used in regenerative medicine?
Fibroblasts, keratinocytes, osteoblasts, endothelial cells, chondrocytes ect.
What kind of stem cells are used in regenerative medicine?
Adult stem cells
- Can be taken from the patient so will be no immune response
- If person has genetic disease this doesn’t help
Embryonic stem cells
- Can differentiate into any cell type
Induced pluripotent stem cells
- Can still be taken from the same person –allogenic
- Can differentiate into any cell type
How are cells cultured in a lab?
- Cells are cultured in growth medium which contains the necessary requirements for cell growth e.g. nutrients, growth factors
- Laminar hood keeps aseptic
- Incubated at 37 degrees
- Lids of the petri dish are not sealed as there needs to be gaseous exchange
What ensures that the cells cultured for patient use are of a good quality?
Good manufacturing practice (GMP) ensures that medicinal products are consistently produced and controlled to the quality standards appropriate to their intended use
Must be free from animal products
What is the role of the ECM?
ECM provides support for cells and contributes to the mechanical properties of the cells. It also provides bioactive cues for cells, acts as the reservoirs of growth factors and potentiates their actions and acts as a scaffold for orderly tissue renewal
What is the composition of ECM?
Fibrous structural proteins – Collagen, elastins
Water hydrated gels – proteoglycans, hyaluronan
Adhesive glycoproteins – fibronectin, laminin
How does the structure of collagen allow its function?
- Unusually high abundance of proline, hydroxyproline and glycine. They twist around each other to form a triple helix.
- These triple helices are aligned side by side to form collagen fibrils.
- These then create collagen fibres which have an incredible tensile strength
Give the structure of proteoglycans?
- Composed of glycosaminoglycan chains linked to a specific protein core
- Very hydrophilic and form hydrated compressible gels
Where are proteoglycans found?
Joints
What is the role of fibronectin?
Allow the attachment of the cell to the ECM
What is a RDG sequence?
arginine, glycine, aspartic acid sequence in fibronectin
- what integrins on the cell surface bind to
How do cells adhere to ECM or biomaterials?
When cells adhere to the ECM there are no chemical bonds but the initial contact with the cell surface or material can activate signalling pathways in the cell through stress activated mechanical channels in the cell
What is the structure of an integrin?
- Form heterodimers and are made up of 19 alpha and 8 beta subunits
- They have extracellular, transmembrane and intracellular domain
Why is the structure of a integrin important?
The structure of the integrins determine which ECM proteins are recognised. Most recognise multiple ECM proteins – there is a lot of redundancy
What are the types of integrin signalling?
- In inside out signalling, there is a ligand which is bound to cytoplasmic tail which leads to conformational change in the integrin and changes its ability to bind a ligand outside the cell
- In outside in signalling, they bind an intrinsic ligand which transduces the signal inside the cell which leads to further assembly of the actin cytoskeleton and activation of signalling pathways
What signalling pathways does integrin binding lead to?
The signalling pathways used by them are mediated through focal adhesion kinase (FAK). This auto phosphorylates and recruits downstream proteins including the focal adhesion complex (a macromolecular assembly of proteins at the site of cell adhesion). This can then activate proliferation and cell signalling
What is mechanotransduction?
The process by which external mechanical stimuli are transmitted into the nucleus
What are biomatierals?
Non-viable materials used in a medical device indented to interact with biological systems. They can be used to develop scaffolds for tissue engineering
What is the main problem. with biomaterials?
The main issue with biomaterials is that the body rejects them through the immune response
When was the first biomaterial that did not lead to rejection discovered?
In the second world war, a man examined the eyes of pilots and noticed that they had splinters in their eyes that did not induce an immune response. He concluded that this material (used to make the cockpits) could be used to create intraocular
Define biocompatibility
Biocompatibility of materials is the ability of a material to perform with an appropriate host response in a specific application
What is meant by biomaterials leading to an appropriate host response?
- Resistance to blood clotting
- Resistance to bacterial colonisation
- Normal healing
What were the first generation of biomatierals?
Called bioinertness
- The body fluid did not interact with the materials and were stable to not induce any major adverse responses
When were biomaterial considered biocompatible?
If it was non-toxic, non-carcinogenic and had adequate mechanical properties
What was the second generation of biomatierals?
Bioactive
- This era started with the bioglass: the first material to seamlessly seal with bone
- Biodegradable materials
- However, non of these materials response to the needs of the body
What was the third generation of biomatierals?
Functional tissue
- Polymers
What are polymers?
