Fabrication of Novel Biomaterials

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15 Νοε 2013 (πριν από 3 χρόνια και 4 μήνες)

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Fabrication of Novel Biomaterials

for Peripheral Nerve Tissue


Engineering Strategies

Archit Sanghvi, David Silva, Kiley Miller, Julie Williams,


Angela Belcher, Christine Schmidt


Motivation


A key goal in the design of new polymeric biomaterials for
biomedical and drug delivery applications is to
specifically control
the interactions of biomolecules and living cells with biomaterial
surfaces



Our major focus is on the
development of materials to promote the
regeneration of damaged nerves

and to overcome drawbacks with
the current clinical use of nerve grafts



Use of normal donor nerve from an uninjured body location
(autograft) is limited by tissue availability, the need for multiple
surgical procedures, and loss of function at the donor site



One possible alternative would be to develop engineered
constructs to replace those elements necessary for axonal
proliferation, including support cells (i.e., Schwann cells), growth
factors, scaffolding material, and matrix proteins


Use of Synthetic Biomaterials


Two key criteria of any implanted biomedical device include:
biocompatibility with the body and ability of material to
function physically in desired capacity



The ability to design, fabricate, and optimize surfaces tailored
for tissue engineering application is crucial for synthesizing
biocompatible materials



One of the more difficult problems in bioengineering science
is to reproduce the properties and functionality of human
tissues and organs using artificial biomaterials

Project Goals

(1) Physical modification of biomaterials


Construct 3D biologically active polymer
-
based nerve guidance
channels (NGCs) for nerve repair application



Design nerve conduits with micron
-
scale precision (spatial
control) to enhance cellular interactions with synthetic materials


(2) Chemical modification of biomaterials



Fabricate polymer
-
based synthetic materials to mimic natural
cellular microenvironments (biomimetics)



Develop ligand
-
based surface modifications onto biodegradable
polymer surfaces



(1)
Physical Modification


3D Nerve Conduit Fabrication (Microfab Inc.)

MediLab
TM
ink
-
jet based Tissue
Engineering Platform

100
m
洠潰瑩捡c 灯汹浥爠睡癥杵楤攠
獰汩s瑥爻r灡瑴敲渠牥灲敳敮瑡e楶攠潦⁡o
扩晵牣慴b搠灡瑨睡p

Y
-
shaped
bifurcated
conduit


Cells adhere to and are viable on
Microfab Surface

24 hr study illustrating
cell viability using
live/dead assay (green
-
live, red
-
dead)

10x

10x

10x

10x

72 hr study illustrating
cell viability using
live/dead assay (green
-
live, red
-
dead

Cells: PC12 (rat adrenal tumor cells) neuron
-
like cells used in study

Material: Copolymer of poly
-
lactic acid (PLA) and poly
-
ethylene glycol (PEG)

Quantitative Data Illustrating PC12 Cell
Adhesion on Microfab Polymer


*Control: Cells were plated on the same surface area using plexiglass wells on polystyrene TC dishes



Conclusion: Results showed excellent potential for NGC development



using Microfab polymer, because in vitro studies demonstrated



comparable PC12 adhesion and viability




Time Study


Control* (cells/ml)


Microfab Polymer (cells/ml)

Before incub.

After incub.

Before incub.

After incub.

24 hr

5.75 x 10
5

4.35 x 10
4

5.75 x 10
5

3.25 x 10
3

48 hr

1.80 x 10
6

2.43 x 10
5

1.80 x 10
6

2.55 x 10
4

72 hr

1.80 x 10
6

3.45 x 10
5

1.80 x 10
6

1.25 x 10
4

Surface Engineering of Microfab
Polymer

Biotin moieties presented at the polymer surface
are used to immobilize tetrameric avidin
molecules. Free biotin binding sites on the avidin
molecules are in turn used to anchor biotinylated
ligands such as,
b
NGF.

SEM of Microfab polymer
(PLA
-
PEG) on glass slide,
illustrating precise
-
control
fabrication

(2)
Chemical Modification


Selection of Peptides with Polymer
Binding Specificity



Specific Objective


Current techniques to modify material surfaces are difficult due to
limited selectivity for binding non
-
biological surfaces


A unique “biopanning” method (described below) would be used to
select for
specific peptides that bind to a range of non
-
biological
materials with high specificity, resulting in direct fabrication of
modified materials



Potential Applications


Nerve Regeneration: Chemical modification of biomaterials to enhance
neuron
-
specific interactions


Electronics: Use in electronic devices to control placement and
assembly to direct nanoscale fabrication (‘bottom
-
up’ approach)


Drug
-
delivery: Site
-
specific delivery via controlled peptide
-
polymeric
vehicle interaction




Determining Peptide
-
Libraries via
“Biopanning” Technique


A library containing ~10
9

unique
phage
-
displayed peptide
combinations is incubated with the
polymer of interest (target)


Unbound phage are washed away,
eluting the specifically
-
bound phage


Eluted phage are amplified and taken
through additional cycles of
panning/amplification to enrich the
pool of phage in favor of the tightest
binding sequences


After 3
-
4 rounds, individual clones
are isolated and sequenced


We are currently researching
specific
-
peptide sequences for two
biomaterials: polypyrrole (PP) and
poly
-
lactic
-
coglycolic acid (PLGA)


Step 1

Step 2

Step 3

Future Work


Incorporation of biotinylated
b
乇N 潮⁐ A
-
biotinylated
-
PEG via avidin


Effects of neurotrophic factor (i.e.,
b
乇N⤠
杲慤楥湴渠獵牦慣r
-
浯m楦i敤e䵩捲潦M戠灯汹浥m
system with PC12 cells


Determining specific
-
peptide libraries for PP and
PLGA to modify polymers for ligand placement





Acknowledgements


National Institutes of Health (NIH) SBIR Grant


Microfab Technologies Inc.


Michael Grove (Chemist, Microfab Inc.)


Dr. Angela Belcher’s Lab


Texas Materials Institute (TMI)