By: Sara Anam, Garni Tatikian

Thousands of people die every year waiting for organs from a donor

Mechanical solutions (kidney dialysis, heart-lung bypass machines, etc.) and synthetic solutions (blood vessels, joint replacements) are not as efficient and don’t last as long as the real organ◦

There’s also a risk of infection, rejection, etc.

The ideal solution is to be able to grow our own organs

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Overview of Tissue Engineering ◦

Role of Scaffolds

Anatomy and Physiology of the Bladder

Approaches to Scaffold Design

Introduction to Dr. Anthony Atala’s artificial bladder project

The Process of Decellularization

The Process of Making Pre-Made Porous Scaffolds

Results of Dr. Atala’s Experiment

The Future of Scaffolds and Artificial Bladders

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Function◦

Collect and store urine from kidneys◦

Holds about two cups of urine for 2-5 hours◦

Releases it out to urethra when it is full

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Location◦

Part of the urinary system◦

Inside pelvis behind the pelvic bone

Other Components of the Urinary System◦

Ureters: Tube that allows urine to run from the kidney to the bladder◦

Urethra: Tube that allows the urine to run from the neck of bladder to the exterior of the body◦

Sphincters: Valves that control flow from the urethra

Internal Sphincter/External Sphincter: Regulate storage and emptying of bladder

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Resembles a balloon

Empty Bladder: Small, like a deflated balloon

Filled Bladder: Rounded shape that rises to the abdominal cavity

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Three Layers of the Bladder1. Mucosa

-composed of transitional epithelium (urothelial cells) -contains no blood vessels -lines bladder and ureters -stretches as bladder fills up

2. Submucosa -composed of connective tissue -contains blood vessels, nerves and glands -supplies mucosa with nutrients

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3. Detrusor Muscle◦

Composed of smooth muscle

◦

Expands to store urine and contracts to expel urine

◦

Contraction activated by release of transmitters from motor nerves

◦

Normally remains relaxed to allow bladder to fill

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• Nerves in the spinal cord carry information between bladder and brain

• When a certain amount of urine is in the bladder, internal pressure becomes strong

• Stretch receptors in the bladder wall become activated and send signals to the brain

• This results in the desire to urinate

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• The brain sends a signal to the spine• The spine forwards the signal to the bladder• Small contractile waves occur in the detrusor muscle,

causing the internal sphincter to loosen• The external sphincter will then loosen as well, and the

bladder empties

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Things can go wrong when there is tissue damage

Tissue damage can be caused by trauma, birth defects, neurological diseases and cancer

It usually results in low volume and high pressure in the bladder

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Birth Defects1) Exstrophy

Rare

Abdominal wall fails to close during fetal development

Posterior bladder sticks out of lower abdominal wall

Part of urinary bladder present outside the body

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Birth Defects

2) Myelomeningocele (Neurological Disorder)

Caused in pregnancy when two sides of spinal cord don’t join together

Incomplete development of lower part of the spinal cord and its coverings

Nerves of spinal cord damaged

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Bladder Cancer

Cells multiply and form an area of abnormal cells

Doctors will try to surgically remove the tumour (this will damage bladder wall)

Advanced stages of cancer: Part or all of the bladder will be removed

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Augmentation Cystoplasty◦

Involves tissue grafts

◦

Can use tissue from any organ attached to the bladder ex. intestine, stomach

◦

Can also use synthetic materials

◦

Result: Larger storage capacity and reduced amount of pressure on the bladder wall

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Enterocystoplasty

◦

Most widely used form of cystoplasty

◦

Patch of small intestine is used

◦

Bladder is cut open◦

Patch of intestine used to augment bladder

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The Extra-Cellular Matrix (ECM) is a protein filled structure that surrounds cells

Most cells secrete their own ECM and are anchored in them

ECM varies based on the tissue it’s in

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Provides a physical environment in which cells grow, migrate, and function

Influences the structural and mechanical properties of the cells that grow in it◦

Consider: Bladders must be elastic since they are constantly expanding and retracting

Provides bioactive signals that regulate the activities of the cell

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Acts as both a resevoir and a deliverer of growth factors

Is biodegradable in the event that changes in vascularization and remodeling of the tissue is required

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To develop an organ that’s identical to the one we want to mimic, we should build it in a

scaffold that’s just like the ECM of its tissue!

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Experimented with the :◦

Collagen Matrix Scaffold:

Scaffold Approach: Decellularizing

the bladder’s submucosa tissue

The resulting ECM is very rich in collagen

◦

Collagen-PGA Scaffolds:

Scaffold Approach: Pre-made porous scaffold technique

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•

Process: Remove the cells from an allogenic or xenogenic tissue, leaving behind the ECM

•

Advantages: Scaffold will have the closest imitation of the natural mechanical properties and composition of the ECM

•

Disadvantages:

Incomplete removal of the cells may trigger an immune response

The ECM is damaged during the production processes

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Goal:Complete this process without

negatively affecting the composition, mechanical

properties, and the biological activity of the ECM!

