College 1 – “An introduction to Tissue Engineering” – 22nd of November 2012



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Stem cells


Definition - Undifferentiated cells with the potential to differentiate and generate a large number of mature cells of one or more lineages.

Features:

  • An unlimited replication potential (for self-renewal)

  • Morphologically indistinct - have a high nucleus/cytoplasmic ratio

  • Commitment to differentiate in culture, slow at first, then at a rapid rate (maximum doubling time 12-14 h.) (also slow; all sorts of tissues)

  • Stem cells systems in rapidly proliferating tissues e.g. skin, bone marrow but also in organs with slow rates e.g. liver, b-islet cells

  • Stem cell commitment initiates organ function, repair and genesis

Sources:

  • Some adult tissues

    • Isolation and growth of stem cells from adult tissues

    • Stem cells found in certain tissue types e.g. bone marrow and peripheral blood, hair, skin, adipose tissues etc.

    • Resulting stem cell lines thought to be capable of differentiating into limited range of tissue - recent research suggests otherwise
       now: push the differentiated cells push them back into an undifferentiated state. Differentiation pathways are directed by specific growth factors (the interactions between the cell and the environment are quite complicated to interpret).

    • Resulting tissue may be genetically compatible or not (donated)

    • Successful transplantation of some stem cells e.g. from bone marrow, has been possible for some years

    • No specific legal restrictions

    • General legal provisions on removal and use of human tissue apply

 Potential from an adult cell is less than from an embryonic cell.

  • Some foetal tissues

    • Cells from aborted foetuses or umbilical cord blood of newborn babies

    • Tissue sources are readily available and current use is restricted only by the need for consent from the mother

    • Tissues rich in stem cells e.g. liver can be extracted and cells successfully grown and concentrated in the laboratory

    • Resulting stem cell lines capable of differentiating but may only be capable of forming some types of tissues but not others
       totipotent  pluripotent  multipotent.

    • Resulting tissue not genetically compatible with the subject being treated unless cord blood stored at birth for future use

    • No specific legal restrictions on use of foetal tissue or cord blood

    • Some regulations which require Research Ethics Committee approval

  • Ethical/religious issues

  • Great potentials!

  • Umbilical cord blood

  • Early embryos
    –created by in vitro fertilisation (IVF)

  • Reprogrammed adult cells (theoretical)

–using cell nuclear replacement techniques
Summary Embryonic Stem Cells

  • ES cells are developmentally transient in the embryo since they only can be generated from 5-7 cay old blastcyst.

  • Biological function lies in development to construct the whole body.

  • They are capable of undergoing an unlimited number of symmetric divisions.

  • ES cells are pluripotent: they can differentiate in every germ layer, tissue or cell.

  • ES cells are capable of colonizing the germ line and giving rise to eggs or sperms.

  • Clonogenic properties: a single cell can give rise to a colony of genetically identical cells, or clones, which have the same properties.

  • ES cells express specific markers.



  • Grown on feeder layers, thus a risk of viral infection exists: also clean separation of animal cells and human technically not solved: however, new techniques exist which allow growth of ES cells without feeder layer.

  • Unlimited proliferation potential after transplantation: risky, since it could result in cancerous growth.

  • People claim that ES cells are a cell culture artifact since there is no natural role for them in regeneration.

  • Are the biological progenitors of adult stem cells but relationship between ES cells and adult stem cells is not clear.

  • ES cells are ethically controversial.

  • There is a reasonable hope that ES cells could be used in clinical applications.


College 3 – “Skin” – 13th of December 2012


Anatomy of skin:
Skin has two layers:

  1. The epidermis (the outer, thinner layer):

    1. Cell types

      1. Keratinocytes (about 90% of total cells)
        (Corneocytes  after keratinocytes go to the outer layers, located in strata corneum)

      2. Melanocytes
         Produce melanin, which colours cells (pigment)

      3. Langerhan cell – immune response

      4. Merkel cells – touch sensitive

    2. Structural features

      1. Five layers (strata)
        Strata -basale, -spinosum, -granulosum, -lucidum and –corneum
         from inside to outside the body

      2. Avascular and alymphatic

      3. No nerve endings

    3. Epithelial tissue

      1. Keratin

      2. Melanin

      3. Lipids

  • The thickness of the epidermis is normally 200μm, but at the hands and feet 2-3mm.
    Best is to measure the skin thickness with ultra sound.

  • Interacts with the outer-world.

  1. Epidermal-Dermal junction
     Molecule transport is possible over the junction, as is information transport.
     The junction is wavy.

  2. The dermis (the inner, thicker layer):

    1. Cell types

      1. Fibroblasts / myofibroblasts

      2. Microvascular endothelium
         to produces blood vessels.

    2. Structural features

      1. Blood and lymph vessels

      2. Hair follicles, serbaceous glands and sweat glands

      3. Nerve

      4. Papillary dermis and Reticulum dermis
        Papillary dermis is positioned above the reticulum dermis. The change between the two dermises is graduate.

