Revision cards Flashcards

Question

What are proteoglycans?

Answer 1

Composed of glycosaminoglycan chains linked to a specific protein core. Very hydrophilic, form highly hydrated compressible gels.

Answer 2

Very diverse e.g. fibronectin contains a RGD sequence (Arg-Gly-Asp). The sequence is recognised by cells as a cell binding domain. If cells will not adhere to biomaterial you can add this sequence onto the biomaterial (structurally integrate).

Answer 3

Cells mechanically interact with ECM before making contact via receptors/signalling parts. Cell flattens out to reach appropriate shape. Can move around and explore their environment.

Answer 4

19a and 8B subunits. Form heterodimers with extracellular domain, transmembrane domain and short intracellular domain. ECD and ICD can be used in inside-out and outside-in signalling. a and B can make up many different combinations, different affinit depending on environment. Both specificity and redundancy allowing a robust system.

Answer 5

-Extrinsic ligand 'outside-in' (collagen, laminin, fibronectin) causes a conformational change in integrin, changing their affinities for specific ECM components, activates signalling pathways. -Intrinsic ligand 'inside-out' (talin, kindins) causes changes which intiates the assembly of the actin cytoskeleton. Mostly mediated by focal adhesion kinase which recruits a number of other proteins to the sites of focal adhesion triggering a cellular response.

Answer 6

Process by which external mechanical stimuli are transmitted into the nucleus. Cells can sense the environment and alter it accordingly.

Answer 7

Macular degeneration clinical trial with 2 patients. Treatment looks promising from Phase 1 trials. Stem cells used were SHEF-1hESC. Using fully differentiated embryonic stem cells placed on a coated, synthetic basement membrane.

Answer 8

Nonviable materials used in a medical device intended to interact with biological systems. Can be used to develop scaffolds for tissue engineering. Developed from the field of medical devices.

Answer 9

Using hair, silk etc for suturing wounds. Issues with infection and immune response. Sir Harold Ridley examined eyes of pilots and saw that they had plastic splinters from the cockpit without an immune response. Developed intraocular lens for the treatment of cataracts. Important development: a material can be integrated into the body without an immune response, making it biocompatible.

Answer 10

Ability of a material to perform with an appropriate host response in a specific application. e.g. haemolyalysis membrane for patients with poor kidney function, the membrane is only in contact with blood for a couple of hours. Urinary catheter is implanted for days or weeks. Hip replacement is needed for life.

Answer 11

Resistance of blood clotting Resistance to bacterial colonization Normal healing

Answer 12

First generation: Bioinertness Determined to be biocompatible if do not induce toxic reaction or carcinogenesis. Second generation: Bioactivity Bioglass is a ceramic implant that you can place in rats. Bonded with bone and could not be separated. Cannot respond to inputs. Third generation: Functional tissue Materials that can induce a cellular response at a molecular level.

Answer 13

'Poly' (many) 'meros' (parts) Large molecules made up of chains or rings of linked monomeric units. Mw 200,000 (compared to h20 18Da). Can be linear, branched or networked. Common polymer biomaterials include Polyethylene, Polytetrafluroethylene, Polypropylene, Polyvinylchloride, Polydimethylsiloxane, Poly (methyl methacrylate). Typically have carbon atoms plus side chains.

Answer 14

``` Can be arranged in different shapes -Homopolymer -Random copolymer -Alternating copolymers -Block copolymer -Graft copolymer It is possible to mix two polymers e.g. blend of homopolymer A and B. We can change their functional properties by changing how they are arranged. ```

Answer 15

Crosslinked polymer networks that are insoluble but swell in aqueous medium. Offer an environment that resembles the highly hydrated state of natural tissues. Can model soft tissue environments.

Answer 16

Already exist in animal or plant tissue. 1) Protein-based natural polymers Collagen - well integrated, highly common, high immunogeneity Gelatin - less immunoggenic, denatured form of collagen Fibrin - major component of blood clots Elastin and Soybean 2) Polysaccharides Chitosan - present in fungi and crustaceans Aliginates - produced by seaweed and some bacteria Hyaluronan - present in umblical cord, skin, cartiliage Chondroitin sulfate - present in cartilage Obtain the plant/animal tissue by extraction. Purify them from the tissue. Concentrate them to get good enough sample.

Answer 17

Synthesise from scrath. Polylactic acid Polyglycolic acid Poly (lactic-co-glycolic) acid

Answer 18

Hybrid molecules made by incorporation of biologically active macromolecules onto the balance of synthetic polymers. e.g. semi-synthetic PEG (polyethylene glycol)-fibrinogen. PEG fives density, stiffness and biodegradability and fibrinogen gives biofunctional domains.

Answer 19

``` Advantages: -Already exist, easily obtainable -Existing bioactivity -Can make the most of naturally-occuring properties Disadvantages: -May be difficult to harvest -Potential ethics -Batch-to-batch variability -Shelf-life -Immune response ```

Answer 20

``` Advantages: -Can be tailored to specific properties -Can control process and structure Disadvantages: -Diffferent structures that may not interact with tissues -Difficult to predict bioactivity ```

Answer 21

- Physical/mechanical - strength, elasticity, architecture - Chemical - degradability, resorbtion, water content - Biological - interactions with cells, release of biologically acitve signals

Answer 22

Degradable materials: Hydrolysis of a polymer require H20 to break down covalent bonds to give degradation products. For it to be biocompatible degradation products should be non-toxic. Resorbable materials: Total elimination of the initial foreign material and its byproducts. Broken down by the body, metabolised leaving no trace.

Answer 23

Strength, toughness, fatigue resistance, stability.

Answer 24

- Chemically or physically altering the atoms/molecules in the existing surface - Overcoating the existing surface with a material having a different composition - Creating surface textures or patterns

Answer 25

Engler et al (2006) Matrix elasticity directs stem cell lineage specification. Mesenchymal stem cells were placed on substrates of different elasticity (soft tissue, muscle, collagenous bone resemblance). The cells were directed to different lineages (neural, muscle, bone) just based on stiffness, can sense mechanical environment,

Answer 26

- Thin surface modifications (3-10nm) to avoid changing the marker properties of the material - Delamination resistance - Surface analysis

Answer 27

Cells do not interact directly with materials. - A later of protein (from growth or plasma) adheres to the surface of biomaterials - Cells recognise these proteins and adsorb to them depending on the surface material properties.

Answer 28

Surfaces that resist adsorption of proteins and/or adhesion of cells e.g. PEG and Zwitterionic. In medical devices this is good because may inhibit bacterial colonization.

Answer 29

Attachment of biomolecules to polymer surfaces | Immobilisation of integrin-binding peptides or entire proteins (RGD domains added).

Answer 30

Cell adhesion to materials occurs through receptors in the cell membrane Arginine-glycine-aspartic acid (RGD) domain in fibronectin and vitronectin Cellular responses can vary with the surface density of RGD peptides immobilised

Answer 31

Can be created using microstamping or microcontact printing (µCP) - Prepolymer is poured onto a structured master - Prepolymer is cured and the stamp is peeled off the mamster - Stamp is cut into smaller pieces using an enzyme - Stamp is inked by soaking in ink solution - Ink printed on suitable surface - A pattern of substrate is obtained, cells adhere to ECM area stamp

Answer 32

Chen et al 1997 It was already known that angiogenesis relies on interplay of chemical and mechanical signals. Endothelial cell growth is also critical, early experiments suggest that increases in spread area are accompanied by an increase in cell proliferation. Hypothesis: cell shape per se controls cell fate of endothelial cells. Approach: Micropatterning of fibronectin islands on a non-fouling surface of lots of different sizes Result: The extent of cell spreading determined whether a cell underwent proliferation or apoptosis. Same GF, same genes, same ECM but different geometry, different fate outcome. Cells can filter the same set of chemical inputs to produce different functional outputs. Warmflash et al (2014) used this to recapitulate gastrulation, Teixeira et al (2006) used microstamping or microcontact printing. Thin lines, cells were perpendicular, thick lines cells were vertical.

Answer 33

Scaffolds provide the support for cells to proliferate and maintain their functions. They deliver and retain cells and bioactive molecules. Therefore when you are tissue engineering you need to develop an artificial extracellular matrix which would usually perform these functions in vivo.

Answer 34

- Material science: is it biodegradable? - Scaffold architecture: does it have adequate mechanical properties? - Scaffold-cell interaction: does it allow cell attachment/function? - Up-scaling production: can it be mass produced to meet clinical need? - 3D - Nutrient supply: is there capability for it to be vascularised to allow for O2 perfusion and tissue integration?

Answer 35

3D scaffold can be added to two types of cells to support the cells. If the scaffold is degradable, it will gradually degrade and the cells put down their own ECM, allowing for stability at all times. The scaffold itself and the degradation process should be non-toxic. Degradation kinetics should be matched to the tissue you are trying to regenerate.

Answer 36

The surface properties of the scaffold must allow for cell attachment. If they don't (e.g synthetic materials) they can be made functional by adding RDG domains which create cell binding domains. The scaffold mustn't hinder the ability of cells to attach to the matrix and start signalling (functionality).

Answer 37

Naturally derived materials Synthetic materials Semi-synthetic materials Acellular tissue matrices

Answer 38

Can be considered as natural. However, all the cellular material is removed from the extracted tissue, either by physical scrapping, although this is often not enough. Chemical methods (acids/bases) or biological methods (enzymes) can be used instead. Removing acellular components also removes antibodies to prevents an immunogenic reaction to seeded cells.

Answer 39

Decellularisation maintains intricate structures that would otherwise be difficult to manufacture. Exploiting the intact 3D structure of the ECM Some are commercially available (alloderm) Up-scaling may be a problem. Acessibility is a potential issue. However it is important to fully decellularize a tissue to avoid initiating the host response, bacteria contamination or crosslinking which will affect the tissue's normal morphology.

Answer 40

Macroscopically the final shape of the tissue engineered is defined by the scaffold. Microscopically human skin and bone are usually very porous for infiltration of O2 and nutrients. However, there may be limited interconnectivity and some pores may not be accessible. Further more too many pores will decrease the mechanical stability of the tissue, however this depends on the tissue being engineered.

Answer 41

Porogen leaching is a method used to create an artificial porous scaffold. It involves mixing a polymer solution with salt particles and placing it into a mold. Solvent evaporation causes polymer to solidify which when placed in water causes the salt to leach out. This is then freeze-dried to create a porous scaffold where the salt particles used to be. You can control the size of the salt particles to control the pore size.

Answer 42

Phase separation is a method used to create an artificial porous scaffold based on properties of a mixed system. It involves mixing the polymer solution with a solvent, placing into a mold. Using temperature, you can separate a polymer-rich phase and a solvent-rich phase. Evaporate the solvent to cause the polymer to solidify. Pores are formed where the solvent used to be.

Answer 43

Electrospinning is a method used to create an artificial porous scaffold. Polymer solution is placed into a syringe. It is linked to a high voltage power supply. An electromagnetic field is created between the tip of the syringe and the grounded collection plate. As the polymer is released from the syringe the electromagnetic field causes the polymer fibres to splay. Very fine micronanofibres are collected onto the grounded plate.

Answer 44