Large molecules made up of linked monomeric units
They have a large molecular weight and can be presented in different structures e.g. linear, branched and network. A car tyre is a network polymer
What are the most common monomeric used units in biomaterials?
PE, PTFE, PVC
What is a homopolymer?
In the simplest form, there is one monomeric unit repeated
What is a copolymer?
Multiple different monomeric units in the same polymer
- Can manipulate the monomeric unit arrangement which will determine the property of the materials
What are hydrogels?
In natural state, polymers are brital and hard but some are naturally hydrophilic so will swell up in an aqueous medium. These are known as hydogels
Give an example for the use of hydrogels
Soft contact lenses
What are the classifications of biomaterials
- Natural
- Synthetic
- Semi-synthetic
Give examples of natural biomaterials
Protein based natural polymers
- E.g. Collagen (found commonly in animals and plants), gelatine, silk (low degradation time), Fibrin (component in blood clots)
Polysaccharides
- E.g. Chitosan, alginates, hyaluronan
Give examples of synthetic biomaterials
Polyactic acid, polygycolic acid
What are semi-synthetic materials?
Hybrid molecules made by the incorporation of biologically active macromolecules onto the backbone of synthetic polymers
Give examples of semi- synthetic biomaterials
Semi synthetic PEG-fibrinogen
- PEG controls the density stiffness and biodegradability
- Fibrinogen presents biofunctional domains
What are the advantages and disadvantages of natural biomaterials?
Advantages
- Built in bioactivity: Integrin recognise RDG domain so cells recognise it
Disadvantages
- Purification cost
- Immunogenicity – induce immune response
- Lack of mechanical properties
- Batch to batch variability – difficult to isolate from same source
What are the advantages and disadvantages of synthetic biomaterials?
Advantages
- Controlled mechanical properties and degradation kinetics
- Easy processability into custom shapes and structures
- Minimal batch to batch variability – synthesised on an industrial scale
- Cost effective
Disadvantages
- Biocompatibility may be difficult to predict
- Immune rejection
What are degradable biomaterials?
- Used as a scaffold and overtime degrades and replaced with tissue. Therefore, biodegradability can be a positive thing.
- This means that there is a hydrolysis of a covalent bond. This results in the formation of by-products which also need to be non-toxic
What are resorbable biomaterials?
These materials can be reabsorbed into the body and removed so that there is total elimination of the initial foreign material and its by-products
What properties should a biomaterial possess to be used for internal sutures?
- Do not want another surgery to remove so they should be biodegradable and the length of biodegradability should depend on the organ as it needs to hold the wound together and heal but if in there to long it will induce a host response
- Used sheep gut but now synthetic used
What properties should a biomaterial possess to be used for soft contact lenses?
- Required to correct material so must be transparent and hold their shape to maintain refractory angle
What properties should a biomaterial possess to be used for artificial hip joints?
- Need to have high mechanical strength
What are the bulk properties of biomaterials?
- Strength
- Toughness
- Fatigue resistance
- Stability
How did Engler et al, 2006 investigate how mechanical properties of biomaterials can influence how cells behave?
Mesenchymal stem cells placed on three different materials. High (represent bone), medium and low (represent blood) stiffness materials. They all lead to different responses on the stem cells showing they can sense their location and respond accordingly
What are surface modifications of biomatierals?
Alter the surface of the biomaterial to achieve the desired properties
- The layer modifying should not be too thick as this would change the bulk properties and prevents delamination resistance
How are surface modifications achieved?
This can be done by
- Chemically altering the atoms in the existing surface
- Over coating the existing surface with a material of a different composition
- Creating surface textures or patterns
How do cells interact with biomaterials?
- The proteins from serum/growth medium adhere to the surface of the glass and the cells adhere to this protein
What are non fouling materials?
Materials that do not allow protein absorption
How can non fouling materials be an advantage?
This is an advantage sometimes in medicine as it will result in no bacteria colonisation
How are non fouling materials a disadvantage?
In tissue engineering it is a disadvantage as want cells to adhere
How can non fouling materials be functionalised?
By chemically adding RDG domains to the surface
- Can also use this to decide where the cells attach to the surface
How can RDG domains be added to non fouling materials?
Using micro contact printing
- A prepolymer is poured on - It is then cured and the stamp peeled off the master
- The stamp is then cut into smaller places and Inked by soaking in ink solution
- The ink is printed bb contacting an inked stamp with a suitable surface
- A patterned substrate is obtained
What did Chen et al, 1997 hypothesise about the role of cell shape?
