Tissue engg.

TISSUE ENGINEERING

Arushe Tickoo

B.Tech Biotech

Tissue Engineering

Tissue Engineering is the in vitro development (growth) of tissues or

organs to replace or support the function of defective or injured body

parts.

Tissue Engineering is using a persons cells to create a new artificial

fully alive tissue or organ that can replace or improve/heal the old

one in the body.

Principle

1. Tissue engineering implies the addition of suitable cell types to a

suitable support matrix, through which an organised and functional

tissue is formed (resembling the tissue engineering)

2. Epithelial and endothelial cell layers organize themselves easily in

vitro, but connective tissues do not form appropriate structures

spontaneously.

3. Simple concept: Building material (e.g., extracellular matrix or

biodegradable polymer), seed it with living cells and bathe it with

growth factors. When the cells multiply, they fill up the scaffold and

grow into three-dimensional tissue, and once implanted in the body,

the cells recreate their intended tissue functions.

Initially approved by FDA for treatment of venous leg ulcers, later on in 2000 for

treatment of diabetic foot ulcers.

Apligraf contains two types of cells – an outer layer of protective skin cells, and

an inner layer of cells contained within collagen. Both types of cells contain

substances similar to those found in human skin. Apligraf does not contain

certain things in skin such as hair follicles, sweat glands or blood vessels.

Pro-osteon is a coralline hydroxyapatite, bone like graft used to fill defects in

bone.

In 1960’s artificial skin was being used to treat burn victims, later on modified

into synthetic fibres for artificial skin grafts.

1n 1990’s pro-osteon coral derived bone graft material was introduced and 1996

integra’s was approved for as an tissue regeneration product.

In 1998 “Apligraf ” approved for treatment of ulcers,

PRO OSTEON

Pro Osteon

Implant 500

Apligraf

(artificial

skin)

Cell substrate and Support material

Nature of the support material depends on the information and

suitability for the adherent cell types.

It is divided into 5 broad classes:

Traditional

Abiotic

materials

(metals and

ceramics)

Bio- prostheses

Natural

materials are

modified to

make them

biologically

inert

Synthetic

Resorbable

polymers are

used

Semi-natural Natural

polymersNatural

material are

conjugated

with synthetic

material

Biomolecule

s such as

proteins and

polysacchari

des are used

BIOINERT RESORBABLE BIOACTIVE

Support material

Bio-Compatibility

1. No material can be totally inert when implanted but the group known as “bioinert”

only provoke the formation of scar tissues (eg: stainless steel in artificial hips)

2. Resorbable materials dissolve when implanted with generation of harmless

dissolution products (eg: polymers like PLLA using suturing)

Poly(L-lactic) acid is a biodegradable thermoplastic a aliphatic polyester derived

from renewable resources, such as corn starch (in the United States), tapioca roots, chips

or starch (mostly in Asia), or sugarcane.

3. Bioactive material stimulate a biological response from the body (eg: synthetic

hydroxyapatite ceramics and bioactive glasses.

Bioprostheses

1. It is formed by the extensive cross-linking of natural tissue eg: porcin heart

valves and tendons

2. These are designed and fabricated primarily to function as long as possible

independently and without modification by surrounding tissue

eg: collagen based connective tissue stabilized by glutaraldehyde, can survive

unchanged for many years.

Traditional support materials are not used because they do not

integrate within reasonable period.

Prosthetic heart

valves

1. Resorbable polymers are used which are hydrolysed and then

phagocytosed, the greatest advantage of such material is their easy

and cheap production in a controllable & reproducible manner at

large scale.

2. Less compatibility than natural polymers

3. Synthetic polymers used are PGA [poly(glycolic acid)],

PLA[Poly(l-lactic acid)], polycarbonate, polycaprolactone.

4. PLA, PGA and PLGA[poly(lactic-co-glycolic acid) are most widely

used, PLA is amorphous and hydrophobic degrading to release lactic

acid.

Semi natural & natural substrate

Natural macromolecules are cross-linked polysaccharides,

chemically cross-linked polysaccharide is mammalian

hyaluronan, stabilized by benzyl esterification of increasing

number of side chains.

Collagen sponges are also used, prepared from various insoluble and

aggregated collagen eg: collagen scaffold in tubular shape, with smooth

muscles and endothelial cells

Tissue engineered

heart valve

Tissue engineered

vascular graft

Types of cells

1. Autologous cells are obtained from the same individual to which

they will be re-implanted. Autologous cells have the fewest

problems with rejection and pathogen transmission, however in

some cases might not be available

2. Allogeneic cells come from the body of a donor of the same

species.

3. Xenogenic cells are these isolated from individuals of another

species. In particular animal cells have been used quite extensively in

experiments aimed at the construction of cardiovascular implants.

4. Syngenic or isogenic cells are isolated from genetically identical

organisms, such as twins, clones, or highly inbred research animal

models

Stem cells are undifferentiated cells with the ability to divide in culture and

give rise to different forms of specialized cells. According to their source

stem cells are divided into "adult" and "embryonic" stem cells, the first class

being multipotent and the latter mostly pluripotent; some cells are

totipotent, in the earliest stages of the embryo.

Scaffolds

Cells are often implanted or 'seeded' into an artificial structure capable of

supporting three-dimensional tissue formation. Scaffolds usually serve at

least one of the following purposes:

1. Allow cell attachment and migration

2. Deliver and retain cells and biochemical factors

3. Enable diffusion of vital cell nutrients and expressed products

Allow the manipulation of

cells to form as correctly

shaped

Structures that are able to

support 3-D cell structures

Scaffold

Stem cells

Undifferentiated cells with ability to divide in culture & give rise to

different forms of specialized cells.