``` Additive manufacturing (aka rapid prototyping or free form fabrication) is a method used to create an artificial porous scaffold. It is a collection of methods. Process of joining materials to make objects from 3D model data usually layer upon layer. 3d scaffold design is converted to particular file that makes virtual slices in 2D. This is then transferred to a rapid prototyping machine. Roller spreads polymer across the table, inkjet head secretes a polymer binder to specific areas according to the shape determined by the computer. ```

Answer 45

High control of architecture Production of scaffolds with precise morphologies Combines medical imagine like MRI to fabricate anatomically shaped implants. Main limitation is that there is a limited number of biomaterials that can be used.

Answer 46

Usually when you combine the artificial scaffold and seeded cells, it may be the case that the cells are not integrated or that they clog the pores. Cell encapsulation provides an alternative way to provide a better integration in 3D printing. In this method, scaffolds and cells are already combined and gaps are free to act as pores.

Answer 47

Kang et al (2016) sought to address the challenge of producing 3D, vascularised cellular constructs of clinically relevant size, shape and structural integrity. Designed the ITOP Integrated Tisuue-Organ Printer to overcome the limitation of currently used bioprinting technologies (structural integrity, mechanical stability, printability). Contains different bioinks: cells combined with hydrogels which are non-toxic and bio-compatible. New idea to print cell-laden hydrogels with sacrificial scaffold to provide initial mechanical integrity.

Answer 48

Small molecules (corticosteroids and hormones) Proteins and peptides Oligonucleotides

Answer 49

Initially extracted from bone matrix. Members of the TGFb family. Made by osteoblasts (bone-forming cells) Osteoinductive - able to recruit osteoblasts and initiate differentiation of mesenchymal stem cells.

Answer 50

The efficient clinical use of BMPs depends on the delivery strategy. BMPs act locally so they easily dissipate. The use of biomaterials that can retain and sequester BMPs will enhance efficacy in bone regeneration.

Answer 51

``` Lutolf et al (2003) Polyethylene glycol (PEG) is non-fouling so it is functionalised using RDG domain. Crosslinked to create a network mesh using MMP substrate sites. Cells in the vicinity recognise sites and bind to the scaffold. Cells secrete MMPs that degrade MMP cleavable bonds serving as crosslinks for the matrix. rhBMP2 is physically entrapped into the pores of the PEG gel by mixing it with PEG precursor before gelation. BMP is liberated from the matrix and can diffuse from the site signalling osteoblast precursor cells to secrete bone marrow and regenerate the bone. ```

Answer 52

3D muscle construct 15mm x 5mm x 1mm dimension containing mouse myoblasts. Designed a fibre bundle structure for muscle organisation. PCL pillars were used to maintain the structure and to induce cell alignment. The printed construct was cross-linked with thrombin solution to induce gelation of fibrinogen and uncross-linked sacrificial material was removed by dissolving in cold medium. TE construct matured into a functional muscle in vivo in rats. High cell viability after the printing process

Answer 53

``` Medical imaging 3D CAD model Visualised motion program 3D printing process 3D bioprinted tissue product ```

Answer 54

First they tested the sensitivity of the gels to proteolysis by cell-secreted MMPs using an in vitro model system for cell invasion. Fibroblasts recognised the cleavage site and could invade. Control: no MMP cleavage site or MMP inhibitor Monitored by BMP release Functional proof cam from testing it on critical injuries (large enough so that the bone cannot regenerate). MMP-sensitive, no BMP MMP-insensitive, BMP present No regeneration however MMP-sensitive with BMP showed good regeneration of the bone.

Answer 55

Classical bioreactors are fermentors used in industry to grow high numbers of eukaryotic and prokaryotic cells for the production of antibiotics, recombinant genes and metabolic products under controlled conditions. In tissue engineering bioreactors enable us to direct, maintain and intiate 3D structures which would be very difficult otherwise.

Answer 56

- The ability to grow 3D tissue structures of relevant clinical sizes, same as in vivo - The growth and 3D assembly of multiple cell types that are requires for more complex functional tissue.

Answer 57

Static culture is usually performed in a culture flask, no special equipment is required, it is relatively cheap and easy. Cells are grown statically without any mixture, causing concentration gradients to occur. Fed with medium every day/few days - no control over pH and environment. Diffusion of O2 and nutrients is inefficient so cells on the inside will become necrotic.

Answer 58

A dynamic culture is the mixing of the cells in culture. Gives rise to homogenous concentrations of nutrients, toxins and other components. Can control environment, pH, temperature etc. Cells are placed in the bioreactors, medium is pumped in and out from medium tank by peristatic pump providing perfusion.

Answer 59

- To establish spatially uniform cell distributions on 3D scaffolds - To overcome mass transport limitations in 3D constructs - To expose the developing tissue to physical stimuli (physical conditioning)

Answer 60

Usually you just place cells of choice on scaffold using a pipette, relying on gravity to pull the cells through the porous structure and distribute through 3D. This is an inefficient process so cells will ususally just stay on the top and many will be lost. This is a problem especially when many biopsies are painful. Therefore you need a high seeding efficiency, short inoculation period and uniform distribution of cells within the scaffold since this is what will determine the biochemical activity and mechanical strength of final TE construct.

Answer 61

Bone marrow stromal cells were seeded onto scaffolds and after 18 hours MTT assay was used. MTT is converted by the mitochondria from a soluble yellow salt into an insoluble purple formazan salt. Used to see if cells are alive and metabolically active and where they are distributed on the scaffold. Compared between static (white area present) and perfused (even distribution).

Answer 62

Once cells are delivered to a scaffold you have to make sure that they are viable and functional. External mass transfer: delivery of nutrients and O2 from the inner bulk to exterior Internal mass transfer: delivery of nutrients and O2 from the exterior to the inner bulk Also involves the removal of metabolites and CO2

Answer 63

Wendt et al 2008 Chrondrocytes seeded using perfusion cell seeding. Cultured for 2 weeks with OR without perfusion. In statically cultured scaffold, there is a large amount of empty space where cells have undergone necrosis. Cells are only at the edges. Whereas the perfused culture has lots of cells all spread across.

Answer 64

Tissues and organs in the body are subject to complex biomechanical environment. These physical forces include hydrodynamic/hydrostatic, mechanical and electrical. The tissue engineered construct needs to be able to maintain these too. Eg. Pulsatile flow in blood vessels causes erthrocytes mature to their appropriate phenotype.

Answer 65

Diversity in bioreactor design reflects the range of signals needed for formation of various tissues e.g. heart, bone, valves. Biomaterials making up bioreactors must not induce toxocity or unwanted effects, they must be biocompatible and non-fouling (don't stick). They must also be sterile so are either single use or can be sterilised in an autoclave. Withstand 37 degrees.

Answer 66

Magnetic stirrer creates dynamic culture, the turbulence can cause damage to cells. Scaffold is dispensed from needles into the bulk media. Internal transfer is still quite limited for centre. Relatively cheap and simple. Mass transfer in the flasks is not good enough to deliver homogeneous cell distribution throughout scaffolds and cells predominantly reside on the construct periphery.

