BMS354 Principles of Regenerative Medicine and Tissue Engineering Flashcards

Question

What kind of stem cells are used in regenerative medicine?

Answer 1

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

Answer 2

- 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

Answer 3

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

Answer 4

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

Answer 5

Fibrous structural proteins – Collagen, elastins Water hydrated gels – proteoglycans, hyaluronan Adhesive glycoproteins – fibronectin, laminin

Answer 6

- 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

Answer 7

- Composed of glycosaminoglycan chains linked to a specific protein core - Very hydrophilic and form hydrated compressible gels

Answer 8

Allow the attachment of the cell to the ECM

Answer 9

arginine, glycine, aspartic acid sequence in fibronectin | - what integrins on the cell surface bind to

Answer 10

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

Answer 11

- Form heterodimers and are made up of 19 alpha and 8 beta subunits - They have extracellular, transmembrane and intracellular domain

Answer 12

The structure of the integrins determine which ECM proteins are recognised. Most recognise multiple ECM proteins – there is a lot of redundancy

Answer 13

- 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

Answer 14

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

Answer 15

The process by which external mechanical stimuli are transmitted into the nucleus

Answer 16

Non-viable materials used in a medical device indented to interact with biological systems. They can be used to develop scaffolds for tissue engineering

Answer 17

The main issue with biomaterials is that the body rejects them through the immune response

Answer 18

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

Answer 19

Biocompatibility of materials is the ability of a material to perform with an appropriate host response in a specific application

Answer 20

- Resistance to blood clotting - Resistance to bacterial colonisation - Normal healing

Answer 21

Called bioinertness | - The body fluid did not interact with the materials and were stable to not induce any major adverse responses

Answer 22

If it was non-toxic, non-carcinogenic and had adequate mechanical properties

Answer 23

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

Answer 24

Functional tissue | - Polymers

Answer 25

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

Answer 26

PE, PTFE, PVC

Answer 27

In the simplest form, there is one monomeric unit repeated

Answer 28

Multiple different monomeric units in the same polymer | - Can manipulate the monomeric unit arrangement which will determine the property of the materials

Answer 29

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

Answer 30

Soft contact lenses

Answer 31

- Natural - Synthetic - Semi-synthetic

Answer 32

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

Answer 33

Polyactic acid, polygycolic acid

Answer 34

Hybrid molecules made by the incorporation of biologically active macromolecules onto the backbone of synthetic polymers

Answer 35

Semi synthetic PEG-fibrinogen - PEG controls the density stiffness and biodegradability - Fibrinogen presents biofunctional domains

Answer 36

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

Answer 37

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

Answer 38

- 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

Answer 39

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

Answer 40

- 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

Answer 41

- Required to correct material so must be transparent and hold their shape to maintain refractory angle

Answer 42

- Need to have high mechanical strength

Answer 43

- Strength - Toughness - Fatigue resistance - Stability

Answer 44

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

Answer 45

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

Answer 46

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

Answer 47

- The proteins from serum/growth medium adhere to the surface of the glass and the cells adhere to this protein

Answer 48

Materials that do not allow protein absorption

Answer 49

This is an advantage sometimes in medicine as it will result in no bacteria colonisation

Answer 50

In tissue engineering it is a disadvantage as want cells to adhere

Answer 51

By chemically adding RDG domains to the surface | - Can also use this to decide where the cells attach to the surface

Answer 52

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

Answer 53

- Hypothesised that cell shape controlled cell survival

Answer 54

- 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

Answer 55

- 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

Answer 56

- 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.

Answer 57

- 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

Answer 58

The process preserves intricate structures that are difficult to replicate outside the body but are important for function

Answer 59

Accessibility – use the dermis of the skin, submucosa of small intestine, pericardium. Some are now commercially available (Alloderm)

Answer 60

- 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

Answer 61

A pore is the could space within a scaffold

Answer 62

The porosity is the number of pores and how well they are connected

Answer 63

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

Answer 64

- Adequate mechanical properties - Biodegradable - Allows cell attachment and function - Accessible - Allows tissue and integration and vascularisation

Answer 65

- Porogen leaching - Phase separation - Electrospinning - Additive manufacturing

Answer 66

- 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

Answer 67

Can change the size if the pores by changing the size of the salt particles but the way they are distributed cannot be changed

Answer 68

- 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

Answer 69

- 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

Answer 70

The process of joining materials to make objects from 3D model date, usually layer upon layer

Answer 71

3D printing

Answer 72

- 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

Answer 73

Advantages - Production of scaffolds with precise morphologies - Combines medical imaging to fabricate anatomically shaped implants Limitations - Limited number of biomaterials can be used

Answer 74

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.

Answer 75

A variation on 3D printing where cells are mixed with the polymer and then printed out

Answer 76

Looked at 3D printing system to produce human scale tissue with structural integrity

Answer 77

- 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

Answer 78

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

Answer 79

- Made ear, bone and muscle Able to show that after removing the scaffold, muscle fibres maintained integrity and would mature in differentiation medium

Answer 80

- Small molecules - Proteins and peptides - Oligonucleotides

Answer 81

- Abundant in bone matrix and made by osteoblasts (bone forming cells) - Members of the TGFbeta family

Answer 82

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

Answer 83

- 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

Answer 84

- 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

Answer 85

- 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

Answer 86

- 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

Answer 87

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

Answer 88

- 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

Answer 89

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

Answer 90

Pharm et al showed that the average pore size of electrospun scaffolds increased with increasing fibre diameter

Answer 91

Disadvantages include involving toxic organic solvents which can be harmful to cells

Answer 92

- 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

Answer 93

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

Answer 94

They are structural proteins which are spun into fibres for use outside the bod

Answer 95

- Silks are fibrous proteins and usually 100s kDa long | - Contains high proportions of glycine and alanine which are biologically cheap amino acids

Answer 96

- 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

Answer 97

The prolines make the protein feedback on its self to form beta sheets

Answer 98

Formed of a combination of order and disordered parts

Answer 99

Formed many fibres which form layers to produce silk

Answer 100

Kevlar is stiffer then spider silk but silk is stronger, it doesn’t break with more force - it bends

Answer 101

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

Answer 102

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

Answer 103

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

Answer 104

Tanks full of medium where can grow cells to a high density under a tight control of conditions. This can maximise the yield produced

Answer 105

- 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

Answer 106

- 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

Answer 107

- 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

Answer 108

- 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

Answer 109

The way the cells attach and distribute will determine the mechanical and functional properties of the construct

Answer 110

- 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

Answer 111

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

Answer 112

Can flow media through the construct meaning that the cells can access entire scaffold leading to a more even distribution

Answer 113

- 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

Answer 114

- 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

Answer 115

- 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

Answer 116

- 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

Answer 117

- 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

Answer 118

- Spinner flask bioreactor - Rotating wall bioreactor - Perfusion bioreactor - Compression bioreactor

Answer 119

- 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

Answer 120

- 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

Answer 121

- 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

Answer 122

Apply mechanical stimulus to the construct

Answer 123

- Complex organs are difficult to decellularise | Can use a perfusion bioreactor

Answer 124

- Compression bioreactor | - Cartilage is a load baring tissue so is under mechanical stress in differentiation

Answer 125

- 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

Answer 126

Tamma et al, 2009 - Culture osteoblasts - There are more osteoblasts produced in near 0 gravity environment which could explain the decreases in gravity

Answer 127

- 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

Answer 128

Patients receiving them need ant-thrombotic drugs which can predispose them to lack of blood clots

Answer 129

- 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.

