A Prospective Pilot Study to Evaluate Wound Outcomes and Levels of Serum C-reactive Protein and Interleukin-6 in the Wound Fluid of Patients with Trauma-related Chronic Wounds

If surgical closure of chronic wounds is an option, choosing an appropriate time to definitely close these wounds remains a challenge. Although the underlying mechanisms of nonhealing are not completely understood, serum C-reactive protein (CRP) and interleukin-6 (IL-6) in wound fluid have been found to be markers of the systemic and local inflammation state of chronic wounds. The purpose of this prospective, descriptive pilot study was to evaluate the effect of debridement, systemic antibiotics, and negative pressure wound therapy (NPWT) on the outcomes of trauma-related chronic wounds and changes in local inflammation responses, measured using CRP and IL-6 levels as indicators of cytokine regulation.   Between June 2012 and May 2013, 20 consecutive patients (14 men, six women, mean age 40 [range 17–56] years) with various trauma-related, nonhealing chronic wounds were enrolled in the study after failing to heal for an average of 8.5 (range 6–16) weeks using a protocol of regular debridement and gauze dressings. Before the start of the study, wounds were cultured, and laboratory values for white blood cell count (WBC), neutrophils, and levels of serum CRP and IL-6 in the wound fluid obtained. Wounds were surgically debrided and NPWT (continuous at 125 mm Hg) applied. All patients were prescribed systemic antibiotics, and mean time interval between NPWT dressing changes was 5 (range 3–7) days. During an average mean NPWT treatment time of 13 (range 5–20) days, CRP and IL-6 concentrations decreased from 66.4 mg/L to 10.4 mg/L and 44.1 pg/mL to 8.6 pg/mL, respectively (P <0.001). The presence/absence of bacteria, WBC, and neutrophil counts did not change. No complications were noted, and all wounds were successfully closed using various surgical procedures. In this study, clinical wound improvement and a significant decrease in wound fluid CRP and IL-6 levels were observed. Studies with a larger sample size and a more robust study design may help elucidate the relationship between inflammatory molecules, infection, and healing outcomes.

Chronic wounds, defined as wounds that fail to heal for at least 6 weeks, most commonly include venous ulcers, arterial insufficiency ulcers, pressure ulcers, and diabetic ulcers,¹ as well as soft tissue injuries and trauma surgery incisions. Trauma patients, such as spinal cord injury patients, are at high risk for developing chronic wounds such as pressure ulcers; a systematic review² showed that 25% to 85% of persons with spinal cord injury were at risk for pressure ulcers.   The exact mechanisms impairing wound healing are not fully understood, especially regarding molecular pathogenesis.³ A literature review and prospective study⁴ highlighted the biochemical differences between healing and nonhealing wounds. All wound healing begins with a pro-inflammatory reaction to tissue injury; in chronic wounds, progress stalls in the pro-inflammatory response, which is much greater in nonhealing compared to healing wounds and in chronic more than in acute wounds.⁵ One review⁶ describes the process as follows: in the healing wound, tissue injury is just a single, transient, and limited stimulus for the inflammatory cytokine, followed by a normal sequence of epithelialization, extracellular matrix (ECM) production, angiogenesis, and scar formation; whereas in the nonhealing wound, tissue injury is recurrent, which might prolong and amplify the pro-inflammatory cytokine cascade to impair healing.

Direct closure for a chronic nonhealing wound usually is not possible due to necrosis, suppuration, or tissue defects. A review of the literature⁷ recommends aggressive debridement to remove necrotic tissue, foreign material, and infecting bacteria, after which the wound is treated until filled with healthy and vascularized tissue. Negative pressure wound therapy (NPWT) is an option for this period. A recent systematic review⁸ found NPWT could facilitate wound healing by increasing local blood flow and the formation of granulation tissue in combination with producing mechanical pressure to promote wound closure. Another review⁹ mentioned that although this kind of therapy was used to treat various wound types, the impetus for devising it was difficult-to-treat chronic wounds. This concept was supported by Moues et al¹⁰; in a randomized controlled trial with 54 patients, NPWT appeared to be effective in improving the wound condition of chronic wounds, showing a quick and steady improvement as opposed to acute wounds that showed a more irregular wound healing progress. A recent meta-analysis¹¹ including 10 trials confirmed the benefit of NPWT over standard wound care (gauze dressings) for chronic wounds regarding change of wound size and time to healing.

