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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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

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States. J Clin Microbiol. Epub ahead of print 26 Dec 2013.

Bacterial Biofilm

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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.

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Jennings. CORR. 2012;470(10)2663-70.

Mecwan. JBMRA. 2011.

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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.

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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