Answer 130

Wanted to mimic the mechanical stimulation and perfusion of heart valves in vitro to improve engineered tissues

Answer 131

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

Answer 132

Concluded that the forces were necessary to mimic what happens in the body to promote mesenchymal stem cell differentiation and tissue formation

Answer 133

- The vocal folds has specific physical stimuli in vivo | - This bioreactor mimics the sound by speakers to create vocal folds

Answer 134

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

Answer 135

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

Answer 136

The skin that is replaced still does not look or function the same

Answer 137

It is needed for protection, regulation and sensation | - The main function is protection of the body from UV light and microbes

Answer 138

Epidermis Dermis Hypodermis

Answer 139

Varies from 0.5 mm on eyelids to 1.55mm on soles of feet

Answer 140

Protect from environmental factors

Answer 141

- No blood vessels, gets from nutrients from the dermis - Mainly Keratinocytes which secrete keratin which gives skin its toughness and creates the water proof layer. Contributes 90% of epidermis - Melanocytes which secrete melanin to protect from the UV light - Merkel cells – involved in sensation of touch - Langerhans cells are antigen presenting cells and are involved in the immune response - Forms a stratified epithelium

Answer 142

Mitotic cells are found in the bottom basal layer and differentiate as they move up the layers. Basal layer also secretes ECM which produces basement membrane which separates the epidermis from the dermis

Answer 143

- Makes the bulk of the skin - Composed of collagen with some elastin and glycosaminoglycans - Cells found are mainly fibroblasts which lay down collagen as well as blood vessels, sweat glands and hair follicles

Answer 144

The bottom layer which functions as a thermal insulator and stores fat as a major source of energy

Answer 145

During wound repair, fibroblasts are recruited and lay down new collagen. Myofiberblasts are similar to fibroblasts and smooth muscle cells and their role is to contract the wound. If they are over stimulated it can lead to over contraction and cannot move the skin

Answer 146

Acute trauma e.g. burns Chronic wounds Surgery Genetic disorders

Answer 147

Epidermal Superficial partial thickness wounds Deep partial thickness wounds Full thickness wounds

Answer 148

- Affect only epidermis and are characterised by erythema and minor pain - They do not require surgical treatment - Do not lead to scarring

Answer 149

- Affect the epidermis and superficial part of the dermis - The wounds are wet and weeping with red to pink blisters - Very painful sue to the exposure of sensory nerves - Heal spontaneously

Answer 150

- Involves greater dermal damage - Moist and white/red in appearance - Result in fewer skin appendages remaining - Scarring is more pronounced

Answer 151

- Complete destruction of epithelial regenerative elements - Wound appearance, dry leathery and rigid - No spontaneous healing

Answer 152

Early excision of dry scab (ESCHAR) - Remove denatured proteins as they can trigger inflammatory response and can harbour microbes. The patients are already immune repressed so this can cause problems Wound closure - reduces scarring and increase mortality Skin grafts - A skin graft is a tissue of epidermis and varying amounts of dermis that is detached from its own blood supply and placed in a new area

Answer 153

Categorised based on how much dermis is taken - Split thickness: the epidermis and part of the dermis is taken - Full thickness: both epidermis and dermis is taken

Answer 154

Removing skin from uninjured part to injured part | Use meshing of skin grafts to cover a larger area

Answer 155

The new cells need to get nutrients from the wound bed. This can keep the cells alive for 2-3 days In the meantime, there is vascular growth within the wound bed. The graft will only ‘take’ if this occurs

Answer 156

For the graft to take, the wound must be prepared properly - The graft must adhere to the wound bed (no bleeding, infection or movement) - The graft needs a thin layer of connective tissue. Cannot graft skin directly onto bone or tendon

Answer 157

- If the trauma is very extensive then sometimes there is not enough skin autologous skin to use for the graft. Then consider skin allografts - The use of cadaveric skin for temporary prevention of fluid loss or wound contamination. However, this can lead to pathogen transmission and immunogenic rejection

Answer 158

- Readily available - Cause no immune response - Cover and protect the wound - Enhance the healing - Lessen the pain - Leave no scars - However, there are no skin substitutes so far which do all of these things

