3/11/2017

1

©2017 National Pressure Ulcer Advisory Panel | www.npuap.org

What’s in that wound bed? Slough, Eschar, or Biofilm?

Linda J. Cowan, PhD, ARNP, FNP-BC, CWS

Disclosures

• Employed as a Research Health Scientist, North

Florida/South Georgia Veterans Health System,

Gainesville, FL.

• Research funding received from:

– VA

– Biomonde

– Healthpoint

– Smith & Nephew

– Hollister

– Medline

• This material is the result of work supported with

resources and the use of facilities at the VA.

3/11/2017

2

Disclaimers

Speaker does not endorse any one •

particular company’s products, is not

employed by industry, has no financial

interest in the listed commercial

companies.

Contents of this presentation do • not

represent the views of the U.S.

Department of Veterans Affairs or the

United States Government

Participants will describe:

• Key characteristics of chronic non-

healing wounds

• Impediments to wound healing

• Characteristics of slough, eschar, and

biofilm in open wounds

• Evidence-based approaches to address

or remove slough, eschar, and biofilm

from open wounds

• Potential antibiofilm treatment strategies

3/11/2017

3

Chronic Wounds vs. Acute Wounds

• All chronic wounds begin as acute

wounds

• Common chronic wounds

– Venous ulcers of the lower extremities

– Diabetic foot ulcers

– Pressure ulcers

– Complex trauma and surgical wounds

Key characteristics of chronic wounds

Imbalanced at microcellular level •

Stuck in inflammatory phase• 1-4

High MMPs / Low TIMPs (inverse correlation)–

High inflammatory cytokines–

Low growth factors–

Fibroblast inhibition– 5

Does • not follow expected pathway to

healing (less than 50% improvement in 4

weeks)6

3/11/2017

4

Percent wound healing in 4 weeks

6Sheehan, P., Jones, P., Caselli, A., Giurini, J.M., Veves, A. (2003). Percent Change in Wound Area of

Diabetic Foot Ulcers Over a 4-Week Period Is a Robust Predictor of Complete Healing in a 12-Week

Prospective Trial. Diabetes Care, 26(6), 1879-1882. Figure courtesy of G. Schultz, taken from:

http://care.diabetesjournals.org/content/diacare/26/6/1879/F1.large.jpg?width=800&height=600&carousel=1

58% of pts

who had >50%

reduction in

wound size at

4 weeks,

healed at 12

weeks6

Only 9% of pts

with <50%

reduction in

wound size at

4 weeks,

healed at 12

weeks6

Intrinsic impediments to wound healing

Physiological, potentially modifiable:•

Nutrition–

Blood sugar control–

Immune compromise (HIV, Sickle Cell)–

Improved management of certain conditions •

Pain, psychological stress–

Edema – – e.g. lymphedema

Tissue viability, perfusion & oxygenation–

Physiological, not modifiable:•

Advanced age –

Certain comorbid conditions (CVA, SCI, –

neurodegenerative, cancer, etc.)

3/11/2017

5

Extrinsic impediments to wound healing - potentially modifiable14

– Medications

• chemotherapy, steroids, anticoagulants

– Persistent or repetitive trauma

• immobility - failure to off-load, inappropriate shoes or

mobility devices, wet-to-dry dressings

– Exposure

• Smoking/nicotine, alcoholism

• Environmental, toxic chemicals, hygiene (personal &

environmental), parasites, pets, etc.

– Physical barriers

• Rolled wound edges, non-viable tissue (slough, fibrin,

eschar)

– Invasion – virulent pathogens (biofilm)

T-I-M-E-(s) Principle for WBP1,8,9

T - remove non-viable Tissue in wound

I - address Infection (prevent, treat,

remove problematic organisms/biofilm)

M – manage Moisture34

E – address wound Edges

S – address Surrounding Skin

3/11/2017

6

Documenting wound assessments

Location•

Suspected etiology, contributing factors•

Size (W X L X D in cm)•

Undermining, tunneling (clock method)•

Exudate (color, amount, odor)•

Wound bed tissue (color, amount viable)•

Wound edges and surrounding tissue•

Last treatments used, compliance, •

wound response, patient/CG education

Describing wound tissue

• Color of wound bed (in percentages)

• Viable (living tissue with good perfusion)

• Non-viable (dead/dying host tissue)