- Hypothesised that cell shape controlled cell survival
How did Chen at al, 1997 test that cell shape controlled survival?
- Used micro stamping of fibronectin islands. Cells attached to these islands and assumes the exact shape of the islands. They then looked at the survival of these shapes. The shape alone affected whether cells proliferated or entered apoptosis
What properties must a scaffold have?
- Provide mechanical and biological functions of ECM
- Porous to allow vasculature bring the oxygen and nutrients needed for the cells covering the scaffold
- Degradable so that they do not need to be removed by surgery.
- The degradation products need to be non-toxic.
- The degradation kinetics need to be carefully designed based on the tissue it is being used in
What are acellular tissue matrices?
- Another material for scaffold fabrication
- Could be considered naturally derived. They are obtained by decellularisaing tissues and organs – remove all cells and antigens resulting a white tissue matrix. This matrix will therefore not induce an immune response so can be used without immunosuppressans.
How are tissues decellularised?
- Usually involves mechanical scraping, acids and bases and enzymes to digest the cellular and nuclear components
- If process not complete or bacterial contamination then it may result in scar tissue formation and an immune response
- Cross linking of ECM can be problematic as the body cannot remodel it so again can induce scar tissue formation
What are the of advantages acellular tissue matrices?
The process preserves intricate structures that are difficult to replicate outside the body but are important for function
What are the of disadvantages acellular tissue matrices?
Accessibility – use the dermis of the skin, submucosa of small intestine, pericardium. Some are now commercially available (Alloderm)
Why is the architecture of scaffolds important?
- The final shape of the tissue engineered structure is defined by the scaffold – can use MRI to find the exact shape needed
- Microscopic architecture also very important. Need to be porous to allow vasculature. When trying to recreate this in vitro need to keep this in mind
What is a pore?
A pore is the could space within a scaffold
What is porosity?
The porosity is the number of pores and how well they are connected
Why is the porosity of a scaffold important?
Some scaffold has 100% accessibility and 100% interconnecting meaning can get to any pore though multiple roots but some are not. This is important because if too porous then there will be a lower mechanical strength
What are the properties of an ideal scaffold?
- Adequate mechanical properties
- Biodegradable
- Allows cell attachment and function
- Accessible
- Allows tissue and integration and vascularisation
What are the methods of scaffold fabrication?
- Porogen leaching
- Phase separation
- Electrospinning
- Additive manufacturing
Outline Porogen leaching
- Uses a polymer dissolved in a solvent. The polymer is mixed with salt particle of a certain size
- This mixture is poured into a mold. Then evaporate the solvent and the polymer will solidify. The salt particles are still dispersed inside it.
- Put this solid polymer into water and the salt will leach out leaving with a porous scaffold where the salt was present in the polymer
Evaluate porogen leaching as a method of scaffold fabrication
Can change the size if the pores by changing the size of the salt particles but the way they are distributed cannot be changed
Outline phase separation
- Polymer is dissolved in a suitable solvent and subsequently placed in a mold that will be rapidly cooled until the solvent freezes
- Solvent is then removed by freeze-drying and pores will be left behind in the polymer
Outline electrospinning
- Commonly used due to its ability to provides very fine fibres (nanofibers).
- Polymer solution in syringe which is attached to a high voltage power supply. This creates an electromagnetic field between the needle and a grounded plate.
- The polymer is slowly injected and due to the electromagnetic field it causes the polymer fibres to spray and the solvent will evaporate and the fibres are collected on the grounded plate
- The parameters (e.g. voltage, distance from plate ect) can be changed which will influence the types of fibres get at the end
What is additive manufacturing?
The process of joining materials to make objects from 3D model date, usually layer upon layer
What is the most common type of additive manufacturing?
3D printing
Outline 3D printing
- Uses a roller which delivers a layer of polymer onto the surface.
- Have cartridges which are filled with adhesive. This is controlled by a computer and the adhesive is only added to certain areas.
- Once one layer is done, the platform moves down and the roller adds another polymer layer and the process is repeated.
- When complete, the adhesive is cured by heating and unwanted polymer is removed
What are the advantages and limitations of 3D printing?
Advantages
- Production of scaffolds with precise morphologies
- Combines medical imaging to fabricate anatomically shaped implants
Limitations
- Limited number of biomaterials can be used
What is the problem with 3D printing as a method of scaffold fabrication?
Material is printed and then the cells are put on the scaffold. This has problems because the cells sometimes do not populate the scaffold properly and may sit in the pores which are needed to be open for vasculature to go through.