Answer 159

``` Layer to be substitute - Epidermis - Dermis - Compound Durability - Temporary - Permanent Product origin - Biologic - Biosynthetic - Synthetic ```

Answer 160

- A key step is isolation of keratinocytes from a skin biopsy and expanding in culture - Cultured keratinocytes are delivered on the wound and form a new epidermal layer

Answer 161

Used as an epidermal substitute - Skin biopsy from the patient, isolate the epidermis and extract the keratinocytes - The keratinocytes are cultured and form colonies which will merge and create a stratified epithelium. This forms cultured epithelial sheets

Answer 162

The graft takes of these vary a lot (from 15-80%). It is difficult to work with something with such a varied success rate

Answer 163

Studies indicated that when the keratinocytes are cultured, if they differentiate so that they don’t produce the correct integrins anymore then when they are placed on the dermis then they will not attach. The cells need to be at the right stage of differentiation

Answer 164

Used as an epidermal substitute - Take a tissue biopsy, culture the cells and deliver back to the patient - Autologous cells used so results in no immune response

Answer 165

Used as an epidermal substitute - Developed by Celltran ltd which was part of the University of Sheffield - Sub confluent autologous keratinocytes. The cells did not fill the entire flask which kept them in the right differentiation stage - They also developed a synthetic silicone delivery membrane which allowed successful delivery to the wound bed - Was successful in treating chronic wounds. Required repeated delivery

Answer 166

- Keratinocytes can be easily expanded in vitro | - Can be autologous

Answer 167

Only contain keratinocytes so can only replace the epidermal layer. If there are deeper injuries then also require a dermal substitute

Answer 168

Needed for deeper skin wounds - Most products are acellular. Idea is to provide a scaffold that will be infiltrated by cells of the host and remodel the matrix

Answer 169

- Dermal layer: made from bovine type 1 collagen and shark chondroitin-6-sulfate. This is xenogeneic (not from a human) - Epidermal layer: made from silicone. This regulated heat and fluid loss. - The collagen-chondroitin matric allows in growth of cells from the wound bed and the artificial silicone epidermis regulates heat and fluid loss

Answer 170

Once the cells infiltrate, the silicone membrane can be removed and replaces with epidermal substitute

Answer 171

Dermal substitue - Acellular human allogenic dermal matrix (decellularised skin) preserved by freeze drying - Full thickness skin burns alloplastic breast reconstruction, abdominal wall reconstruction, rhinoplasty

Answer 172

Entails a two-step process - Put dermal substitute into the wound bed - Add epidermal substitute which delivers the cells of the epidermis

Answer 173

A types of skin graft - Aims to mimic the histological structure to normal skin – contain both epidermis and dermis - Most of these products are based on allogenic skin incorporated (different person) into a dermal scaffold - Enable production of large batches of the product because from a different pattern

Answer 174

Composite skin which consists of bovine type 1 collagen cultures with allogenic male neonatal fibroblasts and keratinocytes - There are no skin appendages, melanocytes, hair cells Allcell: includes cultured allogenic fibroblasts and keratinocytes obtained from the same neonatal foreskin - Fibroblasts are seeded into a bovine type 1 collagen sponge with keratinocytes on top - Cytokines and growth factors from the product promote host cell migration and wound healing

Answer 175

- The average wait time ranges from 3 to 12 weeks after the biopsy is taken. If want to produce autologous skin, it can only happen once the patient arrives at the clinic. It is difficult to produce quick autologous treatments. This is why many use allogenic cells - Only use keratinocytes and fibroblasts. There are many other cell types in the skin which are required for normal function and look. - Some products are considered clinical success but economic failure.

Answer 176

Used gene therapy to genetically modify 7 year olds cells who was suffering with Epidermolysis bullosa (EB) and provide a life saving treatment

Answer 177

- Causes spontaneous skin blistering and is very painful with life threatening complications (skin is a protective barrier so without it can be prone to infections and sepsis). - It causes a high risk of skin cancer (probably due to the skin constantly trying to heal) and affects 1 in 50000

Answer 178

- Epidermis and dermis are separated by the basement membrane and blisters occur in-between these layers - Normally the basement membrane allows the epithelial cells in the basal layer to adhere to the dermis. - This is allowed to happen due to the macromolecular protein complex in the basement membrane. - Keratin and other intermediate filaments such as laminin interact with Alpha6beta4 integrins. - Laminin also interacts with collagen of the dermal layer. The basement membrane is a structured and ordered structure. - If there is a mutation that affects any of these interactions then the dermis and epidermis will come apart

Answer 179

This patient has a mutation in laminin 332 and this results in dysfunctional laminin stopping integrin interaction - Mutation that affects the splicing of laminin 332 within intron 4

Answer 180

- Had denuded and blistering skin and very few areas of non-blistering skin - Treated with antibiotics to stop sepsis and attempted skin transplants to replace denuded skin but this was unsuccessful. Thought that there was no treatment and would have to be put on palliative care

Answer 181

Corrected EB by genetically modifying epidermal stem cells and transplanted into the patient. This was only done on a very small area of skin so was not a life-saving treatment

Answer 182

This study was taken as a proof of concept that this could work and allowed a one off compassionate treatment for this boy as had not been through clinical trials

Answer 183

Taking a skin biopsy and culturing keratinocytes to put back in the patient would not work in this case as it is a genetic disease so this would happen again. - There also is not enough skin

Answer 184

- Took the correct sequence of the mutated gene and put in viral DNA and delivered to the patient cells. They did not correct the gene itself but did deliver a correct version - This was done ex vivo. They took a small skin biopsy and cultured the keratinocytes on feeder layers and expanded. - They were then transduced with the viral vectors. This should result in corrected cells. - Prepared the skin substitutes. They cultured on fibrin cultured grafts - There were 3 transplantations targeting different areas

Answer 185

- To look where in the DNA did the viral genome integrate to ensure that the gene integrated into a safe place and not genes essential for cell function (these would die off before insertion so shouldn’t affect treatment) or into an oncogene (which could lead to cancer if inserted into the patient). - Most of the genes were inserted into introns rather than exons and appeared to be safe to proceed

Answer 186

- Wanted to establish that the regenerated epidermis came from the modified cells. - To ensure that the epidermis had adhered to the dermis - Look at the expression of laminin

Answer 187

- Probed for cadherin to act as a control and for the viral promotor (t-LAMB3). - In normal skin, cadherin was present but no viral promotor and then after 4 months can see the viral promotor is the epidermis of the arm showing it has regenerated from corrected cells.

Answer 188

o . They looked at histological sections of the skin. Follow ups showed no blisters and a clear epidermal-dermal junction

Answer 189

Using immunocytochemistry - After treatment saw clear expression of laminin between the epidermis and dermis which could not be seen before treatment.

Answer 190

The phenotype of EB could be seen when culturing the cells. The mutated cells could not adhere to the flask but the genetically correct keratinocytes were able to stick to the cells

