Secondary Logo

Journal Logo


Understanding Wound Fluid and the Phases of Healing

Hanson, Darlene MS, RN; Langemo, Diane PhD, RN, FAAN; Thompson, Pat MS, RN; Anderson, Julie PhD, RN, CCRC; Hunter, Susan MSN, RN

Author Information

Determining the components in wound fluids, particularly chronic wound fluid, is not an easy task. Through the process of microdialysis1 and other methods, wound fluids are analyzed to determine content for both treatment and research purposes.2 Several recent studies have compared acute wound fluids with chronic wounds to determine differences in how these wounds heal.2-8

Besides microorganisms, what substances are studied and why are they studied? Some investigators have looked at cytokines and growth factors secreted by cell types, such as platelets, macrophages, and fibroblasts,9 while others have examined growth factors from outside the wound,8 such as insulin-like growth factor (IGF-1). Recent promise has also been noted in the area of proteinases and their inhibitors,9 as proteolytic activity in the wound is continually altered during the course of healing. Reactive oxygen species (ROS), a term that encompasses oxygen-free radicals, have also been studied in wound fluids.9

In addition, biochemical markers, such as total protein, lactate, albumin, creatinine, urea, glucose, lactate dehydrogenase concentrations, and metal ions (zinc, copper, and iron), have been measured. Extracellular components, such as fibronectin, hyaluronan, and osteocalcin, have been studied. Collagens also have been examined for their role in wound healing.9

Wound fluid can be collected directly by aspiration with a needle or glass microcapillaries, or indirectly with a wound vacuum device or foam. Differences in collection of the fluid can change the concentration of wound fluid contents. It has been suggested that direct comparisons between different methods of collection may not be possible.2 Timing the collection of wound fluids may also make a difference, as the components of wound fluid could vary according to the phase of wound healing. Standardization in wound fluid collection is recommended.9

Wound Fluid and Healing Phases

How can clinicians simplify and better understand wound fluids? Focusing on the phases of wound healing may help with understanding of the various studies that analyze wound fluid content.

Early in the first phase of the wound healing process, platelets are the key cells responsible for hemostasis. Several researchers have looked at platelet content and platelet-derived growth factors (cytokines) and inflammatory mediators in wound fluids.3,9 Because some growth factors are derived from platelets, researchers have hypothesized that the placement of platelets in the wound bed might have an impact on healing. However, in a recent study, researchers found no significant adjuvant effect on healing when autologous platelets were applied to 15 chronic venous ulcers.10

Drinkwater et al2 reported concerns that fluids in venous ulcers could degrade and inactivate growth factors because the fluids are proteolytic. Trengove et al3 found the proinflammatory cytokines interleukin-1, interleukin-6, and tumor necrosis factor to be present in significantly higher concentrations in nonhealing ulcers than in healing ulcers, and they suggested that healing may be impaired by inflammatory mediators rather than inhibited by a deficiency of growth factors in chronic wounds.

After hemostasis, the inflammatory phase begins. Chronic wounds are thought to be stuck in the inflammatory phase, and this may delay wound healing. During the inflammatory phase, leukocytes and macrophages accumulate for about 4 to 6 days. Bacteria are destroyed by leukocytes, mainly neutrophils. Macrophages do their work a few days later, replacing the leukocytes.

A recent publication4 suggests a possible link between bacterial presence and the release of heparin-binding protein in chronic leg ulcer wound exudate. This link is not found in acute wounds. The observation is important because an earlier study by Gautam et al11 found that heparin-binding protein induced endothelial hyperpermeability and neutrophil efflux.

Granulation tissue is generated during the third, or proliferative, phase of healing. This type of tissue consists of macrophages, fibroblasts, immature collagen, blood vessels, and ground substance. Fibroblasts stimulate the production of collagen in this phase.

In a study related to the proliferative phase of wound healing, Seah et al12 demonstrated that chronic wound fluid inhibits the growth of dermal fibroblasts, at least partly, by decreasing active Ras levels. Ras is a protein that regulates cell growth in eukaryotic cells.13 Although acute wound fluid has previously been demonstrated to stimulate fibroblast and endothelial proliferation, chronic wound fluid inhibits the same activity.

In the fourth phase, epithelialization, keratinocytes migrate from the wound margins, divide, and become contiguous. Matrix metalloproteinases (MMPs), such as collagenase, are critical in helping with this phase; other MMPs are important to healing. The proteins are regulated by a set of inhibitors.

An interesting study related to MMPs by Lateef et al5 reported that all-trans-retinoic acid suppressed MMP activity and increased collagen synthesis in diabetic skin. Collagen gives skin its strength, and elastin its recoil.14 The study by Lateef et al5 suggests that trans-retinoic acid can improve the structure and function of diabetic skin.

The maturation or remodeling phase is the final stage of wound healing. Collagen fibers reorganize in this phase. Fibroblasts, MMPs, and the inhibitors of MMPs play a role in this process, as do some of the growth factors.