• Boggy (wet spongy consistency)

• Fluctuant (moving in waves, movable &

compressible, variable/unstable)

• Friable (bleeds easily with light touch)

• Hypergranulating (overgrowing baseline)

• Pale (anemic looking)

3/11/2017

7

Characteristics of slough in wounds

• What it is13

– Non-viable host tissue (or “avascular fat”)

– Typically it is moist, white, yellow, grey, or tan

dead tissue; loose or adherent; includes white

blood cells, fibrin, and other proteins

– May have “chicken fat” appearance

• What it is not

– Alive – slough by itself is not living tissue

• Slough will not “grow” on dressings

– Biofilm - may have bacteria/biofilm on it

– ?Blood clot, dried exudate, softened scab?

Fibrin35

Fibringen• is a glycoprotein in vertebrates

that helps in formation of blood clots.

Fibrin• is an insoluble, non-globular protein

formed from fibrinogen during the clotting of

blood. It is formed by the action of the

protease thrombin (clotting enzyme) on

fibrinogen which causes it to polymerize.

The polymerized • fibrin, together with

platelets form a hemostatic plug or clot over a

wound site. 35Laurens, N., Koolwijk, P., DeMaat, M/P. (2006). Fibrin Structure and wound healing. Journal

of Thrombosis and Haemostasis, 4:932-939.

3/11/2017

8

Slough

• Best ways to remove

Characteristics of eschar in wounds

• What it is

– Dry, dead host tissue

• What it is not

– Scab / crust (dried exudate)

– Dry Gangrene (condition where tissue dies

caused by ischemia due to underlying illness,

injury, and/or infection). Fingers, toes, & limbs

most often affected.

• When not to remove

– If providing reliable protective barrier

3/11/2017

9

Eschar

• When to remove

– Integrity is compromised (no longer acting as

body “bandaid”)

– Impediment to healing

• Best ways to remove

Biofilm – What is it?

• “Any group of microorganisms in which cells stick to

each other and often these cells adhere to a

surface. These adherent cells are frequently

embedded within a self-produced matrix of

extracellular polymeric substance (EPS).” Wikipedia

• “Van Leeuwenhoek, using his simple microscopes,

first observed microorganisms on tooth surfaces

and can be credited with the discovery of microbial

biofilms.” Rodney Donlan (2002). Biofilms: Microbial

Life on Surfaces. Emerging Infectious Diseases, 8(9),

881-890.

3/11/2017

10

Biofilm – what it is not

• Not the same as surface or “free-floating”

(planktonic) bacteria

• Not typically identified using traditional

culture swab techniques (identifies

mostly planktonic bacteria)

• Not easy to eradicate!

– Exhibits increased tolerance to antimicrobial,

immunological & chemical attack compared to

planktonic bacteria

Biofilms in >80% of Biopsies of Chronic Wounds7

Versus 6% of Acute Wounds

Very likely more prevalent in chronic wounds than we think!7

Panels A & B: G. James, E. Swogger, R. Wolcott, E. Pulcini, P. Secor, J. Sestrich, J. Costerton, P. Stewart. Wound Rep Regen, 16:37-44, 2008

7M. Malone, T. Barjnsholt, A. McBain, G. James, P. Stoodley, D. Leaper, M. Tachi, G. Schultz, T. Swanson, R. Wolcott. Prevalence of biofilms in

chronic wounds: a systematic review and meta-analysis of published data, J wound Care, JWC 2017; 26:20-25.

A B

3/11/2017

11

Characteristics of biofilm in open wounds

• Mostly* unable to see it with naked eye

• Polymicrobial

– Aerobic + non-aerobic bacteria

– gram pos + gram neg

– Fungus + virus

• Hydrophilic polymeric protective coating

• Quorum sensing

• Attached 2mm below wound bed surface

• Grows back in 48 hours

*Hurlow J. Blanz E. Gaddy JA. Clinical investigation of biofilm in non-healing wounds by

high resolution microscopy techniques. J Wound Care. (2016). 25(9). S11-S22.

Scott Dowd, et al. (2008). BioMed Central Microbiology, 8(43). Courtesy of G. Schultz.

Distribution of aerotolerance of bacterial

populations in chronic wounds

Aerobes

Facultative

Anaerobes

Strict

Anaerobes

3/11/2017

12

Why are bacteria in biofilms so difficult to kill?