Answer 191

- Would this work on other patients – clinical trials - Alternative gene editing strategies (CRIPSR) - Patient age, the boy was young so keratinocytes would have high proliferative ability. Would this work in older patients when regeneration has decreased?

Answer 192

Because the colour of the skin is given my melanocytes and the treatment was given with keratinocytes

Answer 193

Developmentally the epidermis and dermis come from different germ layers - Could use ES or IPS cells

Answer 194

Attempted to recreate the skin structures (integumentary organ) - They hypothesised that pluripotent stem cells can be sued to mimic the developmental patterning

Answer 195

Produce IPS cells. They then created embryoid bodies which start to differentiate randomly. They then aggregated inside collagen and transplanted under the skin of a SCID mouse

Answer 196

This is not a very controlled system | - the IPS cells differentiate randomly in embryo bodies

Answer 197

- The approach worked. Hair grown from SCID mice and originated from the IPS cells (due to the hair colour). - Wnt3b improved the hair creating - Took sections of the hair shafts and saw that there were also additional structures of the skin from the IPS cells - Transplanted this into nude mice which lead to outgrowth

Answer 198

Proved that these cells came from the original IPS cells as they were taken from a male mouse and the nude and SCID mice were female. Therefore, could just look for the presence of the Y chromosome

Answer 199

Disease modelling - To reduce the use of animals in experiments - This can lead to drug discovery - For many illnesses such as EB, Psoriasis, Skin cancer and Allergic contact dermatitis Chemical testing - Test environmental factors in the skin - Cosmetic industry

Answer 200

EB has been modelled in vitro (Itoh et al, 2011) - Can take cells and reprogrammed to IPS cells and differentiated back to keratinocytes to see what is wrong with them - Can probe them to see the mutation. In this case, there was no collagen - Creating skin equivalents, we can model which approaches will help alleviate the phenotype

Answer 201

10 million

Answer 202

- The cornea is the transparent convex tissue which together with the Sclera forms the outer shell of the eyeball. The sclera covers 5/6 and the cornea 1/6 - The cornea covers the iris

Answer 203

There are three main layers: corneal epithelium, stroma and corneal endothelium. Separated by Bowman’s membrane and Descemet’s membrane

Answer 204

Aid sight | Protect the eye

Answer 205

- Due to its transparency (99% of incidence light). The light comes through the cornea onto the lens and focus on the retina - It has a major refractive power (2/3s of eyes refractive power)

Answer 206

- Outer most layer of the cornea - Thickness of 50micrometres - Highly innervated

Answer 207

- Prevent fluid loss - Create a barrier to pathogens - Respond rapidly to wounding (needs to establish the permeable barrier)

Answer 208

- There are 2-3 layers of superficial cells which create tight junctions and generates the primary barrier in the epithelium. On the outer surface, there are microvilli which are covered in Glycocalyx which maintains the tear film of the eye - Under the superficial cells there are the wing cells and then basal cells. Basal cells communicate through desmosomes and are attached to Bowman’s membrane

Answer 209

The whole layer regenerates every 5-7 days

Answer 210

Bulk of the cornea with 90% of the corneal thickness | Relatively acellular and contains mainly collagens, proteoglycans and glycoproteins

Answer 211

Functions include strength and transparency

Answer 212

At the microscopic level, the collagen fibrils are arranged in autologous layers. This is important because the geometric organisation of the collagen means that it transmits most the light without scattering so is transparent

Answer 213

Even though mainly acellular, does contain keratocytes which are long, thin and flattened cells which synthesise and maintain ECM

Answer 214

A single layer of cells in the cornea which are metabolically active

Answer 215

Functions are to maintain stromal hydration

Answer 216

Maintains stromal hydration to maintain transparency. To do this it has evolved as a leaky pump - It is avascular so nutrients must be delivered from either the tear fluid or the aqueous humor - It allows solute and nutrients from the aqueous humor but has to maintain stromal hydration to stop the ECM from collapse - They are therefore very metabolic so that the solutes are constantly coming in and then water is being pumped out

Answer 217

The cornea

Answer 218

- Blink reflex - Wound healing - Tear production

Answer 219

Yes highly but avascular to maintain transparency

Answer 220

Through tear fluid and aqueous humor

Answer 221

- Transparent - Refractive - Prevent formation of blood vessels but allow for innervation - Low immunogenicity - Mechanical properties to allow nutrient delivery

Answer 222

Epithelium as it regenerates normally every 5-7 days. This tells us that there are stem cells available which could be utilised for a treatment

Answer 223

Only available to 7% of affected people world-wide so needs for alternative

Answer 224

A medical device to replace the cornea for people who have repeated failed grafts - Optical polymethylmethacrylate to replace the cornea and then a titanium ring to keep it in place

Answer 225

- It is susceptible to inflammation and glaucoma | - Required a life-long range of antibiotics

Answer 226

Limbal Epithelial Stem Cells

Answer 227

In the limbus of the eye - Limbus is sometimes quite pigmented so can see that it is undulated and it is here where the niches are. - It is located in the corneal rim at the border between the sclera and cornea

Answer 228

- The stem cells can divide asymmetrically and the daughter cells moves out and differentiates. - This gives rise to cells within the basal layer which are still proliferating. - Transit amplifying cells will give rise to wing cells which are post-mitotic. - Renewal of the whole tissue comes from the Limbal stem cells as eventually transit amplifying cells will die off

Answer 229

Creates a vascularised tissue as the cells migrate over the cornea – conjunctiva overgrowth - Causes vison loss

Answer 230

Most are due to injury bit there are some conditions such as Aniridia which cause it

Answer 231

Holoclar - Grafts from cultured cells from the non-diseased eye - Biopsy from non diseased eye. Can be culture in vitro to form epithelial cell sheets. Usually detach cells using trypsin but with sheets we want them to remain together so use temperature responsible dishes. At 37 degrees’ cells can attached and at 20 the dishes become hydrophobic so the cells can be lifted off the plate

Answer 232

There was a proof of concept study: Pellegrini et al, 1997 | 75 out of 104 patients showed improved vision

Answer 233

- If someone had a genetic condition then the cells would still be injured. - There will also be a delay as won’t start the making the treatment until know the injury. - Have to take a graft from the healthy eye which could result in damage

Answer 234

Mucosal cells - These cells don’t scar so won’t harm the healthy eye - However, there is a risk of neovascularisation of the cornea - Doesn’t do the same thing as Limbal stem cells so may need to investigate reprogramming

Answer 235

If damaged can create myofiberblasts which would usually apoptose after wound healing. In some pathologies, they continue to secrete ECM which will be an irregular shape and stop the transparency of the cornea

Answer 236

Biomaterials | Cell-based

Answer 237

Fagerholm et al, 2014 - Used recombinant human collagen as a cell free scaffold - Upon implantation, there was endogenous cell recruitment and began to remodel the matrix and lay down new ECM. A new cornea was formed o Because it is acellular, there is no need for immune suppression o There was innervation of the scaffold o The visual acuity wasn’t great so the scaffold didn’t have the correct mechanical properties

Answer 238