Factors in Multiple Phases of Healing

The results of some studies have implications for more than 1 phase of wound healing. For example, researchers have quantified oxidative stress biomarker profiles when looking at wound fluid content.6 Some of these biomarkers include total protein carbonyl content, malondialdehyde content, and the total antioxidant capacities in acute and chronic wound environments. Albumin is a prominent protein oxidized in acute and chronic wounds. Although acute wound fluid had higher protein concentration than chronic wound fluid in the study,6 chronic wound fluid exhibited significantly higher total antioxidant capacities (inhibition of cytochrome c reduction).

The ROS within a wound is an important factor in wound healing. An imbalance in oxidant/antioxidant status leads to an excess of ROS available to cause cellular damage (DNA, lipids, proteins, and cell function), including migration, proliferation, and extracellular matrix synthesis.6,15 Acute wounds are considered to be less inflammatory and more apt to heal than chronic wounds. As a result, they are subjected to less ROS exposure.

The prevalence of chronic wounds in the aging population may partly be related to nonenzymatic antioxidant levels in the tissues and fluids of these patients. A decrease in plasma antioxidants and epidermal and dermal cells is noted in older adults.6

A study by Yeoh-Ellerton and Stacey7 demonstrated major differences in ferritin and transferrin levels in chronic and acute wounds. Chronic wound fluid had a significantly greater ferritin level and a lower transferrin level. A significant reduction in the ferritin level was observed in healing versus nonhealing chronic leg ulcers. The study results suggest that oxidative stress occurs in chronic wounds, and that iron likely contributes to exacerbation of tissue damage and delayed healing.

Further study of wound fluids is needed. Perhaps a better definition of what is in a wound may lead to more products targeted to specific phases of healing and the corresponding components of wound exudate.

Three posters presented at the 2004 Clinical Symposium on Advances in Skin & Wound Care relate to the issue of what is in wound fluid. The following abstracts and results address what is currently known about wound healing and exudate.

The Role of an Oxidized Regenerated Cellulose/Collagen Wound Matrix Dressing in Chronic Wounds of Mixed Etiology

Maeve Curran, PT, and James Osborne, RD, CDE, Mad River Community Hospital Arcata, CA; Christine Herb, RN, CWOCN, Becon Nursing Consultant, Cathedral City, CA; Oscar Paz-Altschul, MD, FACS; Gary Garcia, MD, ABIM; and Katie Esqueda, PTA, Desert Regional Medical Center, Palm Springs, CA

ABSTRACT: Problems often arise in the chemical wound healing cascade with chronic nonhealing wounds. Research has established that nonhealing wounds of various etiologies demonstrate elevated levels of pro-inflammatory cytokines, increased protease activity, and diminished growth factor activity. An oxidized regenerated cellulose-collagen dressing significantly reduced exudates, increased granulation, and enhanced epithelialization, thus promoting advanced healing rate. Wounds were advanced from chronic proliferation phase to remodeling/epithelialization phase of the wound healing cascade. One may conclude that accelerated wound healing was achieved with ORC/collagen via its binding protease activity, and facilitating growth factor activity, resulting in progressive angiogenesis and accelerated wound repair.

Comparison of Different Collagen Preparations Used as an Adjunct with Becaplermin Gel in Chronic Leg Wounds

Stanley Carson, MD, and Carol Ajifu, PT, Fountain Valley Regional Hospital Wound Care Program, Fountain Valley, CA

OBJECTIVES: Improving wound response to becaplermin gel by using collagen and determining in this use if one type of collagen has a greater effect than others used.

ABSTRACT: Comparison of different collagen preparations used as an adjunct with becaplermin gel in chronic leg wounds. It has been noted that using collagen with becaplermin gel appeared efficacious in many chronic wounds. Questions regarding this usage, including the differing, if any, effect of various collagen preparations, remain. To obtain information about differing effects of collagen, we performed a retrospective review of 40 consecutive patients using collagen preparations with becaplermin gel. All patients were diabetic. All wounds were of the lower extremities and not on pressure-bearing areas. Wounds were present for at least 2 months. Patients were comparable in nutrition, HbA1c levels, circulation, and offloading of the affected areas. Patients were previously treated with becaplermin gel for at least 4 weeks without response. Wounds were 9 cm2 to 50 cm2 with an average depth of 5 mm in size. Different collagen preparations (12 SIS, 15 granular collagen, 13 collagen-cellulose) were moistened/applied over becaplermin gel on 40 chronic wounds in 40 patients. Dressings were changed 3 times a week. Maintenance sharp debridement was routinely performed.

At 6 weeks, 10/12 patients healed in the SIS group (average 28 days) 12/15 in the granular collagen group (average 36 days), and 7/13 patients healed in the collagen-cellulose group (average 42 days).

Differences appeared to exist between the different collagen preparations when used with becaplermin in time of healing (SIS shortest), cost of treatment (collagen-cellulose highest), and ease of use (granular collagen and SIS easier).

