Wound bed preparation (WBP) is a care paradigm that considers the whole patient by treating the cause and patient-centered concerns first, followed by addressing the components of local wound care: debridement, infection/inflammation, and moisture balance (Figure 1).1 The WBP was first introduced in 2000 and has been periodically updated.2–6 In 2011, the WBP Consensus document was formulated from 12 authors, which was then tested with an expert consensus of 47 external wound care key opinion leaders using a modified Delphi technique.6 This article explores the WBP paradigm from bench to bedside and discusses 2 new treatment options that address the inflammation and infection component of WBP: (1) reduction of elevated levels of matrix metalloproteases with an ovine collagen extracellular matrix (ECM) dressing and (2) management of infection and critical colonization with an open polyvinyl foam bound with gentian violet and methylene blue in a nonrelease formulation for surface critical colonization.
©Sibbald et al, 20116
BIOCHEMICAL BASIS OF WOUND HEALING
The term biochemistry encompasses all chemical reactions that occur in living organisms.7 The macroscopic phases of normal wound healing are actually occurring as a result of an ordered, interdependent, and overlapping series of cell-mediated biochemical reactions. After an injury, a unique and specific cascade of biochemical reactions occur in order to heal wounds. Local host cells start the healing process by producing and releasing endogenous chemical substances in the wound base in response to injury. These endogenous chemicals alert all types of cells in the immediate vicinity to change both their behaviors (eg, increased proliferation and migration) and their output or production of chemicals that advance the healing process. The bloodstream augments the process by delivering important exogenous chemicals, such as oxygen, glucose, and amino acids that provide both energy and resources to the cells involved in healing activities.
At the cellular level, platelets form a thrombin clot, neutrophils act as scavengers of debris and dead tissues, macrophages provide growth factors, endothelial cells form new blood vasculature, fibroblasts lay down new collagen, and epithelial cells migrate from the periphery to close the wound. Platelet-derived growth factors help in the early stages, with metalloproteases digesting wound debris, then followed by tissue inhibitors of metalloproteases setting the stage for proliferation of granulation tissue. These processes are aided by growth factors, such as vascular endothelial growth factor, transforming growth factor β, and epidermal growth factor, for example.4
CHRONIC WOUNDS: PERSISTENT, ABNORMAL INFLAMMATION
Chronic wounds represent a failure in the normally ordered sequence of wound healing. This means that the sequence of chemical reactions that typically lead to wound closure has been thrown out of balance or sequence for some reason. The general types of conditions that will affect chemical reactions (either speeding them up or slowing them down) include changes in local pH, changes in temperature, and changes in the amounts of chemical reactants. Analyses of the chemical makeup of a chronic versus an acute wound can be made by sampling the wound fluid.8 Such analyses have demonstrated that the same cells and chemicals are present in both types of wound; however, the relative amounts or ratios of important chemicals, such as growth factors, inflammatory mediators, proteases, and proteins, are very different in the chronic versus the acute wound. Some of the differences in these chemical ratios are related to the presence of bacterial cells in the wound at levels greater than the host’s ability to control. The bacteria interfere with the host cells and the cascade of chemical reactions that should lead to wound closure. The foreign bacterial cells produce their own chemicals that are usually tissue destructive, as well as stimulate host cells to produce more and more inflammatory mediators. The persistent presence of bacteria in the wound can be a stimulus for the persistently high levels of metalloproteases being released from inflammatory cells that digest the normal collagen scaffold in the base of a healing wound.9 The altered chemical environment in the chronic wound can also affect the host cells in negative ways. For example, keratinocytes in the wound environment often become unresponsive to cellular signaling and pile up at the wound edge.10 Other cells such as the fibroblast may become prematurely aged, losing their ability to proliferate and produce collagen, a state known as senescence.11 The result of these changes is abnormal healing and persistent inflammation, necessitating treatment with anti-inflammatory agents, either topically (surface compartment) or systemically (deep and surrounding compartment).