- Cell free corneal implants consisting of recombinant human collagen and a synthetic lipid were grafted by keratoplasty into corneas of unilaterally blind patients at high risk pf rejecting donor allografts - Followed for 24 months. Patients with acute disease were relieved of pain 2 weeks post operation. Corneas with immune or degenerative conditions showed no visual improvement - 3 out of 6 patients showed significant vision improvement - Further testing is needed but this study showed that cell free implants are potential safe efficacious options for treating high risk patients

Answer 239

Found that in the limbus there are also Limbal stromal cells which remodel stromal scarring in model animals and supress fibrotic scar formation

Answer 240

Endothelium does not regenerate - Could potentially transform them with oncogenes but this could be problematic in therapies - Could use pluripotent stem cells

Answer 241

- Osteochondral defects affect both the articular cartilage and the underlying subchondral bone and leads to mechanical instability of the joint and the risk of inducing osteoarthritic degenerative changes - To repair these large defects, there must be transplantation of osteochondral units. However, the problems with these transplants include limited material available, donor site morbidity and difficulty in matching the topology of the grafts with the injured sites - 3D tissue grafts of pre-defined size and shaped can be engineered by combining the patient’s own cells with 3D porous biomaterials and can overcome the limitations of transplanting osteochondral units

Answer 242

- Cell seeding is the dissemination of isolated cells within a scaffold. It is the first step in 3D culture and plays a crucial role in determining the progression of tissue formation - The initial distribution of cells within the scaffold after seeding has been related to the distribution of tissue subsequently formed within engineered constructs suggesting that uniform cell seeding could establish the basis for uniform tissue generation

Answer 243

Static loading of cells by micro pipetting is the most common cell seeding method. However, it is often associated with low seeding efficiencies and non-uniform distributions within scaffolds.

Answer 244

- Spinner flask bioreactors can be used which will uniformly distribute cells into scaffold with high porosity and high interconnectivity. If the scaffold is thick or less porous then spinner flask bioreactors also lead to non-uniform cell distribution. - Can also use perfusion bioreactors which enhances the convective transport of the cells throughout the entire scaffold volume. Using perfusion bioreactors for seeding is useful because culturing of the construct can also be performed under perfusion within a single unit

Answer 245

Rotating wall bioreactors can also enhance mass transfer of nutrients. Culturing in a rotating wall bioreactor resulted in cartilaginous constructs having better biochemical and biomechanical properties than static or spinner flask cultures

Answer 246

One major challenge is to translate research scale production models into clinically applicable designs that are reproducible, economically acceptable and maintains good manufacturing practice

Answer 247

- The integumentary organ system has important roles in protecting, waterproofing and thermal regulation and regenerative therapies could provide important treatments for burns and scars as well as models for drug screening - They therefore generated a 3D bioengineered integumentary organ system from IPS cells which included appendage organs such as hair follicles. They used a novel in vivo transplantation model called clustering-dependant embryoid body transplantation

Answer 248

- Epithelial germ layers were induced from iPS cells. Saw that both epithelial and mesenchymal cells were generated in the embryoid bodies through the presence of integumentary markers Sox2 and p63 and neural crest markers Snail and Twist. - Transplanted the EBs into nude mice. Formed a teratoma consisting of all three germ layers. When EBs were transplanted with collagen gel, which contained EBs cultured for 7 days under non-adhesive conditions, organ formation occurred in 4 out of 9 explants

Answer 249

- The bioengineered integumentary organ system was fully functional in nude mice and can be properly connected to surrounding host tissues without tumorigenesis - The bioengineered hair follicles show the proper hair eruption and cycle - Potential uses of this bioengineered organ system include in vitro assay system, animal model alternative and organ replacement therapy

Answer 250

Significant problem in society with 9000 cases in the UK each year

Answer 251

Occurs mainly in the young population - This is unlike other injuries. This is because it occurs mainly in car accidents - Therefore can't work and become a burden

Answer 252

- Axons are surrounded by myelinating Schwann cells and are enclosed by endoneurium (connective tissue) - They are bundled together into fascicles which are bundles together by Perineurium - Epineurium then groups the fascicles creating nerve cable

Answer 253

Elongation - Connective tissues allow 10-20% elongation before structural damage occurs - Severe lesions that disrupt the axon Laceration - 30% of nerve injuries Compression - External mechanical pressure in the conductive membrane

Answer 254

Neuropraxia Axontmesis Neurotmesis

Answer 255

- No/little structural damage and no loss of nerve continuity - Symptoms are transient and reversible - E.g. carpal tunnel syndrome

Answer 256

Complete interruption of the axon and its myelin sheath | Perineurium and Epineurium intact

Answer 257

Nerve and the surrounding stroma are completely disconnected No spontaneity recovery Weakness and atrophy

Answer 258

Wallerian degeneration

Answer 259

- Distal axons come away from the body and start to degenerate to produce debris - Debris is phagocytosed by macrophages - Schwann cells will start to clear up the debris. They proliferate and align themselves in tracts called the bands of bunger to create a scaffold for regenerating axons guiding them to the target site - They produce neurotrophic factors which attract axons

Answer 260

No wallerian degneration - Macrophages infiltrate much more slowly due to the BBB meaning they cannot prevent the inhibitory action of myelin. - Reactive astrocytes also produce glial scars that inhibit regeneration in the CNS

Answer 261

Surgical reconstruction Grafts Nerve conduits

Answer 262

- The severed nerve stumps will suture the individual fascicles back together. This is only possible if the never stumps are close to each other. - If the gap is too large then pulling them together will reduce the blood flow. Stretching by 8% reduced blood flow by 50%.

Answer 263

Autologous - A nerve segment from elsewhere in patient’s body - Advantage is that there is a low risk of immune rejection - Disadvantages include loss of function at the donor site, two surgeries required, limited size and type Allogenic - Same species donor - Advantages include no secondary surgery and no loss of function of donor site - Disadvantages include higher risk of rejection and limit in availability

Answer 264

Hollow structures that nerves are placed in to guide the regenerative nerves. This will increase the concentration of intraluminal proteins (growth factors) as they are trapped within the structure

Answer 265

- When the stumps are placed inside the conduits, the conduit fills with plasma which is full of ECM proteins and fibrin. Fibrin form fibrin cables which from a scaffold allowing Schwann cells to migrate and guide axons. This will allow regeneration. The result will be thinner than the original tissue but regeneration does occur - Whole regeneration process takes years but begins within hours after implantation

Answer 266

- Permeable (porous) – provide nutrients to Schwann cells - Long term degradable – Need regeneration to take place but not have to remove by another surgery - Biocompatible – low immunogenicity - The material needs to be suturable to attach the nerve stumps

Answer 267

Fabricated | Decellularised nerve conduits

Answer 268