Answer 67

Centrifugal forces generated due to the rotation of the cylinder counterbalance the gravitational pull on the scaffolds. As tissue grows in the bioreactor, the rotational speed must be increased in order to balance the gravitational force and ensure the scaffold remains in suspension.

Answer 68

The culture medium continually circulates through the TE construct which stays static. Most mass transfer limitations are mitigated. The effects of direct perfusion can be highly dependent on the medium flow rate.

Answer 69

Used to apply mechanical stimulus to cell-seeded constructs. This can be acting constantly, intermittently or cyclically. Apply mechanical pressure and examine the effects of this.

Answer 70

Complex architecture of tissues makes decelluarising them difficult. Perfusion bioreactor has been used for the heart, lung, liver and pancreas, mostly used for whole organs. Porcine heart The barbed end of the tubing is inserted into the aorta of the native heart. The tubing must be secured with hose clamps or zip ties above the aortic valve to ensure perfusion through the coronary arteries. The hear is then submerged in a water in a 4L beaker and air bubbles must be removed from the tubing using a pump. As solutions are perfused through the coronary arteries, the heart will lose its native colour and become white. It can then be re-seeded with cells again.

Answer 71

Articular cartilage is a load-bearing tissue, it is exposed to cyclical stress. Bioreactors with mechanical loading have been used for tissue engineering of cartilage. Mauck et al (2000) Loading applied in cycles improved biomechanical properties of engineered articular cartilage.

Answer 72

Space flights represent the best environment to investigate near-zero gravity effects but there are major limitations for setting up experimental analysis. Rotating wall bioreactors have been developed by NASA. Tamma et al (2009) studied osteoclasts and the effects of near-zero gravity, found there was a decrease in bone density.

Answer 73

Heart valves are required for directing the flow of blood. May be causes that valves cannot open or close, causing reverse flow. Current treatment options are surgical repair or valve replacement. Prosthetic valve replacement isn't ideal because you need anti-thrombotic drugs and they don't grow with children. Tissue engineering offers the potential of creating a valve that will grow and adapt with growth. Sourcing cells for this is difficult as you need smooth muscle cells and endothelial cells - stem cells may be the main choice. Must sustain the biophysical forces that cells experience in vivo: mechanical strech and hydrodynamic shear.

Answer 74

Developed by Engelmayr et al (2008) Hypothesised that mimicking the mechanical stimulation and perfusion in vitro could lead to further improvement of the engineered tissues. Flex-stretch-slow bioreactor Place a scaffold seeded with cells which is fixed onto 2 posts, one which is fixedd and another which is movable which is linked to a linear motor to drive its movement. Enables flexing and stretching of scaffolds and allows media flow over the scaffold. Culture media can be recirculated within the bioreactor chamber via a magnetically coupled paddle-wheal to provide a laminar flow and associated fluid shear stresses to scaffold specimens.

Answer 75

Mesenchymal stem cells derived from sheep bone marrow were cultured on polyglycolic acid/polylactic acid scaffolds. Test group under mechanical stimuli and hydrodynamic shear had increased collagen content and effective stiffness of engineered valves after 3 weeks in culture compared to controls. By the end of the 3 weeks engineered valves had a modulus comparable to those generated from smooth muscle cells in previous studies. Cyclic flexure and laminar flow can synergistically accelerate MSC-mediated tissue formation.

Answer 76

Nikiason et al (1999) Pulsatile bioreactors/pump mimic pulses found in blood vessels. Vocal fold are subject to mechanical stimuli so bioreactor incorporate speakers to mimic vocal fold maturation.

Answer 77

- Mimicking native cell behaviour - we need further understanding of tissue development and regeneration - Scaling up. Most bioreactors are specialised devices with a low volume output. Time consuming and labour intensive.

Answer 78

Skin substitutes were the first tissue engineered created ex vivo. There is a huge clinical need for skin substitutes and keratinocytes have been well cultured since the mid 1970s.

Answer 79

-Flat e.g. epidermis, cornea One dominant cell type, relatively simple -Tube e.g. blood vessels and urethras Several cell types, serve as conduits -Hollow non-tubular organs e.g. bladder, stomach -Solid organs e.g. kidneys, lungs, heart and liver Complexity in structure, histology, function More complex - more difficult to recreate. Need a good knowledge of their structure and function first.

Answer 80

Largest organ in the body by surface area About 10% of body mass Functions of the skin: -Protection against chemicals, UV light and microbes -Regulation of water, temperature, sweating etc -Sensation, contains sensory nerves 3 main layers: epidermis (neuroectoderm), dermis (mesoderm) and hypodermis.

Answer 81

A thin layer that varies in thickness according to location. Protects the body from environmental factors. Consists only of cells: -Keratinocytes (95%) produce keratin for toughness -Melanocytes - produce melanin, located at the bottom -Langerhans' cells - dendritic cells, antigen properties -Merkel cells - touch sensation, near neurons Mature epidermis is a multilatered epithelium, upper layer is shed. Cells in basal layer are mitotic so will divide and move up and differentiate. Basal cells will secrete ECM matrix to create basement membrane.

Answer 82

Bulk of the skin Composed of the skin with some elastin and glycosaminoglycans (mainly molecular). Main cell types are fibroblasts - important for wound healing. Also contains blood vessels, hair follicles, sebaceous and sweat glands.

Answer 83

A network of adipose cells and collagen | Functions as a thermal insulator and shock absorber and stores fat as an energy reserve.

Answer 84

There is a huge clinical need for skin substitues, namely in 4 instances: - Acute trauma (most common) - Chronic wounds - Surgery - Genetic diseases (bullous conditions) Thermal trauma is one of the most common reasons for skin loss. Burns and scalds cause rapid and extensive wounds. Damaging large areas of skin can lead to death.

Answer 85

The wound healing process involves 3 stages: inflammatory response phase, fibroblastic repair phase and maturation-remodelling phase. The remodelling of new tissue is never as good as the original, so has a detrimental to appearance, function and mechanical properties.

Answer 86

Consequence of the scarring. During the healing process the myofibroblasts the cells which lay down collagen (phenotype similar to fibroblasts and smooth muscle cells) try to contract and close the wound. Overstimulation may lead to contracture whereby the limb cannot be moved.

Answer 87

Skin wounds are classified according to the depth. - Epidermal - Partial thickness (superficial or deep) - Full thickness

Answer 88

``` e.g. sunburn Affects the epidermis Characterised by erythema and minor pain Do not require surgical treatment No scarring ```

Answer 89

Affect the epidermis and superficial part of the dermis. Wound appearance: wet and weeping, red to pink blisters. Very painful due to exposure of sensory nerves. Heals spontaneously.

Answer 90

Involve greater dermal damage. Wound appearance is moist white/red/pink. Results in fewer skin appendages remaining. Scarring is more pronounced.

Answer 91

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

Answer 92

1) An early excision of a dry scab (ESCHAR): dry scab contains denatured proteins that can trigger immune response. Good breeding ground for microbes. 2) Wound closure: reduces mortality from sepsis and morbidity from the scarring. 3) Skin grafts: gold standard treatment

Answer 93

Skin grafts are grafts of tissues of epidermis and varying amounts of dermis that is detached from its own blood supply and placed in a new area. Types of skin graft: -Split thickness - epidermis, some dermis -Full thickness - epidermis, all dermis

Answer 94

Autologous skin grafts or autografts are the gold standards for full-thickness skin wounds. Skin grafts are obtained from a non-injureed site of the patient. Obtained used a dermatome, graft may be meshed to cover a larger area.

Answer 95

Graft take refers to how the skin grafts intergrates to the new site. Remove the graft from its old vascularisation, placed on the wound site. Initially (2-5 days) the graft will survive by diffusion of nutrients from the new site (plasmatic imbibition). Over time, the capillaries on wound bed start to grow and cause revascularisation.

Answer 96

Preparation of the wound bed is the most critical factor for a successful 'graft take'. The graft has to adhere to the wound bed (no bleeding, infection or movement). The graft needs a thin layer of connective tissue that is vascularised.

Answer 97

The use of cadaveric skin for a temporary prevention of fluid loss or wound contamination. Can be obtained from non-profit skin banks. Possibility of pathogen transmission and immunogeneic rejection.

Answer 98

There is a great need for skin substitutes because there is a limited availability of skin grafts, there is often pain and scarring in the donor site area and there may be further pain inflicted on the patient. An ideal skin substitute would be readily available, cause no immune response, cover and protect the wound, enhance the healing, lessen the pain and leave no scars.

Answer 99

A key step is isolation of keratinocytes from a skin biopsy. Small biopsy taken from non-injured site and deattach the epidermis from the dermis. The keratinocytes are then isolated and expanded in culture. 3cm sqaured can be increased by 1000 fold after 3-4 weeks. Cultured keratinocytes are delivered onto the wound and form a new epidermal layer. This formed cultured epithelium sheets e.g. Epicel, EPIBASE, MySkin.

Answer 100

``` Celtran Ltd (Uni of Sheff spin-out company). Subconfluent autologous keratinocytes. Synthetic silicone delivery membrane to enable handling and manipulation. Applications include using it to treat chronic wounds through repeated treatments - very successful. ```

Answer 101

- Epidermal substitutes contain only keratinocytes grown in vitro and can be applied or sprayed onto the wound site - They are effective in treating chronic ulcers - Combination with a dermal substitute is needed to achieve full-thickness wound healing.

