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Microporous Chitosan Sponge Scaffold Keaton Smith July 2013 New Technology Presentation

Microporous Chitosan (v2)

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Page 1: Microporous Chitosan (v2)

Microporous Chitosan Sponge Scaffold

Keaton Smith

July 2013

New Technology Presentation

Page 2: Microporous Chitosan (v2)

Manufacturing MethodsAnd

Morphology Results

Page 3: Microporous Chitosan (v2)

Chitosan Solution + SiO2(s)

Lyophilize

Sponge with Suspended

SiO2(s) Particles

Sponge Neutralization and SiO2 Dissolution

LyophilizeSponge with SiO2 Particle Sized Pores

Page 4: Microporous Chitosan (v2)

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

Page 5: Microporous Chitosan (v2)

SiO2(s)

Page 6: Microporous Chitosan (v2)

SiO2(s)

Page 7: Microporous Chitosan (v2)

Lyophilize

Page 8: Microporous Chitosan (v2)

Sponge with SuspendedSiO2 Particles

Page 9: Microporous Chitosan (v2)

Sponge with SuspendedSiO2 Particles

Page 10: Microporous Chitosan (v2)

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.

Page 11: Microporous Chitosan (v2)

Lyophilize

Page 12: Microporous Chitosan (v2)

Microporous Sponge

Page 13: Microporous Chitosan (v2)

Microporous Sponge

Page 14: Microporous Chitosan (v2)

Microporous Sponge

Page 15: Microporous Chitosan (v2)

Microporous Sponge

Page 16: Microporous Chitosan (v2)

Microporous Sponge

Page 17: Microporous Chitosan (v2)

Standard Sponge‒

Page 18: Microporous Chitosan (v2)

Standard Sponge‒

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‒Standard Sponge‒ Microporous Sponge

Page 20: Microporous Chitosan (v2)

Laboratory Evaluation Results

Page 21: Microporous Chitosan (v2)

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

Page 22: Microporous Chitosan (v2)

‒Standard Sponge‒

Day 3, Live/Dead Staining

Microporous Sponge

Page 23: Microporous Chitosan (v2)

‒Standard Sponge‒

Day 21, Live/Dead Staining

Microporous Sponge

Page 24: Microporous Chitosan (v2)

Microporous Sponge‒

Day 21, Live/Dead Staining

Page 25: Microporous Chitosan (v2)

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

Page 26: Microporous Chitosan (v2)

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

Page 27: Microporous Chitosan (v2)

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

g/m

l)

Page 28: Microporous Chitosan (v2)

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

Page 29: Microporous Chitosan (v2)

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)

Page 30: Microporous Chitosan (v2)

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?

Page 31: Microporous Chitosan (v2)

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

Page 32: Microporous Chitosan (v2)

Similar Research‒

Page 33: Microporous Chitosan (v2)

‒ Similar Research

Page 34: Microporous Chitosan (v2)

‒ 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.

Page 35: Microporous Chitosan (v2)

‒ 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.