EDGE EFFECT AND AN OVERVIEW OF AN OVINE COLLAGEN EXTRACELLULAR MATRIX DRESSING
Successful wound healing involves using a systematic WBP approach to identify and address the underlying causes of a wound and patient-centered concerns. The practitioner must address all the components of WBP to achieve desired outcomes. These local wound care components are moisture balance, debridement, and infection/inflammation control.6 However, for some wounds deemed as healable, addressing these components may still not achieve wound closure. In these cases, a fourth component must be addressed—edge effect.
Edge effect occurs when epithelium fails to migrate across a firm and level granulation base.12 In these cases, the epidermal edge may have a steep cliff-like appearance, rather than the desired tapered edge of advancing purple-pink epithelium sloping into the mature granulation base. Within the WBP paradigm, edge effect is linked with the infection and inflammation component. Elevated protease levels have been linked with edge effect and failure of wound closure. Research also highlights the importance of the ECM, a crucial component of the dermal layer.13,14
New wound technologies have been developed to address the need for ECM replacement and reduction of protease activity. One example is the use of ovine (sheep) forestomach matrix source. When selecting a tissue source, it is important to ascertain patient wishes, as tissues of human, bovine, and porcine origin may be rejected by people of certain cultural/religion backgrounds and compromise the patient-provider relationship.15,16 The use of an ovine forestomach matrix source helps address patient-centered concerns by respecting the cultural/religious preferences of patients who seek to avoid products derived from human, bovine, or porcine sources.
CHRONIC WOUNDS: INFECTION AND BACTERIAL DAMAGE
Infection results from bacterial damage in and around the wound. The superficial critical colonization is represented by a thin layer of soup in a soup bowl that represents the deep and surrounding infection. Bacteria have their own excreted chemicals (eg, metalloproteases), which in turn disrupt the normal ordered sequence of wound healing.17 This can result in a stalled but ultimately healable wound that can be treated with a topical antimicrobial agent or dressing.
When selecting local wound care treatment, there are 3 components of local wound care to be considered: debridement, infection/inflammation, and moisture balance. Common topical agents often used are antibiotic creams or ointments. However, the use of a topical antibiotic cream or ointment instead of a dressing has a number of disadvantages6,18:
* potential for contact allergic reactions
* narrow antimicrobial spectrum that does not address all the organisms often present in a chronic wound
* does not provide moisture balance
* will not provide autolytic debridement
* 1 mutation of a bacteria may result in antimicrobial resistance.19
CHRONIC WOUNDS: USE OF A MODERN WOUND DRESSING
In contrast to a topical antibiotic cream, the form and function of a modern wound dressing, such as a gentian violet and methylene blue absorbent antibacterial dressing, are ideally suited to addressing the WBP component of critical colonization. The dressing contains methylene blue and gentian violet bound to a polyvinyl alcohol (PVA) foam that features a 3-dimensional open-cell structure with interconnected cells. In vitro, PVA foam absorbs up to 12 times its weight in exudate.20 Coupled with its absorption, the methylene blue/gentian violet–bound foam traps and inhibits exudate-associated bacterial growth (ie, inhibiting bacterial cells’ ability to divide by unfavorably altering the redox or oxidation-reduction potential within the bacterial cell). This inhibition of bacteria may assist the host resistance to minimize further bacterial damage and facilitate the proliferative stage of healing. Gentian violet and methylene blue absorbent antibacterial dressings are available in several forms: foam wafer, packing material, and ostomy ring dressings.
Bacteria in the wound also produce endotoxins and other inflammatory mediators, some of which may be associated with pain. Trapping some of the present bacteria may lower local pain levels and improve healing. A particular advantage of gentian violet and methylene blue absorbent antibacterial dressings is compatibility with enzymatic debriding agents (eg, collagenase) that are not compatible with other antimicrobial dressings including silver, iodine, and other antimicrobial agents.21 The nonrelease formulation of the dressing will also not harm wound base–associated growth factors or endogenously delivered biological agents.22 Color change may occur in the dressing, from deep blue to a lighter blue or white caused by bacterial inhibition and depletion of the methylene blue and gentian violet components, which provides an important visual indicator to guide dressing change.