Answer 102

- Provide a dermal alternative to facilitate the process of wound healing. - Acellular - require colonization from the wound bed. - After application to a prepared dermis, these substitutes are colonized and vascularized by the underlying cells. - 2 step process: first applying a dermal substitute followed by an epidermal cover.

Answer 103

Consists of two layers -the dermal layer from bovine type I collagen and shark chondroitin-6-sulfate (xenogenic) -epidermal later made of silicone (pseudoepidermis) The collagen-chrondroitin matrix allows in-growth of cells from the wound bed and the artificial silicone epidermis regulates heat and fluid loss. Semi-permanent as it is delivered to the wound bed, and the cells infiltrate the scaffold over 20 days. After vascularization and formation of the neodermis the membrane can be removed.

Answer 104

Acellular human allogenic dermal matrix preserved by freeze drying. Used for full-thickness skin burns, alloplastic breast reconstruction, abdominal wall reconstruction, rhinoplasty.

Answer 105

- Aim to mimic the histological structure of normal skin - Most of these products are based on allogeneic skin incorporated into a dermal scaffold. - Enable production of large batches of the products 'off the shelf' - Temporary bioactive dressings: allogeneic will not last long and will be rejected. - Relieves pain and promotes wound healing

Answer 106

Includes cultured allogeneic fibroblasts and heratinocytes obtained from the same neonatal foreskin. Fibroblasts are seeded into a bovine type I collagen sponge and keratinocytes on top. Cytokines and growth factors from the product promote host cell migration and wound healing.

Answer 107

- The average wait time ranges from 3-12 weeks after the biopsy is taken - Currently available composite skin substitutes use only 2 cell types: keratinocytes and fibroblasts but in normal skin there are other cells involved too (Merkel, Langerhans, melanocytes). - Clinical success but economic failure!

Answer 108

Composite skin consisiting of bovine type I collagen cultured with allogeneic male neonatal fibroblasts and keratinocytes - resembles normal skin structures.

Answer 109

Hirch et al 2017 7 year old boy admitted to hospital in June 2015 with severe skin injuries (60% loss of epidermis). Suffering from epidermolysis bullosa, a genetic disorder affecting 1 in 50,000 live births. Had spontaneous (or result of minor injury) skin blistering which was painful and life-threatening due to the risk of sepsis. High risk of skin cancer. Had a splice site mutation in intron 14 of LAMB3 (laminin 332) causing poor attachment of epidermis to dermis. Suffered from infection from Staphylococcus and Pseudomonas. Attempts with antibiotic treatment, topical dressings and ointments, skin transplant from father and split thick graft, but nothing worked.

Answer 110

Epidermis and dermis are usually divided by basement membrane, but blisters form when the epidermis comes away from the dermis. Basement membrane is a well-organised structure that contains a number of extracellular proteins. In the epidermis, keratinocytes are inserted with plectins and BPAGs. These interact with a6 B4 integrins and some types of collagens. Across the basement membrane they interact with laminin 332 and other collagens in the dermis. This usually keep them adherent, however, mutations in these components means the protein is absent or non-functional. Different groups of epidermolysis bullosa depending on non-functional protein. German authorities allowed for a one-off compassionate treatments involving genetically modified stem cells. Proved successful, normal skin appearance and function. Procedure took 7-8 months.

Answer 111

Mavilio et al (2006) Italian group specialised in correcting junctional epidermolysis bullosa using genetically modified epidermal stem cells. Deemed successful in small areas. New EU regulations in GMP limited further research.

Answer 112

Alone this wouldn't work as there would still be no wildtype laminin produced, the basement membrane would not be adherent enough and the skin blistering would continue. Needs a change in the genetics.

Answer 113

Gene therapy aims to repair or replace a mutated gene by integrating the correct gene into the patients cells. This can be done in two ways: EX VIVO: Cells taken from the patients, manipulated in culture and then placed back into the patient. IN VIVO: Cells are manipulated within the body. Vectors are used to carry the corrected gene to be integrated into the patient's cells. This can be done using viral or non-viral (e.g. liposomes) methods.

Answer 114

They used ex vivo harvesting of cells whereby a skin biopsy from a safe area was taken and cultured on a feeder layer of fibroblasts. These were passaged and expanded over time. They are then transduced with a retroviral vector carrying the full-length of LAMB3 cDNA. The cells were passaged further so that the gene would incorporate into the patient's DNA. They made epidermal sheets from the cultured cells. For the first skin transplant they compared keratinocytes grown on plastic or fibrin. Fibrin performed better so this was used for second and third transplants.

Answer 115

The cells were characterised and next generation sequencing was performed to identify where the gene has integrated (exons, introns, intergenic, promotors). You can check to see if any of the other genes (tumour suppressors/oncogenes) have been disrupted which may be dangerous. They found that very few landed in exons and none seemed to disrupt major regulatory genes so it was deemed safe to be used for the patient.

Answer 116

Since this was an experimental treatment the patient was followed up by taking various biopsies at different time points to assess them for dangerous markers. Next generation sequencing, clonal analysis, immunoflurescence, histology, in situ hydridisation were all performed.

Answer 117

In situ hybridisation: Looking for the presence of the corrected gene. Used Cdh1 (control) and t-LAMB3 probes to see if the cells have integrated the correct gene. After 4 months post-transplant, the t-LAMB3 was present showing effective transduction. Histology: at admission you can see the epidermis moving away from the dermis. But after the transplant, there is visible better attachment. Immunofluorescene: Using laminin probes. At admission, the boy had no laminin332-B3 expression but 4 months post-transplantation there is good laminin-332 expression resembling the control. Behaviour: laminin332-B3 null keratinocytes came away from the flask edge but laminin332-B3 corrected ketatinocytes stuck and required enzymatic treatment for deattachment.

Answer 118

The epidermis has a high turnover, skin will rectify itself every month. When placed in culture keratinocytes make 3 different clones: holoclones (clones of proliferative, undifferentiated cells), paraclones (clones of differentiated cells) and meroclones (intermediates). The question is... Is the epidermis renewal driven by a restricted number of long-lived stem cells (holoclones) OR equipotent progenitors?

Answer 119

An integration profiles of cells was performed to identify which cells were driving epidermal renewal. The viral integration sites were mapped in holoclones and compared to the integration sites in biopsied cells (4m, 8m). Fewer integration sites were seen at 4 months than in the initial culture but many integration sites were preserved between 4 and 8 month samples. This is because most of the cells differentiated and died and the rest gave rise to differentiated cells (paraclones and meroclones). The remaining contribute to epidermis renewal (holoclones) over the long-term.

Answer 120

- Need to ensure a longer term follow up for the patient to avoid missing any dangerous signs. Particularly looking for skin cancer. - Further and wider clinical studies needed for safety and efficacy. - Alternative gene editing strategies could be used e.g. CRISPR - Does the patient's age have an effect on efficacy? Younger patients have proliferative keratinocytes, would it work in adults?

Answer 121

- Disease modelling - replacements for animal models, ethical and representative. Diseases include epidermolysis bullosa, vitiligo, psoriasis, skin cancer and allergic contact dermatitis. - Drug discovery - Chemical testing - again avoids using animals Better we can mimic in vivo the more accurate these studies/tests will be.

Answer 122

Itoh et al (2011) Disease modelling of EB where collagen 7 is lost. Somatic cells were taken from the patient and were induced to become pluripotent. They can differentiate into any cell (keratinocytes, fibroblasts etc.) WT and EB patient-specific iPSCs were generated and characterised. They were differentiated in keratinocytes and characterised. This has the potential to be used to look for drugs that will alleviate phenotype.

Answer 123

Psoriasis is a chronic inflammatory skin disease. Gives red plaques on the skin with inflamed appearance. Due to keratinocyte hyperproliferation, immune cell infiltration into epidermis and increased angiogenesis.

Answer 124

In vitro 2D: Use patient keratinocytes with addition of cytokines (TNFa, IL-1a, IL-17, IL-22) to mimic secretions of immune cells or via a co-culture model whereby immune cells themselves are integrated. 3D: Keratinocytes and fibroblasts are combined with the addition of cytokines to model the inflammatory response.

Answer 125

15-20% of general population have ACD resulting from environmental chemical exposure. A consequence of a repeated exposure of the skin to an allergen.

Answer 126

Need for vascularisation, immunity, pigmentation, appendages (sweat glands, hair follicles, dermal papillae), hypodermis, innervation. To source these cells you could use tissue-specific stem cells but skin is derived from multiple embryonic germ layers so you need a common population that will give rise to all. Pluripotent stem cells are promising.

Answer 127

Takagi et al (2016) Fabrication of skin and its appendages could contribute to regenerative therapies. Difficult to create in vitro. -Hypothesis: pluripotent stem cells can be used to mimic the developmental patterning. -Experimental approach: Make embryoid bodies. Using iPS cells, cells will begin to organise/differentiate into different clusters. EBs placed in collagen gels and then transplanted further into SCID mice (in vivo organogenesis). -Results: When treated with Wnt10, there was creation of hair which was dark, showing it was iPS cell-derived. iPS cell derived hair follicles had a correct structure and connected with local tissues.

