By: Sara Anam, Garni Tatikian

Transcription

1 By: Sara Anam, Garni Tatikian

2 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 2

3 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 3

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5 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 5

6 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 6

7 Resembles a balloon Empty Bladder: Small, like a deflated balloon Filled Bladder: Rounded shape that rises to the abdominal cavity 7

8 Three Layers of the Bladder 1. 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 8

9 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 9

10 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 10

11 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 11

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13 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 13

14 Birth Defects 1) 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 14

15 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 15

16 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 16

17 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 17

18 Enterocystoplasty Most widely used form of cystoplasty Patch of small intestine is used Bladder is cut open Patch of intestine used to augment bladder 18

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20 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 20

21 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 21

22 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 22

23 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! 23

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25 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 25

26 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 26

27 Goal: Complete this process without negatively affecting the composition, mechanical properties, and the biological activity of the ECM!

28 1) Obtain tissue that contains the desired ECM 2) Physically separate the unwanted tissue structures from the ECM: 1) Methods include: 1) Freezing and Thawing 2) Sonication- agitation of particles using sound energy 28

29 3) The cell remnants are removed from the ECM 4) Optional Step: Tissue is often disinfected and dehydrated 29

30 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! 30

31 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 31

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33 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 33

34 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 34

35 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 35

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37 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? 37

38 The properties of synthetic materials can be modified to suit a particular situation They can be mass produced 38

39 Consider the Poly (α- hydroxyl acid) family: PGA PLLA PLGA A copolymer of PGA and PLLA 39

40 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 40

41 There are four main techniques for preparing these scaffolds. Fiber Bonding Solvent Casting/ Particular Leaching Gas Foaming Phase Separation 41

42 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 42

43 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 43

44 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 44

45 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 45

46 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 46

47 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 47

48 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: % 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 48

49 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 49

50 A bladder biospy sample of 1-2 cm^2 was taken The urothelial cells and the smooth muscle cells were separated and cultured independently 50

51 Number of bladders Collagen Matrix PGA- Collagen Matrix 3 Yes No No Omental Covering 1 Yes No Yes 3 No Yes Yes Omental-membrane that lines the abdominal cavity 51

52 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 52

53 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 53

54 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 54

55 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 55

56 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) 56

57 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 57

58 (2010). Bladder Problems. November 6, [ (2010) Bladder Cancer. November 5, [ Cancer Symptoms] Bladder Augmentation. November 5, [ Augmentation.html] (2010). Spina Bifida. November 5, [ Bladder Augmentation and Incontinence Surgery. November 7, [ National Cancer Institute: Bladder Cancer. November 8, [ Bladder Anatomy and Physiology. November 8, [ physiology] Bladder. (2000). Bladder. November 07, [ The bladder. November 07, [ (2009). The Urinary Bladder. November 06, [ S. G. Kumbar. (September 2008). Electrospun nanofiber scaffolds: engineering soft tissues. (November 6, 2010). [ 58

59 Dolly, Brandon. Emrich, Adam. Groothuis, Sarah. Halenda, Greg. Jankowski, Tito. Tissue Engineering. The Lancet. (April 15, 2006). Tissue-engineered autologous bladders for patients needing cystoplasty. November 4, JN2NY2&_user= &_coverDate=04%2F21%2F2006&_rdoc=1&_fmt=high&_orig=search&_origin =search&_sort=d&_docanchor=&view=c&_searchstrid= &_rerunorigin=scholar.google&_a cct=c &_version=1&_urlversion=0&_userid= &md5=c38e82065d6431b23c665e1d 452a88df&searchtype=a#bib2 Mikos, Antonios; Temenoff, Jeanna; Formation of highly porous biodegradable scaffolds for tissue engineering. November 5, Atala, Antony; Bauer, Stuart; Soker, Shay; Yoo, James; Retik, Alan. (April 4, 2060) Tissue-Engineering Autologous Bladders for Patients Needing Cystoplasty. November 5, &_user= &_coverDate=01%2F31%2F2009&_rdoc=1&_fmt=high&_orig=search&_origin=searc h&_sort=d&_docanchor=&view=c&_searchstrid= &_rerunorigin=scholar.google&_acct=c &_version=1&_urlVersion=0&_userid= &md5=1097ad5eb f0f712a82306fa b7&searchtype=a 59

60 Chan, B.P., Leong, K.W.(December 17, 2008). Scaffolding in tissue engineering: general approaches and tissue-specific consideration. November 5, Badylack, Stephen. (September 19, 2002). The Extracellular Matrix as a Scaffold for Tissue Reconstruction. November 5, &_user= &_coverDate=10%2F31%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=searc h&_sort=d&_docanchor=&view=c&_searchstrid=1 60