WBP: TREAT THE CAUSE
As is the case for all chronic wounds, the first WBP recommendation is to treat the cause. For example, in venous leg ulcers, the possibility of coexisting arterial disease should be assessed, with a positive indication being an ankle/brachial pressure index greater than 0.8. Clinical signs of venous disease also must be documented, including varicosities, pitting edema, dark brown pigment, and lipodermatosclerosis. Compression bandages are best suited to heal a venous ulcer with support stockings to prevent a recurrence.
In the case of diabetic foot ulcer, there are 3 specific components to be addressed, represented by the mnemonic VIP: vascular supply, infection, and plantar pressure redistribution. In this case, ankle-brachial pressure index should be greater than 0.65 or less than 1.3. Noncompressible vessels may result in a falsely elevated reading. In these cases, healability will be determined by measuring the pressure in the large toe. Pressures greater than 55 mm Hg or a transcutaneous oxygen saturation greater than 30 mm Hg are required for healing, but lower levels are often associated with delayed healing, for example, levels of transcutaneous oxygen saturation greater than 40 mm Hg for normal healing.
Deep and surrounding infection can be thought of as a soup bowl with the wound base on the inside bottom attached to the sides of the bowl. Four out of the seven STONEES (Figure 2) criteria reflect the sides of the bowl: size increasing, increased temperature, new areas of breakdown, and erythema/edema = cellulitis. The other 3 STONEES criteria reflect the deep component (bottom of the wound): os for probing to bone, exudate, and smell. Deep and surrounding infection should be diagnosed clinically, and although not always necessary, a bacterial swab may be taken to identify potential pathogens along with their antimicrobial sensitivity and resistance patterns. If a wound is deemed healable but stalled despite appropriate WBP, the edge effect component should be considered. This may necessitate advanced active therapies.10 A case series utilizing a gentian violet and methylene blue absorbent antibacterial dressing was performed on lower-extremity chronic wounds with signs of critical colonization and follows this article on page 9.
The clinical mnemonic STONEES defines 7 clinical signs of deep and surrounding wound infection (Figure 2):
aWhen noting increased exudate and smell, clinicians need an additional STONEES criteria for systemic treatment. ©Sibbald, 2009.
* Size: Expanding wounds invades the sides of the wound or develops a deeper base through destruction of the wound base or both.
* Temperature: Evidence shows a 3°F (1.7°C) temperature difference between the wound margin and mirror image skin was 8 times more likely to be associated with deep and surrounding infection. Infrared thermometry is underutilized in clinical practice and should be encouraged. In a study of leg ulcers, temperature was able to identify deep and surrounding infection in 19 of 22 subjects when used as a sole criterion.23
* Os: Latin word for “bone.” Exposed or probing to bone has been documented in previous studies to be a reliable and valid clinical test for osteomyelitis.24,25
* New breakdown: As wounds deteriorate, they expand laterally. Often, small islands of tissue break down in the wound margins, and then they join up to become confluent with the original wound.
* Exudate: The body’s response to injury.
* Erythema and edema: Otherwise known as cellulitis. Although not the most common feature present in chronic wounds, this is an important criterion for the diagnosis of deep and surrounding infection.
* Smell: Usually indicates the invasion of gram-negative and anaerobic organisms after the wound has been primarily compromised with the more virulent gram-positive organisms.