Answer 128

The study produced fully-functional iPSC-derived explants including hair follicles and sabaceous glands with proper connections to the epithelium, dermis, fat, aneator pili muscles and nerve fibres. A big step towards complete reconstruction of the skin.

Answer 129

Together with the other tissues of the eye, the cornea facilitates sight. The sclera and the cornea make up the spherical outer shell. The sclera covers 5/6th of the eye whereas the cornea covers the iris (1/6th).

Answer 130

- Aiding sight: it is transparent to 99% of incident light. Has refractive power (2/3) along with the lens to focus light onto the retina. - Eye protection: barrier to external environment protecting against dust and microbes.

Answer 131

Made up of three layers with 2 membranes. - Corneal epithelium - Bowman's membrane - Corneal stroma - Descemet's membrane - Corneal endothelium

Answer 132

Outer most layer with a thickness of 50μm (10% of cornea). Highly innervated with nociceptors. Functions include prevention of fluid loss, a barrier to pathogens and production of a rapid response to wound healing. 5-7 cell layers, entirely cellular. Stratified and non-keratinized squamous epithelial cells. Innermost layer is basal cells which are attached to bowman's membrane via hemidesmosomes and to each other via desmosomes. Basal cells are mitotic and will give rise to wing cells which will give rise to superficial cells. Superficial cells have tight junctions to make the cornea impermeable to dust and glycocalyx present on microvilli to retain tea fluid on the eye.

Answer 133

Similar to the dermis of the skin. 90% of corneal thickness. Contains collagens, proteoglycans and glycoproteins. Relatively acellular but does contain keratocytes which are long thin flatterned cells. They synthesise and maintain ECM proteins in the stroma. To keep the shape correct and the appropriate distance between collagen fibres. Functions: -Provide strength, holding the shape of the eye, important for refractory power. -Allows transparency due to the ordered collagen structure (bundled together in lamella). Distance between them and their location is important.

Answer 134

A single layer of cells. Acts to maintain the stromal hydration. Can't be too hydrated or the collagen fibre structure will be disrupted by the water molecules, causing swelling. Acts as a leaky pump, allows solutes and nutrients in to stroma from aqueous humor but also pumps out water from stroma using various channels and pumps. Highly metabolically demanding process.

Answer 135

The cornea is the most densely innervated body structure. Filled with nociceptor endings from the ophthalmic branch of the trigeminal nerve. Important for blink reflex, wound healing and tear production.

Answer 136

It is AVASCULAR If it were to have blood vessels, it would no longer be transparent. For the diffusion of oxygen and delivery of nutrients the tear layer and transport from the humor is important.

Answer 137

- The cornea is avascular - Collagen is organised into a specific way in the stroma - The stroma is kept in a dehydrated state - Very few cells present in the stroma and certainly no pigmented cells.

Answer 138

Corneal disease/injury is the second leading cause of vision loss (first in bacterial infection in 3rd world countries). 10 million people suffer from vision loss due to corneal disease (e.g. trachoma) or injury (e.g. acid/base burns). Traditionally treated by corneal transplantation whereby the cornea is exised and a new, usually cadaveric is sutured in to replace faulty version. But there may be immune rejection, viral/disease transmission and the sourcing/logistics of storing and transporting corneas is difficult. Need for regenerative therapy that will completely restore function of the eye.

Answer 139

A synthetic medical device known as keratoprosthesis. Used for patients with repeated failed organs. Require life-long regime of antibiotics. Medications needed to control inflammation and glaucoma (due to uncontrolled pressure in the eye). Need for regenerative therapy that will completely restore function of the eye.

Answer 140

If the cornea epithelium is injured it can regenerate quite well without scarring. This is due to the limbal epithelial stem cells. They are located on the corneal rim at the border between the sclera and the cornea. Found in undulated niches. Have the ability to undergo asymmetric division, some will stay in the limbus and others will give rise to daughter cells. These will migrate outwards and give rise to trans-amplifying cells (divide rapidly in the basal layer), undergo differentiation and become post-mitotic (wing cell layer) or terminally differentiated cells (squamous cells).

Answer 141

When numbers of limbal stem cells fall below a certain threshold due to injury/disease. Unable to regenerate. May be due to congenital conditions such as aniridia or internal/external injury. In the absence of LSCs cells from conjunctiva migrate to the cornea and create opaque and vascularised tissue (vision loss).

Answer 142

If the corneal disease is unilateral the easiest approach is to take part of the limbus from the other eye and transplant it directly or expand it to create a corneal epithelial sheet on amniotic layer/synthetic scaffold.

Answer 143

Pellegrini et al 1997) performed studies using expanded limbal stem cells which demonstrated that the ocular surface in patients with ocular burns could be restored. 2015: Approval for stem cell therapy using limbal stem cells was given in Europe. Holoclar was developed.

Answer 144

Stem cell therapy using limbal stem cells. Patient's healthy eye has the limbal area explanted and shipped to a production facility whereby cells are cultures and expanded. Can be frozen at this stage or used directly. Cell culture is placed in a fibrin matrix to help deliver the cells to the patient.

Answer 145

75 out of 104 patients studied showed: - Stable corneal surface with no surface defects - Little or no ingrown blood vessels - Reductions in pain and inflammation - Improvements in vision No immune rejection issues because it is an autologous approach. But if both eyes are diseased, it cannot help. And you may damage the eye from which you are removing the limbal cells.

Answer 146

In cases when autologous sourcing of limbal stem cells is not possible you can take cells from the muscosa tissue. It doesn't cause scarring in the eye or the mucosa but there is a risk of neovascularization of the cornea.

Answer 147

When collagen fibrils are disrupted they are very hard to recreate. When the stroma is injured the wound healing process begins. The keratocytes will differentiate into fibroblasts which will become myofibroblasts to close the wound. Then they should undergo apoptosis be in some conditions they become overactive leading to scarring. Cornea will no longer be transparent.

Answer 148

They must be acellular at the time of implantation. They are expected to promote repopulation by the host's cells and reinnervation. A range of materials have been tested for this purpose including decellularised cornea, collagen and recombinant collagen and self-assembling peptides.

Answer 149

Fagerholm et al (2014) Produced recombinant human collagen cell-free implants. Showed successful endogenous cell recruitment, regenerated neo-corneas that were stably integrated and nerve and stromal cell repopulation. There is no need for immunosuppression. Visual acuity could be improved by using better materials and the scaffold didn't have appropriate mechanical properties. Islam et al (2018) Used recombinant human collagen reinforced with a synthetic lipid. Tested in pigs and in 7 patients. Implants were shown to improve vision and relieve pain.

Answer 150

Take cells that usually make stroma to secrete a deposit ECM proteins. ECM sheet are produced populated with stromal cells. Cells secrete their own ECM that can be remodelled. Can be used with autologous cells. Allows for cell amplification and relatively large amounts of the fabricated tissue. Obvious source of cells would be keratocytes. They can be isolated from the stroma, amplified in vitro, in some cases they will change their phenotype to become more fibroblastic but under serum and ascorbic acid they will lay down ECm proteins. Others will change from keratocytes to fibroblasts to keratocyte-like cells. Or you can use stem cells.

Answer 151

-Human corneal endothelium does not regenerate! -Endothelial cells have a finite life span leading to a decrease in density with age. At birth 3500-4000 cells/mm2 but by 85 only have 2300 cells/mm2. Necessary to have 500 cells/mm2 to be functional. -If endothelial cells are put in culture they don't regenerate and if they are immortalised they lose their phenotype which has implications for research and clinical use. Recent development suggests that endothelial cells may be able to be derived from pluripotent stem cells.

Answer 152

It should allow for adequate mass transport, prevent angiogenesis, have tensile strength/mechanical properties, have low immunogenicity, support nerve regrowth. For epithelium: continuous replacement of cells, maintenance of barrier integrity and optical transparency. For stroma: high tensile strength, optical transparency.

Answer 153

Patients with corneal transplants have limited re-innervation yet innervation is essential for normal function of cornea. Can we use growth factors to encourage re-innervation?

Answer 154

9000 cases in the UK per year Mainly in young people (working population) Main cause in peace time is car accidents. Brings financial, healthcare and social burden.

Answer 155

The nervous system is divided into central nervous system (brain and spinal cord) and peripheral nervous system (paired, cranial, spinal nerves) which stem from the CNS and relay electrical signals to and from. Cellular composition includes neurons and glial supporting cells but it varies across the CNS and PNS.

Answer 156

Axons are surrounded by mylinating Schwann cells and are enclosed by endoneurium. Individual axons are bound together by perineurium to form fascicles.

Answer 157

- Elongation: the connective tissue of nerves allows 10-20% elongation before structural damage occurs. Severe lesions can disrupt the axon. - Laceration: 30% of nerve injuries - Compression: external mechanical pressure on the conductive membrane. If it's persistent it leads to nerve injury.