What is Cell encapsulation?
A variation on 3D printing where cells are mixed with the polymer and then printed out
What did Kang et al, 2016 do to further the use of 3D printing?
Looked at 3D printing system to produce human scale tissue with structural integrity
What did Kang et al, 2016 use to produce human scale tissue?
- A new 3D printer called an integrated Tissue-Organ Printer (ITOP).
- Common problem faced was killing the cells during in printing. To overcome the issues they were having, this printer had several cartridges which dispense different things.
- Cells were encapsulated in a polymer already (with polymer that is not toxic to cells).
- They used a sacrificial material that allowed them to keep the mechanical structure of the construct while it was being printed
What sacrificial material did Kang et al, 2016 use?
Used two types of sacrificial materials: PCL and pluronics (only in place during printing). Printed Cell-laden hydrogels and sacrificial scaffold to provide initial mechanical integrity to the cells
What did Kang et al, 2016 achieve using cell encapsulation?
- Made ear, bone and muscle
Able to show that after removing the scaffold, muscle fibres maintained integrity and would mature in differentiation medium
What biomolecules have been explored for inducing tissue regeneration?
- Small molecules
- Proteins and peptides
- Oligonucleotides
What are BMPs?
- Abundant in bone matrix and made by osteoblasts (bone forming cells)
- Members of the TGFbeta family
What is meant by BMPs being osteoinductive?
If ectopically implant BMPs then it will form ectopic bone. This ability is believed to be due to their ability to recruit osteoblasts and cause tissue specific stromal cells to differentiate into osteoblasts
How did Lutolf et al, 2003 investigate the role of bioactive molecules in scaffolds?
- It was clear that BMPs were only osteoinductive if they are locally delivered and retained to have the correct concentration
- They hypothesised that they could use scaffolds to module the effect of BMPs and to enable them to be delivered locally and maintained at the correct concentration to have the osteoinductive affect
How did Lutolf et al, 2003 prepare the gel for the scaffold they used?
- They prepared gel formed of the non-fouling PEG which was then functionalised using RGD domains. They then crosslinked PEG and added a site for cleavage by matrix metalloproteinases.
- These are enzymes that the cells use to remodel the matrix. They entrapped BMP2 inside these gels.
- The pore sizes are large enough to hold BMP but small enough that it doesn’t just diffuse through the gel
How did Lutolf et al, 2003 show that the gel they produced could be cleaved and degraded?
- Did functional studies to show gel worked. Used 3D fibroblasts and see if they can degrade the matrix using matrix metalloproteinases which they could. The scaffold could therefore be degraded and the cleavage sites could be cut by cells
- They then checked if BMP2 can be released from scaffolds. Saw a gradual release of BMP over time
How did Lutolf et al, 2003 show that their scaffolds with BMP entrapped could promote bone regeneration?
- Tested this in vivo. The idea is that if can bring BMP to the site of injury then it should induce tissue regeneration in the bone.
- Took rats with critical size bone defect. Used a control scaffold with no BMP which showed no repair as well as a control that had BMP but no cleavage sites added.
- In scaffolds with BMP and cleavage sites resulted in bone regeneration in the rat
How could the scaffold from Lutolf et al, 2003 result in bone regeneration?
This occurred as responding cells adhere to RGDs in matrix. Cells secrete MMPs that degrade MMP cleavable bonds serving as crosslinks for the matrix. BMP liberated from matrix diffuses from site, signaling osteoblast precursor cells. Osteoblasts secrete bone matrix and bone is regenerated
What are the limitations of the conventional scaffold fabrication techniques?
- E.g. salt leaching, gas forming, phase separation and freeze drying
- They do not enable precise control of internal scaffold architecture or the fabrication of complex architectures
- Using toxic solvents in these techniques can lead to cell death if not correctly removed
What are the limitations of phase separation?
Limitations include the addition of organic solvents such as ethanol inhibits the incorporation of bioactive molecules or cells during scaffold fabrication and the small pore sizes obtained
How can the pore size in scaffolds be altered in electorspinning?
Pharm et al showed that the average pore size of electrospun scaffolds increased with increasing fibre diameter
What is a disadvantage of electrospinning?
Disadvantages include involving toxic organic solvents which can be harmful to cells
How can the disadvantages of electrospinning be overcome?
- This can be overcome using melt electrospinning which doesn’t involve organic solvents and instead heats the polymer to its melting temperature.