If 3 or more STONEES criteria are present, treatment with a systemic agent should be initiated. Gram-positive coverage may be required if wounds are present for less than 1 month and the patient is immune competent. Broad-spectrum coverage with gram-positive, gram-negative, and anaerobic antimicrobial agents is usually required in the presence of diabetes mellitus or other forms of immune suppression, as well as for wounds with durations of a month or longer.6
Most people with diabetes will develop at least some peripheral neuropathy over time.26,27 The presence of a callus indicates increases in plantar pressure that is undetected because of loss of protective sensation. Blisters are due to surface friction indicating that the foot is moving in relation to footwear. Appropriate deep-toed shoes are indicated for every person with diabetes and neuropathy. When an ulcer develops, the criterion standard for pressure offloading is a contact cast for forefoot ulcers or a removable cast walker made irremovable.28
It is important to address patient-centered concerns. One of the most common and pressing concerns is pain. A numerical rating scale (such as 0–10) can be used to assess patient pain. When doing so, it is important to anchor the patient response; for example, tell the patient that a level “10” pain rating is equivalent to slamming a car door on a finger and that a level “5” is equivalent to a bee sting. Most individuals can tolerate up to a level “3” pain. Higher pain levels usually require treatment to effectively perform activities of daily living. If pain is stimulus dependent, it is often described as gnawing, aching, tender, or throbbing (nociceptive pain). The World Health Organization pain ladder for nociceptive pain includes acetaminophen (especially for individuals aged ≥65 years), nonsteroidal anti-inflammatory drugs, and narcotics.29 Pain that is not stimulus dependent or spontaneous is often described as burning, stinging, shooting, and stabbing (a sign of neuropathic pain). In these cases, treatment is best accomplished with low-dose tricyclic antidepressants, gabapentin, pregabalin, or carbamazepine.30,31 Smoking and smoking cessation are another patient-centered concern of tremendous importance to wound healing, in particular, vascular impairment.32 There is also the problem of accessing care, particularly for individuals living in rural or remote areas and individuals with low income.
HEALABILITY, LOCAL WOUND CARE: DEBRIDEMENT, MOISTURE BALANCE
Once the cause of the wound has been corrected, the patient-centered concerns have been addressed, and there is adequate blood supply to heal, this is termed a healable wound. However, not all wounds are healable. The terms maintenance wound and nonhealable wound define conditions that will result in delayed healing or even further deterioration. A maintenance wound occurs when there is adequate blood supply, but 1 of 2 conditions exists: (1) the patient refuses to adhere to the treatment outlined; for example, he/she will not wear compression bandages for venous disease; (2) the healthcare system does not cover a needed component of care, such as plantar pressure redistribution devices for the treatment of diabetic neuropathic foot ulcers.
A nonhealable wound by contrast does not have adequate blood supply to heal, often from distal disease that cannot be bypassed or dilated, or when the patient is in a negative protein balance due to overwhelming disease, such as a terminal cancer.
Debridement is the removal of necrotic debris or devitalized tissue. Conservative surgical debridement is the preferred practice for the nonhealable maintenance wound. Conservative surgical debridement is the removal of slough, but not vital tissue and should not induce postprocedure bleeding. Other debridement methods include autolytic methods with dressings (such as calcium alginates, hydrogels, and hydrocolloids), mechanical debridement, enzymatic (collagenase), and biologic (maggots). The wet-to-dry method of mechanical debridement is discouraged because of painful disruption of granulation tissue, but newer ultrasound methods do have some clinical utility. Sharp surgical debridement should be reserved for wounds with the ability to heal. Debridement contraindications include uncontrolled pain, greater risk of bleeding, and inadequate skill of the available healthcare professionals.
LOCAL WOUND CARE
Local wound care starts with documentation. It is important to record the location and the longest length and widest width of the wound at right angles. The characteristics of the base, edge, exudate, and odor must also be recorded.
Moisture balance is another important consideration since the classic papers of Winter33 with animal data and Hinman and Maibach34 with human subjects. Moisture balance is accomplished with dressings that create a moist wound surface for a healable wound. Modern superabsorbent dressings are similar to diapers in that they wick moisture away by capillary action, while also locking away fluid. A foam dressing wicks fluid away from the wound and, depending on the pore structure, also transfers fluid back when there is an accumulation of more fluid in the dressing than on the wound surface. Calcium alginate dressings are bioreabsorbable through the conversion of the fibers to a hydrogel by donating calcium and binding sodium. The donated calcium will decrease surface blood and prevent crust formation, which can act as a proinflammatory stimulus. Hydrogels donate water and provide a viscous format that facilitates adhesion to the wound surface. Hydrocolloid wafers will absorb some moisture (due to a carboxyl terminal) but also have a hydrophobic terminal (methylcellulose). These hydrocolloid dressings also contain gelatin, pectin, and an adhesive on a backing that is usually polyurethane. Films are protective dressings that can absorb a small amount of water through the moisture vapor transmission rate of the dressings (for wounds usually around a figure of 750 g/m2/24 hr) and facilitate less moisture transfer than dressings designed for intravenous sites that may have a much higher evaporation rate with a moisture vapor transmission rate closer to 3000 g/m2/24 hr.