Answer 158

Seddon, 1942 NEUROPRAXIA No/little structural damage, no loss of nerve continuity. Symptoms are transient and reversible. Includes entrapment neuropathies like carpal tunnel syndrome and 'Saturday Night Palsy' (radial nerve). AXONOTMESIS Complete interruption of the axon and its myelin sheath. Perineurium and epineurium remain intact. NEUROTMESIS Nerve and the surrounding stroma are completely disconnected. No spontaneous recovery. Weakness, atrophy and poor prognosis.

Answer 159

When axons are severed, axon distal to site of injury are now disconnected to the cell body and will begin to degenerate due to proteases and metabolic activity disconnect. This leads to cell debris which is cleared up by macrophages. Schwann cells take up regenerative phenotype. They proliferate and align into tracts called bands of Bungrer. They serve as a conduit for growing axons. Takes about 1-3mm a day.

Answer 160

Macrophages are crucial in nerve regeneration but in CNS macrophages infiltrate much more slowly because there is a blood spine barrier that macrophages cannot infiltrate. This delays the removal of inhibitory myelin. Astrocytes become 'reactive astrocytes' which produce glial scars that inhibit regeneration. -In PNS: repair of damage is actively promoted -In CNS: repair of damage is inhibited CNS and PNS require different regenerative medicine strategies. More success in PNS.

Answer 161

Surgical reconstruction Grafts Nerve conduits

Answer 162

Surgical reconstruction has been used historically. Suturing the stumps of the nerves back together. Can only be used if the stumps are proximal, if the gap is larger than 1mm, it cannot be used because it will cause elongation injury. Blood flow reduces by 50% when the nerve is stretched 8%, complete ichaemia is caused at 15%.

Answer 163

When the gap between the nerve stumps is too big, the gold standard are grafts. Can be autologous: a nerve is taken from somewhere else in the patient's body (usually sensory nerve). Gives low risk of immune rejection but causes loss of function at the donor site, two surgeries are required and it is limited by the size and type of nerve they can use. Or can be allogeneic which is from the same species but different individual. Good because there is no secondary surgery and no loss of function at donor site. However, there is a higher risk of rejection and limited availability in cadavers.

Answer 164

Nerve conduits are devices where 2 stumps of a severed nerve are placed inside a conduit. It guides regenerating axons, prevents infiltration of scar tissue and increases concentration of intraluminal proteins. Conduit fills with plasma from the severed nerves (hours). The plasma is filled with fibrin precursoes which will form a fibrin cable. There is promoted proliferation and migration. The resulting tissue is notably thinner. Can take months/years.

Answer 165

- Good mechanical strength to prevent collapse - Not too tough, shouldn't affect function or surrounding soft tissue - Porous to nutrients and oxygen, prevents fibroblasts entering - Rate of degradation should match tissue - No immunogeneiticty - Bio-compatible - Suturable - Able to be sterilised to avoid bacterial infection

Answer 166

"Top down approach" Take a nerve which already exists in nature and stripping it down to its bare bones. Complete scaffold for the physical properties as seen in vivo. Aim is to retain the ECM architecture and remove antigens. Less immunogenic and hollow compared to an allograft. -Provides a 3D scaffold to support nerve regeneration -Clean pathways allow cell migration and axonal regeneration -Axon regeneration is well distributed through nerve thickness -Functional incorporation of the nerve conduit Off the shelf: 'Avance'

Answer 167

- Decellularised nerve conduits | - Fabricated (biological, semi-synthetic, synthetic)

Answer 168

"Bottom up approach" Use biomaterials to fabricate scaffold. We are in control of production, more flexibility to change biodegradability/porosity, biochemical signals, incorporation of support cells, electrical activity, intraluminnal channels and oriented nerve substratum.

Answer 169

Many natural materials have been used e.g. chitosan, collagen, fibrin, fibronectin, gelatin, keratin and silk fibroin (biodegradable, biocompatible, structural integrity). Synthetic materials can also be used. Biodegradable materials like poly(lactic) acid, poly(lactic-co-glycolic) acid, poly(caprolactone), poly(ethylene glycol). Electrically active materials which can contribute to the function of the nerve like Piezoelectric. Non-biodegradable like silicone and Gore-tex can be used but must be removed from the body.

Answer 170

The chance of successful regeneration with nerve guides is reduced once an injury gap reaches a certain value. At short gap lengths, the fibrin cable is robust enough to provide a platform for regeneration. At longer lengths thinning restricts regeneration. No fibrin cable at large lengths.