``` Natural - Collagen - Silk Synthetic - Biodegradable materials - Electrically active materials Non-biodegradable materials - Semi-synthetic ```

Answer 269

More in control of their structure

Answer 270

- Retains the ECM architecture and removes antigens (stops immune response) - It is hollow so not in the way of regenerative axons and less immunogenic than allogenic approaches

Answer 271

The chance of successful regeneration with nerve guides is reduced, once an injury gap reaches a certain value (critical gap length) - At short gap lengths, the fibrin cable is robust enough to provide a platform for regeneration - At longer lengths, thinning restricts regeneration

Answer 272

This is the length at which regeneration occurs 50% of the time

Answer 273

- Creating conduits that are no longer hollow but instead contain ECM components, intraluminal supports, neurotrophic factors and cells which promote regeneration - Approaches use one or more of these (combinatorial approaches)

Answer 274

- Effective matrices are weak, viscoelastic hydrogels with a high-water content (high concentrations would prevent axonal penetration) - Matrigel (ECM proteins produced by mouse sarcoma cells) promotes nerve regeneration. Not very robust as there as a large batch to batch variably as come from mouse sarcoma cells. Therefore, not ideal for clinical use

Answer 275

- Tunnels within hollow tube to recreate the natural scaffolding of the nerve - This has been used successfully in clinic. Post-operational tests showed that nerve functionality increased

Answer 276

- Support axonal growth - Migration and proliferation of Schwann cells - Increase neuroprotection modulation of intrinsic signalling pathways - Need for controlled release of these factors - NGFs have little influence over motor neurons

