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Microporous Chitosan Sponge Scaffold
Keaton Smith
July 2013
New Technology Presentation
Manufacturing MethodsAnd
Morphology Results
Chitosan Solution + SiO2(s)
Lyophilize
Sponge with Suspended
SiO2(s) Particles
Sponge Neutralization and SiO2 Dissolution
LyophilizeSponge with SiO2 Particle Sized Pores
Chitosan Solution + SiO2(s)
Sponge Solution Formulation (Jan 2013)• 2% (w/v) Chitosan (HMC 95/3000)• 1% (v/v) acid
– 3:1 ratio of lactic:acetic
• 12.5% (w/v) of SiO2
– 35 to 70 µm– Sigma Aldrich, Davisil grade 643, 200-425 mesh
SiO2(s)
SiO2(s)
Lyophilize
Sponge with SuspendedSiO2 Particles
Sponge with SuspendedSiO2 Particles
Sponge Neutralization and SiO2 Dissolution
SiO2(s) + 2NaOH(l) Na2SiO3(l) + H2O(l)
12M“Liquid Glass”
Other Possible Reactions:
Na2SiO3(l)+ 2HA(l) → H2SiO3(l) + 2NaA(l)
Chitosan + Na2SiO3(l) → No Reaction Expected
Chitosan + NaOH(l) Chitosan DDA
Heat
Heat
Silicic Acid
Reffitt, DM et al. Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 2003 Feb;32(2):127-35.
Lyophilize
Microporous Sponge
Microporous Sponge
Microporous Sponge
Microporous Sponge
Microporous Sponge
Standard Sponge‒
Standard Sponge‒
‒Standard Sponge‒ Microporous Sponge
Laboratory Evaluation Results
Direct Contact Biocompatibility (n=5)‒
HMC 95/3000 Sponge with SiO2 Sized Pores0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
120%
130%
140%
One Day Treatment Three Day Treatment
Pe
rce
nt
Ce
ll V
iab
ility
Standard Sponge Microporous Sponge
‒Standard Sponge‒
Day 3, Live/Dead Staining
Microporous Sponge
‒Standard Sponge‒
Day 21, Live/Dead Staining
Microporous Sponge
Microporous Sponge‒
Day 21, Live/Dead Staining
Four Day, Weight-Based, Enzymatic Degradation (n=3)
‒
HMC 95/3000 Sponge with SiO2 Sized Pores90%
91%
92%
93%
94%
95%
96%
97%
98%
99%
100%Chart Title
Pe
rce
nt
Re
ma
inin
g
Standard Sponge Microporous Sponge
Swelling Ratio (n=3)‒
Antibiotic Uptake (n=3)‒
• The standard sponge holds:16.85 ± 1.12 X its weight in water
• The microporous sponge holds:19.09 ± 1.04 X its weight in water
• The standard sponge can adsorb :113.23 ± 18.74 µg vancomycin / mg of chitosanfrom a ~5mg/ml vancomycin solution
• The microporous sponge can adsorbs:125.67 ± 23.36 µg vancomycin / mg of chitosanfrom a ~5mg/ml vancomycin solution
Antibiotic Elution (n=3)‒
1 3 6 12 24 48 720
200
400
600
800
1000
1200
Standard Sponge (HMC)
Microporous Sponge
Time (hours)
Va
nc
om
yc
in C
on
ce
ntr
ati
on
(µ
g/m
l)
Energy-Dispersive X-Ray Spectroscopy (Elemental Analysis, n=3)
‒
Starndard Sponge
Element Weight % Atomic %
C 54.13 60.13
N 9.50 9.10
O 36.18 30.34
Na 0.10 0.06
Si 0.10 0.05
Ca 0.00 0.00
Microporous Sponge
Element Weight % Atomic %
C 54.74 61.07
N 9.82 9.39
O 35.09 29.39
Na 0.03 0.02
Si 0.19 0.09
Ca 0.13 0.05
X-Ray Diffraction‒
5 10 15 20 25 30 35 400
500
1000
1500
2000
2500
3000
3500
4000
4500Raw Chitosan
Standard Sponge
Microporous sponge
°2θ Cu Kα
Dif
fra
cte
d X
-Ra
y In
ten
sit
y (
co
un
ts)
Future Direction and Questions‒
• Establish uses, or purpose (cell scaffold and drug delivery)• Would an altered chitosan chemistry be more useful?