1. Extracellular polymeric substance (EPS) of

biofilm • Dense matrix impairs diffusion of large antibodies

• EPS materials chemically react (neutralize) microbicides

• Negative charges of polysaccharides and DNA bind

cationic molecules like Ag+, antibiotics, PHMB+

2. Persister bacteria have low metabolic activity• Antibiotics only kill metabolically active bacteria

3. Oxygen diffusion to center of biofilm is limited• Promotes growth of anaerobic bacteria

4. Synergism between different bacteria• MRSA secrete resistance proteins

• Pseudomonas secrete catalase that destroys H2O2

Slide material: Courtesy of G. Schultz, PhD

Biofilm

• When to remove

– When biofilm presence has negative

consequences

– When interferes with wound healing

– When especially virulent (β hemolytic

streptococci)

– To prevent re-growth

• Best ways to remove

– DEBRIDEMENT

– Total kill

3/11/2017

13

Evidence for debridement methods

• Sharp

– Scalpel, scissors, curette

• Enzymatic (collagenase)

– Pros (gentle) Cons (slow)

• Autolytic (exudate/MMPs)

– Pros (gentle) Cons (slow)

• Ultrasonic (low and high frequency)

– With and without forced water

• Mechanical (debriding gauze, wet-to-dry)

– Surfactants (w/wo mechanical wiping)

• Larval/biological – medicinal maggots

Ultrasonic debridement: PA biofilm

3 day old PA biofilm 3 day old PA biofilm after low

frequency non-contact, non-

thermal ultrasound

3/11/2017

14

Gauze debridement of biofilm bacteria on pig skin explants

Slide material: Courtesy of G. Schultz, PhD

Effect of wiping only: total and biofilm PA bacteria

Wiping with gauze only

Yang Q, Larose C, Porta AD, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing can reduce

bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Slide material courtesy of G. Schultz, PhD

3/11/2017

15

Yang Q, Larose C, Porta AD, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound dressing

can reduce bacterial biofilms in a porcine skin explant model. Int Wound J 2016; Slide material courtesy of

G. Schultz, PhD

Wiping + Surfactant

Daily wiping + surfactant gel vs. total & biofilm PA bacteria

Effect of Daily Wiping + Surfactant gel on PA

Bacteria

Yang Q, Larose C, Della Porta AC, Schultz GS, Gibson DJ. A surfactant-based wound

dressing can reduce bacterial biofilms in a porcine skin explant model. Int Wound J

2016; Slide material courtesy of G. Schultz, PhD

3/11/2017

16

©2017 National Pressure Ulcer Advisory Panel | www.npuap.org

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

Before Wiping After Wiping Day 1 Day 2 Day 3

Via

ble

Bacte

ria (

CF

U)

Total Bacteria Biofilm Bacteria

Wiping only time course: A. baumannii

Slide material courtesy of G. Schultz, PhD

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

1.0E+08

Before Wiping After Wiping Day 1 Day 2 Day 3

Via

ble

Bacte

ria (

CF

U)

Total Bacteria Biofilm Bacteria

Wiping + surfactant gel: A. baumannii

Not impressive

Slide material courtesy of G. Schultz, PhD

3/11/2017

17

1.E-01

1.E+00

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

0 24 48 72 96

Hours

Gauze Surfactant Gel

Surfactant Gel with 2% Mupirocin Surfactant Gel with 8.5% Mafenide Acetate

Co

lon

y Fo

rmin

g U

nit

s

Total Acinetobacter baumannii treated withSurfactant gel + antibiotics made daily

LDT Evidence?• Gray, M. (January 01, 2008) Systematic Review14

– Is maggot debridement effective for removal of necrotic tissue from chronic wounds?

– January 1960 to February 2008: 4 studies (pooled n=193);

• 3 studies <60 subjects

– Compared MDT (LDT) to autolytic/other debridement

• pressure ulcers, leg ulcers, burn wounds

– Concluded: “evidence base for the efficacy of maggot debridement therapy (MDT) in the management of necrotic wounds is sparse.”

– “Even though clinical evidence supporting the use of MDT for debridement of wounds is lacking, clinical experience strongly suggests that this technique is an effective and safe method of debridement for selected patients.”

• Increased evidence from 2008 to 2017

– more than 300 studies!

