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Tissue Engineering and Regenerative Composite Scaffolds, Common…
Tissue Engineering and Regenerative Composite Scaffolds
Basic Terms
Extracellular Matrix
ECM
can be produced artificially or through decellularization of living tissue
artificial Scaffolds do not support bioactive support
a structure that can be populated with cells to create a tissue or organ to be transplanted
a type of scaffold
Decellularization
The removal of cells from tissue to leave behind the structural support of a tissue
Methods
physical
can damage scaffolding
mechanical fragmentation
pulverizing
Freezing and thawing cycles
more pores form from the formation of ice
osmotic
chemical
sodium dodecyl sulfate
SDS
sodium lauryl sulfate
Ethylenediamine Tetraacetic Acid
EDTA
Triton X-100
Tris-HCl
Standard Chemicals
Sodium hydroxide
ethanol
guanidine hydrochloride
sodium acetate
Hydrogen Peroxide
Enzymes
DNases
RNases
Xenogeneic
Comes from an animal
Can carry risks for human use currently
residual immunogenicity
contaimination from biological agents
Used mostly for running pre human trails
Allogenic
comes from humans
most ideal
tissues and organs can be acquired through donation
Recellularization or repopulation
reintroducing cells from a person into the scaffolding to make a viable tissue/organ
Three methods
seeding
Adhering cells to a surface causing them to spread out along the surface
Methods vary between suspended and adherent cells
injecting
using a needle to inject cells into a scaffold
profusion
using the circulatory or lymphatic system to deliver fluid
Tissue Autographs
come from patient
gold standard
can have many limitations
led to the need for allternatives
in vivo
inside of a living organism
in vitro
outside of a living organism
artificial environment
Proof of concept
Acellular Skin
Common and Commercially available
Current goals
reduce risk of immune response
not cause an infection
succeed in a high rate of decellularization or removal of DNA
keep scaffolding intake
high rate of recellularization (for most types)
met demands for transplants or grafts
make process efficient and take a short period of time
methods by tissue/organ type
Cartilage
Need for: disease, trauma, aging degeneration, and congenital alteration
Difficult to accomplish
successes in articular, meniscal, laryngeal, tracheal and nasoseptal
Three Types
Hyaline
various methods being studied for optimization
Best protocol determined out of 24
FT cycles followed by hypotonic buffer (tris-base, pH 8), 0.1 M HCl, 1 g/L pepsin and peracetic acid
multisteps
may not guareentee complete cellular removal
sometimes physical means that damage the scaffold to increase surface area
reduces exposure to decellularization agents
preserve microstructure
increased permeation of agents
Cartilage must be reconstructed afterwards
Ex. After being in 10% acetic acid solution (2.5 M) to homogenize. It was freeze dried and lyophilized the ECM suspension.
It then can be crossed linked
Larynx
setup similar to Trachea with detergent enzymatic method
4% sodium deoxycholate, dH2O, 2000 kunitz units (KU) Dnase-1 in 1 M NaCl followed by washes of dH20
Changes in Protocols
Dnase treatment. Then Freezed in liquid nitrogen and rinsed in solution of 2 mM Cacl2 and 1.3 mM MgSO4.
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Dorsal Augmentation
previously used costal cartilage (connects sternum to ribs)
use human derived nasal cartilages (Cadaver)
osmotic treatment in dH20 and standard chemicals
no enzymatic digestion
cost effective and easy to produce
best proposed
Vocal Fold augmentation
nasoseptal cartilage (cadaver)
Tris/EDTA with protease inhibitor, 1% triton x-100 or 2% SDS
DNase/RNase
recellularization of articular cartilage
Cytocompatibility tests
human adipose-derived stromal cells
MSCs or primary chondrocytes
Trachea
Previously populated
epithelial cells (bronchi) or inferior turbinate mucosa and bone marrow MSCs
Proposed
Seed MSCs onto scaffold with autologous epithelium patches
reduce delays and risk of malignancy
Repair
sourced from adipose derived SSCs
proposed
debate if nessary
animal studies: same survival rate
Successful human cases (two)
increase in life expectancy
detergent enzymatic method and repopulated with autologous cells and tissue patches
Fibrocartilage
also menisci
Decellurization
Method A
solution concentration of 2% for 2 Weeks
complete cell removal
Method B
incubates in acetone, hydrogen peroxide and osmotic solutions
Method C
24 h enzymatic digestion in 0.05% trypsin EDTA and 48 h incubation 2% triton X-100 and 1.5% peracetic acid
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proposal of homogenizing approaches
Recullurization
Difficult
dense ECM and few interstitial spaces
Proposals
chemotactic cell seeding techniques
recombinant human bone morphoology
Needle punching
1 mm spacing, 28G microneedle
Chondrogenic differentiation of synovial fluid derived MSCs
Needs Exogenesis growth factor supplementation
TGF-β3 and IGF-1
already established methods
rat chondrocytes
human adipose derived stem cells
human synovial fluid derived MSCs
Elastic Cartilage
bone
Skeletal muscle
Tendons
Adipose Tissues
Heart
Vessels
Gingivia
Dental Pulp
Schneiderian Membrane
Intestine
Liver
Pancreas
Kidney
Bladder
Male Reproductive System
Female Reproductive System
Female Reproductive System
Cornea
Vocal Folds
Peripheral Nerves
Complex Composite Structures
Ex. An entire Limb
my goals
use scaffolding to create a viable process to create a universal donor blood from composite scaffolding and ex-vivo organ perfusion
multiple steps need to be established and furthuring of technology for this to happen
first step is to establish what organ and supporting tissues will be needed
bone and bone marrow
Cell Sources or additional sources for scaffolding
Umbilical Cord
Placenta
Amniotic Membrane
From patient
Common Abbreviations for Reference
ECM
EDTA
dH2O
SDS
Sodium dodecyl sulfate
KU
Kunitz Units
MSCs
Mesenchymal Stem Cells
distilled water
Ethylenediamine Tetraacetic Acid
Extracellular Matrix