Answer 171

Length at which regeneration occurs 50% of the time. Mouse 5mm Rat 10mm

Answer 172

``` Aim is to bridge this gap so it is no longer an issue using... -ECM components -Intraluminal support -Cell grafts -Neurotrophic factors Or combinatorial approach ```

Answer 173

- Matrices generally increase the critical gap length - Effective materials are weak, visoelastic hydrogels with a high water content (high concentrations would prevent axonal penetration). - Matrigel promotes nerve regeneration but not suitable for clinic because it comes from mouse sarcoma cells - Laminin, fibronectin and collagen involved

Answer 174

Relies on scaffold generation of conduit to have intraluminal guidance, micro-grooved luminal design, electrospun fibrous outer conduit, variations in conduit design, surface functionalisation and combinatorial approaches.

Answer 175

Neurotrophic factors can be placed within the tube. -Support axonal growth -Migration and proliferation of Schwann cells -Increase neuroprotection modulation of intrinsic signalling pathways Nerve growth factors and Neurotrophin-3 show potential.

Answer 176

Challenge is to release neurotrophic factors in a controlled way to avoid dissipation. Could use a polymer to release them which will then biodegrade. Means of delivery could include diffusion-based release, suspension, affinity-based delivery, microsphere encapsulation. Gradients rather than isotropic presence. Regenerative medicine is trying to recrete gradient of laminin 1 and growth factors across the nerve conduits to guide axon growth.

Answer 177

Can deliver cell grafts with the nerve conduits. May be neurotrophic factors, schwann cells, differentiated or undifferentiated stem cells or geneticallt modified cells. All experimental. Schwann cells are critical for sucessful nerve regeneration could be delivered along with nerve conduits. They create bands of bungner, secrete neurotrophic factors and proliferate (4-7x cells).

Answer 178

Development of biometric micro-patterned device incorporated with neurotrophic gradient and schwann cells. - Used a micro-patterned surface that can directionally guide the axon as physical cue. - Neurotrophic gradient membrane that can continually attract axon outgrowth from proximal to distal stump as chemical cue. - Schwann cells that can support the growth of neurite and form myelin sheath around axon as biological cue.

Answer 179

Blood vessels. Upper airway tubes like trachea and larynx. Urogenital tissues. Most will have a layer of endothelium, a layer of muscle, connective tissue. Best approach so far has been to used existing organs, decellularise them and then recellularise with patient's autologous cells, and then transplant back in.

Answer 180

Biological bottlenecks Vascularization -In vitro: nutrients and oxygen flow through the media -In vivo: must have an immediate blood supply or necrosis Host response: inflammatory and immune response Regulation and commercialisation.

Answer 181

Most tissues in the body contain a vasculatory network. Most cells are located 100-200um from capillaries but there are differences in cells' sensitivity to oxygenation. Islet cells are particularly sensitive whereas cartilage cells can maintain viability even in 1mm TE construct. In vitro this is overcome by using bioreactors. -Avoid graft necrosis -Generate thicker tissues -Help graft innveration -Improve graft function

Answer 182

Folkman (1971) Hypothesis: Tumour growth is angiogenesis-dependant. Patient with retinoblastoma with a highly vascularized tumour. Tumour had a number of metastases with about 1mm diameter. Metastases only contained a rim of viable cells on the outside but central cells were necrotic due to a lack of vascularization. Therefore tissues require vascularization when diffusion is no longer sufficient.

Answer 183

There are macrovessels (arteries and veins), microvessels (arterioles and venules) and capillaries which mediate the exchange of nutrients due to differences between blood pressure and interstitial fluid. Thick layer of connective tissue surrounding the vessel, several layers of smooth muscle. Inner layer is single layer of endothelium, lining the lumen separated from the SM by basal lamina. Exact composition depends on anatomy and function. e.g. capillaries only contain endothelium surrounded by pericytes.

Answer 184

- Vasculogenesis: de novo blood vessel formation that occurs in embryogenesis, formation from progenitor cells. - Angiogenesis: new blood vessel formation via extension/remodelling of existing blood vessels. - Arterogenesis: maturation of blood vessels via increasing in the lumen.

Answer 185

Embryo is small so oxygen and nutrients can diffuse to support the life of cells. When embryo reaches a certain size it requires vascularization. Mesoderm produces precursors called Hemangioblasts. These differentiate and create tubes which create a primary capillary plexus.

Answer 186

Sprouting new blood vessels from the existing ones. The major drive of this process is hypoxia. Hypoxia-induced factors (TF) will initiate cascade of evenets to lead to new blood vessels sprouting. Angiogenesis stimulus VEGF is produced and signals from hypoxic area to endothelial cells. Endothelial cells become active and secrete enzymes like matrix metalloproteinases. These enzymes digest the basal lamina and create a hole. Endothelial cells will start moving through the holes and create vascular sprouts. These will proliferate and moce towards VEGF signal. Lumen forms and recruitment of pericytes and SMC allows it to mature. Blood starts flowing through and oxygen is delivered. Physiologically takes place in wound healing and ovarian cycle.

Answer 187

- Vascular Endothelial Growth Factor (VEGF) promotes endothelial cell proliferation, migration and differentiation. - Hypoxia inducible factor (HIF) stimulates transcription of VEGF, PDGFR, TGF-a, EGF and erythropoietin. - Platelet derived growth factor (PDGF) recruits and encourages proliferation of pericytes and SMCs. - Angiopoietin regulates endothelial cell survival, sprouting, pericyte recruitment. - Matrix metalloproteinases. Basal lamina degrades, ECM remodelling.

Answer 188

Family of growth factors with preferences for different receptors. VEGF-A, VEGF-B, PlGF will preferentially bind to VEGFR-1 and are involved in vasculogenesis, Whereas VEGF-C and VEGF-D will bind to VEGFR2 and important for angiogenesis.

Answer 189

Blood vessels remodelling as a response to fluid shear stress (can be caused by occlusion). Increase in shear stress causes endothelial cells to release GFs (like TGF-B). Causes proliferation of endothelial and smooth muscle cells and matrix remodelling.

Answer 190

- Strategies for facilitating vascular ingrowth (scaffold design or functionalization) - Prevascularization (in vitro or in vivo)

Answer 191

Background: angiogenesis is a multistep process. VEGF is the initiator of angiogenesis but not sufficient to induce mature vessels. PDGF promotes maturation of blood vessels. Hypothesis: Dual delivery of VEGF and PDGF can direct the formation of a mature vasculature. VEGF is mixed into a scaffold and released to stimulate the growth of immature vessels. PDGF is encapsulated into PLG microspheres to facilitate maturation of nascent vessels. SUCCESSFULLY creates a mature vascular network.

Answer 192

Time-consuming process because microvessel growth rate i about 5um/h. May not be sufficient to prevent necrosis in 3D contructs after implantation.

Answer 193

The TE constructs is cultured in vitro to build prevascularized structure with endothelial cells. The perivascular network can create a connection with the existing blood vessels in the tissue (transplant site) by anastomising. Endothelial cells spontaneously self-assemble into capillary-like structures. This is faster than new blood vessels formation but is not as mature or well-spaced as would like. Also issues with where the cells will be sourced from.

Answer 194

A scaffold is implanted into easily accessible and well vascularized tissue. Subcutaneous tissue or muscles pouches for example. Microvessels ingrow from the host. After vascularization the implant is transferred to the defect site. Requires 3 surgeries.

Answer 195

Prevascularization method. A scaffold is implanted into a muscle flap. Microvessels ingrowth from the host. After vascularization the entire flap is transferred to the site in need of repair. The vascular pedicle of the flap is surgically anastomosed to host vessels. Major morbidity to the donor site.

Answer 196

Arteriovenous loop technique. Uses a vein or synthetic graft to form a shunt loop between an artery and vein. Protected with a plastic chamber. AV loop leads to spontaneous sprouting of vessels. When vascularized it can be transferred to the repair site. Allows tissue vascularization but the tissue is not embedded in the surrounded muscle tissue. No major morbidity at the donor site.

Answer 197

- Versatile strength, easy to develop, easy to translocate to multiple tissues. - Relies on vessel ingrowth, limited result if used alone, can introduce cell seeding problems.

Answer 198

- Doesn't rely on ingrowth, no extra surgery | - Complex strategy, varies from tissue to tissue, vessel maturation in vitro needs attention, anastomosis is slower.

Answer 199

- Direct perfusion after microsurgery, mature and organised vasculature - Extra implantation/surgery, identification of location with vascular axis, scaffold might be filled with fibrosis tissue.

Answer 200

- Angiogenic factors have been effective, possibility to tune vascularization - Still relies on vessel ingrowth, factors might have a negative effect, release profile of these factors is critical.

Answer 201

Host response is very complex and occurs at various levels. Local interactions: how the TE construct interacts with the local environment. Systemic interactions: through blood and immune system, it can communicate with the rest of the body. Outcome of both of these determines the functional integration or complications of the TE construct. Body responds to biomaterial via the inflammatory response and to cells via the immune response. The is known because of the response to medical devices and cell replacement strategies. There is cross-talk between them.

Answer 202

The effects of biomaterial on body include changes to wound healing, infection, toxicity, tumourigenicity, particle embolization, hypersensitivity reactions. Effects of body on biomaterial include enzymatic degradation, calcification, abrasion and corrosion.

Answer 203

3 major phases: inflammatory, proliferative, remodelling. Cute blood vessels bleed into the wound, blood clot forms and leukocytes clean the wound. Blood vessels regrow and granulation tissue forms, epithelium regnerate and scar forms. If the stimulus is removed (acute) cell death occurs but regeneration and repair can occur. If the wound is persistent (chronic) fibrosis occurs leading to a tissue scar. In TE construct there is a response similar to wound healing.

Answer 204

Similar to wound healing. Implantation of TE construct injures the tissue causing a flow of blood to enter. Proteins from the blood will coat the surface of biomaterial. This creates a transient matrix, which serves as an attachment site, upon which neutrophils attach first. Monocytes also attach and differentiate into macrophages. Macrophages cannot clear the debris because biomaterial is too large. Cue frustrated phagocytosis

Answer 205

Similar to wound healing. Implantation of TE construct injures the tissue causing a flow of blood to enter. Proteins from the blood will coat the surface of biomaterial. This creates a transient matrix, which serves as an attachment site, upon which neutrophils attach first. Monocytes also attach and differentiate into macrophages. Macrophages cannot clear the debris because biomaterial is too large. Cue frustrated phagocytosis. This leads to macrophages fusing creating foreign body giants. If the material is still too big, they can secrete ROS and other enzymes which can degrade the surface of the material.

Answer 206

Tissue response to implanted biomaterials. Abundant deposition of extracellular matrix/collagen. Isolation of biomaterial from local tissue environment. This hinders tissue construct integration - cannot interact with surrounding tissues.

Answer 207

Implanted microelectrodes are implanted to intiate transmission of electrical signals. Encapsulation prevent this transmission. Vascularization cannot penetrate fibrous capsule. Diffusion barrier - some devices rely on their interaction with blood to measure levels but encapsulation prevents this.

Answer 208

Very common when related to medical devices. Joint prostheses have fewer infections. Initially localized to the implant site, but may cause sepsis. When the skin barrier it's easy to see how bacterial infection can occur. But in urinary catheters, the skin barrier is not broken but bacterial infections still occur. When coated with antibiotics (didn't reach systemic) the bacterial infections decreased. Suggested bacterial biofilms.

Answer 209

Communities of bacteria grown on surfaces of abiotic materials and host tissues. The bacteria embed themselves in a matrix known as 'extracellular polymeric substance' or aka slime. Ancient adaptation. Allows for coordinated behaviour and enhanced survival at the population level.

Answer 210

Planktonic bacteria begin to attach to surfaces, secrete slime and proliferate. The adhesion becomes irreversible. They also maturate, causing some of the bacteria to leave the colony, become planktonic and establishing new attachments.

Answer 211

Staphylococci are particularly proven to create biofilms. Worldwide there is an increas ein multidrug-resistant methicillin-resistant strains. S. aureus (MRSA) and S.epidermis (MRSE). Biofilm infections can occur on surgical repair mechanisms (staples, sutures, mashes). Can occur on orthopedic prosthetics. May take months to develop and difficult to access.

Answer 212

Cells do not interact directly with materials. A layer of protein (from growth media or plasma) adheres to the surface. Protein adsorption is affected by the surface properties of the material. The race to the surface refers to whether the cells or the bacteria can make it to the surface first. If there are cells already attached to the surface there is less room for the bacterial cells to attach or create biofilms.

Answer 213

Surfaces that resist adsorption of proteins and/pr adhesion of cells. Poly(ethyl glycol) and zwitterionic polymers. In medical devices, inhibits bacterial colonization. To functionalize, add RGD so cells can adhere.

Answer 214

Using peptides that can self-assemble into 3D network. These matrices allow attachement of mammalian cells but not bacterial cells due to topology. Labelled bacterial cells have been shown not to 'SaNet'.

Answer 215

By-products of physical/chemical wear can enter the bloodstream and cause side-effects in other parts of the body. Example: metal-on-metal hip replacements, releasing colbalt and chromium in the blood. Can bind to proteins and present to the immune system.

Answer 216

Exposure of blood to an artificial material can cause thrombosis, embolization and consumption of platelets and plasma coagulation. e.g. stent thrombosis Blood in contact with metal stent. Rare but serious complication. Death (20-48%) // MI (60-70%) Combatted with anticoagulant drugs clinically.

Answer 217

Has not been experimentally proven but it may be the case that you can say that tumours developing in the region of the medical device may be causal, althought no evidence. Mechanisms not clearly understood.

Answer 218

- Humoral immunity: mediated by soluble antibodies produced by B lymphocytes. - Cellular immunity: mediated by T lymphocytes

Answer 219

- Topography: some will attach others won't - Stiffness: influences cell behaviour - Non-fouling coatings - Functionalization with biomimetic coatings - Delivery of anti-inflammatory agents - Encapsulation of anti-inflammatory agents - Gene delivery systems: modifying cells to secrete certain factors - The use of embedded immune cells secreting pro-angiogenic and pro-regenerative cytokines

Answer 220

Type 1 diabetes is an autoimmune disease where there is a loss of insulin-secreting islet cells. The Edmonton protocol shows sucessful islet transplantation from cadavers, but there is a need for immunosuppression. Immunoisolation of islet cells could give the secretion of insulin using a selectively permeable membrane, wouldn't require immunosuppression.

Answer 221

Performing clinical trials using immunoisolation of islet cells to treat type 1 diabetes. Using human embryonic stem cells. Active area of research with lots of different approaches.

Answer 222

- Hypopituitarism - Geneticallt engineering cells to secrete neurotrophic or angiogenic factors - Enhancing tumour-specific cellular immunity

Answer 223

- Elixir Sulfanilamide incident (1937): Used for a Staphylococcus infection. Developed for children and added raspberry flavour but was toxic to those who were given the drug (aka antifreeze). - The Nuremberg Code (1946): Volunteers must consent to testing. - The Softenon (thalidomide) incident (1956). Mothers given drug for morning sickness but babies born with deformed limbs. Need for analysis of long-term. This led to the Declaration of Helsinki (1964): A statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data.

Answer 224

- Discovery: in research lab in academia/pharma shows safety and promise of drug - Pre-clinical: done in vitro in suitable assays, then animal models - CT Phase I: small group of healthy people. Looking at safety. - CT Phase II: larger group. Looking at efficiency and safety. - CT Phase III: much larger group. If there is an existing drug will involve comparison to this. Risk/benefit ratios. - Authorization

Answer 225

- European Medicines Agency (EMA) which includes the EMA Committee for Advanced Therapies - Food and Drug Administration (FDA) in USA - Ministry of Health, Labour and Welfare in Japan To unit these: The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for human use.

Answer 226

Advanced Therapies Medicinal Products Which is either: -A gene therapy medicinal product -A somatic cell therapy medicinal product -A tissue engineered product (also a combination product because it contains cells and scaffold)

Answer 227

- May survive in the tissue potentially for the lifetime of the recipient - May respond to their environment and interact with other cells and humoral factors in ways which are not encounted with other medicines - May have the capacity to replicate or mature in vivo thus evolving their functionality after they have been administered to the patient - May migrate and distribute in tissues or organs other than the one intended - Cells must be free from microbiological contamination and be of sufficient identity, purity and potency - Difficult to sterilize - Must have good storage protocols (master/working cell banks)

Answer 228

No standard set of preclinical tests. The diversity and inherent properties of TE products require a case by case consideration. An overarching set of general considerations available.

Answer 229