Choosing an appropriate time to definitely close chronic wounds remains a challenge for clinicians. According to a systematic review,¹² clinicians mainly depend on consistent observation of the changes in area, depth, and granulation rate of the wound over a period of several weeks to predict chronic wound healing. More objective and convenient tools are required. Other studies^13-17 note uncontrolled and sustained inflammatory response could prevent chronic wounds from healing; local and systemic components play important roles. In most of these studies,^13-15,17 serum C-reactive protein (CRP) and interleukin-6 (IL-6) in wound fluid were shown to be sensitive and reliable markers in reflecting the systemic and local inflammation state of chronic wounds and in predicting their healing.

The purpose of this prospective study was to evaluate the effect of debridement, systemic antibiotics, and NPWT on trauma-related chronic wound outcomes and changes in local inflammation responses, measured using the biomarkers CRP and IL-6.

Study population. Inpatients at the investigators’ trauma center with trauma-related chronic wounds (ie, wounds that failed to heal or frequently recurred after traditional gauze dressing change therapy and repeated debridement for at least 6 weeks) needing NPWT were consecutively recruited to this prospective study between June 2012 and May 2013. The Ethical Committee of Tongji Hospital affiliated with Huazhong University of Science and Technology, Wuhan, China, approved the research. Written informed consent was obtained from all patients. Patients with comorbidities that might delay wound healing, including venous/arterial insufficiency, diabetes, and compromised nutritional status, were excluded, consequentially excluding chronic wounds related to their conditions.

Study protocol. Samples of peripheral blood and wound fluid were collected before the initial surgical debridement (resecting necrotic and infected tissues) and application of NPWT, as well as at each dressing change and at the last day of NPWT. White blood cells counts (WBC), neutrophils, and serum concentrations of CRP were measured in blood samples. Subsequently, NPWT (VSD Inc, Wuhan, China) consisting of a foam, semi-occlusive dressing and drainage pipe connected to a negative pressure device was applied. The sterile polyurethane foam was trimmed to fit the wound cavity and drain adequately and then sealed with a transparent drape. Continuous negative topical pressure of 125 mm Hg was applied using vacuum pumps.¹⁸ The foam dressing was changed once to twice a week. NPWT was provided until the wound was filled with granulation tissue.

The canister collecting the exudate from the wound was changed once a day; wound fluid was collected before the dressing change to minimize potential contamination from blood during dressing change. Wound fluid was analyzed for IL-6 level. Tissue biopsy samples were obtained using a scalpel, taking viable tissue from the center of the wound when surface exudate was removed during the initial debridement and at the last day of NPWT. Tissues were tested to identify but not quantify bacteriology culture. As NPWT began, empirical antibiotic therapy targeting the bacteria most commonly found in chronic wounds was provided and regulated according to the culture results and clinical follow-up. All samples were tested by the Central Clinical Laboratory of Tongji Hospital; reference ranges for CRP and IL-6 levels also were derived in this manner (CRP = 0.1–3.0 mg/L; IL-6 = 1.5–7.0 pg/mL).

The endpoint of the trial was reached when the wound was freshly and fully granulated and could be definitively closed by direct suture, split-thickness skin graft, or muscular flap reconstruction. Closure time and type were judged by two surgeons and based on the local condition of the wound and laboratory test results. Patients also were monitored for complications, such as bleeding, fistula, osteomyelitis, or sepsis.