Characteristic Features:

1. They are capable of dividing & renewing themselves for long

periods

2. They are unspecialized

3. They can give rise to specialized cell types.

Pluripotent

cellsTotipotent

cells

Multipotent

cells

Stem cells could be:

1. Embryonic stem cells

2. Adult stem cells

Embryonic stem cell lines are cultures of cells derived from epiblast

tissue of inner cell mass of a blastocyst or earlier morula stage

embryos — approximately 4 to 5 days old in humans & consisting of

50–150 cells. ES cells are pluripotent & give rise during

development to all derivatives of 3 primary germ layers:

1. ectoderm,

2. endoderm &

3. mesoderm.

Embryonic stem cells

Totipotent stem cells can differentiate into embryonic & extra embryonic

cell types. Such cells can construct a complete, viable organism. These cells

are produced from fusion of an egg & sperm cell. Eg: Fertilized egg

Multipotent stem cells give rise to a limited range of cells within a tissue

type. Eg: Hematopoietic stem cells

Pluripotent stem cells are descendants of totipotent cells & can

differentiate into nearly all cells, but cannot give rise to an entire

organism. i.e. cells derived from any of three germ layers

Unipotent cells can produce only one cell type, their own, but have the property

of self-renewal, which distinguishes them from non-stem cells. E.g. muscle stem

cells.

Stem Cells (Hematopoietic)

Mesenchymal

Mesenchymal stem cells, or MSCs, are multipotent stromal cells

that can differentiate into a variety of cell types, including:

osteoblasts (bone cells), chondrocytes (cartilage cells), and

adipocytes (fat cells).

There are three basic steps in tissue engineering.

1. The first step is actually getting the base cells to work with.

2. The second step is putting the altered cells into a scaffold in order to

incubate the cells.

3. The final step is to put the newly created cells or organ into use.

STEPS INVOLVED:

.

TISSUE ENGINEERING

95% of the cells in the epidermis are keratinocytes. These cells are

found in the basal layer of the stratified epithelium that comprises

the epidermis, and are sometimes referred to as basal cells, or basal

keratinocytes.

Tissue Engineered Skin

Tissue engineering of skin became feasible in 1975 with the

demonstration that sheets of human keratinocytes could be grown in

the laboratory in a suitable form for grafting. This was a simple,

cohesive sheet of cells cultured from the donor on a feeder layer of

fibroblasts .

The epithelial component is able to regenerate in culture, since the

cells grow as a continuous sheet over a suitable surface, producing a

continuous layer which progresses to form cornified layers.

Keratinocytes cells in skin

Though both scar tissue and normal skin are made with collagen

proteins, they look different because of the way the collagen is arranged.

In regular skin, the collagen proteins overlap in many random directions,

but in scar tissue, they generally align in one direction. This makes the

scar have a different texture than the surrounding skin. Scar tissue is also

not as flexible as normal skin, and does not have a normal blood supply,

sweat glands, or hair

Various forms of implantable skin substitutes

1. Integra consists of insoluble bovine collagen type I and the

glycosaminoglycan chondroitin sulfate. This can be covered in a

keratinocyte sheet at the time of implantation.

2. Dermagraft consists of PGA polymer mesh of suitable pore size,

seeded with human dermal fibroblasts from neonatal foreskins.

3. Apligraf consists of human dermal fibroblasts seeded into a type I

collagen gel and allowed to contract under tension.

Artificial skin

Human urothelial cells and bladder smooth muscle cells can be

cultured.

The criteria are that the final structures need to form elastic tubes

or bladders, and the implant should not allow crystal formation

from urine or harbour local infections.

Support materials tested have included resorbable polymers

[polyglycolic acid) and poly(lactic-co-glycolic acid) co-polymer:

PGA and PLGA and cross-linked collagen sponges.

Urothelial and bladder muscle cells seeded onto PGA scaffolds

formed urothelium-like, vascularized bilayered tissues when

implanted

Tissue Engineered Urothelium

Tissue engineered

vascular graft

Engineered bladder tissue

ARTIFICIAL BONE GRAFTS: PRO OSTEON

Safe, strong, and cost effective bone grafts are now performed

using synthetic material known as Pro Osteon Implant 500.

It is sterile, biocompatible (meaning the body’s immune system

does not reject it), and it is easily sculpted to fill a defect in

fractured bones.

Pro Osteon mimics the internal structure of human bone. This

synthetic material is made by subjecting a common, non-decorative

form to coral to a patented chemical process which converts the

coral to hydroxyapatite, the same mineral content of human bone.

The porous, interconnected structure of the coral remains intact,

providing an ideal matrix through which new bone tissue can grow.

Using Pro Osteon on a long bone, the surgeon determines the amount of bone

graft he needs and shapes the Pro Osteon block to fit into the damaged area.

The graft area is stabilized with a metal plate, screws, or some other form of

internal fixation.

pro-osteon

complex

Bioengineered Tissue Implants Regenerate Damaged Knee Cartilage

ScienceDaily(July 5, 2006)

Cartilage was removed from 23 patients with an average age of 36 years.

After growing the cells in culture for 14 days, the researchers seeded them

onto scaffolds made of esterified hyaluronic acid, grew them for another 14

days on the scaffolds, and then implanted them into the injured knees of the

study patients.

Cartilage regeneration was seen in ten of 23 patients, including in some

patients with pre-existing early osteoarthritis of the knee secondary to

traumatic injury. Maturation of the implanted, tissue-engineered cartilage was

evident as early as 11 months after implantation

Tissue engineered organs

(A)(B)

(C)

(A): tissue engineered heart valve

(B): tissue engineered vascular graft

(C): tissue engineered human ear

Tissue engineered

artificial skin

Ear growing on the

mouse