– What is the best acid? Does this matter when using 15M NaOH?– Should we increase DDA?– Should we increase MW?– What’s the best concentration or ratio of SiO2 to chitosan during
manufacture?
• Tighter control on SiO2 particulate size? What size is optimal?• Other options:
– Chitosan sponge coatings with cell-sized pores.– Enhanced bilayered chitosan sponge with standard top layer, and
micropored, close-packed bottom layer.• Silicic Acid – will it dissolve and/or degrade chitosan and stimulate
bone growth?
Patent Search‒
• Process for preparing an absorbent foam …– Palani Raj Ramaswami Wallajapet et al– http://www.google.com/patents/US5948829– Patent number: 5948829
Filing date: Nov 25, 1997Issue date: Sep 7, 1999
• Composite sponge wound dressing made of …– Jui-Sheng Lee et al– http://www.google.com/patents/US6693180– Patent number: 6693180
Filing date: Apr 4, 2002Issue date: Feb 17, 2004Application number: 10/115,007
• Method of producing chitosan scaffold having high tensile strength and ...
– Chun-Ho Kim et al– http://www.google.com/patents/US20080242850– Application number: 12/157,120
Publication number:US 2008/0242850 A1Filing date: Jun 9, 2008
• Wound dressing and method for controlling severe, life-threatening bleeding
– Kenton W Gregory et al– http://www.google.com/patents/US7482503– Patent number: 7482503
Filing date: Jun 14, 2002Issue date: Jan 27, 2009Application number: 10/480,827
• Wound dressings, apparatus, and methods for controlling severe, life ...
– Kenton W Gregory et al– http://www.google.com/patents/US7820872– Patent number: 7820872
Filing date: Oct 31, 2007Issue date: Oct 26, 2010Application number: 11/981,111
• Highly porous chitosan bodies– Peter D. Unger et al– http://www.google.com/patents/US5525710– Patent number: 5525710
Filing date: Sep 12, 1994Issue date: Jun 11, 1996
Similar Research‒
‒ Similar Research
‒ Similar Research (examples from Ratner group)
Osathanon, et al. Microporous nanofibrous fibrin-based scaffolds for bone tissue engineeering. Biomaterials 29 (2008)4091-4099.• Using sphere-templated porous, nanofibrous fibrin scaffolds with incorporated nanocrystalline
hydroxyapatite to promote bone formation.
Fukano, et al. Epidermal and dermal integration into sphere-templated porous poly(2-hydroxyethyl methacrylate) implants in mice. J Biomed Mater Res Part A: 94A: 1172-1186,2010.• Using sphere-templated porous poly(2-hydroxyethyl methacrylate) to stimulate epidermal and
dermal proliferation.
Galperin, et al. Degradable, Thermo-Sensitive Poly(N-isopropyl Acrylamide)-Based Scaffolds with Controlled Porosity for Tissue Engineering Applications. Biomacromolecules 2010, 11, 2583-2592.• Biodegradable poly(N-isopropyl acrylamide) hydrogel with controllable pore size and highly
interconnected porous structure using a sphere-templating technique.
Underwood, et al. Quantifying the effect of pore size and surface treatment on epidermal incorporation in to percutaneously implanted sphere-templated porous biomaterials in mice. J Biomed Mater Res Part A. 2011:98A;499-508.• Using sphere-templated porous poly(2-hydroxyethyl methacrylate) to establish optimal pore size.
‒ Similar Research (examples involving chitosan)
Hsieh et al. Morphology and characterization of 3D micro-porous structured chitosan scaffolds for tissue engineering. Colloids and Surfaces B: Biointerfaces 57 (2007) 250-255.• Created microporous chitosan scaffold for cell culture and tissue engineering scaffold by a
foaming and liquid hardening technique. Pore sizes range from 200 to 500µm.
Park et al. Cellular and Soft Tissue Compatibility to High Interconnecttivity between Pores of Chitosan Scaffold. Macromolecular Research. 20(4)2012;397-401.• Created microporous chitosan scaffold for cell culture and tissue engineering scaffold by a
thermally induced phase-separation process. Pore sizes range from 4 to 100µm. Had good fibroblast proliferation.
Zhang et al. Channelled scaffolds for engineering mycoardium with mechanical stimulation. J Tissue Eng Regen Med 2012;6:748-756.• Created chitosan-collegen scaffolds with micropores (un controlled around 40 - 100µm) and
parallel channels (200µm diameter) using for cardiac tissue engineering.