    3. Extracellular matrix

      1. Extracellular water

      2. Collagen

      3. Elastin

      4. Proteoglycans
        Glycosaminoglycans (GAGs),
        hyaluronan, dermatin sulphate, chondroitin-6-sulphate, heparin sulphate
         A lot of water is in the tissue because of the interaction between the sulphate-groups and the hydrogen atoms of water.

Function of Skin

  • Regulation of body temperature (homeostasis)

  • Sweating

  • Changes in flow of skin blood flow

  • Burning wounds will give problems with the cooling down of the body.
    (Most burning wounds with people younger 10 years old or elderly people.)

  • Protection of underlying tissues/organs

  • Physical barrier against abrasion, bacterial invasion (chemical), dehydration and UV radiation

  • Hairs and nails also offer protection

  • Total area of skin is 2 m2.

  • Sensation

  • Excretion

  • Small amounts of water, salts and organic compounds are excreted via sweat glands

  • Immunity

  • Langerhan cells fend off foreign invaders of the body
     Langerhan cells will set up antibodies.

  • Synthesis of Vitamin D

  • Initiated by UV exposure – aids in the absorption of Ca &P from the GIT to the blood

Mechanical Properties of Skin

  • Testing modalities

Tension, Biaxial, Torsion, Shear and Compression Suction testing

  • In vivo versus in vitro testing
     In natural state the skin is under pretension (due to elastin and collagen (critical orientation).
    While testing the skin should be stretched and kept moist.

  • Directional aspects – Concept of Langer lines
     In natural state skin is anisotropic, this is because the collagen fibers have a preferred direction.
     The surgeon will cut the patient so that the wound will rather close than open (Langer Lines).

  • Changes with age – increase in collagen cross- linking
     The skin will stiffen with age due to this, also because the elastin production decreases.


Specific Wound Types

  • Acute
    – Elective wounds
     patient has chosen for it (e.g. surgery)
    – Surgical wounds - generally repair
    – Burns - due to their potential mortality -TE an obvious option but market is not predictable ?
     Major burns >20% BSA (body surface area)
    Severe burns >60% BSA
     Treatment options: surgical skin transplantations involving split skin graft and mesh
    autografting.
    - Often results in scar formation and wound contraction – hence poor cosmesis and limited
    joint mobility (functional effect).
     Burn wounds can extend through the epidermis into the dermis. Therefore the ideal TE
    product would act as a total skin equivalent (comprising both epidermis and dermis). If we
    want to develop engineered skin; fibroblasts and keratinocytes should inserted.

  • Chronic
    – Venous leg ulcers and arterial ulcers
     Venous ulcers are caused by venous return (not pressure!).
     Arterial ulcers are due to a lack of supply.
    – Diabetic ulcers
     Diabetic people don’t feel pain in e.g. feet, that is why they get these ulcers.
    – Pressure ulcers (doorligplek)
     Their incidence increase with age and represent the future goal of TE technology
     10% of the people in a hospital get a pressure ulcer. In 2009 in the USA a law has been
    introduced that obliges the hospital to give a compensation if the patient gets a pressure
    ulcer. So it is getting more important in the programs.
     It can go up to the bone and the wounds are smelly.
     Patients can die from it (Actor Superman).


Potential Approaches to TE

  1. Epidermal replacements - consisting of keratinocytes grown either alone, on the surface of a tissue culture flask) or in close association with a carrier vehicle such as a polymeric film or bioresorbable matrix

  2. Dermal replacements - consisting of a structure able to support infiltration, adherence, proliferation and neomatrix production by fibroblasts and possible endothelial cells.
  3. Skin substitutes - a combination of 1 and 2.




Formation of Support Structures

  • support cell ingrowth

  • provide a suitable substrate for adherence

  • facilitate cell proliferation and production of ECM

  • resorb from wound site in a controlled manner
     or breakdown with time

  • minimal toxicity

  • low immunogenecity

  • mechanical properties similar to uninjured tissues

  • Candidate materials - collagen (bovine or porcine sources) fibrin, fibronectin, chitin/chitosin, chondritin-6-SO4, basement membrane proteins, hyaluronan, PLA, PGA
     a lot of proteoglycans

  • Natural and synthetic tissues are used by the companies.


Historical Perspective

  • Eugene Bell found that fibroblasts could infiltrate a collagen gel and turn it into a fibrous living matrix. (also by diffusion)

  • Yannas and Burke developed a dermal component of bovine dermal type I collagen crosslinked with C-6-S (sulphate) on a silicone backing sheet.

  • Handsborough noted that when allogenic fibroblasts were seeded into a PLA/PGA matrix, many components of ECM are synthesised including collagen (types I,III,IV), elastin, fibronectin and decorin.

Commercial Perspective

  • The Organogenesis story

  • A skin equivalent construct, Apligraf TM

  • Transcyte TM and Dermagraft TM

  • See the articles.

  • One offered their product too cheap  not enough profit
    One too expensive
    Both their first attempt did not exceed to make profit.



Professors Harry Navsaria and Irene Leigh (Queen Mary)


 Skin Tissue Engineering Myth or Reality?
 Can we produce quick epidermal damage?

Improvements in Keratinocyte Technology

  • Culture conditions

  • Serum free media
     Culture media is critical (expensive…).