Data collection. Patient age, wound type and etiology for becoming chronic, primary injury mechanism, NPWT course, number of foam dressing changes, and type of wound closure were recorded by the first author; patient anonymity was maintained.

Statistical analysis. Due to nonparametric distribution, all variables were expressed as median and interquartile range (IQR). Nonparametric, one-way repeated measure analysis of variance (ANOVA) was used to determine differences among the various time points. A two-tailed P value <0.05 was considered statistically significant.

A total of 20 patients (14 men, six women, mean age 40 [range 17–56] years) met the study criteria. Surgical incision, pressure ulcer, and injured soft tissue were the three most common wound types in this study in 11 (55%), three (15%), and four patients (20%), respectively. The other two wounds (10%) were a chronic sinus and chronic fistula.   Of the 20 wounds in the study, 11 were caused by trauma (eg, surgery performed to repair primary trauma such as internal fixation of bone fracture). Primary trauma mechanism and wound type and etiology are shown in Table 1 and include infection, foreign bodies, long-term immobility (bedridden), and tuber ischiadicum cysts. Before the study, wounds had remained unhealed for an average of 8.5 (range 6 –16) weeks. Primary closure for these wounds was impractical because of skin defects after the initial sharp debridement or soft tissue conditions necessitating prolonged observation. During the treatment, NPWT dressings were changed twice for 17 patients and once for three. The mean time interval between dressing change was 5 (range 3–7) days and the median duration of NPWT was 13 (range 5–20) days (see Table 1).

Cultured bacteria were present in 15 cases. The most frequently cultured bacteria species were Staphylococcus aureus (15 samples, 53.6%), Escherichia coli (nine 32.1%), Acinetobacter baumanii (seven 25%), and Pseudomonas aeruginosa (one, 3.6%) among 28 samples with positive cultures. Vancomycin, linezolid, meropenem, and aztreonam were the most common antibiotics used. The intravenous therapy lasted for 5 to 7 days and then oral regimen continued as a combined therapy. Nonquantitative bacterial culture was performed in two cases where microbiology results changed from positive to negative by days 11 and 14 of NPWT treatment, respectively. At the end of NPWT, bacteria were still present in 13 (65%) of the wounds (see Table 2).

Before initial debridement and applying NPWT, the median CRP and IL-6 concentrations were 22 and four times higher than the reference range, respectively, which were 66.4 mg/L (range IQR, 41.3–122.9 mg/L) and 44.1 pg/mL (range IQR, 31.9–68.7 pg/mL). Similar trends toward decrease and significant drop off in level of CRP (P <0.001) (see Figure 1a) and IL-6 (P <0.001) (see Figure 1b) were detected over time during the treatment. The median CRP and IL-6 concentrations were 10.4 mg/L and 8.6 pg/mL, respectively, at the last day of NPWT.

Preoperatively, median WBC and neutrophil counts were 7.1 (range IQR, 5.8–9.0) 10⁹/L and 5.3 (range IQR, 3.7–6.8) 10⁹/L, respectively. Elevated values of WBC and neutrophils were observed in three patients, and by the end of the study, two had normal levels, changing from 12.84 to 6.59 10⁹/L and 11.18 to 5.69 10⁹/L, respectively. No statistically significant change was noted for overall counts of WBC and neutrophils (P >0.05) as the trial progressed (see Figure 2).

Once the wound was filled with granulation tissue, wounds were closed using split-thickness skin grafting (five patients), muscular flap reconstruction (two cases), or sutures (11 patients). The remaining two wounds were managed using once-a-day gauze dressing change for 1 week and then sutured. Postoperative complications such as bleeding, fistula formation, osteomyelitis, or sepsis did not occur. Three-month follow-up observation revealed all study wounds remained healed.