- However, as the polymer melts it has a lower charge density meaning that the fibres produced are thicker than those produced from electrospinning
Why is silk better then industrial produced replacements?
Industry makes polymeric fibres such as Kevlar which are high performing and take a lot of energy to break. Nature makes natural fibres such as silk from spiders/snails and due to natural selection, it is much stronger, cheaper, lower energy and biodegradable then anything we can make in industry
Define silk
They are structural proteins which are spun into fibres for use outside the bod
What is the primary structure of silk?
- Silks are fibrous proteins and usually 100s kDa long
- Contains high proportions of glycine and alanine which are biologically cheap amino acids
How does the primary structure of silk make they tough?
- The fact that the chains can move slightly due to the prolines producing kinks meaning that they are tough and can dissipate energy
- Protein hydration and hydrogen bonds are key to structure and strength
What is the secondary structure of silk?
The prolines make the protein feedback on its self to form beta sheets
What is the structure of silk at a nanoscale?
Formed of a combination of order and disordered parts
What is the structure of silk at a hacroscale?
Formed many fibres which form layers to produce silk
Is kevlar (commerically produced) or silk stronger?
Kevlar is stiffer then spider silk but silk is stronger, it doesn’t break with more force - it bends
How can we change the mechanical properties of the silk produced?
Silks are spun to move at the natural speed of the animal. Spiders can slightly change this speed. Can take silk and change the speeding conditions and can change the mechanical properties of the silk. Can therefore replicate any of the fibres in a better and environmental way
Why is silk useful in biomedical research?
Can be turned into any structure so can be used to recreate any apart of the human body e.g. neural implants, collagen, lasers. Silk will degrade in the body and the rate of degradation can be controlled by controlling the beta sheets
Give examples of what silk has been used to in regenerative medicine?
Peripheral nerve repair
- Put fibres inside a straw to form a nerve conduit
Vascular repair
- Vascular graft for haemodialysis – stop vein collapse
Orthopaedic implants
- Treatments of meniscus and cartilage damage in the knee – this is a common
- Can use an artificial meniscus which can be put in between the cartilage on the knee. The mechanical properties work due to its biological basis
What are bioreactors?
Tanks full of medium where can grow cells to a high density under a tight control of conditions. This can maximise the yield produced
What are the current challenges in tissue engineering?
- Trying to create not just the cells but a 3D structure with cells in an appropriate spatial orientation. Growing to relative clinical sizes is difficult
- The growth and 3D assembly of multiple cell types that are required for more complex functional tissue
What is the problem with normal tissue culture?
- The media is static on top of the cells in the cell culture dish. This can result in a concentration gradient across the flask changing the cell density and the behaviour of the cells
- Only know the pH at the time of feeding, after that have no control
What are dynamic culture systems?
- Designed to mix the media and give control over the environment
- A medium tank is connected to a peristaltic pump which feeds the media to the cells. Can also have a control of gas pH to monitor or control
- This results in homogeneous concentrations of nutrients, toxins and other components
What are the three roles of bioreactors in tissue engineering?
- To establish spatially uniform cell distributions on 3D scaffolds
- To overcome mass transport limitations in 3D culture
- To expose the developing tissue to physical stimuli
Why is the way that a scaffold is seeded important?
The way the cells attach and distribute will determine the mechanical and functional properties of the construct
What is needed when seeding cells on a scaffold?
- Need a high seeding efficiency to stop the loss of rare and important cells. There may not be huge amounts of cells which can be used so cannot waste them. This process needs to be maximised
- Want to achieve a uniform distribution of cells across the scaffold
What is the problem with seeding cells in a static way?
Can deliver the cells in a static way by pipetting the cells into the scaffold and by gravity they will attach to the scaffold. This can lead to cells just attaching to the top and not moving through the scaffold
How can the problem of static cell seeding be overcome?
Can flow media through the construct meaning that the cells can access entire scaffold leading to a more even distribution
Describe the assay used to see how the seeded cells are distributed on a scaffold?
- MTT assay – indicated were cells are and if they are alive. It is a purple die that will turn purple from yellow if high metabolic activity meaning the cells are dying
- Statically seeded cells accumulate at the top. High density can lead to negative effects
What are the transport limitations of 3D cultures?
- Once the cells are inside the scaffold, they need oxygen
- Mass transfer of external and internal mass and removal of metabolites and CO2
- There is a maximum distance between cells and capillaries is 200micrometres. This is the same in vitro meaning that cells need to be close to the medium
How did Wendt et al, 2008 overcome the transport limitations of 3D culture?