1)

Obtain tissue that contains the desired ECM

2)

Physically separate the unwanted tissue structures from the ECM:

1)

Methods include:1)

Freezing and Thawing2)

Sonication-

agitation of particles using sound energy

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3) The cell remnants are removed from the ECM

4) Optional Step: Tissue is often disinfected and dehydrated

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Dehydration:◦

Loss of Water During Decellularization

Negative Results: Collapse of collagen fibers, changes in degradation rate, and less cellular attachment

Positive Results: Less leaching of growth materials, increased shelf life, and modified strength and mechanical properties

◦

Tradeoff: Sometimes processes like vacuum pressing are used to encourage dehydration despite the negative affects!

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5) The scaffold is terminally sterilized ◦

Methods of sterilization used on all of Dr. Atala’s bladders, including the pre-made porous ones, included UV light and ethylene oxide

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•

Process:

Uses natural and synthetic biomaterials in an attempt to create a scaffold that mimics the ECM

•

Advantages: Lots of control over the materials used and the architecture and microstructure of the scaffold

•

Disadvantages: Time consuming

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Biocompatibility with the cells being grown and the tissue it will be implanted in

Stability (before and after implantation)

Sterilizable

Eventually Dissolves ◦

Products of breakdown can’t be toxic ◦

The degradation rate upon implantation should equal the growth rate of the host tissue

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Large surface area for cells◦

Usually porous

The pores should be large enough to host cells and should be interconnected so that nutrients and wastes can be exchanged between the cells

Should include growth factors, cell adhesive lignands, and certain topography that encourages growth in a particular pattern

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Naturally existing materials such as and collagen and alginate

Concerns:◦

The properties of these material’s can’t be easily modified for specific applications◦

Can enough of this material be harvested?

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The properties of synthetic materials can be modified to suit a particular situation

They can be mass produced

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Consider the Poly (α-

hydroxyl acid) family:◦

PGA ◦

PLLA◦

PLGA

A copolymer of PGA and PLLA

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Advantages of the Poly (α-

hydroxyl acid):

They are FDA approved

They are sufficiently stable

Degradation◦

Degrade when undergoing hydrolysis ◦

Fibers can be modified to control their degradation rate◦

The product of their degradation can be metabolized and excreted by cells

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There are four main techniques for preparing these scaffolds. ◦

Fiber Bonding◦

Solvent Casting/ Particular Leaching◦

Gas Foaming◦

Phase Separation

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Scaffold fibers are immersed in a PLLA solution

The solution is evaporated leaving PLLA covered fibers

The product is heated. PLLA will melt first, filling empty spaces in the fibers.

Once the fibers melt, they won’t collapse. They’ll instead start to weld where fibers cross

The PLLA is removed

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Advantages:◦

Large surface area◦

81% porosity◦

Up to 500 micrometers pores

Disadvantages:◦

Uses toxic chemicals that must be completely removed from the scaffold using vacuum drying◦

Overheating/Toxic agents make it hard to incorporate growth factors during scaffold processing

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Dissolve fibers in chloroform or methylene chloride

Add a water soluble compound (Ex. Salt)

Evaporate the solvent

Place the resulting product in water for 2 days until the salt comes off

The salt particles will create pores in the fiber

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Advantages: ◦

High interconnectivity and biocompatibility ◦

Works well with a large range of cells◦

Amount of porosity can be controlled based on the amount of salt added

Disadvantages:◦

Use of organic materials risks adding pharmacological agents to the scaffold

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Here, gas is used as the porogen

Solid discs of the fibers are placed in chamber

They are initially exposed to CO2 at high pressures

The CO2 pressures drop to atmospheric level over three days

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Advantages: ◦

93% porosity ◦

Pore sizes of up to 100 micrometers◦

No organic solvents involved

Disadvantages:◦

Not enough interconnectivity ◦

Extreme processing conditions prevent incorporation of growth factors into the scaffold during processing

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Technique 1: Liquid-

Liquid Phase Separation◦

Fibers are dissolved in a liquid with a low melting point◦

They are then cooled and vacuum dried

Technique 2: Freeze-

Drying◦

Fibers are placed in a solution of methylene oxide and water

◦

They then undergo freeze-drying

Advantages:

90-95 % porosity

Disadvantages:◦

Use of harsh organic solvents (Technique 1 hasn’t even been tested for biocompatibility)

◦

Technique 2’s pores are too small for effective use, but it’s believed this can be improved

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The Candidates

7 Patients, ranging from 4-19 years of age, were given bladder transplants

All had myelomeningocele

They had poor bladder compliance

Had not responded to other medical treatments

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A bladder biospy sample of 1-2 cm^2 was taken

The urothelial cells and the smooth muscle cells were separated and cultured independently

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Number of bladders

Collagen Matrix

PGA- Collagen Matrix

Omental Covering

3 Yes No No

1 Yes No Yes

3 No Yes Yes

Omental-membrane that lines the abdominal cavity

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Smooth cell muscles were used to line the exterior of the scaffold

The urothelial cells were added to the interior of the scaffold

After being placed in a container with a growth medium, the scaffold was placed in an incubator for 3-4 days

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An incision is made into the midsection of the patients

Dr. Atala augmented the artificial bladder with the native bladder using polyglycolitic sutures and fibrin glue