NERDS CRITERIA FOR CRITICAL COLONIZATION AND SUPERFICIAL WOUND INFECTION TREATMENT
All chronic wounds are contaminated and become colonized when bacteria attach to the tissue and begin to multiply. Critical colonization occurs when bacteria damage tissue and delay or prevent healing. As mentioned previously, this is similar to a thin layer of soup in the soup bowl. The diagnosis of critical colonization is made clinically based on 3 or more of the 5 NERDS criteria (Figure 3):
aWhen noting increased exudate and smell, clinicians need an additional criterion for the NERDS criteria for topical treatment. ©Sibbald, 2009.
* Nonhealing: Wound is not 30% smaller over the last 4 weeks.
* Exudate: Body’s response to injury.
* Red friable granulation: Due to bacterial stimulation of vascular endothelial growth factor, an excess of blood vessels forms over collagen, resulting in red friable bleeding tissue upon dressing removal.
* Debris: Dead cells on the surface of the wound represented by yellow or brown-tinged slough.
* Smell: Represents gram-negative and anaerobic tissue damage.
TREATMENT OF CRITICAL COLONIZATION
Treatment of critical colonization requires combining the antimicrobial agent(s) with moisture balance and, when required, autolytic debridement. Currently available topical antimicrobial agents include:
* silver, combined with foams, alginates, contact layers, hydrogels, and other forms of dressings with a potential for anti-inflammatory action;
* iodine, in a cadexomer molecule for absorption and autolytic debridement along with a slow-release formulation in a polyethylene glycol mesh available in the United Kingdom, Canada, and Australia;
* polyhexamethylene biguanide, a chlorhexidine derivative in a nonrelease foam or gauze/packing ribbon formulation;
* honey (Manuka medical grade), combined with alginates or a hydrogel (honey has formal regulatory approval as an antibacterial dressing in some countries, including Canada);
* methylene blue and gentian violet bound to a polyvinyl foam.
One of the ways in which bacteria become resistant to antimicrobial treatment is to form a biofilm. Planktonic bacteria adhere to a surface and by using quorum sensing begin to form a biofilm by secreting a glycocalyx. This biofilm then protects the bacteria within. This community of bacteria has some organisms in the resting state, which may not be susceptible to antimicrobials, whereas other organisms are periodically released from the biofilm. These free or planktonic-state organisms can then create new infections.35 Biofilm formation can also stimulate a host inflammatory response.
Inflammation is itself an important component of WBP. Inflamed wounds may have coexisting systemic diseases. They may also exhibit unique clinical characteristics, including palpable purpura, livedo patterns, focal necrosis areas, and satellite areas of tissue breakdown. Erythema is localized, and wound pain is constant with the onset of new lesions. In the inflamed chronic wound, periwound skin temperature is normally not elevated.
Diagnostic tests for wounds with suspected underlying systemic inflammatory disease (eg, vasculitis or pyoderma gangrenosum) often focus on biopsies, patch testing for contact allergy, and investigations for autoimmune disease. However, increasing evidence has suggested that there are components within the biochemical environment of the wound that may have a profound impact on healing. Proteases are enzymes that are involved in physiologic reactions that can be either beneficial, such as in digestion, or detrimental, such as the destruction of ECM. Elevated levels of certain proteases have been associated with reduced levels of growth factors and destruction of the ECM.36
Modern topical wound dressings incorporate technologies to reduce abnormally elevated levels of matrix metalloproteases. This in turn decreases superficial wound inflammation and may allow the wound to heal where it would otherwise be impossible. One example of this is a recently developed ovine collagen ECM dressing that features a decellularized ECM component and effects broad-spectrum matrix metalloprotease reduction.37
Other advanced or active therapies that have the potential to heal a stalled chronic wound include biological agents, such as growth factors, skin substitutes (cellular/acellular); complementary treatments, such as electrical stimulation or ultrasound; hyperbaric oxygen therapy; and negative-pressure wound therapy.