3/11/2017

18

Evidence for larval debridement

Multiple actions of • Lucilia sericata larvae in hard-to-heal wounds: Larval

secretions contain molecules that accelerate wound healing, reduce

chronic inflammation and inhibit bacterial infection.

Cazander, G., Pritchard, D. I., Nigam, Y., Jung, W., & Nibbering, P. H. (– 2013).

Bioessays, 35(12), 1083-1092.

A • randomized controlled trial of larval therapy for the debridement of leg

ulcers: Results of a multicenter, randomized, controlled, open, observer

blind, parallel group study.

Mudge– , E., Price, P., Neal, W., & Harding, K. G. (2014). Wound Repair and

Regeneration, 22, 1, 43-51.

Selective • Antibiofilm Effects of Lucilia sericata Larvae Secretions /

Excretions against Wound Pathogens. Bohova, Jana, Majtan, Juraj,

Majtan– , Viktor, & Takac, Peter. (2014). Hindawi Publishing Corporation.

Antimicrobial • peptides expressed in medicinal maggots of the blow fly

Lucilia sericata show combinatorial activity against bacteria.

– Poppel, A. K., Vogel, H., Wiesner, J., & Vilcinskas, A. (2015). Antimicrobial Agents and

Chemotherapy, 59(5), 2508-14.

LDT – mechanisms of action

• Larval enzymes: protease, collagenase, ammonia, allantoin

and urea, lysozymes20,22,25,26,28,33

• Increase in alkalinity - breaks down necrotic tissues26-28,31,33

• Antimicrobial action of LDT secretions: peptides (diptericins,

lucifensin); chymotrypsin disrupts protein adhesion-

mediated biofilm formation; crude methanol extract21-25, 29-31

• Improved antibiotic effectiveness (re-susceptibility to

antimicrobials observed after LDT)29

• Stimulate fibroblast proliferation and promote fibroblast

motility; may improve angiogenesis (amino acid derivatives),

vascular perfusion, and tissue oxygenation; may reduce

scarring; reduces inflammation20, 26-28, 32, 33

3/11/2017

19

Larval Debridement

PA01 biofilm culture

24 hours after LDT

PA01 biofilm culture

before LDT

Potential antibiofilm strategies

• Prevention – reduce risk factors

• Debridement

• Selecting suitable topical products

• Selecting suitable systemic products

• Combined approaches

– “one-two” punches

– LDT + advanced therapies / skin grafts

– Ultrasonic treatments + antimicrobials

– Surfactant gels + mechanical disruption

3/11/2017

20

What is in that chronic wound bed?Slough, Eschar, Biofilm?

Summary

• Examine – not only with naked eye

• Determine - what is it?

• Address – targeted treatment

• Evaluate treatment effectiveness, wound progress

• Prevent regrowth

Special Thanks

To our US Veterans and their families

Micah Flores, PhD

Gregory Schultz, PhD

Dan Gibson, PhD

Qingping Yang, MS

Josh Yarrow, PhD

Gary Wang, MD, PhD

Randall Wolcott, MD

Cynthia Garvan, PhD

Alessandra Della Porta (UF Student)

Meg Kincaid, BS

Casey Bopp, RN

3/11/2017

21

Questions?

• Contact information:

– linda.cowan@va.gov

– ebpdoc@gmail.com

Specific References

1. Schultz, G. S., Sibbald, R. G., Falanga, V., Ayello, E. A., Dowsett, C.,

Harding, K., Romanelli, M., ... Vanscheidt, W. (2003). Wound bed

preparation: a systematic approach to wound management. Wound Repair

and Regeneration: Official Publication of the Wound Healing Society [and]

the European Tissue Repair Society, 11, 1-28.

2. Trengove, N.J., Stacey, M.C., Macaulley, S., Bennett, N., Gibson, J.,

Burslem, F., Murphy, G., Schultz, G. (1999). Analysis of the acute and

chronic wound environments: the role of proteases and their inhibitors.

Wound Repair and Regeneration, 7, 442-452.

3. Demidova-Rice, T., Hamblin, M.R., Herman, I.M. (2012). Acute and Impaired

Wound Healing: Pathophysiology and Current Methods for Drug Delivery,

Part 1: Normal and Chronic Wounds: Biology, Causes, and Approaches to

Care. Adv Skin Wound Care, 25(7), 304–314.