``` Since the product has been regulated safely you must adhere to this protocol each time it is made to avoid risks. Since TE is biological, cells can change, age and have genetic changes. In scaffolds, must consider: -Material sourcing Adventitious agents -Toxicity of process additives -Sterility and Sterilization ```

Answer 230

Using embryonic stem cell-derived retinal pigment epithelium patches in Macular degeneration. - Engineering of RPE patch: pluripotent stem cell culture allowed to spontaneously differentiate. RPE cells are exised and expanded and placed on a scaffold. At every stage there are tests of appearance and the karotype. - Preclinical trials: Considerations of safety, functionality, engraftment, immune response in animal models. Was tested in pigs due to human eye similarity. - Clinical trials: Phase I not appropriate to test on healthy volunteers due to surgery, instead this is a very small exploratory trial.

Answer 231

- A tool ensuring that the situation at the start of the trial is identical in different groups studied (the treatment group and comparator) - Every participant has the same chance of receiving the treatment studied - Standardized and indepdent of the investigator - Issues/solutions for TE products - double blind trials are very difficult when administration involves surgery

Answer 232

Important for determining the efficacy. Either one person follows up all the patients or all care givers should be appropriately trained. The trial should be designed to minimize the effect of the procedure on patient care.

Answer 233

When treatment is given before it has been clinical trials. ATMPs exempt from the centralized marketing authorization procedure. Individual case studies based on risk-benefit assessment.

Answer 234

The valley of death is the gap between discoveries in the lab and therapeutic leads that enter clinical trials. Research often doesn't leave academia because big pharma companies are adverse to the big risk. - Financial rewards are not guaranteed - 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 235

1990s Advanced Tissue Sciences made products such as Transcyte and Dermagraft. Funded by enthusiastic researchers, promising market for burns victims and products were praised by doctors. But ATS never made a profit and went bankrupt in 2002. Started clinical trials for Dermagraft in 1991 and applied for FDA approval as a medical devise. Expected approval in 1995 but unanticipated delays meant that FDA did not approve until 2001 - could not recover.

Answer 236

Mistakes: -Regulatory approval and caused delay in releasing product to market -Manufacturing was challenging -Funding was raised to build a manufacturing facility which could not be paid off Lessons: -Management: financial planning, R and D costs, clinical trials costs, strategic focus -Regulators: importance of communication -Investors: reg. med. therapies are high risk investment

Answer 237

- Biological bottlenecks: vascularization and host response, difficult to become functionally integrated - Complex regulatory/commercialisation landscape, less well-established, expensive, risky, lengthy

Answer 238

World's population is rapidly increasing - expected to reach 10 billion by 2050. Meat is the most valuable livestock product since it contains all the essential amino acids, highly biodegradable minerals and vitamins. Incredibly resource-dependent and environmentally taxing. 24% greenhouse gases, 37% landmass, 70% water withdrawal. Animals are farmed close together, giving a breeding ground for diseases and antibiotic resistance. Current meat production is unsustainable - need for engineered food.

Answer 239

Churchill in 1932 proposed that we would be able to grow parts of a chicken rather than growing whole ones and wasting most of it. NASA cultured muscle tissues from the common goldfish in 2002 (Benjamin et al). First lab-grown burger in 2013, PR opened the world's eyes to this possibility.

Answer 240

Myofibres bundle into muscle fibers which bundle into fascicles which are attached to bone via ligaments. Satellite cells are quiescent cells (low-turnover) and regenerate muscles upon injury. In this instance they become activated and proliferate. Give rise to myoblasts which develop into myotubes leading to de novo myofibers. Satellite cells are used in TE because they have the ability to proliferate and have well-established differentiation cues.

Answer 241

Take an adult biopsy from an animal and isolate satellite cells using enzymatic treatment to remove other things. Culture the cells in a proliferative phase. 30 doublings max (not infinite). The medium is not chemically defined and you're still using serum from animals. Then use known signals/factors/transcription factors to induce differentiation into appropriate cell.

Answer 242

- Embryonic or induced pluripotent stem cells: unlimited proliferation but need for differentiation optimization. - Alternative adult stem cells e.g. mesenchymal stem cells

Answer 243

- Edible and non-toxic material - Non-allogenic biomaterial - Supportive of full differentiation/maturation Porous collagen microspheres could be a potential scaffold and can be placed in bioreactors for scale-up. But this does not produce a highly organised structure. 3D printing could solve this problem.

Answer 244

- Scale: global meat production is 293 million tons/year. Requires 1 bioreactor per 10 humans (significant footprint) and culture medium would be vast. - Efficiency: optimisation of scale-up and differentiation. Needs to be cost-effective. - Customer acceptancy: Needs to be as good as current meat. Plus addition of 'healthier' 'reduced fat' 'increased vitamins/linoleic acid' (anti-inflammatory and anti-tumourogenic).

Answer 245

Fat from pre-adipocytes to give taste. Milk from mammary gland cells?! Leather from skin samples taken from animal, induced collagen production, layered sheets fused together, finished, dyed and conditioned (good that it won't have hair).

Answer 246

Severe limitations of current culture systems. - Many cells in vitro lose their in vivo phenotype in 2D culture. - Model organisms are more relevant but don't have single cell analysis tools So organ-on-a-chip technology was developed as a middle ground between experimental tractability and physiological relevance.

Answer 247

``` Device for culturing cells in continuously perfused micro-metersized chambers. Incorporates minimal units that mimic tissue and organ level functions. Incorporates microfabrication (creating the chips themselves) and microfluidics (how fluids behave at a micro-metersized level). Can be made in the lab using polydimethylsiloxane whic begins as a liquid but sets as a solid due to the high temperature, leaving a channel down the middle (silicon mould can be 3D printed for specific structure). Can use channels to create gradients of different molecules. ```

Answer 248

Lung on a chip: already established. Porous membrane with a channel whereby lung cells are fixed and the air flows above them and below is a capillary cell layer and blood flow. The vacuum surrounding the membrane changes the pressure, giving a stretching motion. Mimics the basic functional unit of the alveolus. - Bacteria can be introduced and WBCs move from the blood layer to attack - Pulmonary oedema can be modelled by introducing IL-2 and analysing water retention. Body-on-a-chip systems holds promise to mimic multiple tissues and organs and how they link together and communicate.