Selection of advanced therapies should be based on a matching between the wound needs and the precise function of the advanced therapy. This is similar to the choice of an antimicrobial dressing that requires a similar form and function relationship within the evidence informed framework of WBP.
Wound bed preparation continues to be a holistic approach to persons with wounds including the treatment of the cause, patient-centered concerns and the 3 components of local wound care (debridement, infection/inflammation, and moisture balance) along with the edge effect. The authors have incorporated the role of a methylene blue and gentian violet foam dressing for critical colonization and moisture balance along with ovine extracellular matrix dressing for the edge effect to manage stalled but healable wounds.
* Wound bed preparation is a whole-patient paradigm where providers must address the cause of the wound and patient-centered concerns before moving onto local wound care.
* There are 4 components to local wound care: debridement, moisture balance, infection and inflammation, sometimes followed by the related edge effect.
* Utilize the NERDS criteria for superficial critical colonization and the STONEES criteria for deep and surrounding infection.
* There is evidence that demonstrates a positive response to the gentian violet and methylene blue absorbent antibacterial dressing for challenging and stalled lower extremity chronic wounds. Further research is required to support this result.
* Some advanced topical wound dressings incorporate technologies that provide broad-spectrum matrix metalloprotease reduction, such as an ovine collagen ECM dressing.
* Consider matching the form and function of the dressing to the wound’s condition.
1. Sibbald R, Williamson D, Orsted H, et al. Preparing the wound bed: debridement, bacterial balance and moisture balance. Ostomy Wound Manage 2000; 46 (11): 14–22,24-8,30-5.
2. Falanga V. Classifications for wound bed preparation and stimulation of chronic wounds. Wound Repair Regen 2000; 8: 347–52.
3. Sibbald RG, Orsted H, Schultz GS, Coutts P, Keast D. Preparing the wound bed 2003: focus on infection and inflammation. Ostomy Wound Manage 2003; 49 (11): 24–51.
4. Schultz GS, Sibbald RG, Falanga V, et al. Wound bed preparation: a systematic approach to wound management. Wound Repair Regen 2003; 11 (Suppl 1): S1–28.
5. Sibbald RG, Orsted HL, Coutts PM, Keast DH. Best practice recommendations for preparing the wound bed: update 2006. Adv Skin Wound Care 2007; 20: 390–405.
6. Sibbald RG, Goodman L, Woo KY, et al. Special considerations in wound bed preparation 2011: an update. Adv Skin Wound Care 2011; 24: 415–36.
8. Trengove N, Stacey M, McGechie D, Mata S. Qualitative bacteriology and leg ulcer healing. J Wound Care 1996; 5: 277–80.
9. Armstrong DG, Jude EB. The role of matrix metalloproteinases in wound healing. J Am Podiatr Med Assoc 2002; 92 (1): 12–8.
10. Tomic-Canic M, Ayello EA, Stojadinovic O, Golinko MS, Brem H. Using gene transcription patterns (bar coding scans) to guide wound debridement and healing. Adv Skin Wound Care 2008; 21: 487–92.
11. Telgenhoff D, Shroot B. Cellular senescence mechanisms in chronic wound healing. Cell Death Differ 2005; 12: 695–8.
12. Woo K, Ayello EA, Sibbald RG. The edge effect: current therapeutic options to advance the wound edge. Adv Skin Wound Care 2007; 20: 99–117.
13. Schultz GS, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen 2009; 17: 153–62.
15. Enoch S. Informed consent should be obtained from patients to use products (skin substitutes) and dressings containing biological material. J Med Ethics 2005; 31 (1): 2–6.