Improving Hyperbaric Oxygen Outcomes for Diabetic Foot Ulcer Patients Using an Ionic Silver Powder

Judy Lajoie, RNC, CDE, ACHRN, Kingsbrook Jewish Medical Center, Brooklyn, NY

OBJECTIVES: 1. Discuss the role of hyperbaric oxygen as an adjunct to wound healing. 2. Discuss the mechanism of action of ionic silver in reducing bioburden. 3. Explain the synergistic effect of oxygen and silver on chronic wound healing, especially in diabetic ulcers.

ABSTRACT: Hyperbaric oxygen therapy (HBO) has been shown to stimulate and support wound healing for multiple complex conditions. The benefits from HBO can be especially beneficial to ischemic wounds as a result of trauma, diabetes, or radiation. Diabetic ulcers often become chronic wounds, and the treatment course is plagued with infection and delayed closure time. The addition of a silver powder to the wound bed stimulated granulation, decreased wound bed depth, and promoted wound closure.

METHODS: Random case selection. All patients had diabetic foot ulcers and met the CMS classification of a Wagner level 3. The prescribed course of treatment was 5 times a week HBO for 8 weeks. The silver powder was applied once daily after the HBO treatment and once daily on the weekend by the visiting nurses. After week 8, the silver powder was continued daily by the wound care staff, the family, or the visiting nurse.

DISCUSSION: Oxygen drives phases of healing, including collagen maturation, endothelial development, and promotion of granulation tissue especially important for ischemic wounds. Silver is a known antimicrobial agent. In a controlled release ionic formula, it can reduce bioburden and is not cytotoxic.

CONCLUSIONS: Patients and staff reported satisfaction with the product. It was noted that the wounds that received the silver powder all closed within a total of 16 weeks, whereas those that did not receive the silver powder did not close in the 16-week time frame. Delivering the silver in conjunction with HBO had an impact on patient care outcomes. Recommendations were made to adopt, as its new standard of care, inclusion of the silver powder when HBO was initiated for diabetic foot ulcers to reduce infection and odor and increase time to healing.


1. Clough G, Noble M. Microdialysis-a model for studying chronic wounds. Int J Low Extrem Wounds 2003;2:233-9.
2. Drinkwater SL, Smith A, Burnand KG. What can wound fluids tell us about the venous ulcer microenvironment? Int J Low Extrem Wounds 2002;1:184-90.
3. Trengove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen 2000;8(1):13-25.
4. Lundqvist K, Herwald H, Sonesson A, Schmidtchen A. Heparin binding protein is increased in chronic leg ulcer fluid and released from granulocytes by secreted products of Pseudomonas aeruginosa. Thromb Haemost 2004;92:281-7.
5. Lateef H, Stevens MJ, Varani J. All-trans-retinoic acid suppresses matrix metalloproteinase activity and increases collagen synthesis in diabetic human skin in organ culture. Am J Pathol 2004;165:167-74.
6. Moseley R, Hilton JR, Waddington RJ, Harding KG, Stephens P, Thomas DW. Comparison of oxidative stress biomarker profiles between acute and chronic wound environments. Wound Repair Regen 2004;12:419-29.
7. Yeoh-Ellerton S, Stacey MC. Iron and 8-Isoprostane levels in acute and chronic wounds. J Invest Dermatol 2003 October;121:918-25.
8. Wagner S, Coerper S, Fricke J, et al. Comparison of inflammatory and systemic sources of growth factors in acute and chronic human wounds. Wound Repair Regen 2003;11:253-60.
9. Moseley R, Stewart JE, Stephens P, Waddington RJ, Thomas DW. Extracellular matrix metabolites as potential biomarkers of disease activity in wound fluid: lessons learned from other inflammatory diseases? Br J Dermatol 2004;150:401-13.
10. Senet P, Francois-Xavier B, Benbunan M, et al. Randomized trial and local biological effect of autologous platelets used as adjuvant therapy for chronic venous leg ulcers. J Vasc Surg 2003;38:1342-8.
11. Gautam N, Olofsson AM, Herwald H, et al. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med 2001;7:1123-7.
12. Seah CC, Phillips TJ, Howard CE, et al. Chronic wound fluid suppresses proliferation of dermal fibroblasts through a Ras-mediated signaling pathway. J Invest Dermatol 2005;124:466-74.
13. Lowy DR, Willumsen BM. Function and regulation of ras. Annu Rev Biochem 1993;62:851-91.
14. Hess CT, Kirsner RS. Orchestrating wound healing: assessing and preparing the wound bed. Adv Skin Wound Care 2003;16:246-57.
15. Moseley R, Leaver M, Walker M, et al. Comparison of the antioxidant properties of HYAFF-11p75, AQUACEL and hyaluronan towards reactive oxygen species in vitro. Biomaterial 2002;23:2255-64.
© 2005 Lippincott Williams & Wilkins, Inc.