Dissertation Presentation (v2)
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Transcript of Dissertation Presentation (v2)
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
Parvizi. President Acceptance Talk. 2012 MSIS Annual Meeting. August 2012.
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icasualties.org
http://www.huffingtonpost.com/dan-froomkin/iraq-soldiers-wounded_b_1176276.html
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Bloom, B. et al., AAOS, 1992.
Lifesaving, Debridement and Irrigation
Systemic vs. Local Antibiotic Delivery
Systemic Delivery
Musculoskeletal
Wound Site
Local
Delivery
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.
Bacterial Biofilm
http://woundsinternational.files.wordpress.com/2011/02/schematic-representation-of-polymicrobial-biofilm-formation.jpg
Pandalus borealis
http://www.redorbit.com/news/science/1685380/warm_ocean_waters_could_lead_to_shrimp_decline/
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
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
Degradation
Drift
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
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)
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
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)
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
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
× ×
×
Gelfoam (10 Days) Neutral Chitosan (10 Days)
5.6 pH Chitosan (10 Days) 4.6 pH Chitosan (10 Days)
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
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
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
Biocompatible
Full Wound Coverage
Diffusion Properties
Drug Delivery
Biodegradable
Adhesive
1.
2.
1. Chitosan Sponge 2. Novel Chitosan Paste
Injectable
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
Paste In Vitro Characterizations Differences
Scanning Electron Microscopy (SEM)
X-ray Diffraction (XRD)
Attenuated Total Reflectance Fourier Transform
Infrared Spectroscopy (ATR-FTIR)
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
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)
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)
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
Cell Culture Insert for Paste Biocompatibility
80:20, 0.05 M acetic acid to neutral chitosan paste
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
* *
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
50:50, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
*
30:70, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
30:70, 0.05 M Acetic Acid Chitosan to
Neutralized Chitosan Paste
4.6 pH, 0.25 M Acetate Buffered
Chitosan Sponge
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 ×
50:50, 0.05 M Acetic Acid Chitosan to Neutralized Chitosan Paste, not injectable at 0.5 mL
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)
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
Thank You