16. Easterbrook C, Maddern G. Porcine and bovine surgical products: Jewish, Muslim, and Hindu perspectives. Arch Surg 2008; 143: 366.
17. McCarty SM, Cochrane CA, Clegg PD, Percival SL. The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing. Wound Repair Regen 2012; 20: 125–36.
18. Smart V, Alavi A, Coutts P, et al. Contact allergens in persons with leg ulcers: a Canadian study in contact sensitization. Int J Low Extrem Wounds 2008; 7: 120–5.
19. Lipsky BA, Hoey C. Topical antimicrobial therapy for treating chronic wounds. Clin Infect Dis 2009; 49: 1541–9.
20. Hollister Inc. Hydrofera Blue Antimicrobial Dressings Monograph. Hollister Inc. 2013.
21. Shi L, Ermis R, Kiedaisch B, Carson D. The effect of various wound dressings on the activity of debriding enzymes. Adv Skin Wound Care 2010; 23: 456–62.
22. Ramsay S, Jelf C, Aust D, Roche E. Comparison of the effects of antimicrobial wound dressings on cell viability, proliferation, and growth factor activity. Poster Presentation. Toronto–World Union of Wound Healing Societies Congress; June 4-8; 2008.
23. Fierheller M, Sibbald RG. A clinical investigation into the relationship between increased periwound skin temperature and local wound infection in patients with chronic leg ulcers. Adv Skin Wound Care 2010; 23: 369–79.
24. Grayson M, Gibbons G, Habacher W, et al. Use of ampicillin/sulbactam versus imipenem/cilastatin in the treatment of limb-threatening foot infections in diabetic patients. Clin Infect Dis 1994; 18: 683–98.
25. Lavery LA, Armstrong DG, Peters EJG, Lipsky BA. Probe-to-bone test for diagnosing diabetic foot osteomyelitis: reliable or relic? Diabetes Care 2007; 30: 270–4.
26. Low PA, Benrud-Larson LM, Sletten DM, et al. Autonomic symptoms and diabetic neuropathy: a population-based study. Diabetes Care 2004; 27: 2942–7.
27. Park TS, Park JH, Baek HS. Can diabetic neuropathy be prevented? Diabetes Res Clin Pract 2004; 66 (Suppl 1): S53–56.
28. Wu SC, Jensen JL, Weber AK, Robinson DE, Armstrong DG. Use of pressure offloading devices in diabetic foot ulcers: do we practice what we preach? Diabetes Care 2008; 31: 2118–9.
30. Saarto T, Wiffen PJ. Antidepressants for neuropathic pain. In: Saarto T, ed. The Cochrane Collaboration. Cochrane Database Syst Rev [Internet]. Chichester, UK: John Wiley & Sons, Ltd; 2007. http://doi.wiley.com/10.1002/14651858.CD005454.pub2
. Last accessed December 30, 2013.
31. Huizinga MM, Peltier A. Painful diabetic neuropathy: a management-centered review. Clin Diabetes 2007; 25 (1): 6–15.
32. McRobert J. Smoking and its effects on the healing process of chronic wounds. Br J Community Nurs 2013; (Suppl): S20–3.
33. Winter G. Formation of the scab and the rate of epithelisation of superficial wounds in the skin of young domestic pig. Nature 1962; 193: 293–4.
34. Hinman C, Maibach H. Effect of air exposure and occlusion on experimental human skin wounds. Nature 1963; 200: 377–8.
35. Bjarnsholt T. The role of bacterial biofilms in chronic infections. APMIS Suppl 2013; 136: 1–51.
36. Wiegand C, Schönfelder U, Abel M, Ruth P, Kaatz M, Hipler UC. Protease and pro-inflammatory cytokine concentrations are elevated in chronic compared to acute wounds and can be modulated by collagen type I in vitro. Arch Dermatol Res 2009; 302: 419–28.
37. Negron L, Lun S, May BC. Ovine forestomach matrix biomaterial is a broad spectrum inhibitor of matrix metalloproteinases and neutrophil elastase. Int Wound J 2012 Oct; 1–8.