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Persistence of bioluminescent bacteria on and around degradable and non-degradable surgical meshes in a murine model BioInterface 2012, Dublin, october 23-25 Jelmer Sjollema University Medical Center Groningen dep. Biomedical Engineering Groningen The Netherlands

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Outline

•  Clinical background •  Objectives •  Materials and methods •  Results •  Discussion

Accepted for publication in Acta Biomaterialia

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~ 60% repaired with mesh graft

Application of Mesh-grafts

[Engelsman et al, 2007]

Abdominal wall defect

introduction

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Biomaterialen

Infections on commonly used meshes

Engelsman et al. Biomaterials, 2007

Monofilament polypropylene 1 - 4 % (Dunne 2003, Andersson 2003, Lal 2003, Sohail 2004, Neumayer 2004, Jezupros 2006)

Multifilament polypropylene 2 % (Jezupros 2006)

Monofilament polyester 3 - 4 % (Chevrel 1990, Bonnamy 1999, Marchand 1999, Hamy 2003)

Multifilament polyester 7 - 16 % (Leber 1998, Machairas 2004)

ePTFE 0 - 9 % (Leber 1998, Balen 1998, Gonzalez 1999, Bauer 1999, Petersen 2001)

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Stratified approach to repair

Grade 3: Permanent synthetic repair material generally not recommended; potential advantage to biologic repair material Grade 4: Permanent synthetic repair material not recommended; biologic repair material should be considered

Grade 1: Choice of repair material by surgeon preference and patient factors Grade 2: Increased risk for surgical site occurrence suggests additive risk of permanent synthetic repair material, and potential advantage for appropriate biologic reinforcement Breuing et al. Surgery, 2010, Volume 148, Number 3

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Biologic repair material

The ideal mesh would provide: • Strength • Flexibility • Scaffold for tissue incorporation • Resistance to infection. • Resorb

•  Grafts are available from both human cadaveric (Alloderm®) and porcine (Permacol®, Surgisis®) sources.

•  These grafts are all composed of an acellular collagen scaffold to provide tissue incorporation and neovascularization.

•  Tissue ingrowth and neovascularization are thought to give biosynthetic grafts some inherent resistance to infection.

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Hypothesis and objective

•  Immune system is frustrated around a biomaterial

•  A rapid restoration of the efficacy of the immune system may be anticipated once the biomaterial has fully been degraded.

Hypothesis

•  It is hypothesized that dissolution of the biomaterial will yield clearance of biofilm bacteria as well as bacteria in surrounding tissue.

The objective of this study is: to compare staphylococcal persistence on and around a degradable and non-degradable surgical mesh

Method: Bioluminescence imaging - In vitro - In vivo Culturing Histology

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Materials

Use of commercially available materials Prolene®

–  Ethicon Inc., Sommerville, New Jersey, USA –  non-degradable, doubly filamented

polypropylene, –  surface area of 2.46 cm2cm-2

Surgisis Biodesign™ –  Cook Biotech Inc., Bloomington, IN, USA –  degradable fleece –  porcine small intestinal submucosa –  high surface area

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Growing biofilms and biofilm evaluation

Cryopreserved bioluminescent S. aureus Xen29

Plating Culture on blood agar plates

preculture Inocculate 5ml TSB with one coloniy Appr. 24 hrs incubation

Biofilm assay Add 100µl to 50 ml TSB Insert mesh on stainless steel hook Incubate for 72 hrs

Shaking 30 rpm

Biofilm assay 1 Dislodge biofilm Sonication Fluorescence Microscopy Live-Dead staining : SYTO9 and Propidium-iodide Fluorescence Microscopy

Biofilm assay 2 Intact biofilm In vitro Bioluminescence Imaging Subcutaneous

implantation In mice

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Implantation

4 groups of mice •  In each group mice are implanted with in-occulated meshes or a sterile mesh •  Meshes are:

–  Surgisis Biodesign –  Prolene

Anesthesia With isoflurane/O2

Pain treatment With Bu-prenorfine (Temgesic) 1/2 hr prior to surgery, shave area of mesh implant

Make 1 cm incision and prepare 2 cm deep pocket

Anesthesia With isoflurane/O2

Pain treatment With Bu-prenorfine (Temgesic) 1/2 hr prior to surgery, shave area of mesh implant

Make 1 cm incision and prepare 2 cm deep pocket

2x3

2x9 Implant inocculated mesh

Implant sterile mesh

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Imaging during course of infection

Longitudinal imaging: following each animal in time

•  11 times in 28 days •  Results in terms of

amount of light produced (Bioluminescence flux in photons/second)

•  Sacrifice after 28 days •  Explantation for CFU

counting of mesh and surrounding tissue

•  Histology

Days post implantation

Bioluminescence imaging

12 mm biopsy

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Sample preparation

Cut sample in half

Fixation in 10% buffered formaldehyde, store at 4ºC

50%

Histological Investigation

Histology

50%

Culturing Investigation

Save in 300 µl RTF

Culturing

Separate tissue and mesh

carefully from biopsy

Save in 10 ml isotonic saline

Culturing

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Biofilm formation in vitro • Numbers of viable cells on degradable meshes were higher than on non-degradable meshes. • Bioluminescence from bacteria show the same significant difference.

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Bioluminescence in vivo

•  For degradable meshes bioluminescence decreases in time

•  For non-degradable meshes bioluminescence stays constant

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CFU ex vivo

•  For degradable mesh bioluminescence decreases in time

•  For non-degradable mesh bioluminescence stays constant

•  All mice with non- or partly degraded meshes were culture positive

•  Only 50% of the tissue samples from mice with fully degraded meshes were culture positive.

Non-degraded or partly degraded mesh

Fully degraded mesh

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Histology

•  Degradable mesh: No difference between contaminated and non-contaminated meshes.

•  Non-degradable mesh: Abundant presence of neutrophils and macrophages in the areas where the implant likely had been present.

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CFU ex vivo

•  For degradable mesh bioluminescence decreases in time

•  For non-degradable mesh bioluminescence stays constant

•  All mice with non- or partly degraded meshes were culture positive

•  Only 50% of the tissue samples from mice with fully degraded meshes were culture positive.

Non-degraded or partly degraded mesh

Fully degraded mesh

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Bioluminescence in vivo

•  For non-degradable mesh bioluminescence stays constant

•  For fully degraded meshes bioluminescence drops top undetectable levels

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Bioluminescence in vivo

•  For non-degradable mesh bioluminescence stays constant

•  For fully degraded meshes bioluminescence drops top undetectable levels

•  For partially degraded meshes bioluminescence stay constant or even slightly increase

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Conclusions

•  The persistence of a bioluminescent S. aureus strain on and around surgical meshes develops differently for degradable than for non-degradable mesh materials.

•  Degradation of an implant appears to create the conditions in which the immune responses are sufficiently effective again to clear the surrounding tissue from infecting staphylococci.

•  Bacteria in the peri-implant region seem to need a biomaterial to survive.

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Acknowledgements

This research forms part of the Project P4.01 NANTICO of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation.

Acknowledgements

UMCG Biomedical Engineering Mojtaba Daghighi René Dijkstra Gooitzen van Dam Henny van der Mei Henk Busscher Acedemic Medical Center, University of Amsterdam Sebastiaan Zaat Valory de Boer Leonie Jaspers