dissertation presentation (v2)
TRANSCRIPT
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Dissertation Defense
Keaton Smith
Monday, January 27, 2014
An Evaluation of Chitosan Paste as an
Injectable and Adhesive, Adjunctive
Therapy for Musculoskeletal Wound
Infection Prevention
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Parvizi. President Acceptance Talk. 2012 MSIS Annual Meeting. August 2012.
Corso. Inj Prev.2006;12(4):212-8.
Spinner, J., The Washington Post, 2007
Boxma H et al. Lancent 1996 http://socialnewsdaily.com/tag/boston-marathon-cell-phones-not-working/
http://www.defense.gov/home/features/2009/1209_marineassault/hires/230767.jpg
icasualties.org
http://www.huffingtonpost.com/dan-froomkin/iraq-soldiers-wounded_b_1176276.html
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http://www.unboundedmedicine.com/2005/11/08/open-fractures-classification-and-its-clinical-manifestations-3/
Bloom, B. et al., AAOS, 1992.
Lifesaving, Debridement and Irrigation
Systemic vs. Local Antibiotic Delivery
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Systemic Delivery
Musculoskeletal
Wound Site
Local
Delivery
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https://medium.com/@FERNnews; Imagining the Post-Antibiotics Future
Bacterial Antibiotic Resistance
• Limbago. Report of the 13th Vancomycin-resistant Staphylococcus aureus from the United
States. J Clin Microbiol. Epub ahead of print 26 Dec 2013.
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Bacterial Biofilm
http://woundsinternational.files.wordpress.com/2011/02/schematic-representation-of-polymicrobial-biofilm-formation.jpg
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Pandalus borealis
http://www.redorbit.com/news/science/1685380/warm_ocean_waters_could_lead_to_shrimp_decline/
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CH2OH
HO
NH
O O
C O
H
O
C
H H
H
H
H
H
H
H HO
NH2
H
CH2OH
CH3
O
2-amino-2-deoxy-β-D-glycopyranose β-(1-4) linked 2-acetamido-2-deoxy-β-D-glucopyranose
Deacetylated Unit Acetylated Unit
Chitosan
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Smith. CORR. 2013.
Megan. JBMRA. 2013.
Reves. JBMRB. 2013;101(4)630-9.
Norowski. J Tis Eng Reg Med. 2012. Zugravu. J Biomater Appl. 2012.
Parker. JBMRB. 2013;101(1)110-23.
Jennings. CORR. 2012;470(10)2663-70.
Mecwan. JBMRA. 2011.
Norowski. Implant Dent. 2011;20(1)56-67.
Stinner. J Orthop Trauma. 2010;24(9)592-7.
Smith. JBMRB. 2010(1)203-11.
Noel. CORR. 2010;468(8)2074-80.
Kim. Biomater. 2010;31(14)4157-66.
Reves. JBMRB. 2009;90(1)1-10.
Chesnutt. Tissue Engr A. 2009;15(9)2571-9.
Majd. JBMRB. 2009;90(1)283-9.
Kim. JBMRB. 2009;90(1)145-55.
Greene. CORR. 2008;466(7)1699-704.
Noel. CORR. 2008;466(6)1377-82.
Chesnutt. JBMRA. 2009;88(2)491-502.
Yuan. JBMRB. 2008;86(1)245-52.