The omental membrane was added after the suturing process

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Follow up: Ranging from 22 to 61 months

The frequency of urination leakage post-surgery was:◦

1.5 to 3.5 hours for patients with the collagen bladder◦

2.5 to 4 hours for the patient the collagen bladder that was covered with omental wrap

◦

3 to 7 hours for the patients implanted with the collagen-

PGA bladders with omental wraps

The collagen-PGA bladders were deemed the best option

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The patients did not experience metabolic problems

The new bladder performed better than the other surgical options, without all of the negative side effects (ex. mucous production and absorbing material)

Biopsies that the bladder had the proper triple layer structure

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Neo-bladders for Patients With Neurogenic Bladders

Dr. Atala is conducting phase II of his study

Recruited 10 patients with spinal cord injuries and failing bladders◦

The patients are between 3 to 21 years of age◦

Haven’t responded to other medical treatments◦

Haven’t had any other augmenting procedures

Using their neo-bladders to further study and improve this treatment

Objective: ◦

To measure the safety and efficiency◦

To improve the compliance (pressure vs. volume)

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Nanofibers

Fibres within the diameter on the order of nanometers

Has ultrafine continuous fibers and high porosity

Being explored for scaffold building

Similar properties to that of ECM

Better at regenerating tissues

Need to improve mechanical properties to match preferred tissue properties

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(2010). Bladder Problems. November 6, 2010. [http://biomed.brown.edu/Courses/BI108/BI108_2007_Groups/group10/problems.html]

(2010) Bladder Cancer. November 5, 2010. [http://www.emedicinehealth.com/bladder_cancer/page3_em.htm#Bladder Cancer Symptoms]

Bladder Augmentation. November 5, 2010. [http://www.surgeryencyclopedia.com/A-Ce/Bladder-

Augmentation.html]

(2010). Spina Bifida. November 5, 2010. [http://kidshealth.org/parent/system/ill/spina_bifida.html#]

Bladder Augmentation and Incontinence Surgery. November 7, 2010. [http://www.med.wayne.edu/urology/DISEASES/bladderaug.html]

National Cancer Institute: Bladder Cancer. November 8, 2010. [http://training.seer.cancer.gov/bladder/anatomy/layers.html]

Bladder Anatomy and Physiology. November 8, 2010. [http://www.wellbladder.com/bladder_anatomy__physiology]

Bladder. (2000). Bladder. November 07, 2010. [http://www.mamashealth.com/organs/bladder.asp]

The bladder. November 07, 2010. [http://www.cancerhelp.org.uk/type/bladder-cancer/about/the-bladder]

(2009). The Urinary Bladder. November 06, 2010. [http://education.yahoo.com/reference/gray/subjects/subject/255]

S. G. Kumbar. (September 2008). Electrospun nanofiber scaffolds: engineering soft tissues. (November 6, 2010). [http://iopscience.iop.org/1748-605X/3/3/034002/]

58

Dolly, Brandon. Emrich, Adam. Groothuis, Sarah. Halenda, Greg. Jankowski, Tito.

Tissue Engineering.http://biomed.brown.edu/Courses/BI108/BI108_2007_Groups/group10/tissue_engineering.html

The Lancet. (April 15, 2006).

Tissue-engineered autologous bladders for patients needing cystoplasty. November 4, 2010.http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1B-

4JN2NY2&_user=1067350&_coverDate=04%2F21%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin

=search&_sort=d&_docanchor=&view=c&_searchStrId=1527763858&_rerunOrigin=scholar.google&_a

cct=C000051241&_version=1&_urlVersion=0&_userid=1067350&md5=c38e82065d6431b23c665e1d

452a88df&searchtype=a#bib2

Mikos, Antonios; Temenoff, Jeanna;

Formation of highly porous biodegradable scaffolds for tissue engineering. November 5, 2010. http://www.ejbiotechnology.info/content/vol3/issue2/full/5/index.html#29

Atala, Antony; Bauer, Stuart; Soker, Shay; Yoo, James; Retik, Alan. (April 4, 2060)

Tissue-Engineering Autologous Bladders for Patients Needing Cystoplasty. November 5, 2010. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B7GHW-4TK92GS-

1&_user=1067350&_coverDate=01%2F31%2F2009&_rdoc=1&_fmt=high&_orig=search&_origin=searc

h&_sort=d&_docanchor=&view=c&_searchStrId=1527847381&_rerunOrigin=scholar.google&_acct=C0

00051241&_version=1&_urlVersion=0&_userid=1067350&md5=1097ad5eb4673100f0f712a82306fa

b7&searchtype=a

59

Chan, B.P., Leong, K.W.(December

17, 2008).

Scaffolding in tissue engineering: general approaches and tissue-specific consideration. November 5, 2010.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2587658/

Badylack, Stephen. (September 19, 2002).

The Extracellular Matrix as a Scaffold for Tissue Reconstruction.

November 5, 2010. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WX0-46T3XWD-

9&_user=1067350&_coverDate=10%2F31%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=searc

h&_sort=d&_docanchor=&view=c&_searchStrId=1

60