  • Exclusion of xenogenic material

  • Delivery systems (pre-confluent)

    • membranes ( hyaluronan, Collagen, PLA etc)

    • microcarriers / beads
       Where cells grow on outside.

    • Sprays
       force should not kill the cells, controlled spray technic is needed.

Convential keratinocyte grafting: wait untill 100% confluence is totally covered.


Pre-confluent keratinocyte grafting: not directly grown on the glass but on a film, wait untill 70% full. So it is a short-time-process.
How do you evaluate wound product?

  1. Take on the back of a pig the skin away (pigs of the same breath and age).

  2. Put the engineered skinmodel (autologous cells) back in the hole.

  3. Look at how the hole will heal.

Results after 6 weeks:


7 groups: 3 pairs and 1 single.
Conclusions: 1. Medium of group 1 is the highest  Keratinocytes and dermis have an interaction.

2. It is big range.

Study with allogeneic dermis: the effec of using allogeneic dermis is less significant.


  • Fromation of neo-dermis and vasculatrisation is complete only takes places under areas of epthelial cover.

  • Normal innervation is achieved only in the presence of epidermis.

  • Optimal attachement, proliferation and differentiation is only oissible in the presence of dermis.


Hyaluronan (Hyaluronic acid) – Fiddia (Italian company)

  • Endogenous part of extracellular matrix

  • High levels in foetal tissue
     we are trying to imitate what happens in the embryo.

  • Chemotactic to mesenchymal cells

  • Increases collagen deposition in vivo

  • Pro-angiogenic

  • Enhances Cultured Epithelial Autograft (CEA) take

  • Tested on the back of pigs. The results showed that the product enhances the wound healing to be more quickly. In the section of the wound bed could be seen that there were more fibers and epidermal-dermal junction in the HA-treated wound than in the control-group after 6 weeks.

Do allogeneic fibroblasts survive transplantation?


After 7 days in vivo, less than 0.01% of cell population derived from allogenic fibroblasts. Is it usefull? We do not know, but there is evidence that they are usefull in the initial kick off.
Post-Grafting Complications

  • Globally 6 million patients require extensive grafting

  • Contraction occurs with conventional skin grafts

  • Estimated 30% of all conventional skin grafts (thin split thickness skin grafts) for extensive thickness burns injuries or traumatic skin loss

  • Prevention by short-term immobilisation, splinting of grafts and wearing of pressure garments (worn for > 1 year)
     Compression to try to minimize the contraction.

  • May be preconditioning will help.


Scars:

  • Scar Prevention

    • Potential problem in tissue engineering

    • Interfacial problem - integration of graft with minimal scarring at the edges e.g. wound repaired with graft pieces - resembles “an array of postage stamps” with excess scarring at the interfaces.

      • You want to integrate the TE product with the healthy tissue for interaction.

  • Wound healing
    Scarring is an overproduction of wound tissue, too much remodeling on the edge of the tissue.

    • Chronic wounds - fail to heal

    • Hypertrophic scarring - important in severe burns

    • Keloids - important in minor injuries where scar tissue outgrows the boundary of the injury. Very common in Afro- Caribbeans, Chinese and Japanese populations.

  • Extent of Problems involving Scarring

    • Skin - surgery, bites and associated with burns

    • Eyes - chemical burns/ blunt trauma the production of connective tissues that are opaque to the cornea.
       overproduction of collagen so it is not transparent anymore, this will lead to blindness.

    • Adhesions - gut, intestine and tendons

    • CNS (central nerve system) injury - glial scarring can prevent reconnections of nerve endings

    • Fibrotic disorders e.g. liver sclerosis

      • E.g. overproduction will stop normal movement and so the functioning.

  • Traditional Therapy for Scarring (Largely palliative)

    • Nursing

    • Compressive bandaging and garments

    • Oils and massage

    • High pressure water

  • TGFβ’s Role in Wound Healing
    (Transforming Growth Factor)

    • Seconds after wounding there is a release of TGFβ’s, predominantly TGFb1 from stores in degranulated platelets. This release is independent of signals associated with gene transcription and translation.

    • TGFβ’s are chemotactic to:

      • Endothelial cells stimulating angiogenesis

      • Macrophages leading to the release of more TGFβ and other cytokines …..

      • Fibroblasts stimulating ECM synthesis and inhibiting degradation.

    • TGFβ 1 and 2 are more common in adult tissue, but in foetal tissue TGFβ 3 is most common and less TGFβ 1 and 2 are present.
      In foetal tissue the wound always heals up, therefore to minimize scarring TGFβ3 is needed.
      Also evidence from experimental data (he even tested it on himself)

  • Current direction:

    • Genetics of Scarring

      • Use of knock-out mice( with over/under expression of cytokines/growth factors) which help to identify candidate polymorphisms/ genes which render susceptibility to keloid scarring

      • Examine DNA of families with established scarring
         is it genetic??

      • Tissue profiling of chronic wounds, scars – mRNA technology

    • Tissue Engineering
      The development of an anti-scarring therapy in association with TE products

  • We will also have to look at post effects.



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