2009
2010
2011
2012
2013
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Degradation
Drift
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Chitosan Powder Material
0.17 M Acetic Acid
and Chitosan Solution
Dehydrated Acetic Acid,
Chitosan Product
Neutralization
and Lyophilization
Hydration and Application
4.6 pH Acetate Buffered
Chitosan Sponge (Dehydrated)
IN VITRO CHARACTERIZATION:
Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Attenuated Total
Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Gel
Permeation Chromatography (GPC), Differential Scanning Calorimetry (DSC)
IN VITRO FUNCTIONALITY:
Degradability, Biocompatibility
IN VIVO FUNCTIONALITY:
Rat Intramuscular Degradability and Biocompatibility Model
Dissolution
Lyophilization
SPONGE FABRICATION:
Hydration and Application
5.6 pH Acetate Buffered
Chitosan Sponge (Dehydrated)
Neutralized Chitosan
Sponge (Dehydrated)
5.6 pH, 0.25 M
Acetate Buffer Wash
and Lyophilization
4.6 pH, 0.25 M
Acetate Buffer Wash
and Lyophilization
Hydration and Application
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Sponge In Vitro Characterizations Differences
Scanning Electron Microscopy (SEM)
X-ray Diffraction (XRD)
Attenuated Total Reflectance Fourier Transform
Infrared Spectroscopy (ATR-FTIR)
Gel Permeation Chromatography (GPC)
Differential Scanning Calorimetry (DSC)
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Lyophilization Dissolution Neutralization and/or Buffer Wash
Sponge Fabrication
Formulations Tested:
1. Neutral Chitosan Sponge
2. 5.6 pH, 0.25 M Acetate Buffered Chitosan Sponge
3. 4.6 pH, 0.25 M Acetate Buffered Chitosan Sponge
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0
20
40
60
80
100
0 1 2 3 4 5 6 7 8 9 10
Perc
ent
of th
e R
em
ain
ing S
ponge (
%)
Day
Neutral Chitosan Sponge
pH 5.6 Buffered Chitosan Sponge
pH 4.6 Buffered Chitosan Sponge
Sponge Enzymatic Degradation (n = 3)
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0
20
40
60
80
100
120
NeutralChitosanSponge
pH 5.6BufferedChitosanSponge
pH 4.6BufferedChitosanSponge
NeutralChitosanSponge
pH 5.6BufferedChitosanSponge
pH 4.6BufferedChitosanSponge
1 Day 3 Days
Perc
ent
Cell
Via
bili
ty (
%)
Sponge Direct Contact Biocompatibility (n = 5)
Polyurethane Control = 100% Viability
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Sponge In Vivo Biodegradability and Biocompatibility
×
• Chitosan Paste Versions Used:
• Gelfoam Gelatin Sponge (n = 10)
Degradable Control
• Neutralized Chitosan Sponge (n = 10)
• 5.6 pH, Acetate Buffered Chitosan
Sponge (n = 10)
• 4.6 pH, Acetate Buffered Chitosan
Sponge (n = 10)
• 4 site per rat
• 2 time points (4 and 10 days)
• 20 Sprague-Dawley rats total
× ×
×
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Gelfoam (10 Days) Neutral Chitosan (10 Days)
5.6 pH Chitosan (10 Days) 4.6 pH Chitosan (10 Days)
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0
5
10
15
20
25
30
35
40
GelatinSponge,n = 10
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,n = 10
pH 4.6BufferedChitosanSponge,n = 10
GelatinSponge,
n = 9
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,
n = 9
pH 4.6BufferedChitosanSponge,
n = 9
4 Days 10 Days
Perc
ent
Impla
nt A
rea P
er
Defe
ct A
rea (
%)
Sponge Histological Analysis
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0
10
20
30
40
50
60
70
80
90
100
GelatinSponge,n = 10
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,n = 10
pH 4.6BufferedChitosanSponge,n = 10
GelatinSponge,
n = 9
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,
n = 9
pH 4.6BufferedChitosanSponge,
n = 9
4 Days 10 Days
Perc
ent
Fib
rous T
issue A
rea P
er
Defe
ct A
rea (
%) Sponge Histological Analysis
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0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
GelatinSponge,n = 10
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,n = 10
pH 4.6BufferedChitosanSponge,n = 10
GelatinSponge,n = 10
NeutralChitosanSponge,n = 10
pH 5.6BufferedChitosanSponge,n = 10
pH 4.6BufferedChitosanSponge,n = 10
4 Days 10 Days
Inflam
ma
tory
Response
Sponge Histological Analysis
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Biocompatible
Full Wound Coverage
Diffusion Properties
Drug Delivery
Biodegradable
Adhesive
1.
2.