Answer 277

``` Schwann cells - Critical for successful nerve regeneration - Bands of Bungner - Secrete neurotrophic factors - Proliferate Stem cells - Used for grafting ```

Answer 278

- NGF plays an important role in the survival of sensory neurons and in the extension of the axons. It is therefore being used in nerve conduits - Incorporating NGF results in an increase in functional recovery compared to control groups - It influences the proliferation and migration of Schwann cells and facilitates regeneration

Answer 279

Folkman et al, 1971 - Tumour growth dependants on the angiogenesis - Patient had a tumour of the eye which was well vascularised. Around it there were several small metastases which had a periphery of alive cells but the centre was necrotic. These metastases did not have vasculature meaning the centre cells could not be nourished - Small tumours can be fed by diffusion but when it grows it requires vasculature to keep the cells alive - This knowledge could offer a potential treatment for tumours – block the nourishment of the cells

Answer 280

- Avoid graft necrosis - Generate thicker tissues - Help graft innervation - Improve graft function

Answer 281

- Macro vessels: Arteries and veins - Micro vessels: arterioles and venules - Capillaries

Answer 282

Responsible for the transfer of the nutrients. This is achieved through the difference of pressure in the capillaries and the interstitial fluid

Answer 283

Single layer of endothelial cells surrounding the lumen which secrete anti-thrombotic factors to stop blood clotting

Answer 284

- Vasculogenesis - Angiogenesis - Arteriogenesis

Answer 285

- De novo blood vessel formation from progenitor cells - The embryo becomes large enough to need vasculature. Formed mesoderm precursors which differentiate into hemangioblasts which move to certain sites in the embryo to form tubes and then the primary capillary plexus

Answer 286

New blood vessel formation via extension of existing blood vessels

Answer 287

- When there is a lack of oxygen (hypoxia). VEGF signals to endothelial cells which secrete enzymes, matrix metalloproteinases, which act on the basement membrane and create punctures. - The endothelial cells proliferate and create vasculature sprouts which elongate towards the hypoxic signal and form a new lumen - Mature by the recruitment of smooth muscle cells and pericytes - This will result in the place being less hypoxic meaning that the hypoxia inducible factor decreases then angiogenesis is turned off (negative feedback loop)

Answer 288

- Occurs during wound healing and ovarian cycle

Answer 289

Vascular endothelial growth factor (VEGF) – Involved in endothelial cells proliferation, migration and differentiation angiogenesis factor

Answer 290

Hypoxia inducible factor (HIF) – stimulation of transcription of VEGF, PDGFR, TFG-alpha, EGF and erythropoietin in response to hypoxia

Answer 291

Platelet-derived growth factor (PDGF) – Recruitment and proliferation of smooth muscle and pericytes - this matures developing vessels

Answer 292

Involved in angiogenesis | - Regulation of endothelial cell survival, sprouting, pericyte recruitment

Answer 293

Secreted in response to hypoxia and involved in angiogenesis - Basal lamina degradation, ECM remodelling

Answer 294

- VEGFR2 involved in angiogenesis, VEGFR1 involved in Vasculogenesis and VEGFR3 in lymphangiogenesis - VEGFR2 activation results in endothelial cell proliferation, migration and survival

Answer 295

Maturation of blood vessels via increasing the lumen

Answer 296

Occlusion of an artery which prevents blood flow from an artery which will exert certain amounts of sheer stress onto its endothelial cells. This releases TGFbeta which causes degradation of ECM and promotes remodelling through the proliferation of endothelial and smooth muscle cells. This increases the lumen sizes and forms an artery

Answer 297

- Vasculogenesis doesn’t require any pre-vasculature - The key signals are different: Vasculogenesis – growth factors in the embryo, Angiogenesis – hypoxia, Arteriogenesis – sheer stress of the vessel

Answer 298

Scaffold design - Different ways to make porous scaffolds which doesn’t compromise mechanical properties Scaffold functionalisation - Can use different molecules to promote vascularisation e.g. VEGF, PDGF, bFGF - It is crucial to have controlled release of growth factors – the timing is important

Answer 299

They created a polymeric system for dual growth factor delivery - VEGF is the key initiator of angiogenesis but the vessels would not be mature without PDGF. They hypothesised that both factors are required and should be delivered in a certain way - Delivered by two stage release of growth factors

Answer 300

Results showed the formation of a mature vascular network when there was the dual release of VEGF and PDGF. This was not seen in just one of the growth factors - The distinct kinetics of delivery allowed mimicking of the natural process - Highlighted the importance of multiple growth factor delivery for engineering artificial tissues

Answer 301

- Time consuming - Slow growth rate - May not be sufficient to prevent necrosis in 3D constructs

Answer 302

Prevascularisation strategies - In vitro prevascularisation - In vivo prevascularisation

Answer 303

- The tissue engineering construct is cultured in vitro to build prevascularised structure - The network can create a connection with the existing vessel in tissue (anastomose). This is faster than the new blood vessel formation

Answer 304

There is a problem with how cells would be sources in the clinic and issues with using mature endothelial cells

Answer 305

- Angiogenic ingrowth - Flap technique - Arteriovenous (AV) – loop technique

Answer 306

- Place scaffold in enriched vascularised network and micro vessels will ingrowth from the host. After this occurs, can remove and transfer to the defect site - This method involves three surgeries so is not likely to be used

Answer 307

- A scaffold is implanted into a muscle flap which is then vascularised from the host - The whole flap is then transferred to the site in need of repair. This allows larger vessels to be transferred - Problem is that you are sacrificing the while muscle flap

Answer 308

- Uses a vein or synthetic graft to form a shunt loop between an artery and vein which leads to spontaneous sprouting of vessels - The loop is placed in a protected chamber and can then be transferred to repair site - This doesn’t have donor site morbidity

Answer 309

Injury of the body triggers reaction that begins with local interactions and then becomes systemic due to the immune cells spreading around the entire body. The systemic interactions determine the success of the transplant and can lead to rejection

Answer 310

The knowledge of how the body may react comes from medical devices for biomaterials and the inflammatory response. Medical devices usually contain living cells. The way the body responds to living cells is based on the immune response

Answer 311

Effect of biomaterial on the body | Effects of body on biomaterial

Answer 312

- Changes wound healing - Infection - Toxicity - Tumourigenicity - Particle embolization - Hypersensitivity reactions

Answer 313

- Enzyme degradation - Calcification - Abrasion - Corrosion

Answer 314

The foreign body response

Answer 315

- Transplantation results in tissue injury - Blood vessels damage and blood will flow out. ECM proteins such as fibrin lay down a scaffold on the material which enable cells such as monocytes to adhere to the material - Macrophages are phagocytic and clear up the debris - Biomaterial is so large that the macrophages can’t digest then they undergo frustrated phagocytosis. It attracts other macrophages to the site. - Macrophages fuse with other macrophages (granuloma) and forms a foreign body cell and release agents such a super oxides and free radicles which damage the surface of the material - Fibrous encapsulation then occurs

Answer 316

- Fibroblasts lay down collagen and ECM components which leads to isolation from biomaterial to the local tissue environment. - The granuloma ages and increases vascularisation. Endothelial cells form tubes which form into capillaries. - This is an evolutionary response to protect the body but now causes problems with medical devices

Answer 317

Larger than 10 micrometres

Answer 318

- The way the body responds is hard to predict but some biomaterials induce larger responses - Minimal foreign body reaction leads to optimal healing so need to identify the appropriate biomaterial for each application to reduce this

Answer 319

- Implant infections are common and are sometimes located to the implant site but can be more serious and lead to sepsis - There are however some materials that rarely cause infection

Answer 320

- Communities of bacteria grown on abiotic materials. This is an issue for infection - Thought to be an ancient evolutionary response that allow bacteria to behave like multicellular organism by embedding themselves in extracellular polymeric substance (slime) and allows them to increase survival - They adhere to the material and secrete slime allowing them to adhere to each other and irreversibly adhere to the material. - They proliferate and form multi-layer structures. They can become planktonic again and an colonies other sites so that the film is propagated.

Answer 321

Not usually

Answer 322

- Staphylococci (lives on the skin) are particularly prone to creating biofilms. This is an issue when the skin is damaged and can then access the biomaterial - This problem is enhanced due to the development of the anti-biotic resistant strains

Answer 323

Orthopaedic prosthetic joint infections also have this problem and it is difficult to diagnose. Typically treated with systemic antibiotics or remove the biomaterial

Answer 324

There is a kind of race between the bacteria and host cells to reach the surface and whichever reaches first populates the surface

Answer 325

Cells do not interact directly interact with materials, instead a layer of protein adheres to the surface and is absorbed by the material. Could therefore use non-fouling surfaces which are resistant to protein absorption to stop all cells adhering. Then micro stamp it with RGD domain to allow cells to attach

Answer 326

Differentially instructive extracellular protein micro-nets (Farugui et al, 2014) - Used peptide assembled into structures (SaNet) with differential instructive matrix which enhance mammalian cell adhesion but resist bacteria adhesion - This is achieved due to its similarities to native extracellular matrices. This similar protein expression allows it to maintain the biofunctional characteristics: anti-microbial carpets and filopodia recognised adhesion points. This therefore blocks bacteria colonisation and enhances mammalian cell binding - This was shown using SaNet in comparison to collagen and fibronectin. Bacteria cells did not adhere at all

Answer 327

- Wear and tear of the biomaterial can lead to the release of by-products. In metal-on-metal hip replacements caused the release of cobalt and chromium into the blood. - This doesn’t cause an immune reaction but bind to other proteins in the blood which causes them to be perceived as foreign by immune response. This results in failure of the replacement

Answer 328

Stents - An artery clot will be cured using a stent insertion. The stent is covered in anti-coagulant cells. This is rare with severe clinical consequences

Answer 329

Whether a tumour is caused by an implant is difficult to prove. Animal models have not been useful and the mechanism are not clearly understood

Answer 330

- Autologous cells to stop the immune response and remove the need for immunosuppressants. - This is sometimes not possible due to genetic diseases in the cells or lack of supply. Sometimes necessary to use allogenic strategies

Answer 331

Treatment for diabetes | - Showed islet cells can be transplanted obtained from cadavers. This requires immunosuppression otherwise rejection

Answer 332

- Do not need a structure just the cells so in theory it doesn’t matter where the cells are placed as long as it leads to the release of insulin. - Therefore, investigated the immunoisolation approach

Answer 333

- Islet cells are placed in a device which has a semi-permeable membrane - Can receive nutrients through the membrane and secrete insulin but T cells and antibodies cannot enter and kill the cells - This appears like a viable approach that is in clinical trials

Answer 334

Viacyte is using hES cells to derive the beta cells and placing them in a semi-permeable membrane

Answer 335

- For this to be viable, fibrous encapsulation must be prevented as insulin would not reach the blood stream and the cells in the device could not be nourished. There are some allogenic materials that prevent this form occurring. - Could use genetically modified cells

Answer 336

Road traffic accidents, work accidents and domestic accidents

Answer 337

A single neuron can be very long (metre) – makes it difficult to treat

Answer 338

Suturing the proximal and distal ends | Autografting

Answer 339

Micro-sterolithography - Used UV light to produce a photocatalytic reaction and give a sharp point for which to direct a beam. Can move this through a material - Take a liquid version of a material that is biocompatible (PEG, PLA, PCL) which are inert. Use a photo catalyst. - Move the stage with the liquid through the UV beam to create a solid – this gives precise controls over the physical dimensions – can get down to 50micrometres wall width.

Answer 340

Added Guidance scaffolds | - Used aligned polycaprolactone fibres made by electrospinning to create fibres of different diameters

Answer 341

Took some neuronal cells and grew on the fibres and looked for neurites that grew from the cells. Found that there was a correlation between the length of the neurite and the diameter of the fibre

Answer 342

Dorsal root ganglion cultures - Coated in laminin - Label for nuclei, axons and Schwann cells. The Schwann cells almost always physically contact the axons. - Took the fibres and looked if axons and Schwann cells grow along them. They did and Schwann cells were in contact with the axons

Answer 343

- Need to make sure the conduit is not to expensive so must limit the complexity but still make it clinically viable - Problem of cell sourcing: can’t be from a donor so then it must be autologous. This is the same problem as the start (grafting) - Took long to culture and often became containated

Answer 344

- They express the enzyme D-amino acid oxidase which secretes an amino acid from the valine form. Contaminating fibroblasts do not have this enzyme so if culture in a medium with D-Valine then they will die. - Removed many of the components of the medium making it fit the clinical need.

Answer 345

Evolved over time as a result of incidents - Elixir sulphanilamide incident (1937): Treat streptococcal infections for children. They dissolved safinamide in anti-freeze and raspberry flavour and caused the death of over 100 children - The Nuremberg Code (1946): esting should be done only with informed consent - The thalidomide incident (1956): Caused severe developmental defects of the pregnant women’s children so have to look at long term safety now Result of this is Good Clinical Practice which ensures that the data from clinical trials will be reliable and robust

Answer 346