Chronic wound care guidelines¹⁹ published in 2006 emphasize that correctly identifying the etiology of chronic wound and factors that might be contributing to poor wound healing was the key to successful wound treatment. Various factors prevented healing of the trauma-related chronic wounds in this study through the same mechanism that acted as a stimulus to trigger persistent chronic inflammatory reaction. Eliminating (through exclusion criteria) the complex comorbidities related to conditions such as vascular problems and diabetes enabled researchers to study the inflammation response in trauma-related chronic wounds.

 The role of IL-6 and CRP. A review of the literature²⁰ indicated that although normal sequence of inflammation events is an important part of the wound healing process, a continuously high level of pro-inflammatory mediators plays a negative role in initiating the proliferation of fibroblasts and keratinocytes necessary for wound closure and remodeling. Additional review²¹ found chronic inflammation was associated with a vicious cycle where inflammatory cells secreted cytokines that, in turn, attracted more inflammatory cells. It appears, at least in part, that wound healing can be mediated by resolving an excessive inflammation state and cytogenic disequilibrium. This may explain why the treatment approach in these trauma-related chronic wounds that did not heal using traditional gauze dressings was successful.

IL-6 is closely associated with normal wound healing process. A literature review²² found IL-6 increased the adhesion of neutrophils in the wound environment to maintain the inflammatory response. A prospective study investigating the healing phase of chronic leg ulcers by Trengove et al²³ revealed IL-6 was released by epidermal keratinocytes and fibroblasts, directly mitigated in turn by IL-6. Additionally, IL-6 was found in experimental study²⁴ to modulate growth factors or their receptors to promote collagen deposition and angiogenesis. Other laboratory studies revealed wound healing in aged human fibroblasts with lowered IL-6 expression²⁵ and in IL-6-deficient transgenic mice.²⁶ However, high IL-6 levels lasting for an extended period of time can have a detrimental impact on wound healing. A prospective report studying biochemical composition of fluid from chronic wounds²⁷ provided evidence that a persistently high level of IL-6 was correlated with foot ulcer pathogenesis through microvascular and inflammatory mechanisms. A prospective study¹⁷ of 45 patients showed elevated IL-6 levels could be a useful diagnostic marker for high bacterial load, polymicrobial infection, or infection with Pseudomonas spp in chronic ulcers. The elevated baseline levels of IL-6 in the wound fluid of patients in the current study indicated these chronic wounds remained inflamed after the failure of moist gauze dressing therapy. Following debridement, initiation of antibiotic treatment, and NPWT, the IL-6 concentration decreased progressively and eventually reached a relatively normal level, and the wound improved to a point where it could be surgically closed.

CRP is secreted by macrophages, adipocytes, and hepatocytes in response to a wide range of acute and chronic inflammatory events. Although CRP mainly indicates systemic inflammatory state, its value in evaluating the state of chronic wounds has recently been recognized. In a prospective investigation¹⁵ involving 60 participants, instead of treating patients directly with major reconstructive surgery that usually caused extensive hospitalization and patient morbidity, investigators chose to use NPWT, followed by surgical closure guided by the plasma CRP level. This strategy was found to be reliable in patients with postoperative deep sternal wound infection. Another prospective study13 focusing on CRP trends following local and free-tissue reconstruction for chronic wounds revealed persistently elevated levels of CRP increased the likelihood of nonhealing or osteomyelitis. Therefore, based on the increased inflammatory state evident in both local and systematic symptoms, patients in the current study were assumed to be at high risk of clinical failure for wound healing. Fortunately, all wounds healed by the end of trial, and serum CRP level decreased markedly, as did levels of IL-6 in wound fluid.