- Chondrocytes seeded using perfusion cell seeding
- Cultured the cells statically and by perfusion
- In the statically cultured cells, the cells only survive at the edges as that is where the nutrients could get to the cells
- In the perfusion cultured scaffolds, the cells were well nourished and created well organised structures
Why do developing tissues have to be exposed to physical stimuli?
- Tissues and organs in the body are subject to complex biomechanical environment
- There are many forces inside the body including hydrostatic, mechanical and electrical
- To engineer something in vitro, the cells need these pressures – they have evolved in a way to need this
- Bioreactors allow this
What are the design considerations that need to be taken into account when bioreactors?
- Diversity in bioreactor design reflects the range of signals needed for formation of various tissues
- They need to be biocompatible and sterile to stop bacterial infections
What are the types of bioreactor?
- Spinner flask bioreactor
- Rotating wall bioreactor
- Perfusion bioreactor
- Compression bioreactor
What are spinner flask bioreactors?
- The magnetic stirrer stirs the solution to help with the mass transfer of medium to the cells. This helps with the external transfer
- Very accessible and cheap
What are rotating wall bioreactor
- Flask spins around and the scaffold are suspended. Gravity pulls the scaffolds down and the forces of the flask rotating are pulling in the opposite direction. These forces cancel each other out. The scaffold is suspended in medium
- As the cells expand, the forces need to be increased to counterbalance gravity
What is a perfusion bioreactor?
- The culture medium continually circulates through the construct
- Most mass transfer limitations are mitigated
- The effects of direct perfusion and be high dependant on the medium flow rate
What is compression bioreactor?
Apply mechanical stimulus to the construct
What type of bioreactor would be used to decellularise organs?
- Complex organs are difficult to decellularise
Can use a perfusion bioreactor
What type of bioreactor would be used to engineer articular cartilage?
- Compression bioreactor
- Cartilage is a load baring tissue so is under mechanical stress in differentiation
What type of bioreactor would be used to study the effects if near 0 gravity?
- Rotating wall bioreactors
- These were designed by Nasa and there is evidence that in space peoples bone mass decreases. Can therefore study bone density in a 0-gravity environment using these rotating wall bioreactors
Give a study into investigating the effects of 0 gravity?
Tamma et al, 2009
- Culture osteoblasts
- There are more osteoblasts produced in near 0 gravity environment which could explain the decreases in gravity
What is a common problem with heart valves?
- Valves may not open or close entirely. This is a huge problem world-wide but options are limited. Often involves surgical repair or valve replacement
What are the problem with heart valve replacements?
Patients receiving them need ant-thrombotic drugs which can predispose them to lack of blood clots
What is the problem with creating heart valve replacements that grow with the developing body?
- Autologous leads to no rejection but unlikely to get the right cells needed.
- To differentiate these cells appropriately, they need to withstand forces from the moment they enter the body. Animal model studies have shown that they can’t do this unless they have been exposed to the forces outside the body.
How did Engelmayr et al, 2008 use a bioreactor to design a construct for a specific function?
Wanted to mimic the mechanical stimulation and perfusion of heart valves in vitro to improve engineered tissues
What did Engelmayr et al, 2008 do to mimic mechanical stimulation of heart valves?
Made a flex stretch bioreactor
- Can drive movable post from a flex to a stretch position. This can mimic the role of a heart valve in the body
- To mimic blood flow over the valves, they have the medium flow over the scaffold. This exposes the cells to hydrostatic pressure
Used mesenchymal cells derived from sheep bone marrow and cultured in PGA/PLA (synthetic) scaffolds. Then treated with and without the bioreactor
What did Engelmayr et al, 2008 conclude?
Concluded that the forces were necessary to mimic what happens in the body to promote mesenchymal stem cell differentiation and tissue formation
How have bioreactors helped to engineer vocal folds?
- The vocal folds has specific physical stimuli in vivo
- This bioreactor mimics the sound by speakers to create vocal folds
What are the challenges in bioreactor design?
Mimic native cell behaviour
- Further understanding of tissue development and regeneration
Scale up
- All small scale equipment so to generate on a large scale for clinical application may be difficult
Why was skin the first organ to be fabricated?
Is the first organ to be fabricated due to its relatively simple structure and the huge clinical need. It is also because skin cells were the first cells to be cultured outside the body
What is the main problem with skin replacements?
The skin that is replaced still does not look or function the same
What percentage of body mass does the skin account for?
10%