4. Schultz, G. (2014). Molecular and cellular regulation of wound healing: What

goes wrong when wounds fail to heal or heal too much?. London: Henry

Stewart Talks. http://hstalks.com/lib.php?t=HST186.3834&c=252.

5. Harding, K. G., Moore, K., & Phillips, T. J. (2005). Wound chronicity and

fibroblast senescence - implications for treatment. International Wound

Journal, 2(4), 364-368.

3/11/2017

22

Specific References

Sheehan6. , P., Jones, P., Caselli, A., Giurini, J.M., Veves, A. (2003). Percent

Change in Wound Area of Diabetic Foot Ulcers Over a 4-Week Period Is a

Robust Predictor of Complete Healing in a 12-Week Prospective Trial.

Diabetes Care, 26(6), 1879-1882.

Malone7. , M., Barjnsholt, T., McBain, A.J., James, G.A., Stoodley, P.,

Leaper, D., Tachi, M., Shultz, G., Swanson, T., Wolcott, R.D. (2017). The

prevalence of biofilms in chronic wounds: a systematic review and meta-

analysis of published data. Journal of Wound Care, 25(12), 1-5.

Schultz8. , G. S., Barillo, D. J., Mozingo, D. W., & Chin, G. A. (April 01, 2004).

Wound bed preparation and a brief history of TIME. International Wound

Journal, 1(1), 19-32.

Leaper9. , D. J., Schultz, G., Carville, K., Fletcher, J., Swanson, T., & Drake,

R. (January 01, 2012). Extending the TIME concept: what have we learned

in the past 10 years? International Wound Journal, 9, 1-19.

Granick10. , M., Boykin, J., Gamelli, R., Schultz, G., & Tenenhaus, M.

(January 01, 2006). Toward a common language: surgical wound bed

preparation and debridement. Wound Repair and Regeneration, 14.

Specific References

11. Cowan, L., Phillips, P., Stechmiller, J., Yang, Q., Wolcott, R. & Schultz, G.

(2013). Antibiofilm Strategies and Antiseptics (Chapter 4) in Antiseptics in

surgery: Scientific basis, indications for use, evidence based

recommendations, vacuum instillation therapy; Willy, C., & Alt, V. (editors),

33 tables. Berlin: Lindqvist Book Publ.

12. Cowan, L., Phillips, P., Liesenfeld, B., Mikhaylova, A., Moore, D., Stechmiller,

J., & Schultz, G. (June 01, 2011). Caution: When Combining Topical Wound

Treatments, More Is Not Always Better. Wound Practice & Research: Journal

of the Australian Wound Management Association, 19(2), 60-64.

13. Swanson, T., Hurlow, J., Schultz, G., & Fletcher, J. (2014). Slough: What is

it? How do we manage it? International Wound Infection Institute.

http://www.woundinfection-institute.com/wp-

content/uploads/2014/11/Slough_AWMA_2014.pdf

14. Gray, M. (2008). Systematic Review: Is maggot debridement effective for

removal of necrotic tissue from chronic wounds? Journal of Wound, Ostomy,

and Continence Nursing, 35(4).

3/11/2017

23

Additional Resources & ReferencesWound Bed Preparation:

15. Doughty, D. & McNichol, L. (2016). Wound, Ostomy and Continence Nurses

Society® Core Curriculum: Wound Management 1st Edition. Chapter 2:

Wound healing (Janice Beitz). Lippincott Williams & Wilkins, Philadelphia, PA.

16. Enoch, S., Harding, K. (2003). Wound Bed Preparation: The Science Behind

the Removal of Barriers to Healing. Wounds, 15(7).

http://www.medscape.com/viewarticle/459733_5

17. Moffat, C., Falanga, V, Vowden, P. European Wound Management Association

(EWMA). Position Document: Wound Bed Preparation in Practice. London:

MEP Ltd, 2004. Available at:

http://www.woundsinternational.com/media/issues/87/files/content_49.pdf

Larval Debridement Therapy:

18. Cowan, L. J., Stechmiller, J. K., Phillips, P., Yang, Q., & Schultz, G. (2013).

Chronic Wounds, Biofilms and Use of Medicinal Larvae. Ulcers, 2013, 1, 1-7.