Unlike the ability of CRP and IL-6 to indicate concerns regarding the inflammatory stage, WBC and neutrophil counts were normal at the beginning in most patients and did not change significantly during the trial. This might suggest these laboratory values were less useful in monitoring treatment outcome and guiding wound closure for chronic wounds, a concept supported by other prospective studies.^14,15

Infection. Erythema, tumefaction, pain, suppuration, and increased temperature of the wound are signs of infection. However, these acute responses might not be observed in a long-term, chronically infected wound, as noted in a prospective study evaluating the validity of clinical signs to identify chronic wound infection.²⁸ In addition, coverage of the wound with NPWT foam limits the opportunity for frequent inspection. Thus, more objective and sensitive indicators were needed to monitor chronic infection. Several literature reviews^29-31 emphasized the important role of bacteria and the resulting inflammatory response in the etiology of chronic wounds. Two additional prospective clinical trials^32,33 in which NPWT was used to treat tissue defects in 45 patients and soft tissue infection in 21 patients, respectively, supported that NPWT could decrease bacterial load in the wound with concomitant decline in acute phase reactants release. The data regarding cultured bacterial species in the present study are consistent with the results of two systematic reviews,^30,34 which found chronic wound infection often involved polymicrobial organisms, including S. aureus, Enterobacteriaceae, and anaerobes. Surprisingly, despite sharp decline in both the systemic and local inflammatory cytokines during this study, bacteria still were cultured in more than half of cases. This may be partly explained by the methodology of the bacterial test employed. Nonquantitative bacterial culture used in this study cannot reflect the exact number change in bacterial load during treatment.

Debridement. At the beginning of this study, an initial surgical debridement was performed before the application of NPWT, which was consistent with protocol in previous studies.^14,15,34 The initial debridement may have affected changes in CRP and IL-6 levels; however, even though the wounds of all participants in the study had been previously debrided on several occasions (in addition to gauze dressing use), their wounds still persisted in a chronic inflammatory state and failed to heal. In their 35-participant prospective clinical trial, Song et al³⁵ demonstrated that despite various advantages of NPWT, it should be adopted as a bridge between debridement and definitive closure rather than a replacement for surgical debridement in treating chronic wounds. Judicious debridement is characterized by the appearance of healthy, well-vascularized tissue; a decrease in bacterial contamination; and the absence of all foreign bodies and nonviable tissue.⁷ Moreover, a prospective study³⁶ exploring specific gene expression profile in different regions of nonhealing wounds found debridement also had the benefit of converting chronic wounds to acute ones that were more sensitive to wound-healing stimuli.

The sample size in this study was relatively small and limited to exclude certain comorbidties, and there was no comparison group. Also, all healed wounds demonstrated a significant decline in serum CRP and IL-6 in wound fluid, which raises the question of how these wounds would have progressed if levels of these two markers rose, remained stable, or even changed inconsistently. Furthermore, all patients were started on systemic antibiotics that may have affected wound, serum CRP, and IL-6 outcomes. Because this was a descriptive study, it is not possible to draw firm conclusions about the relationship between inflammatory molecules and infection. Further studies with larger samples and a more robust study design may help elucidate this relationship.

Trauma-related chronic wounds that failed to heal after traditional gauze dressing therapy exhibited symptoms of inflammation as characterized by elevated levels of serum CRP and IL-6 in the wound fluid. Sharp debridement, systemic antibiotics, and use of NPWT led to a favorable wound healing process and successful surgical wound closure. At the same time, it was observed that serum CRP and IL-6 levels decreased significantly. This small pilot study may provide the impetus for larger studies to investigate the relationship between biochemical markers and clinical wound outcome.

Dr. Bai’s efforts were supported by the Ministry of Health Industry Sector Funds (Item Number. 201002014), the National 12th Five-support program — Research and Integrate New Technology Demonstration of Trauma (Item Number. 2012BAI11B00), Hubei Science and Technology Key Project (Item Number. 2013CFA075), and Hubei Province’s Outstanding Medical Academic Leader Program. Dr. Yi received support from the National Nature Science Foundation of China (Grant No. 81271348).

Dr. Liu and Dr. Yang are trauma surgeons; Dr. Li and Dr. Yi are professors in trauma surgery; and Dr. Bai is a professor of trauma surgery and Director, Department of Traumatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Please address correspondence to Jun X. Bai, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hangkong Road 1095, Wuhan, China; email: baixiangjun@hotmail.com.