1. Chitosan Sponge 2. Novel Chitosan Paste
Injectable
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Chitosan Powder Material
0.05 M Acetic Acid and Chitosan
Solution
Lyophilization
0.15 M Acetic Acid and Chitosan
Solution
Dehydrated 0.05 M Acetic Acid,
Chitosan Product
Dehydrated 0.15 M Acetic Acid,
Chitosan Product
Neutralization and Lyophilization
Dissolution
Dehydrated, Neutralized
Chitosan Product Grinding and
Combination
80:20, 0.05 M Acetic Acid to
Neutralized Chitosan Paste Device
(Dehydrated)
Hydration and Application
80:20, 0.15 M Acetic Acid to
Neutralized Chitosan Paste Device
(Dehydrated)
IN VITRO MATERIAL CHARACTERIZATION:
Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) Attenuated Total
Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)
IN VITRO FUNCTIONALITY:
Acidity, Absorbency, Injectability, Adhesivity, Degradability, Antibiotic Elution,
Activity, Biocompatibility
IN VIVO FUNCTIONALITY:
Rat, Intramuscular, Degradability and Biocompatibility Model
Mouse Subcuteaneous Catheter Infection Prevention Model
Dissolution
Lyophilization
PASTE FABRICATION:
Grinding and
Combination
Hydration and Application
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Paste In Vitro Characterizations Differences
Scanning Electron Microscopy (SEM)
X-ray Diffraction (XRD)
Attenuated Total Reflectance Fourier Transform
Infrared Spectroscopy (ATR-FTIR)
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Test Group
(p-values)
Adhesive Strength
(kPa), n = 9
(p=0.045)
Maximum Ejection Force
(N), n = 3
(p=0.121)
80:20, 0.05 M Acetic Acid to
Neutralized Chitosan Paste 12 ± 2 128 ± 22
80:20, 0.15 M Acetic Acid to
Neutralized Chitosan Paste 9 ± 1 84 ± 20
Empty Syringe 6 ± 0
Clinically Relevant Ejection Force 330
Paste Adhesivity and Injectability Analysis
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0
10
20
30
40
50
60
70
80
90
100
3 6 9 12 15 18 21 24 27 30
Perc
ent
Rem
ain
ing (
%)
Hours
80:20, 0.05 M Acetic Acid toNeutralized Chitosan Paste
80:20, 0.15 M Acetic Acid toNeutralized Chitosan Paste
Paste Enzymatic Degradation (n = 5)
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0
100
200
300
400
500
600
700
800
900
1 3 6 12 24 48 72
Vancom
ycin
(µ
g/m
L)
per
g C
hitosan P
aste
Hours
80:20, 0.05 M Acetic Acid toNeutralized Chitosan Paste
80:20, 0.15 M Acetic Acid toNeutralized Chitosan Paste
Antibiotic Loaded Paste Elution (n = 5)
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Antibiotic Eluate
Analysis using
Disk Diffusion
1 Hour 72 Hour
PBS Loaded,
0.05 M acid, Paste PBS Loaded,
0.15 M acid, Paste
Vancomycin
Loaded, 0.15M
acid, Paste
Vancomycin
Loaded, 0.05M
acid, Paste
Antibiotic Activity (n = 3) vs. Staphylococcus aureus
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Cell Culture Insert for Paste Biocompatibility
80:20, 0.05 M acetic acid to neutral chitosan paste
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0
5000
10000
15000
20000
25000
70:30 60:40 50:50
Cell
Num
ber
Acidic (0.05 M Acetic Acid) : Neutralized Chitosan Paste Ratio
CellTiter-Glo for cell viability of NHDFs against chitosan paste formulations in transwell plates after 24 and 72 hrs (n = 5, compared to
NHDFs on tissue culture plastic)
24 hours 72 hours
* *
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Paste In Vivo Biocompatibility
×
• Chitosan Paste Versions Used:
• 1 mL of 50:50, 0.05 M Acetic Acid
Chitosan to Neutralized Chitosan
Paste (n = 3)
• 1 mL of 30:70, 0.05 M Acetic Acid
Chitosan to Neutralized Chitosan
Paste (n = 3)
• Historical Chitosan Sponge Controls
• 1 site per rat
• 1 time point (10 days)
• 6 Sprague-Dawley rats total
• Slight to Moderate Immune Response
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50:50, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
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*
30:70, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
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30:70, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
4.6 pH, 0.25 M Acetate Buffered
Chitosan Sponge
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Paste In Vivo Infection Prevention
(Biofilm Forming Staphylococcus aureus)
×
• Chitosan Device Versions Used:
0.5 mL of 50:50, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
• 4 mg/mL Vancomycin Loaded (n = 8)
• 1 X PBS Loaded (n = 8)
8 mm Diameter Neutralized
Chitosan Sponge
• 4 mg/mL Vancomycin Loaded (n = 8)
• 1 X PBS Loaded (n = 8)
• 2 sites per rat
• 16 NIH Swiss mice total ×
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50:50, 0.05 M Acetic Acid Chitosan to Neutralized Chitosan Paste, not injectable at 0.5 mL
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100
1000
10000
100000
1000000
10000000
PBS LoadedSponge
VancomycinLoadedSponge
PBS LoadedPaste
VancomycinLoaded Paste
Avera
ge c
olo
ny f
orm
ing u
nits (
cfu
) /c
ath
ete
r Infection Prevention Analysis (n = 8)
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Description of the
Device’s Ability
In vitro
Chitosan
Sponge
In vivo
Chitosan
Sponge
In vitro
Chitosan Paste
In vivo
Chitosan Paste
Acid Modified Device
Injectable N/A N/A
Adhesive N/A N/A
Biocompatible
Biodegradable N/A
Antibiotic Delivery
Summary Comparison
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Thank You