19. Sherman et al. (2013). Chapter 2 (Maggot Therapy) in Biotherapy-History,

Principles, and Practice: A Practical Guide to the Diagnostics and Treatment

of Disease Using Living Organisms, Springer Publishers: Netherlands.

20. Cazander, G., Schreurs, M. W. J., Renwarin, L., Dorresteijn, C., Hamann, D.,

& Jukema, G. N. (2012). Maggot excretions affect the human complement

system. Wound Repair and Regeneration, 20, 6, 879-886.

21. Kawabata, T., Mitsui, H., Yokota, K., Ishino, K., Oguma, K., & Sano, S. (2010).

Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an

infected environment. Medical and Veterinary Entomology, 24, 4, 375-381.

22. Harris, L. G., Nigam, Y., Sawyer, J., Mack, D., & Pritchard, D. I. (2013). Lucilia

sericata chymotrypsin disrupts protein adhesion-mediated staphylococcal biofilm

formation. Applied and Environmental Microbiology, 79, 4, 1393-5.

23. Teh, C. H., Nazni, W. A., Lee, H. L., Fairuz, A., Tan, S. B., & Sofian-Azirun, M.

(2013). Antibacterial activity and physicochemical properties of a crude methanol

extract of the larvae of the blow fly (Lucilia cuprina). Medical and Veterinary

Entomology, 27, 4, 414-420.

24. Bohova, Jana, Majtan, Juraj, Majtan, Viktor, & Takac, Peter. (2014). Selective

Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound

Pathogens. Hindawi Publishing Corporation.

25. Valachova, I., Takac, P., & Majtan, J. (2014). Midgut lysozymes of Lucilia sericata -

new antimicrobials involved in maggot debridement therapy. Insect Molecular

Biology, 23, 6, 779-787

26. Cazander, G., Pritchard, D. I., Nigam, Y., Jung, W., & Nibbering, P. H. (2013).

Multiple actions of Lucilia sericata larvae in hard-to-heal wounds: Larval secretions

contain molecules that accelerate wound healing, reduce chronic inflammation and

inhibit bacterial infection. Bioessays, 35, 12, 1083-1092.

27. Li, P.-N., Li, H., Zhong, L.-X., Sun, Y., Yu, L.-J., Wu, M.-L., Zhang, L.-L., ... Lv, D.-C.

(2015). Molecular events underlying maggot extract promoted rat in vivo and human

in vitro skin wound healing. Wound Repair and Regeneration, 23, 1, 65-73.

3/11/2017

24

Bexfield28. , A., Bond, A. E., Morgan, C., Wagstaff, J., Newton, R. P., Ratcliffe, N. A.,

Dudley, E., ... Nigam, Y. (2010). Amino acid derivatives from Lucilia sericata

excretions/secretions may contribute to the beneficial effects of maggot therapy via

increased angiogenesis. British Journal of Dermatology, 162, 3, 554-562.

Arora, 29. Shuchi, Baptista, Carl, & Lim, Chu. (2011). Maggot metabolites and their

combinatory effects with antibiotic on Staphylococcus aureus. BioMed Central Ltd.

Beasley, W. D., & 30. Hirst, G. (2004). Making a meal of MRSA—the role of biosurgery

in hospital-acquired infection. Journal of Hospital Infection, 56, 1, 6-9.

Bohova31. , Jana, Majtan, Juraj, Majtan, Viktor, & Takac, Peter. (2014). Selective

Antibiofilm Effects of Lucilia sericata Larvae Secretions/Excretions against Wound

Pathogens. Hindawi Publishing Corporation.

Grassberger32. , M. (2013). Biotherapy-- History, principles and practice: A practical

guide to the diagnosis and treatment of disease using living organisms. Dordrecht:

Springer.

Horobin33. , A. J., & University of Nottingham. (2004). Maggots and wound healing:

The effects of Lucilia sericata larval secretions upon interactions between human

dermal fibroblasts and extracellular matrix proteins. Nottingham: University of

Nottingham.

Sibbald34. , R.G., Elliott, J.A., Ayello, E.A., Somayaji, R. (2015). Optimizing the

moisture management tightrope with wound bed preparation 2015. Advances in

Skin & Wound, 28(10), 466-476.

Laurens35. , N., Koolwijk, P., DeMaat, M/P. (2006). Fibrin Structure and wound

healing. Journal of Thrombosis and Haemostasis, 4:932-939.