Lecture 3 - Decellularized Scaffold Fabrication

Question 1

Decellularized Extracellular Matrix

Answer 1

• ECM templates already assembled by biological tissues as a scaffold to engineer new tissue carriers with substantial advantages
• Created by removal (or attempted removal) of cells from tissue
• Avoids immunological responses to foreign cells
• Hopefully preserves the majority of ECM components and overall tissue structure
• Ideally preserves growth factors present in original tissue and helps modulate synthesis of these growth factors when appropriate cells are present

Question 2

Harvesting of Decellularized ECM

Answer 2

• Harvested from dermis of skin, submucosa of small intestine, urinary bladder, pericardium, liver and tendon
• Decellularized ECM often used to engineer tissue it is derived from

Question 3

Decellularization Process

Answer 3

Effective process should include:

• Use of reagents that will leave remaining ECM biocompatible and biomechanically stable
• Careful characterization of reagent residuals
• Careful characterization of biocompatibility
• Careful characterization of biomechanical strength of the resulting ECM
• Assessment of function and durability in large animal model
• Validation of manufacturing procedures to reproducibly decellularize the tissue

Question 4

Decellularization Approaches

Answer 4

• Enzyme-based decellularization
• Hypotonic lysis
• Detergent-mediated decellularization
• For entire organ, perfusion decellularization

Question 5

Enzyme-based Decellularization

Answer 5

• Based upon controlling enzymatic degradation of constituent cells so as to not negatively affect the ECM
• Most often employed combination is trypsin and ethylenediaminetetraacetic acid (EDTA)
• Decellularization process: incubate tissue with solution of trypsin and EDTA at 37C, sequential washing of tissue to remove residual agents, followed by cryopreservation of tissue

Question 6

Trypsin

Answer 6

• Utilized during routine cell culture to release adherent cells from bottom of flask
• protease that cleaves proteins that link cells to cells
• Collagen ECM is generally not as susceptible to trypsin degradation

Question 7

EDTA

Answer 7

Employed to inactivate the intracellular proteases that may be released as cells are being trypsinized since these proteases can degrade the ECM

Question 8

Advantages of Enzyme-based Decellularization

Answer 8

• Convenient

- Does not physically destroy the ECM (might be chemical effect)

Question 9

Disadvantages of Enzyme-based Decellularization

Answer 9

• If trypsin concentration too high, can degrade ECM
• If not enough EDTA, may not completely inhibit the proteolytic activity of the intracellular proteases released from cells (3D structure)
• Biomechanical integrity of ECM adversely affected by first two

Question 10

Hypotonic Lysis

Answer 10

Currently utilized to produce decellularized pulmonary allograft heart valves
Decellularization procedure:
- Multiple incubations of tissue in hypotonic solution (tris(hydroxymethyl)aminomethane, MgCl2, CaCl2, dithiothreitol, EDTA) over course of two weeks
- DNAse (enzymes that hydrolyze DNA) and RNAse (enzymes that hydrolyze RNA) incubation of tissue to degrade DNA and RNA, sequential
- Sequential washing of tissue to remove both residual hypotonic solution and DNAse and RNAse
- Cryopreservation of tissue

Question 11

Disadvantages of Hypotonic Lysis

Answer 11

• Decellularization process requires tissue to be in solution for extended period of time. Could result in hydrolysis of collagen fibers that negatively affect biomechanical integrity of tissue
• Lack of agent that facilitates cell remnant removal, could result in cellular remnants being left in tissue, potentially causing inflammatory response
• RNAse and DNAse are usually derived from bovine source and thus there exists the ever-present risk of prion disease transmission

Question 12

Detergent-mediated Decellularization

Answer 12

• Detergents can penetrate into ECM and cell membranes

- Two main classes of detergents: non-ionic and anionic

Question 13

Non-ionic Detergents

Answer 13

• Triton X-100

- Lack sufficient detergent strength to fully decellularize tissues

Question 14

Anionic Detergents

Answer 14

• Stronger solubilizing agents than non-ionic detergents due to charged moiety present on their hydrophilic head
• Ex: sodium dodecyl sulphate (SDS) —> may denature proteins if concentration too high, may reduce biomechanical strength
• Ex: N-lauroyl sarconisinate (NLS) —> does not denature ECM, possesses bactericidal properties which have led to use in toothpast, cosmetics, and shampoo, can be viewed as another means by which tissue can be further sterilized

Question 15

Denature

Answer 15

• Losing biological activity

- Gelatin is denatured form of collagen

Question 16

Disadvantages of Detergent-mediated Decellularization

Answer 16

• If correct detergents not selected for decellularziation process, will result in incompletely decellularized ECM (non-ionic detergents)
• Use of overly aggressive detergents (SDS) may negatively affect biomechanical integrity of tissue

Question 17

Application of Decellularized ECM

Answer 17

Can be used either as an implant (directly implanted in body) or as a tissue engineering scaffold to populate cells inside

Question 18

Decellularized ECM as Implant

Answer 18

• Decellularized porcine heart valves and human dermis currently used
• Decellularized porcine ECMs have been successfully used for repair of numerous tissues including lower urinary tract reconstruction, vascular reconstruction, and repair of full and partial thickness skin wounds

Question 19

Decellularized ECM as Engineering Scaffolds

Answer 19

• Cells seeded in decellularized scaffolds aiming to repopulate scaffolds