``` Lab discovery Pre-clinical studies - In vitro: Cell lines - In vivo: Animal models Clinical trials - Phase 1: Done on healthy volunteers to ensure safety - Phase 2: Small group of people with the disease - Phase 3: Larger group ```

Answer 347

EMA, FDA ‘The international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use’ ensures that there are not major differences between different countries so the whole population can benefit

Answer 348

ATMP is a medicinal product which is either - A gene therapy medicinal product - A somatic cell therapy medicinal product - A tissue engineered product

Answer 349

Use of cells - Survival: The cells can survive for the life of the patient and could acquire genetic and epigenetic changes - Evolving functionality: Cells may evolve to continue proliferation and form a tumour - Migration: Cells can bio distribute and end up in the wrong environment. This would interact differently and could have adverse effects No standard set of preclinical tests - Each application is very different and has never been done before - Need case by case considerations

Answer 350

The establishment of manufacturing process and control - Product consistency: Needs to be reproducible so trained in the same way with the same tools and products used Evidence for safety and efficacy in preclinical models - Involves testing in animal models to model human conditions and to consider safety and functionality. - Aware of limitations of the model used Clinical trials on human participants

Answer 351

Exploratory trial - Application requires surgery so using healthy volunteers is not appropriate - Primary concern is safety but is carried out on actual patients Dosage - Not as straight forward as in small molecule drugs Defining the comparator - Usually compare to the gold standard drug – is it better - Harder in regenerative medicine as it requires surgery which can be done differently depending on the skill of the surgeon making comparison difficult Randomisation - Important that it is independent from the investigator so that the results are objective - Cannot do a blind study where some get treatment and some get control in this aspect

Answer 352

- Depends on the health and age of the patient and other treatments available. Has to occur on a case to case bases - Hospital exemption – experimental therapies can be used with consent of the hospital and patients

Answer 353

- The gap between discoveries of therapeutic leads in the labs that never meet the patient - Early phase therapeutic development is carried out in universities but they are not equipped to carry out the preclinical or clinical trials. Usually investors or pharmaceutical companies do this e.g. Big pharma. - However, some companies do not invest in tissue engineering products as there is no guarantee that it will be successful and will therefore not make any money

Answer 354

- Financial rewards are not guaranteed - The government is the highest payer so the industry is heavily influenced by politics - Case by case regulatory pathway - Manufacturing and scale up is complex and expensive - Distribution and storage are complicated by the biological nature of the products - No unique business model

Answer 355

- Need to use model systems to model diseases and processes but it is difficult to find models that are physiologically relevant and can be used - As the physiologically relevance increases, the experimental tractability decreases

Answer 356

- Companies screen 5-10000 compounds. Usually on one of these found to be approved. This translates a huge time to lab to the market and a huge cost in development - Drugs end up being withdrawal from market e.g. VIOXX lead to 88 thousand people having heart attacks and 38K died - The idea would be that the models used would be more predictive to the outcomes in humans

Answer 357

In vitro models - Primary cells: Not representative to the entire body and difficult to source them and every source will have batch-batch variability - Immortalised cell lines: May change the phenotype of the cells and change their behaviour – may test drugs on something that doesn’t reflect in vivo In vivo models Animal models: Even primates do not necessarily give the correct outcome E.g. Monoclonal antibody – 6 patients died in phase 1. It was tested in primates but in humans it caused major organ failure.

Answer 358

There isn’t a good control on spatial relationship between cells – organoids self-organise so we are not on control. Organs on chips were brought in to overcome this

Answer 359

- Not aiming to replace an organ but instead to be used in vitro. They mimic the minimal units to model the organ function

Answer 360

- Polymers (PDMS) mixed with crosslinking agent and pour onto the mould and heat up to solidify - Can then peal this off and place on a glass slide - PDMS is see through meaning can minor cells grown under the microscope and is biocompatible - Puncture several holes to allow the liquid in and out –perfusion system

Answer 361

- How fluid behaves in very small volumes | - They follow laminar flow rather than turbulent flow. This gives more control over the fluid and create gradients

Answer 362

- Geometrical confinement and patterning (Control over where the cells are places) - Presence of flow - Environmental control (pH and temperature) - Sensors and physiological readouts

Answer 363

- Lungs are used for gas exchange and must have a large surface area to volume ratio. This achieved by branching structure and alveolar epithelium. - The alveolus is lined by alveolar epithelium. - Due to breathing, there is constant cyclical stretching of the alveolus. This is important because cells need to encounter these in vivo functions to develop

Answer 364

- Epithelium cells of alveoli and placed on ones side of the membrane and on the other site endothelium cells to represent capillaries. This produces the alveoli capillary interphase and flow air in the top chamber and media in the bottom (represent blood). This creates the air blood interphase - Modelled cyclical stretching by having vacuum chambers are on either side of the two layers

Answer 365

When air flows through the top chamber – this increased surfactant from epithelial cells – mimicking in vivo

Answer 366

- Can be a life threatening disease and acute or chronic - Cytokines TNF-alpha released form epithelium and activates endothelium which start to express cell adhesion antigens (ICAM-1). When this happens, the leukocytes starts to attach to the endothelial cells and allow neutrophils to enter the alveoli to digest the invaded bacteria

Answer 367

- Put TNF-alpha in upper chamber and measure the effects of the endothelium to them. Can measure using immunohistochemistry - Add leukocytes in the lower chamber and can see if it passes the membrane - They then used E.coli labelled with GFP instead of TNF-alpha to see bacterial infection and found that neutrophils invade them

Answer 368

- Excess build up of fluid in the lungs and causes respiratory failure - Can occur as a side effect of cancer therapies e.g. systemic administration of IL-2

Answer 369

Added IL-2 to the blood compartment - Noticed that cells become more permeable and were letting liquid enter the air chamber - This gets worse and worse overtime until the whole chop is filled with liquid as breathing (mimicked) increased - Also caused blood clots

Answer 370

- Drug Ang-1 previously shown to stabilise junctions between endothelial cells and Ang-2 is an antagonist and destabilises the endothelial barrier so was used of a control - Found that Ang-1 reduced permeability

Answer 371

Body-on-a-chip - Linking all the relative organs for drug development Personalised medicine - Can take other things than just cells – breathing patterns, exercise patterns, plasma biomarkers. Can add environmental markers relevant for the patient and can use to accurately predict the response to drugs

Answer 372

By the year 2050 expect to have human population of 10 billion so need a massive increase to cover this Meat production - Responsible for more greenhouse gas emissions then the entire transportation system and is the biggest use of water - Antibiotic use is also a massive issue – develop antibiotic resistance - Current meat production is not sustainable

Answer 373

Follows the basic principles of creating any tissue: the building blocks of tissue engineering put together in a bioreactor to create skeletal muscle

Answer 374

Satellite cells - Take a small biopsy and isolate satellite cells and put them in culture (not immortal) - Proliferation and differentiation phase which can lead to myoblast and myotube formation IPSCs - Unlimited proliferation ability - Need to optimise differentiation - Can include multiple cell types

Answer 375

- Edible - Non-allergenic - Inert - Needs to support full differentiation and maturation

Answer 376

``` Bioreactors - Grow large numbers of cells - Put on porous collagen microspheres - For muscles to form in vitro they need mechanical and electrical stimulation – could design bioreactors 3D printing - Allow a much finer structure ```

Answer 377

Lead to commercial companies - They patent their discovery= can’t build upon research Scale - Global meat production is 293 million tons/year. - Need 1 bioreactor per 10 humans – own carbon footprint Efficiency - Optimisation of scale up - Optimisation of differentiation Customer acceptancy of food tissue engineering - Need taste and texture - Ethics and attitude

Answer 378

``` Healthier meat - Added vitamins - Reducing fat - Increasing levels of conjugated linoleic acid Control over texture and flavour Creation of new products ```

Answer 379

- Meat - Dairy - Leather (skin culture)

BMS354 Principles of Regenerative Medicine and Tissue Engineering Flashcards

(407 cards)